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Improvement of platelet quality in stored platelet concentrates by inhibition of complement activation Chahal, Sarabjit 2000

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I M P R O V E M E N T OF P L A T E L E T Q U A L I T Y IN STORED P L A T E L E T CONCENTRATES B Y INHIBITION OF C O M P L E M E N T A C T I V A T I O N  by SARABJIT  CHAHAL  M . S c , Punjabi University Patiala, India, 1993 A THESIS SUBMITTED IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF  M A S T E R OF SCIENCE in THE F A C U L T Y OF G R A D U A T E STUDIES (Department of Pathology and Laboratory Medicine) We accept this thesis 3$^6jiforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A March 2000  © Sarabjit Chahal  In  presenting this  degree at the  thesis  in  University  of  partial  fulfilment  British Columbia,  of  the  requirements  I agree that the  for  an  advanced  Library shall make it  freely available for reference and study. I further agree that permission for extensive copying of  this thesis for scholarly purposes may be granted by the  department  or  by  his  or  her  representatives.  It  is  understood  that  publication of this thesis for financial gain shall not be allowed without permission.  Department of The University of British Columbia Vancouver, Canada  DE-6 (2/88)  head of my copying  or  my written  ABSTRACT The storage of platelets under routine blood bank storage conditions leads to a loss of viability and responsiveness known as platelet storage lesion. Potential causes, which may produce the storage lesion, include increased metabolism of activated platelets, activation of blood enzyme pathways by contact with the polymer surfaces of blood collection packs, and aging of platelets. Complement activation occurs in platelet concentrates (PCs) during collection, processing and storage, which may contribute to the platelet storage lesion; however, no direct evidence supports this hypothesis. If complement contributes to the storage lesion then the inclusion of specific complement inhibitors in PCs should reduce the extent of the platelet storage lesion. The project was designed to determine whether the addition of a complement inhibitor prevents complement activation in PCs, whether that complement inhibition is related to an improvement or deterioration in the quality of stored platelets and thirdly, whether the inhibitor remains active throughout the 5-day storage period of PCs. Concentrates were split into mini-units and treated with the specific complement inhibitors N-acetyl-aspartyl-glutamic acid ( N A A G A ) or N A A G A - N a , E D T A or buffer. Samples were collected on day 0, 1, 3 and 5 or alternatively, daily during storage. Complement activation was monitored by measuring C3a and SC5b-9 levels. Platelet quality was assessed by morphology, hypotonic shock response (HSR), and platelet activation marker expression. Stability of the inhibitor was determined by monitoring its residual complement inhibitory activity in the bag over the storage period using a modified hemolytic assay.  ii  N A A G A treatment in the first study demonstrated a decreased C3a generation and delayed generation of membrane attack complex, C5b-9 (MAC) (p<0.05). By morphology and HSR, complement inhibition by N A A G A improved the platelet quality (p<0.05). N A A G A slowed the rate of platelet activation, but did not prevent it. N A A G A - N a treatment in the second study also decreased C3a levels and delayed M A C generation (p<0.05). However, by morphology scores and HSR, there was no difference in the two treatments. N A A G A - N a was stable throughout the storage period as determined by the modified hemolytic assay (p<0.05). The first study provides direct evidence that complement activation contributes to the platelet storage lesion; however, complement activation is clearly not the sole cause of the storage lesion. Although in the second study complement inhibition was not related to an improvement of platelet quality, we cannot exclude the possibility that complement is one of the likely candidates that may have an impact on the extended storage of PCs.  iii  TABLE OF CONTENTS Abstract  iI  Table of Contents  iv  List of Tables  viii  List of Figures  ix  Abbreviations  xi  Acknowledgements  xiv  Dedication  xv  CHAPTER 1 INTRODUCTION  1  1.1 Platelets  2  1.1.1  Platelet Structure  2  1.1.2  Role of Platelets in Hemostasis  4  1.2 Platelet Storage Lesion 1.2.1  5  Factors Influencing the Generation of Platelet Storage Lesion  5  1.2.1.1 Effect of Preparative Technique  5  1.2.1.2 Effect of Storage Temperature  7  1.2.1.3 Effect ofpH  8 iv  1.2.1.4 Effect of Residual WBCs  9  1.2.2  Evidence of Platelet Activation in Stored PCs  10  1.2.3  Potential Mechanisms of Platelet Activation  12  1.2.4  Interaction between Complement and Platelets  15  1.2.4.1 Complement Activation  15  1.2.4.2 Complement and Platelet Storage Lesion  18  1.3 Rationale and Objectives  C H A P T E R 2 M A T E R I A L S AND M E T H O D S  19  21  2.1 Materials  21  2.2 Preparation of Reagents for Experiments  23  2.2.1  Preparation of Normal Human Serum and Heat Inactivated Serum  23  2.2.2  Preparation of Platelet Poor Plasma and Heat Defibrinogenated  23  2.2.3  Preparation of PCs  24  2.3 Study Design  26  2.3.1  Study 1  26  2.3.2  Study 2  28  2.4 Techniques  29  2.4.1  Assessment of Complement Activation  29  2.4.2  Hypotonic Shock Response Measurements to Assess the Responsiveness of  Platelets  30  2.4.3  31  Oil Phase Contrast Microscopy to Examine Platelet Morphology  2.4.4  Fluorescence Flow Cytometry to Assess Platelet Activation State and Levels  of Platelet Microparticles  31  2.4.5  34  Functional Complement Assay  2.4.5.1 Comparison of N A A G A and N A A G A - N a  34  2.4.5.2 Titration of N A A G A and N A A G A - N a  35  2.4.5.3 Effect of C a  35  2+  Concentration on the Activation of Complement  2.4.5.4 Determination of Stability of Complement Inhibitor in PC CHAPTER 3 RESULTS  36 37  3.1 Study 1  37  3.1.1  Complement Inhibition by N A A G A  37  3.1.2  Effect of N A A G A on the Quality of Stored PCs  39  3.1.3  Improvement of Platelet Morphology in Stored PCs Following Incubation  With N A A G A  40  3.1.4  Improvement of HSR in Stored PCs Following Incubation With N A A G A . 41  3.1.5  Effect of N A A G A on the Expression of Platelet Activation Markers  3.2 Study 2,. 3.2.1  42 45  Stability of N A A G A - N a in PCs  45  3.2.1.1 Comparison of N A A G A and N A A G A - N a  45  3.2.1.2 Effect of C a  48  2+  Concentration on the Activation of Complement  3.2.1.3 Measurement of Residual Activity of N A A G A - N a  50  3.2.2  Complement Inhibition by N A A G A - N a  50  3.2.3  Effect of N A A G A - N a on the Quality of Stored PCs  53  vi  C H A P T E R 4 DISCUSSION  60  Bibliography  70  vii  List of Tables Table 1. Functional Properties of Platelet Membrane Glycoprotein  3  Table 2. Constituents of Alpha and Dense Granules of Platelets  3  Table 3. Change of platelet size during storage of PCs as measured by M P V (fL)  39  Table 4. GPIb-IX on platelets during storage detected by monoclonal anti-GPIb-IX and flow cytometry  44  Table 5. Change of platelet size during storage of PCs as measured by M P V (fL)  viii  54  List of Figures Figure 1. Activation pathways of complement  17  Figure 2. Methodology used to prepare PCs in two studies  25  Figure 3. Experimental design used in study 1 and study 2  27  Figure 4. The effect of treatment with N A A G A on complement activation in stored PCs. 38 Figure 5. Platelet morphology scores of concentrates stored in the presence of N A A G A . 41 Figure 6. Effect of N A A G A on the platelet hypotonic shock response  42  Figure 7. The effect of N A A G A on the expression of platelet activation markers CD62 and CD63 on platelets  43  Figure 8. Comparison of complement inhibitory activity of N A A G A and NAAGA-Na.46 Figure 9. Titration curve of N A A G A  47  Figure 10. Titration curve of N A A G A - N a  48  Figure 11. The effect of C a  49  2+  concentration on the activation of complement  Figure 12. Measurement of the residual activity of N A A G A - N a over the storage period. 51 Figure 13. The effect of treatment with N A A G A - N a on complement activation in stored PCs  52  Figure 14. Effect of N A A G A - N a on the platelet hypotonic shock response  55  Figure 15. Effect of N A A G A - N a on platelet morphology  56  Figure 16. Microparticle generation in PCs treated with N A A G A - N a  57  ix  Figure 17. Expression of platelet activation markers CD62, CD63, GPIIb-IIIa and the phospholipid PS on platelets collected from PCs treated with or without N A A G A Na  x  Abbreviations ADP  adenosine diphosphatate  ATP  adenosine 5'-triphosphate  P-TG  pMhromboglobulin  CP2D  citrate phosphate double dextrose  DTS  dense tubular system  EA  antibody-sensitized sheep red blood cells  EDTA  ethylene diaminetetraacetic acid  ELISA  enzyme-linked immunosorbent assay  FITC  fluorescein isothiocynate  FL2  red fluorescence channel  FPA  fibrinopeptide A  FS  forward scatter  GMP-140  (CD62)-alpha-granule membrane protein-140, P-selectin  GP  glycoprotein  GPRP  glycyl-L-prolyl-L-arginyl-L-proline  HDP  heat defibrinogenated plasma  HEPES  N-2-hydroxyethyl Piperazine-N-2-ethane sulphonic acid  HIS  heat inactivated serum  HSR  hypotonic shock response  IL  interleukin  MAC  membrane attack complex, C5b-9  MPV  mean platelet volume xi  NAAGA  N-acetyl-aspartyl-glutamic acid  N A A G A - M g magnesium salt of N A A G A NAAGA-Na  sodium salt of N A A G A  NHP  normal human plasma  NHS  normal human serum  OCS  surface-connected open canalicular system  PBS  phosphate-buffered saline  PC(s)  random-donor platelet concentrate(s)  PDGF  platelet derived growth factor  PE  phycoerythrin  PF4  platelet factor 4  PGE1  prostaglandin E l  PPP  platelet-poor plasma  PRP  platelet-rich plasma  PS  phosphatidyl serine  PVC-TOTM  polyvinyl chloride containing tri (2-ethyl-hexyl) trimellitate plasticizer  SC5b-9  soluble lytically inactive stable complex of C5b-9  SD  standard deviation  SS  side scatter  TGF-P  transforming growth factor-P  TNF  tumour necrosis factor  TXA2  thromboxane A2  xn  VBS  veronal buffered saline, (1.8 m M sodium barbitone, 1.56 m M sodium barbituric acid, 145 m M NaCl, pH 7.4)  VBS  + +  V B S , 0.15 m M CaCl , 0.5 m M M g C l , pH 7.2 2  vWF  von Willebrand factor  WBC(s)  white blood cell(s)  2  xm  Acknowledgements I would like to thank every single member of the Devine lab: Derek Sim, Katherine Serrano, Maria Issa, Vicky Monsalve, Nadine Brockmann, Elisabeth Maurer, Elena Levin, and Mitra Samiei for their support, useful discussions, and friendship throughout this project. Special thanks to Dr. Elena Levin and Dr. Maryam Nikhabat Sangari for helping me with the flow cytometry. I would also like to thank Dr. Elisabeth Maurer for the time she devoted for platelet morphology studies. Thanks to my committee members for their input to the thesis. I would like to thank Dr. Cedric J. Carter for the statistical advice. Sincere thanks to my supervisor, Dr. Dana V . Devine, for her excellent supervision and encouragement throughout this project. I would also like to acknowledge Dr. Jacques Luyckx (Transphyto-Industrie Pharmaceutique, Clermond-Ferrand, France) for providing the sodium salt of N A A G A for the completion of this project. Thanks to Jim Sibley for procuring a computer for me on which I was able to write my thesis at home and saved an enormous amount of travelling time. Thanks to the Canadian Blood Services and the generous volunteer donors for providing platelet concentrates for this project. In addition, many thanks to my family members for their support and encouragement throughout my project. M y special and sincere thanks to my beloved husband, Harjinder Chahal, for his understanding, patience, support, and encouragement that helped me to survive through the hard times during my project.  xiv  Dedication  To my beloved Dad.  xv  CHAPTER 1  INTRODUCTION  Under routine blood bank storage conditions the storage of platelets at 22 °C for 5 days causes a progressive loss of platelet function and integrity that makes this blood product less efficient for transfusion. A number of potential causes of the platelet storage lesion have been proposed but the exact processes contributing to the storage lesion and the mediators of platelet activation have not been clearly defined. Complement becomes activated in platelet concentrates (PCs) during the earliest stages of blood collection and continues during storage. Since complement is known to have a negative effect on platelets, this may play a major role in the generation of storage lesion. Two independent studies were performed to study whether complement contributes to the storage lesion. The first study reported in this thesis was performed on non-leukoreduced PCs and used complement inhibitor N A A G A at 5 m M final concentration. As the national standards for PC preparation changed in January 1998 in Canada to prestorage leukoreduction, PCs for the second study were prepared using an in-line leukoreduction filtration system. The second study was performed on leukoreduced PCs and used complement inhibitor N A A G A - N a at 10 m M final concentration.  1  1.1 Platelets  1.1.1  Platelet Structure Circulating blood platelets are membrane-encapsulated cytoplasmic fragments of  bone marrow cells called the megakaryocytes. In vivo, the unstimulated platelet is a disc shaped cell with a diameter of 2-3 jam. The platelet plasma membrane is characterized by extensive invaginations into the platelet cytoplasm, that form the surface-connected open canalicular system (OCS). Canals of OCS serve as conduits through which granular components of the platelet escape into the surrounding plasma during the release reaction. The second membrane system in platelets is the dense tubular system (DTS), a modified endoplasmic reticulum that stores calcium and is the site for cyclooxygenase activity. Below the membrane actomyosin microfilaments are present that cause the platelet to contract. The discoid shape of a resting platelet is maintained by a circumferential band of microtubules, which is present just below the platelet surface. The platelet membrane also contains several important glycoprotein (GP) receptors (Table 1). These receptors mediate adhesion to subendothelial components, followed by platelet activation, aggregation with other platelets and formation of a firm platelet plug at the site of the vascular break (Kuter, D. 1991, Kunicki, T. 1996).  2  Table 1. Functional Properties of Platelet Membrane Glycoprotein  GP complex  Adhesive protein ligand  GPIb-IX GPIIb-IIIa  vWF Fibrinogen, Fibronectin, vWF & Vitronectin Collagen Fibronectin  GPIa-IIa GPIc-IIa  Three different types of granules are present in the cytoplasm: dense granules, alpha granules and lysosomal granules (Table 2), which are released to varying degrees during platelet activation by physiological agonists such as adenosine diphosphate (ADP), collagen and thrombin. Dense granules contain non-protein agonists (ADP, adenosine triphosphate (ATP), calcium and serotonin). The alpha granules contain a wide range of proteins including adhesive proteins, procoagulants, platelet specific proteins, and a number of important growth factors. Lysosomal granules contain a wide variety of hydrolytic enzymes (Kuter, D. 1991, Kunicki, T. 1996).  Table 2. Constituents of Alpha and Dense Granules of Platelets.  Alpha Granules  Dense Granules  vWF Fibrinogen Fibronectin Vitronectin Thrombospondin P-selectin Factors V, XI, XIII PF 4 p-TG TGF-p PDGF  ADP ATP Serotonin Calcium  3  1.1.2 Role of Platelets in Hemostasis The hemostatic mechanism is an integrated system that involves the interaction between endothelium, platelets and soluble clotting factors. Platelets play several important roles: they maintain the integrity of the endothelium, arrest bleeding in injured blood vessels and provide a phospholipid surface for the initiation of the coagulation pathway (Kuter, D. 1991, Kunicki, T. 1996). In vivo, platelets exist as non-adherent discoid cells. When platelets encounter an area of denuded endothelium, they adhere to exposed collagen and this is followed by a change in shape, from discoid to a spheroidal shape and extention of long spiky pseudopods. These changes are due to cytoskeletal rearrangement, which also causes centralization of granules, and the release of granular contents (degranulation or release reaction) into the OCS (White, J. 1981). Platelet activation causes a conformational change in GPIIb-IIIa receptor that increases its binding affinity for fibrinogen which serves as a molecular bridge between activated platelets. Platelet aggregation occurs as platelets are recruited from the circulation by the released ADP to the site of damage. Also, the activation of the plasma coagulation system results in the generation of thrombin, a potent agonist of platelets. The synergistic effect of these agonists promotes further adhesion, degranulation and aggregation of platelets. Finally, fibrin strands, generated from fibrinogen by the action of thrombin, solidify the platelet plug and causes the bleeding to cease.  4  1.2  Platelet Storage Lesion Under current blood-banking practices the storage of PCs for transfusion is  limited to 5 days at 22 °C, which leads to the outdating of a significant number of units. The primary reason for the 5-day restriction is that there is a progressive loss of platelet integrity and function when they are stored for more than 24 hours (Lazarus, H. 1982). Thus the platelet storage lesion encompasses all the changes in platelet morphology, structure and function following their collection, preparation and storage as PCs for use in clinical transfusion practice (Seghatchian, J. and Krailadsiri, P. 1997). The cause of the storage lesion is not known exactly. The other concern is the growth of bacteria, which occasionally may contaminate blood during the collection process, at room temperature.  1.2.1 Factors Influencing the Generation of Platelet Storage Lesion The list of recognized factors influencing the rate of generation of platelet storage lesion during collection, preparation and storage of PCs is very long. In this section, only the effects associated with the preparative technique, storage temperature, pH and the residual white cells (WBCs) are described.  1.2.1.1 Effect of Preparative Technique Conditions under which PCs are prepared from the units of whole blood (WB) can influence platelet viability and function during preparation and storage. Platelet-rich plasma (PRP) harvested from W B is the predominant starting component for the 5  preparation of PCs for transfusion. The shear stresses associated with the first "soft" spin (2200 x g) for 3-4 minutes to prepare PRP and the second "hard" spin (3000 x g) for 5-10 minutes to pellet the platelets induce deleterious changes in the platelets (Bode, A. 1990, Moroff, G. and Holme, S. 1991). The centrifugation forces used in the second spin for pelleting the platelets against each other and the plastic surface of the container wall induce strong platelet activation confirmed by the occurrence of P-TG release into the plasma and the expression of CD62P on the platelet membrane surface (Rinder, H. et al 1991, Snyder, E. et al 1981). Furthermore, the addition of inhibitors of platelet activation such as PGE1 and theophylline to the primary anticoagulant has been reported to preserve in vitro platelet function after preparation and storage (Bode, A . and Miller, D. 1988). The close contact of platelets in the pellet greatly promotes the generation of thromboxane A2 (TXA2) that magnifies the effects of other weak agonists on the activation of platelets. Also, low concentration of C a  2+  makes platelets more responsive  to A D P (Packham, M . et al 1987). The platelet pellet at this point cannot be easily resuspended because the platelets in the pellet are reversibly aggregated due to the binding forces of adhesive molecules such as fibrinogen and fibronectin on platelet receptors. The routine blood banking-practices usually require a "resting" period during which platelets disaggregate, although not optimally. However, the buffy coat approach used to prepare the PC, which is processed with a soft spin harvesting step and which allows the platelets to pellet on a cushion of red cells rather than the plastic surface, reduces the extent of undesirable activation and yields a better platelet product (Washitan, Y . et al 1988).  6  1.2.1.2 Effect of Storage Temperature Considering viability/function issues of platelets, the optimal platelet storage temperature range appears to be 20-24 °C (Slichter, S. and Harker, L . 1976, Filip, D. and Aster, R.1978). To maintain platelet function and viability during storage, PCs must be stored at 20-24 °C; temperatures that are also conducive to the propagation of bacteria, which limits the platelet storage to 5 days. The exposure of platelets to cold temperatures affects metabolic, functional and morphologic characteristics of platelets. Cold storage of platelets markedly reduces posttransfusion platelet viability, which is evaluated as percentage recovery and survival time (Murphy, S. and Gardner, F. 1969). Cold-induced storage lesions include irreversible platelet disc-to-sphere transformation (loss of discoid shape), which is caused by an irreversible loss of the circumferential band of microtubules that maintains the cell's discoid shape (Slichter, S. and Harker, L . 1976, Filip, D. and Aster, R.1978, Gottschall, J. et al 1986). Twenty degree centrigrade is considered as the lower optimum of the room temperature range as the storage of platelets at 18 °C and 19.5 °C has demonstrated reduced mean life spans after transfusion (Gottschall, J. et al 1986). However, recently it has been demonstrated that the storage of PCs, treated with second messenger effectors and enzyme inhibitors, at 4 °C allows platelets to maintain in vitro functional and viability characteristics (Connor, J. et al 1996, Currie, L . et al 1997). Storage at temperatures above 20-24 °C seems to be inferior as it is associated with higher metabolic rate that leads to a substantial increase in oxygen consumption and a decline in pH, which is deleterious to PC quality (Holme, S. et al 1997).  7  1.2.1.3 Effect ofpH There is ample evidence that platelets in standard PCs are activated which leads to the higher metabolic rate of stored platelets and the generation of hypoxic conditions (Murphy, S. et al 1994). During storage platelets consume glucose from plasma and accumulate lactate if there is insufficient oxygen in the storage container. The pH will remain stable as long as the concentration of plasma bicarbonate is sufficient to cope with the production of lactic acid and the walls of the storage container are permeable to allow sufficient gaseous exchange. When bicarbonate is exhausted a quick fall in pH occurs. A fall in pH below 6.2 is associated with release of granular contents, irreversible morphological changes (disc-to-sphere transformation) and loss of viability as measured by post transfusion recovery and survival (Moroff, G. and Holme, S. 1991, Murphy, S. et al 1970, Murphy, S. and Gardner, F. 1975). This fall in pH is roughly proportional to the platelet count in the PC. The concentration of bicarbonate in plasma is sufficient to maintain pH of PC only for 5-7 days, when 50-65 ml of PC is stored in more gas permeable storage containers. So, the depletion of plasma nutrients due to increased glycolytic rate, and the lactate accumulation may present significant barriers to storage beyond 5-7 days (Kilkson, H . et al 1984). As higher metabolic rate of platelets during storage is secondary to platelet activation, it has been demonstrated that the addition of inhibitors of platelet activation to the primary anticoagulant in conjunction with a reduced S/V ratio of the storage container results in a substantial reduction in the metabolic demands (glucose consumption, lactate output and pH fall) in PC (Bode, A . and Miller, D. 1989) and reduces the loss of in vitro platelet function and integrity. A n excessively alkaline pH, >7.6, which results from a  8  low platelet count in PC, is associated with cell damage and reduced post transfusion recovery (Rinder, M . and Snyder, E. 1992, Murphy, S. and Gardner, F. 1975).  1.2.1.4 Effect of Residual WBCs Contaminating WBCs present in PCs cause several negative effects on platelets and also, their presence in stored PCs is associated with a number of direct adverse effects of platelet transfusions. During storage, the uncontrolled activation or fragmentation of WBCs leads to the production of several substances that affect platelets. Destructive lysosomal enzymes released by neutrophills are known to digest various platelet proteins. For instance, elastase, is an enzyme capable of cleaving GPIb on the platelet surface (Aziz, K. et al 1995). WBCs also release vasoactive substances and biologically active cytokines, such as interleukin (IL)-l, IL-6, IL-8 and tumour necrosis factor (TNF), which are believed to produce several adverse effects on platelets during storage (Lumadue, J. et al 1996). WBCs also compete with platelets for nutrients in the plasma. Increased levels of cytokines generated by activated WBCs in stored PCs are able to produce symptoms of febrile nonhemolytic transfusion reactions (Mayelle, L. et al 1993, Stack, G. and Snyder, E. 1994). Prestorage W B C reduction has been shown to decrease the level of biologically active cytokine production over the storage period (Aye, M . et al 1995, Wadhwa, M . et al 1996), which in turn results in a reduction in the rate of febrile nonhemolytic transfusion reactions (Federowicz, I. et al 1996). The removal of WBCs by filtration, although clinically important in reducing some side effects, may cause other negative effects upon the exposure of PCs to W B C reduction filters. WBCs present in blood play an important role in the bactericidal 9  capacity of the donated blood (Hogman, C. et al 1993). Their removal by prestorage W B C reduction may make the PC more prone to the proliferation of bacteria, which may occasionally contaminate the donated blood at the time of venepuncture. The activation of contact system upon exposure of blood plasma to negatively charged material has been reported (Shiba, M . et al 1997); this is in contradiction with other studies (Devine, D. et al 1999). The generation of thrombin by contact system activation could lead to platelet activation. Some filters have been reported to remove already formed activated complement proteins (Geiger, T. et al 1997, Shimizu, T. et al 1994).  1.2.2 Evidence of Platelet Activation in Stored PCs Studies have suggested that platelet activation may play a significant role in the development of the platelet storage lesion (Bode, A. 1990, Snyder, E. 1992, Snyder, E. et al 1981). Stored platelets demonstrate evidence of activation during storage. The activation of platelets in storage causes a decrease in the efficacy of this blood product when it is transfused (Rinder, H. et al 1991). A variety of markers have been employed to examine platelet activation during storage of PCs. Morphological features include loss of discoid shape, pseudopod formation and degranulation upon exposure of platelets to a wide variety of stimuli during collection, preparation and storage of PCs. These changes are due to cytoskeletal rearrangement, which also causes centralization of granules, and their release to the OCS (White, J. 1981). Activation of platelets also causes budding and vesiculation of platelet membrane into microparticles (Bode, A. et al 1991). Loss of membrane asymmetry is another marker of platelet membrane activation whereby a negatively charged phospholipid, 10  phosphatidylserine (PS) normally confined to the inner leaflet of the platelet membrane becomes translocated to the outer leaflet of the platelet membrane (Zawaal, R. and Schroit, A. 1997). This migration forms the platelet surface highly thrombogenic. The activation markers associated with platelet granule release are expressed to a higher degree on the platelet membrane as well as in the supernatant plasma of stored PCs. Alpha granule proteins, P-thromboglobulin (P-TG) and platelet factor 4 (PF4), accumulate in the plasma of PC during preparation and storage (Snyder, E. et al 1981, Shimizu, T. et al 1985). Among the activation-dependent surface markers expressed on the platelet membrane, the best characterized are P-selectin {also known as CD62P, GMP-140 (granule membrane protein-140)} (Fijneer, R. et al 1990) and the 53 kDa protein from lysosomal granules (also known as CD63) (Nieuwenhuis, A . et al 1987). These molecules are sequestered on the granule membrane in resting platelet. With platelet activation and the fusion of the granule membrane with the platelet membrane these glycoproteins are expressed on the platelet surface. These molecules are extensively used as markers for platelet activation. The membrane glycoproteins GPIb (Michelson, A. et al 1988) and the GPIIb-IIIa complex (Minno, G. et al 1983), which are important for platelet function, also change with activation during storage. With storage platelet surface GPIb decreases initially and then recovers due to its migration from intraplatelet pool to the platelet surface. Stored platelets demonstrate increased surface expression of GPIIb-IIIa probably as a consequence of platelet activation and granule release (George, J. et al 1988). As stored platelets are activated in concentrates, they increase their glycolytic rate and lactate output thereby accelerating the formation of hypoxic conditions that may arise  11  during storage (Murphy, S. et al 1994). Therefore pH measurement is an important and useful marker to use in assessing platelet quality. The demonstration of improved preservation of platelet function and structural integrity over an extended storage period when inhibitors of platelet activation, such as prostaglandin E l (PGE1) and theophylline, are used during the preparation of PCs also supports the idea that platelet activation contributes to the platelet storage lesion (Bode, A . and Miller, D. 1988). These agents cause an elevation in platelet intracellular, cyclic adenosine monophosphate (cAMP) thereby inhibit platelet activation. The beneficial effect of these inhibitors on stored platelet quality appeared to be due to a lowering of the metabolic rate thereby preventing the formation of hypoxic conditions during storage (Bode, A . and Miller, D. 1989).  1.2.3 Potential Mechanisms of Platelet Activation During collection, preparation and storage of PCs, platelets are exposed to a variety of activating substances and influences, which impair their quality and function. There is an increasing body of evidence that stored platelets are activated, but the biologic mechanisms of this activation are poorly understood. Starting from the collection of WB from the donor to the administration of a PC to a patient, platelets exist in an activating environment. In vivo, a normal circulating platelet exists in a state of minimal activation and low metabolic rate, probably due to the relative absence of activating substances and influences. The postulated biologic mechanisms of platelet activation include the substances generated by plasma enzyme systems, such as C3a, thrombin and plasmin, and the effects of microenvironment in PCs.  12  Citrated anticoagulants routinely used in the preparation of PCs for storage in blood banks chelates most, but not all, of the ionized calcium thereby slowing, not completely blocking, the activity of blood enzyme systems. Blomback, M . et al (1984) demonstrated the enzymatic activation of the coagulation, fibrinolysis and contact activation systems in platelet-poor plasma (PPP) stored at 6 °C. The polymers and plasticizers of the tubing and containers encountered during collection of W B and processing and storage of PCs provide a surface that may cause the activation of blood enzyme pathways, particularly the fibrinolytic, coagulation and complement systems (Andrade, J. et al 1985, Sevastianov, V . et al 1985). As the non-anticoagulated blood leaves the antithrombotic environment provided by endothelial-lined vessels elaborating prostacyclin and courses through the plastic tubing and enters the collection bag, increased levels of fibrinopeptide A (FPA), a marker of thrombin activity, have been demonstrated (Skjonsberg, O. et al 1986). In such an environment, platelets are exposed to any generated thrombin, a potent physiologic agonist of the platelets, and also to the T X A 2 , provided by the other activated platelets (Packham, M et al 1987), between the collection and the preparation of a PC. Therefore, it would appear that these activating influences are present even before the processing of a PC is begun. As mentioned before, the low concentration of ionized calcium in the anticoagulated blood makes platelets more responsive to A D P , a usually weak agonist of platelets (Packham, M . et al 1987), which is released from activated platelets. The refractoriness of platelets to new in vitro stimuli during processing and storage of PC reflects prior activation and release reaction of platelets to the activating influences. The enzymatic activity from the fibrinolytic and complement systems is also evident in  13  citrated plasma of stored PCs (Bode, A. et al 1989). At high concentrations, plasmin can cleave GPIb on the platelet surface and affects the survival of platelets upon transfusion (Greenberg, J. et al 1979). The supplementation of citrate anticoagulant with inhibitors of platelet activation and thrombin inhibitor, hirudin, has demonstrated an improved preservation of platelet function and structural integrity over the storage period relative to untreated controls (Bode, A. and Miller, D. 1988). The inhibition of plasmin through the addition of aprotinin to PCs has been shown to reduce the degree of storage lesion (Bode, A . and Miller, D. 1989). In addition, the storage of platelets using either reduced plasma storage procedures or artificial media has been somewhat efficacious in reducing the degree of storage lesion (Rock, G. et al 1991, Murphy, S. et al 1991, Holme, S. et al 1988). These approaches all support the idea that the presence of certain plasma proteins contributes to the platelet storage lesion. As mentioned earlier, the shear stresses associated with the processing of PC and the close cell contact of platelets in the pellet after the "hard" spin also greatly enhance platelet activation and release reaction to occur (Snyder, E. et al 1981, Shimizu, T. et al 1985, Packham, M . et al 1987). Also, the high concentration of platelets in a PC and the continuous agitation of the storage container, to optimize gaseous exchange and maintain platelets in a state of suspension over the storage period, favor interactions of platelets with each other as well as with the potentially thrombogenic surface of the storage container, which amplify the response of platelets to activating stimuli. Thus, the collection of blood, and preparation and storage of PC at 22 °C for transfusion exposes platelets to the activating substances produced by the blood enzyme pathways and other  14  influences, which may affect platelets directly or synergistically to produce platelet storage lesion.  1.2.4 Interaction between Complement and Platelets The complement system is a group of plasma and membrane proteins whose normal immune activities include cell lysis, opsonization of bacteria for phagocytosis and the manifestation of an inflammatory response. Complement can also mediate pathogenic processes, such as anaphylaxis, intravascular hemolysis of transfused blood cells, and the activation of platelets.  1.2.4.1 Complement Activation The complement system is an activated enzyme cascade, which like other cascades, is characterized by proteins that normally circulate in an inactive form, the zymogen. Activation of the complement system triggers a sequence of biochemical reactions in which one component activates another component in a cascade fashion. Activation of human complement occurs by two different pathways, classical and alternative (Figure 1); with the activation of C3 (the third component of complement), these pathways converge to form M A C , C5b-9 (reviewed in Devine, D.1994). On activation of the classical pathway, the first component, C I , specifically the C l s subunit, cleaves the next two proteins in the pathway, C4 and C2. The C2 molecule binds to a molecule of C4b, that is already bound to a cell surface, in a magnesium-dependent manner. C2 is then also cleaved by C l s and the cleavage results in the generation of a  15  bimolecular complex, C4b2a, the C3 convertase of the classical pathway that cleaves C3 and C5. A similar C3 convertase is generated by the activation of the alternative pathway of complement, in which the initiating molecule is C3b generated during the spontaneous hydrolysis of C3. In a magnesium-dependent manner, C3b then binds to factor B (FB). Once bound FB is then cleaved by factor D to Bb. The bimolecular complex, C3bBb, is the C3 convertase of the alternative pathway that cleaves C3 and C5. The cleavage of C3 by C3 convertase generates C3b, and the deposition of C3b onto the cell surface is the primary opsonization step of the complement pathway. The generation of a C5b molecule by either C3 convertase is the first step in the formation of M A C , which involves non-enzymatic protein-protein association of C5, C6, C7, C8 and C9 to form a potentially cytolytic complex.  16  Figure 1. Activation pathways of complement. Complement activation may occur by the classical pathway or the alternative pathway. Zymogen forms of complement proteins are cleaved by activated complement proteins that have serine protease activity (bold arrows). Once cleaved, they act on the next protein in the pathway. The membrane attack complex is produced by nonenzymatic protein-protein interactions (Devine, D.1994)  17  1.2.4.2 Complement and Platelet Storage Lesion Complement activation is able to cause platelet activation by the interaction of platelets with the activated proteins of the complement system at several levels (reviewed in Devine, D. 1992). The exposure of platelets to activated complement proteins may generate physiological responses from the platelets ranging from activation (Devine, D. and Rosse, W. 1987, Polly, M . and Nachman, R. 1983), to microparticle formation (Sims, P. et al 1988). A subunit of the first complement protein, C l q , has been demonstrated to bind to platelets in W B via C l q receptor, which results in the activation of GPII-IIIa (Peerschke, E. et al 1992, Peerschke, E. et al 1993). In a washed platelet system, Polley, M . and Nachman, R. 1983 reported that the exposure of human platelets to C3a alone does not induce platelet aggregation but enhances their response to a weak platelet agonist such as ADP. Despite this report that suggested the presence of a putative C3a receptor on human platelets, subsequent experiments with purified C3a have failed to provide a conclusive evidence for direct stimulation of human platelets (Fukuoka, Y . and Hugli, T. 1988, Meuer, S. et al 1981). In purified systems, gel-filtered platelets and purified terminal complement components, the interaction of platelets with the end product of complement activation, M A C , leads to the shedding of platelet microparticles (platelet dust or vesicles) from the platelet surface and an increased prothrombinase activity (Sims, P. et al 1988). Also, activation of platelets by M A C results in the release of granular contents and an activation-associated but functionally incompetent conformation for fibrinogen binding to GPIIb-IIIa (Ando, B . et al 1988). The storage of platelets as PCs is also associated with the activation of complement in the plasma compartment of the concentrates. The activation of  18  complement occurs in PCs during the earliest stages of blood collection and continues during storage (Gyongyossy-Issa, M . et al 1994). The role of complement system in the platelet storage lesion has remained speculative. The activation of the complement system in PCs produces both the anaphylactic peptides of complement and the membrane attack complex (Milletic, V . and Popovic, O. 1993, Schleuning, M . et al 1994).  1.3  Rationale and Objectives There is ample evidence that complement is activated during blood collection,  processing and storage of PCs and in the settings other than platelet storage containers, complement activation is known to have significant effects on platelets including the release of platelet granules, the formation of platelet microparticles and the lysis of platelets, all features of the platelet storage lesion. So, considering the significant level of complement activation in PCs over time in storage and the significant effects that activated complement has on the physiology of platelets, we hypothesized that complement activation is, in part, responsible for the generation of platelet storage lesion and the inhibition of complement activation could help to maintain the platelet quality in stored PCs. In the studies described here, we modulated complement activation by the addition of the specific complement inhibitor N A A G A (Etievant, M . et al 1988) or N A A G A - N a to the PC in order to test directly whether complement activation contributes to the platelet storage lesion. N A A G A is a naturally occurring compound originally isolated from mammalian brains in the 1950's (Tallan, H. et al 1956). N A A G A and N A A G A - N a are potent inhibitors of the cleavage of C3 by either the alternative or 19  classical pathway convertase complexes. N A A G A - M g has been clinically used in the setting of treatment for perennial allergic rhinitis. N A A G A - N a has been shown to be as effective as N A A G A - M g in inhibiting C3 cleavage (Feuillard, J. et al 1991). N A A G A is thought to act on formation and/or function of C3 convertase of both pathways of complement; however its precise mechanism of action is not well defined. For these studies, we chose N A A G A to test the effects of complement inhibition on PC quality as it has been successfully used in humans and as such is not likely to be immunogenic; and it is generally available commercially.  Overall Objective The general objective of this project was to determine whether the inhibition of complement in PCs will improve the quality of stored platelets over the storage period. The overall objective is divided into three specific objectives.  1)  To determine whether the addition of the C3 converting enzyme inhibitor N A A G A inhibits complement activation in PCs.  2)  To determine whether inhibition of complement in PCs improves the quality of stored platelets.  3)  To assess whether the complement inhibitor remains stable in PCs for the 5-day storage period.  20  CHAPTER 2  MATERIALS AND METHODS  2.1 Materials Antibodies For flow cytometry studies, fluorescein-5-isothiocyanate (FITC)-conjugated monoclonal antibodies for CD62P and CD63, and a phycoerythrin (PE)-conjugated antibody for platelet marker GPIb-IX were obtained from Immunotech, Marseille, France. Monoclonal antibody for activated GPIIb-IIIa (PAC-1-FITC) was obtained from Beckton Dickinson, San Jose, C A . Annexin V-FITC used to detect the percentage of cells undergoing apoptosis was from PharMingen, Mississauga, ON.  Other Materials N A A G A used in the first study was obtained from Sigma Chemical Co., St. Louis, M O .  o  o  C H — C — N H — C H -— C — NH 3  CH  2  COO  coo CH CH  2  CH  2  COO  -  N-acetyl-L-aspartyl-glutamic acid  21  It had a purity of 99% and solvent content of 6.3% ethyl alcohol and 4.2% isopropanol. N A A G A - N a used in the second study was a generous gift from Dr. J. Luyckx, Transphyto-Industrie Pharmaceutique, Clermond-Ferrand, France. C3a enzyme immunoassay and SC5b-9 enzyme immunoassay were obtained from Quidel, San Diego,CA. Sheep blood and hemolysin (rabbit IgM anti-sheep red blood cell antibody) used to sensitize sheep red blood cells were obtained from Cedarlane (Hornby, ON). Vacutainer tubes for blood collection and Vacutainer brand SST tubes were obtained from Becton Dickinson, San Jose, C A .  22  2.2  Preparation of Reagents for Experiments  2.2.1 Preparation of Normal Human Serum and Heat Inactivated Serum The normal human serum (NHS) pool was prepared from venous blood from at least 15 healthy individuals distributed evenly over males and females using serum tubes with clot activator. The blood was allowed to clot at room temperature for 30 minutes and centrifuged at 2000 x g at 4 °C for 15 minutes to remove red and white cells and platelets. The serum was then aliquoted and frozen at -70 °C. Heat inactivated serum (HIS) was prepared by keeping NHS at 56 °C for 40 minutes and then ice-cooled for 10 minutes. Precipitated fibrinogen was removed by centrifugation at 2000 x g for 15 minutes at room temperature.  2.2.2 Preparation of Platelet Poor Plasma and Heat Defibrinogenated Plasma One millilitre of each PC sample was centrifuged at 2000 x g for 10 minutes at 4 C and 30 p.1 aliquots of platelet poor plasma (PPP) were frozen at-70 °C for complement studies in the first study. In the second study, 1.5 ml of each sample was centrifuged and 20 ul aliquots of PPP for C3a assay, 70 ul for SC5b-9 assay and 350 ul for the measurement of residual activity of complement inhibitor were frozen at -70 °C. As a result of the centrifugation step, intact platelets with surface associated complement or complement degradation products were not expected to be present in these frozen samples. 23  Heat defibrinogenated plasma (HDP) was prepared by keeping PPP (prepared above) at 56 °C for 40 minutes and then ice-cooled for 10 minutes. Precipitated fibrinogen was removed by centrifugation at 2000 x g for 15 minutes at room temperature. Using this method, HDP from fresh citrated W B was prepared by using PPP prepared from W B .  2.2.3  Preparation of P C s Random-donor fresh PCs for the studies reported here were obtained from either  the Canadian Red Cross Society (CRCS) Blood Services or the Canadian Blood Services (CBS), Vancouver Centre. Blood in single units was collected from healthy volunteer blood donors who had passed screening and who provided consent for donation according to the Blood Centre's standard operating procedures. For the first study reported here, blood was collected into Pall triple-pack collection harnesses in which the initial collection bag was made of polyvinyl chloride and contained citrate phosphate dextrose (CP2D) anticoagulant. Platelets were stored in the attached C L X platelet storage container composed of polyvinyl chloride containing tri (2-ethyl-hexyl) trimellitate plasticizer (PVC-TOTM). For this study, PCs were prepared using standard "soft spin, hard spin" methodology which isolated the packed red cells from PRP by the first soft spin and plasma from the PC by the second hard spin (Figure 2). For the preparation of all PCs, prestorage W B C reduction was introduced in Canada in January 1998. So, for the second study, PCs were prepared using an in-line WBC-reduction filtration system (Leukotrap P L System, Pall Corp., Covina, CA).  24  Figure 2. Methodology used to prepare PCs in two studies. In the first study non-leukoreduced PCs were prepared using "soft spin, hard spin" methodology. In the second study leukoreduced PCs were prepared using an in-line leukoreduction filtration system, in which before the second hard spin PRP was passed through the leukoreduction filter before storing platelets.  f Study  If*Study  Spin  Hard  V  V  25  Briefly, W B bags were centrifuged at 1210 x g for 4.5 minutes and PRP was expressed through the filter within 10 minutes of centrifugation. Filtered PRP was then centrifuged at 4100 x g for 5 minutes to pellet the platelets. Unfiltered PCs used in the first study were prepared by the same centrifugation regimen. After the plasma was expressed, the resulting PCs were rested for 2 hours before placement in a 22 °C platelet incubator with agitation. A l l concentrates were prepared and handled according to the Blood Centre's standard operating procedures, then stored at 22 °C under controlled temperature in a standard blood bank platelet incubator with agitation for 5 days.  2.3  Study Design  2.3.1  Study 1  Six units of fresh PCs were used and the plasma attached to each unit was aliquoted and frozen at -70 °C for later use. Each unit was further split into three miniunits (Figure 3). As C L X containers are designed to allow gas exchange across the plastic surface and also, as the change in surface to volume ratio could alter the pH of the concentrate, the mini-bags used for the first study were created by folding two-third of the bag between two Plexiglas strips and fixed with four bulldog clips. Then approximately 17 ml of PC was injected into each mini bag using sterile technique. These were then treated with 450 pi of either PBS, as a vehicle control, 0.5 M EDTA, as a negative control for activity of divalent cation dependent enzymes, added to a final concentration of 15 m M or the specific complement inhibitor, N A A G A suspended in  26  Figure 3. Experimental design used in study 1 and study 2. 6 units of PCs were used in each study. Using sterile technique each unit was further split into three miniunits. One unit was treated either with complement inhibitor, N A A G A in study 1 or N A A G A - N a in study 2 , and the other two units served as the controls. Each sample was processed for complement studies and the remainder of each sample was retained for hypotonic shock response, morphology and flow cytometry studies.  WB Soft-spin  Hard-spin •  5 days @ 22°C  PC  6 Units  PC  PC  PC  Buffer  NAAGA / NAAGA-Na  EDTA  Samples  Flow cytometry  C3a SCb-9  Hypotonic Shock  Activity of inhibitor  Morphology  27  PBS and added to a final concentration of 5 mM. This concentration of N A A G A was found to effectively inhibit complement activation in a different set of preliminary experiments using PPP samples stored in identical containers and under the same conditions as the PCs. Platelet samples were collected from each unit on day 0, as well as on day 1, 3 and 5 of the storage period. Samples collected on day 0 were taken 2 hours after the last centrifugation step of the concentrate preparation and before the splitting of each unit into 3 mini-units. This 2 hours rest is provided to allow the platelets to disaggregate and to recover. Using sterile technique in a laminar flow hood, 4 ml sample aliquots were removed through the access port in the platelet bag using a 19 gauge needle and 5 ml syringe. In order to maintain constant surface to volume ratio (S/V) over the time of incubation, the platelet bags were clamped to proportionally reduce the size after sampling. To monitor sterility, at each sampling, one drop of PC was spread on a blood agar plate and incubated for one week at 37 °C under 5% CO2. No bacterial growth was detected.  2.3.2 Study 2 For this study also six units of PCs were used. The mini-bags used in this study were created by sealing the platelet bags using an Impulse Foot Sealer (American International Electric, Whittier, CA). Each unit was further split into three mini-units (Figure 3). In two mini-units, 20 ml each and in the third mini-unit 5 ml of PC was injected under sterile conditions. The first two were then treated with 200 pi of V B S ^ , 4  28  as a vehicle control and 200 ul of N A A G A - N a added to a final concentration of 10 mM. The third mini-unit was treated with E D T A as a negative control for the activity of divalent cation dependent enzymes and added at a final concentration of 15 m M . Platelet samples from each subunit were collected on day 0 as well as daily during storage. In this study, 3.5 ml sample aliquots were removed from the platelet bag. Sterility was monitored by blood agar cultures and also, the size of each bag was reduced by clamping at each sampling point. For both studies platelet count and mean platelet volume (MPV) was measured in each sample after mixing one part of PC and two parts of autologous PPP. These tests were performed using an automated blood cell counter (STKR, Coulter, Hialeah, FL). The pH was also measured in all the samples collected. Each sample was processed for complement studies (see section 2.2.2) and the platelet count of the remainder of each sample was adjusted to WB concentration (2xl0 /ml) with autologus PPP and retained 8  for HSR measurements, flow cytometry and morphology studies described below.  2.4 Techniques  2.4.1 Assessment of Complement Activation The degree of complement activation in frozen plasma samples was assessed by measuring the levels of the activation fragment C3a as well as the levels of the terminal complex of the complement, SC5b-9. C3a levels were assessed using a commercially available enzyme-linked immunosorbent assay (ELISA). The C3a assays do not  29  distinguish between C3a and its inactivated form C3a des arg. SC5b-9 levels were also measured by ELISA. Complete sets of plasma samples were thawed quickly at 37 °C and analyzed in batches.  2.4.2  Hypotonic Shock Response Measurements to Assess the Responsiveness of Platelets As a measure of the extent of the platelet storage lesion, the HSR was assessed for  all the samples collected at indicated time points. HSR measures the ability of platelets to recover their normal volume after swelling when exposed to hypotonic solutions. When platelets swell their refractive index increases resulting in an increase in light transmission. Light transmittance changes of PCs exposed to hypotonic shock were measured in a ChronoLog WB Lumiaggregometer (Havertown PA) by using the method of Holme, S. et al (1987). The method is a modification of the procedure described by Valeri, C. et al (1974). Briefly, 500 pi of diluted (2xl0 /ml) and prewarmed (37 °C) PC 8  was stirred in the aggregometer at 900 rpm for 2 minutes at 37 °C. As a control, 250 pi of normal saline (0.9% NaCl) was added to PC sample that produced a dilutional increase in light transmittance (X). To another 500 pi sample (test), placed in another channel, 250 pi of prewarmed distilled water was added which produced a maximum increase in light transmittance due to both cell swelling and dilution effect (Y). This was then followed by a variable recovery associated with return of normal cell volume and a decrease in light transmittance (Y'). The HSR was calculated by expressing the decrease in light transmittance occurring during recovery as a percentage of the total increase in light transmittance corrected for dilution as follows: % H S R (% Recovery) = Y ' x 100% / Y - X  30  2.4.3 Oil Phase Contrast Microscopy to Examine Platelet Morphology Platelet morphology was assessed by a modification of the method described by Kunicki (Kunicki, T. et al 1975), which gives weighted values to disks, spheres, dendritic and balloon forms. The individual performing the microscopy was blinded to the identity of the samples. Oil phase-contrast microscopy was performed on a Nikon Labophot 2 microscope using 100 x objective. Two ul of platelet suspension from each bag was dropped onto a glass slide coated with dichlorodimethylsilane (Sigma Chemical Co., St. Louis, MO). Coated glass slides were used in order to avoid adhesion and spreading of platelets. Samples were allowed to settle for exactly 2 minutes. One hundred platelets were counted in each sample and categorized as discoid, expressing pseudopods, balloon forms, and spread platelets. The phase-contrast images were recorded with a digital camera (Pixera, CA).  2.4.4 Fluorescence Flow Cytometry to Assess Platelet Activation State and Levels of Platelet Microparticles Fluorescence flow cytometry was used to measure the surface expression of the platelet activation antigens CD62P and CD63 as well as GPIb-IX using monoclonal antibodies. No washing and centrifugation steps were performed to avoid platelet activation. Samples were prepared by adding 5 ul of platelet suspension at 2xl0 /ml to 8  50 ul of 1 m M N-2-hydroxyethyl Piperazine-N-2-ethane sulphonic acid (HEPES), pH 7.4, containing a saturating concentration of a FITC-conjugated monoclonal antibody to CD62P, CD63 or GPIb-IX.  31  In the second study, using fluorescence flow cytometry samples collected from each PC were analyzed by two-color fluorescence for platelet degranulation markers CD62P or CD63, platelet activation dependent marker GPIIb-IIIa complex. The migration of negatively charged PS from the inner leaflet to the outer leaflet of the platelet cell membrane was studied by annexin V binding. Annexin V is a 36.5 kDa protein that binds tightly to negatively charged phospholipids in the presence of calcium ions. In this study, samples were prepared by adding 5 ul of platelet suspension (2xl0 /ml) to 50 ul of 1 m M HEPES, pH 7.4, containing a saturating concentration of a 8  mix of two antibodies: a FITC-conjugated activation-dependent antibody and PEconjugated antibody to the platelet marker GPIb-IX (CD42b). FITC-conjugated antibodies used included antibodies against CD62P and CD63 and against activated GPIIb-IIIa complex (PAC-1). For positive control, 5 ul of PRP prepared from fresh citrated W B was added to 50 ul of 1 m M HEPES and activated with lunit/ml of thrombin in the presence of 2.5 m M glycyl-L-prolyl-L-arginyl-L-proline (GPRP) and a mix of the appropriate FITC- and PE-conjugated antibodies. Annexin V-FITC was used to detect percentage of cells that had exposed PS on the cell surface. As annexin V-FITC binding to PS requires C a , HEPES used for 2+  annexin V assay contained 2 m M CaCb and the tubes containing platelets, HEPES and CD42-PE were incubated at room temperature for 15 minutes and then annexin V-FITC was added at a saturating concentration and the tubes were incubated for additional 15 minutes in dark. For positive control for annexin V binding assay, 5 ul of fresh PRP was added to 50 ul of 1 m M HEPES containing CD42b-PE. Platelets were activated with calcium ionophore A23187 (5 uM) in the presence of 2.5 m M GPRP for 15 minutes at 32  room temperature before the addition of annexin V-FITC. Formal saline fixation was avoided intentionally in this case because we learned that somehow after fixation annexin V binding becomes negative. Instead samples were diluted with 500 ul HEPES. Also, since annexin V binding spontaneously increases over time, the diluted samples were analyzed as quickly as possible. However, for other markers, after a 30-minute incubation at room temperature in dark, samples were diluted and fixed with 500 ul saline containing 2% paraformaldehyde to stabilize the surface-bound antibodies. The fixed samples were kept in cold and dark until fluorescence analysis was performed. Samples were analyzed with the EPICS Profile II flow cytometer (Coulter, Hialeah, FL) in the first study and using EPICS X L - M C L flow cytometer (Coulter) in the second study. The light scatter and fluorescence channels were set at logarithmic gain. The flow cytometer was calibrated daily. FITC and PE signals were compensated to avoid the overlap of one population with the other. Platelets were identified on the basis of forward scatter (FS) and side scatter (SS) of laser light as well as their immunoreactivity with anti-CD42b. Control cells stained with non-reactive isotypematched antibody were used to set the regions to define positive population in flow cytometry histograms, less than 2% of negative control cells fell into this region. In the second study, the same sample was analyzed for the presence of these markers both on platelets and microparticles. Platelets and platelet-derived microparticles were discriminated on the basis of FS and SS, and a second bitmap was defined for particles smaller than platelets. Platelet derived microparticles were resolved from electronic noise and background light scatter by gating on red fluorescence channel (FL2) so as to include only those particles distinctly positive for GPIb (CD42-PE). Ten  33  thousand PE-positive particles from each sample were analyzed for FS and SS and FITC and PE fluorescence intensities.  2.4.5 Functional Complement Assay A modified hemolytic assay was used in the second study to determine the stability of complement inhibitor, N A A G A - N a , in the PC over the storage period. This was determined by measuring the residual activity of complement inhibitor over the storage period.  2.4.5.1 Comparison of N A A G A and N A A G A - N a To compare the complement inhibiting activity of N A A G A and N A A G A - N a , 5,7, 9 and 13 m M final concentration of both were chosen. Forty microliters of diluted NHS (1:30) was mixed with 50 pi of VBS containing 0.15 m M CaCl and 0.5 m M M g C l 2  2  (VBS ). Then HIS containing peptide/salt was added to give 5, 7, 9 and 13 m M final ++  concentration. The complement inhibiting activity of peptide/salt treated serum was then measured by adding 60 pi of antibody sensitized sheep red blood cells (EA cells) to the peptide/salt-serum mixture. Briefly, washed sheep red blood cells were incubated with rabbit IgM anti-sheep red blood cell antibody (hemolysin) at a ratio of 1:500 for 45 minutes at 37 °C. The sensitized cells were suspended at a concentration of 10 /L. Then 12  60 pi of these E A cells were used for the above reaction. Color blanks were run in parallel with the test samples and consisted of buffer and E A cells. After the addition of E A cells the tubes were incubated for 1 hour at 37 °C. The hemolytic reaction was 34  stopped by addition of normal saline. Unlysed cells were pelleted by centrifugation and the amount of hemoglobin released was quantitated spectrophotometrically at  A54o  nm  using Reader 340 A T T C (SLT Labinstruments, Austria). The percent lysis of E A cells was calculated for each concentration of complement inhibitor as follows:  (Mean  OD540 for test sample ~ O D | r blank) / (OD540 for 100% c o  0  -  O D l o r blank) X co  100  2.4.5.2 Titration of N A A G A and N A A G A - N a Using above hemolytic assay, the peptide was titrated from 1-5 m M final concentration. N A A G A - N a was titrated from 2-8 m M final concentration in order to find the optimal dose of salt that was used to study the stability of salt in PC over the storage period and also, to study the effect of inhibition of complement activation on stored platelets in the second study.  2.4.5.3 Effect of C a  2+  Concentration on the Activation of Complement  The hemolytic assay developed above was further modified in order to measure the residual activity of complement inhibitor in the PC over storage. Instead of HIS, HDP was used. Using HDP in the absence of exogenous C a  2+  NHS was unable to lyse  E A cells in the presence or absence of complement inhibitor. So, the effect of C a  2+  concentration on the activation of complement was studied by titrating CaCb from 2-12 m M and then from 1-3 m M final concentration in HDP.  35  2.4.5.4 Determination of Stability of Complement Inhibitor in PC HDP prepared (as described in Section 2.2.2) from each sample collected on day 0 as well as daily over the storage period was used to determine the stability of N A A G A - N a in the PC. The volume of all reagents was reduced to one fifth of the original volumes used in a standard CH50 hemolytic assay. Forty microliters of diluted NHS (1:15) was mixed with 50 pi of VBS++. Then 150 pi of HDP containing 2 m M CaCb was added to the mixture. As the total volume of the mixture in each tube was double that of the standard CH50 hemolytic assay, double the amount of NHS (1:15 instead of 1:30) was used to lyse E A targets. The residual activity of the complement inhibitor was then determined by adding E A cells to the inhibitor/serum mixture. Controls run in parallel were color blanks consisting of buffer + E A cells, 100% lysis control consisting of buffer + E A cells + water, and normal control consisting of buffer + NHS (1:15 diluted) + E A cells. The hemolytic reaction was stopped after incubating all the tubes at 37 °C for 45 minutes with normal saline except the 100% lysis controls in which equal volume of distilled water was added to stop the reaction. The amount of complement remaining in each tube was compared with that of serum incubated in the absence of complement inhibitor (100% lysis control). The percent lysis of E A targets was calculated for each sample collected over the storage period using above formula. The activation of complement in the presence of samples from PCs containing complement inhibitor was compared to the control samples containing buffer.  Statistical analysis was performed on all data sets using A N O V A for repeated measures using Systat. Critical values were determined at a significance level of 0.05.  36  CHAPTER 3 3.1  Study 1  3.1.1  Complement Inhibition by N A A G A  RESULTS  In this study our approach to inhibiting complement activation in PCs was to add N A A G A suspended in PBS to 6 mini-units of PCs at a final concentration of 5 m M and compare them to 6 different mini-units treated with the same volume of PBS alone. In order to avoid donor to donor variability in the behavior of platelets and plasma proteins, a 'split bag' design was used in both studies in which each unit acted as its own control. The activation of complement in stored PCs was demonstrated by increasing levels of both the activation peptide of complement, C3a, and the membrane attack complex, SC5b-9 over time in storage. The inclusion of N A A G A in freshly prepared PCs inhibited complement activation throughout the storage period as evidenced by a statistically significant decrease in C3a at day 3 (p<0.05) and day 5 (p<0.001) of storage relative to PBS controls (Figure 4, Panel A). When SC5b-9 levels were used to assess the extent of complement activation, concentrates containing PBS alone demonstrated maximum SC5b-9 generation by day 1 and then the complex degraded; however, N A A G A treatment delayed the maximum SC5b-9 generation to day 3 of storage (Figure 4, Panel B). In this study, one arm contained concentrates treated with E D T A at 15 m M final concentration at day 0. As expected, little complement activation was seen in these concentrates owing to chelation of both calcium and magnesium ions that are required for complement activation.  37  Figure 4. The effect of treatment with NAAGA on complement activation in stored PCs. PCs were divided into three mini-units immediately after preparation from WB units. Mini-units received 5 mM final concentration NAAGA suspended in PBS (squares), 15 mM final concentration EDTA suspended in PBS (triangles), or PBS alone (circles). C3a (Panel A) and SC5b-9 (Panel B) were measured in plasma samples collected from the concentrates at the days of storage indicated. Values represent the means of 6 units ± 1 SD. The difference between two treatments was significant at day 3 and 5 of storage (p<0.05).  (A)  5000  o c o o  en U  0  1  3  d a y s in storage  (B)  I  in U  C/3  0  1  3  d a y s in storage 38  3.1.2 Effect of NAAGA on the Quality of Stored PCs The quality of platelets in stored PCs was assessed by several in vitro assays. The pH of PCs did not change appreciably over the 5 day storage period; pH in concentrates containing N A A G A was 7.47 ± 0.03 at day 0 and 7.33 ± 0.02 at day 5 while pH in PBS controls was 7.47 ± 0.03 at day 0 and 7.5 ± 0.09 at day 5 of the storage period. M P V s as measured by STKR automated cell analyzer did not change between the two treatments (Table 3).  Table 3. Change of platelet size during storage of PCs as measured by MPV (fL). 1  Baseline DayO  NAAGA  Control  2  3  7.10 ±0.45*  Day 1  6.90 ± 0.494  6.90 ± 0.561  Day 3  7.03 ± 0.532  7.03 ±0.612  Day 5  7.30 ± 0.672  7.32 ± 0.484  * = + 1 SD 1 n = 6 units in each group 2 = PCs stored in the presence of 5 mM final concentration N A A G A 3 = PCs stored in the presence of PBS alone  39  Both treatments showed the same trend for PCs stored in the presence or absence of complement inhibitor. Initially the platelets are large, probably due to the shape change by partial activation during processing of WB. Then they recover their discoid shape and smaller volume. As they deteriorate with time in storage they cannot maintain an osmotic gradient across the membrane and their volume slowly increases and they become bigger. In association with this measurement of M P V and cell deterioration, platelet count declined in both treatments; from the baseline levels of 1209 x 10 /L to 9  1116 x 10 /L in PCs treated with N A A G A and to 1071 x 10 /L in PBS controls and this 9  9  drop in the platelet count was not statistically significant.  3.1.3 Improvement of Platelet Morphology in Stored PCs Following Incubation With NAAGA The examination of platelet morphology by oil immersion-phase contrast microscopy was used to determine the platelet morphology scores. Morphology score is a measure of platelet shape change that occurs when platelets are activated or when cellular membrane integrity is lost. The morphology score was calculated using the formula: (4 x disks + 2 x spheres +1 x dendrites + 0 x balloons) and a score of 400 represents 100% disks, the most superior morphology. A l l PCs showed some decline in morphology scores over the storage period. By the third day of storage, there was a significant reduction of morphology scores for both groups; however, the concentrates containing N A A G A demonstrated significantly better morphology scores than PBS controls (p<0.05) on day 5 of storage (Figure 5).  40  Figure 5. Platelet morphology scores of concentrates stored in the presence of NAAGA. Platelet morphology was assessed by oil-phase contrast microscopy and the morphology score calculated for 100 platelets. Morphology scores were determined at the days of storage indicated. Values are means of 6 samples + 1 SD either treated with NAAGA (squares) or buffer alone (circles). The difference between two treatments was significant at day 5 of storage (p<0.05).  400  0 H  1  1  0  1  3  1  5  days in storage  3.1.4  Improvement of HSR in Stored PCs Following Incubation With  NAAGA Studies have demonstrated that platelet aggregations are not a particularly informative test after the first day or two of storage (Murphy, S. et al 1994), so aggregation assays were not performed in the studies described here; instead, hypotonic shock recovery was assessed. Thus, the responsiveness of platelets from untreated samples and samples containing N A A G A was assessed by measuring the HSR. PCs stored in the presence of N A A G A demonstrated a superior HSR throughout the storage 41  period (Figure 6) compared to PBS controls with significantly better recovery on day 3 of storage (p=0.001).  Figure 6. Effect of NAAGA on the platelet hypotonic shock response. 6 PCs were split into identical storage containers and treated either with 5 mM NAAGA in PBS (squares) or PBS alone (circles). At the days of storage indicated, aliquots were removed from the concentrates and platelets were tested for recovery from HSR. Values are reported as means of 6 samples ± 1 SD. The difference between two treatments was significant at day 3 of storage (p<0.05).  100 i  1  80 H  0  H  r—  1  0  1  1  1  3  .5  days in storage  3.1.5 Effect of NAAGA on the Expression of Platelet Activation Markers The surface expression of platelet activation dependent markers over the storage period was assessed by flow cytometry. The surface expression of platelet activation markers, CD62 and CD63 increased over the storage period in all the concentrates stored in the presence or absence of N A A G A (Figure 7). N A A G A was unable to lessen 42  activation marker expression compared to PBS controls. Surface expression of the platelet membrane GPIb-IX, a von Willebrand factor receptor, was also monitored by flow cytometry. On day 0, almost all platelets in all concentrates were GPIb-IX positive. With time in storage both controls and concentrates stored with N A A G A did not show an appreciable decline in the expression of this complex on the platelet surface (Table 4).  Figure 7. The effect of NAAGA on the expression of platelet activation markers CD62 and CD63 on platelets. The expression of CD62 (A) and CD63 (B) was monitored by fluorescence flow cytometry on samples from PCs stored in the presence (grey bars) or absence (open bars) of NAAGA. Platelets samples were collected from PCs on day 0 and after 1, 3 and 5 days of storage. The percentage of platelets expressing CD62 or CD63 was determined using FITC-conjugated monoclonal antibodies against these markers. Each value represents mean of 6 units + 1 SD.  (A)  43  (B)  Q  U  > </5  o a  <u O  a. 0  1  3  days in storage  Table 4. GPIb-IX on platelets during storage detected by monoclonal anti-GPIb-IX and flow cytometry. Baseline Day 0  NAAGA  Control  3  97.7 ± 1.50  Day 1  97.7 ±0.95  97.2 ± 1.07  Day 3  97.3 ± 1.10  96.6 ± 1.09  Day 5  94.9 ± 0.96  93.1 ± 1.30  ** = percentage positive for GPIb-IX * = ± 1 SD 1 n = 6 units in each group 2 = PCs stored in the presence of 5 m M final concentration N A A G A 3 = PCs stored in the presence of PBS alone 44  3.2  Study 2  To determine the stability of complement inhibitor in PC, its residual complement inhibitory activity was monitored over the storage. In the previous study the pH of samples stored with N A A G A did not change over a 5-day storage period. During the titration of N A A G A in order to find its optimum dose to be used for assessing its stability in PC over the storage period, N A A G A being an acid had a pH effect at higher concentrations. Also, it was very hard to adjust the pH of this compound to the physiological pH (7.35). Therefore, a different compound, N A A G A - N a at 10 m M final concentration, instead of N A A G A was used in this study. N A A G A - N a being a salt was not expected to have such an effect. This study was specifically designed to assess the length of time that N A A G A - N a remains active in the PC over the storage period. In this study our approach to determine the stability of complement inhibitor over the storage period was to add N A A G A - N a suspended in V B S  + +  to six mini-units of PCs at 10 m M and compare them to six different  mini-units treated with the same volume of V B S ^ alone. To answer this question, a modified hemolytic assay was developed using HIS and HDP. The assay was developed using both N A A G A and N A A G A - N a .  3.2.1  Stability of N A A G A - N a i n P C s  3.2.1.1 Comparison of N A A G A and N A A G A - N a Different concentrations of peptide and salt of N A A G A were compared to each other. As expected with increasing concentration of peptide/salt there was less lysis of 45  E A targets, which means there was greater inhibition of complement activation. However, the peptide was more effective inhibitor of complement at low concentrations; its inhibiting activity was more than twice that of the salt form (Figure 8). However, at higher concentration they showed similar effects. The peptide and its salt were then titrated for comparison and to find out the optimal dose of the N A A G A - N a that was used in this study. Peptide was titrated from 1-5 m M (Figure 9) and the salt was titrated from 2-8 m M final concentration (Figure 10). As expected, with increasing concentration there was a corresponding increase in the inhibition of the complement activation. N A A G A - N a showed almost 50% inhibition at 5 mM.  Figure 8. Comparison of complement inhibitory activity of NAAGA and NAAGA-Na. HIS containing NAAGA or NAAGA-Na at the concentrations indicated was compared using a modified hemolytic assay. The complement inhibiting activity of NAAGA (circles) or salt (squares) was compared to the relative control for NAAGA (triangles) or salt (diamonds). HIS alone (open triangle) was run in parallel to see the effect of complement proteins from the HIS. The complement activation was measured as percent lysis of EA targets. Values are reported as means of duplicates.  120  •I  80  £  40 0  inhibitor concentration (mM) 46  Figure 9. Titration curve of NAAGA. HIS containing different concentrations (1-5 mM) of NAAGA suspended in VBS" " (circles) was compared 1-1  to corresponding vehicle controls containing VBS""" (triangles) using a modified hemolytic assay. The inhibition of complement activation by each concentration of NAAGA was measured as percent lysis of EA cells. With increasing concentration there was a corresponding increase in the inhibition of complement activation as compared to buffer controls; and NAAGA showed 50% inhibition at 4 mM final concentration. Values are reported as means of duplicates.  47  Figure 10. Titration curve of NAAGA-Na. HIS containing NAAGA-Na at concentrations indicated (squares) was compared to corresponding VBS""" controls (diamonds) using a modified hemolytic assay. The inhibition of complement activation was assessed as percent lysis of EA cells. With increasing concentration of salt there was a parallel increase in the inhibition of complement. Values are reported as means of duplicates.  •S3  80  <  concentration of salt(mM)  3.2.1.2 Effect of Ca  Concentration on the Activation of Complement  During the development of this hemolytic assay effect of calcium concentration on the activation of complement was studied. To study the calcium effect, HDP was made from fresh W B as explained in the Materials and Methods section. The effect of calcium concentration on the complement activation was studied from 2-12 mM. For each concentration, corresponding controls containing the same volume of VBS** as  CaCl2 were made. With increasing concentration of calcium there was a corresponding 48  inhibition of complement activation (Figure 11). Then it was further titrated from 1-3 m M CaCb. Using the above hemolytic assay it was compared to the buffer controls. At physiologic concentration of calcium, 2 mM, there was an increase in the lysis of E A targets (data not shown). So, this concentration of CaCb was chosen to assess the residual activity of complement inhibitor over the storage period.  Figure 11. The effect of C a  2+  concentration on the activation of complement.  Using a modified hemolytic assay HDP prepared from fresh citrated WB and containing concentrations of CaCl2 indicated (circles) was compared to the corresponding controls (squares) containing V B S ^ to study the effect of CaCl concentration on the activation of complement. With increasing concentration there was 2  a corresponding inhibition of complement activation as measured by percent lysis of EA cells. Values are reported as means of duplicates.  49  3.2.1.3 Measurement of Residual Activity of N A A G A - N a The residual complement inhibiting activity of N A A G A - N a was measured, using a modified hemolytic assay and HDP, to determine its stability in the PCs over the storage period. N A A G A - N a present in HDP samples, which were prepared from the samples collected from PCs over the days of storage, inhibited the complement activation thereby inhibited the lysis of E A cells in the presence of 2 m M exogenous CaCh from day 1 through day 5 of storage as compared to VBS" " controls (Figure 12). Although the 1-1  error at day 0 is quite big but the difference between two treatments was statistically significant (p < 0.05).  3.2.2  Complement Inhibition by N A A G A - N a The activation of complement in PCs stored in the presence or absence of  complement inhibitor was demonstrated by increasing levels of the activation peptide of complement, C3a. The storage of freshly prepared PCs in the presence of N A A G A - N a demonstrated a statistically significant inhibition of C3a generation throughout the storage period (p < 0.01) relative to V B S  + +  controls (Figure 13). When SC5b-9 levels  were used to assess the extent of complement activation, the concentrates containing VBS  + +  alone demonstrated maximum SC5b-9 generation by day 1 and then this complex  degraded; however, the inclusion of N A A G A - N a in PCs delayed the maximum SC5b-9 generation to day 5 of the storage period. In the concentrates treated with 15 m M E D T A little complement activation was seen as measured by C3a levels. Interestingly, SC5b-9 levels were high in these concentrates.  50  Figure 12. Measurement of the residual activity of NAAGA-Na over the storage period. 6 PCs were split into mini-units immediately after preparation from fresh WB units and treated either with 10 mM final concentration NAAGA-Na suspended in V B S ^ (squares) or VBS"^ alone (circles). Samples for the measurement of residual activity of NAAGA-Na were collected from the mini-units after the 2 hours rest period (day 0) and every day of the storage period. The residual activity of NAAGA-Na in the HDP samples (squares) prepared from the PC samples was measured using a modified hemolytic assay. Values are reported as means of 6 units ± 1 SD. The difference between two treatments was significant (p<0.05).  100 80 "  days in storage  51  Figure 13. The effect of treatment with NAAGA-Na on complement activation in stored PCs. PCs were divided into mini-units immediately after preparation from WB units. Mini-units received 10 mM final concentration NAAGA-Na suspended in V B S  ++  (squares), 15 mM final concentration EDTA  (triangles) or V B S ^ alone (circles). C3a (Panel A) and SC5b-9 (Panel B) were measured in plasma samples collected from PCs after the 2 hours rest of the PC (day 0), and every day of the storage period. Values represent the means of 6 units ± 1 SD. The difference between two treatments was significant (p<0.05).  (A)  12000 10000 -  0  1  2  3 days in storage  52  4  (B)  400 " f  300 -  days in storage  3.2.3  Effect of N A A G A - N a on the Quality of Stored P C s Other parameters along with the measurement of complement levels were also  measured in this study. The quality of stored platelets was assessed by several in vitro assays. The pH of PCs did not change much over the 5-day storage period. At every day point, the pH of both treatments was > 7.25. pH in concentrates containing N A A G A - N a was 7.29 ± 0.09 at day 0 and 7.28 ± 0.12 at day 5 while pH of V B S ^ controls was 7.29 ± 0.09 at day 0 and 7.41 ± 0.12 at day 5 of the storage period. Platelet count in concentrates containing N A A G A - N a was higher than the controls for the first two days of storage; then at day 3 the count of both treatments was the same and after that point the count went down in both treatments. MPVs as measured by STKR were different between two treatments. Platelets were bigger at day 0 probably 53  due to partial activation, after that M P V of control platelets stayed almost the same whereas the M P V of platelets in concentrates treated with N A A G A - N a slowly went up after day 1 of the storage period (Table 5).  Table 5. Change of platelet size during storage of PCs as measured by MPV (fL). 1  Baseline  Day 0  NAAGA-Na  Control  2  3  7.05 ± 0.43*  Day 1  6.78 ±0.48  6.77 ± 0.58  Day 2  6.90 ± 0.34  6.76 ± 0.44  Day 3  7.01 ±0.35  6.76 ± 0 . 4 1  Day 4  7.08 ± 0.44  6.67 ± 0.37  Day 5  7.13 ±0.37  6.70±0.41  *  = ± 1 SD 1 n = 6 units in each group 2 = PCs stored in the presence of 10 m M final concentration N A A G A - N a 3 = PCs stored in the presence of V B S  alone  54  When HSR was used to assess the responsiveness of platelets the addition of N A A G A Na to PCs had no significant effect on the recovery of platelets (Figure 14).  Figure 14. Effect of NAAGA-Na on the platelet hypotonic shock response. 6 PCs were split into identical storage containers and treated either with 10 mM final concentration NAAGA-Na (squares) or V B S  ++  alone (circles). Platelet samples were collected after the 2 hours rest of  the PC (day 0), and every day of the storage period and tested for recovery of platelets from HSR. Values are reported as means of 6 units; error bars represent ± 1 SD.  100 i  1  80 H  days in storage  The platelet morphology was examined by oil immersion-phase contrast microscopy to determine platelet morphology scores. A l l PCs demonstrated a decline in the morphology scores up to day 3 of storage relative to baseline values but there was no statistically significant difference between the two treatments over the storage period (Figure 15). 55  The surface expression of the membrane phospholipid, PS and the activation dependent markers CD62, CD63 and GPIIb-IIIa was monitored by two-color fluorescence flow cytometry. Same sample was analyzed to detect platelet-derived microparticles and the presence of these markers on platelets. Platelet derived microparticles are very tiny, about one-tenth of the size of a platelet, and it is very hard to capture them. Although we resolved the microparticles from electronic noise and background light scatter by gating on FL2, still the assay used to detect platelet microparticle generation was very noisy. The addition of N A A G A - N a to PCs had no significant effect on microparticle formation (Figure 16).  Figure 15. Effect of NAAGA-Na on platelet morphology. 6 PCs were split into identical storage containers and treated either with 10 mM final concentration NAAGA-Na (squares) or V B S  ++  alone (circles). Platelet samples were collected after the 2 hours rest of  the PC (day 0), and every day of the storage period and assessed for platelet morphology by phase contrast microscopy. Values are reported as means of 6 units; error bars represent + 1 SD.  days in storage  Figure 16. Microparticle generation in PCs treated with NAAGA-Na. Fluorescence flow cytometry was used to identify platelet-derived microparticles in samples from PCs stored in the presence of NAAGA-Na (squares) or V B S  ++  alone (circles). Platelet samples were collected  on day 0 and every day during the storage period. Values are reported as the count of microparticles for 10,000 events and are means of 6 units ± 1 SD.  800  0  i  1  1  0  1  1  2  1  1  3  4  5  days in storage  The surface expression of activation markers CD62 (Figure 17A) and CD63 (Figure 17B) increased dramatically over the storage period in all concentrates when compared to the baseline levels. Surprisingly, the platelets stored in the presence of N A A G A - N a demonstrated significantly higher expression of these markers on their surface relative to controls. The expression of GPIIb-IIIa complex on platelets as  57  detected by PAC-1 binding increased up to day 4 of storage; and then it came down in all concentrates (Figure 17C). The surface expression of PS as measured by annexin V binding increased over the storage period in both treatments (Figure 17D). Less than 8% of platelets positive for annexin V were detected in concentrates treated with or without N A A G A - N a . However, all concentrates were 100% positive for annexin V binding on day 11 of storage. Positive controls at each time point were >90% positive for annexin V binding.  58  Figure 17. Expression of platelet activation markers CD62, CD63, GPIIb-IIIa and the phospholipid PS on platelets collected from PCs treated with or without NAAGA-Na. PCs were stored in the presence of 10 m M final concentration of N A A G A - N a (squares) or VBS" " alone 1-1  (diamonds). Platelets collected from the concentrates were incubated with a mix of two antibodies; a PEconjugated antibody to the platelet marker GPIb-IX and a FITC-conjugated activation dependent antibody against CD62 (Panel A), CD63 (Panel B) and GPIIb-IIIa complex (Panel C). Annexin V - F I T C was used to detect the expression of PS on the platelet surface (Panel D). The percentage of platelets expressing CD62, CD63, GPIIb-IIIa and PS on their surface was monitored by flow cytometry. Platelet samples were collected on day 0 as well as daily during storage period. Values are reported as means of 6 units ± 1 SD.  59  CHAPTER 4  DISCUSSION  Studies on platelet storage recognize that when platelets are removed from circulation, they demonstrate a gradual decline in viability and a loss of in vitro functional activity and post transfusion survival. The storage lesion is induced during the collection of W B and continues through the preparation, processing and storage of PC and is, in large part, a consequence of both the metabolic activity of platelets at 22 °C and their activation due to the absence of in vivo modulators (Bode, A . 1990, Miller, T. and Bode, A . 1988). However, the processes contributing to the generation of storage lesion and the biologic mechanisms of platelet activation are not well defined. Several studies have shown that complement is activated during the stages of blood collection, processing and storage of PCs (Gyongyossy-Issa, M . et al 1994, Miletic,V. and Popovic, O. 1993, Schleuning, M . et al 1994, Schleuning, M . et al 1992, Bode, A . et al 1989). Because the interaction of platelets with activated complement proteins at several steps of the pathway, from C l q receptor occupation (Peerschke, E. and Ghebrehiwet, B . et al 1987) to the terminal complex, C5b-9, may induce platelet activation and/or vesiculation (Sims, P. et al 1988, Polly, M . and Nachman, R. 1983, Devine, D. and Rosse, W. 1987), a causal relationship has been postulated between complement activation and the generation of platelet storage lesion (Miletic,V. and Popovic, O. 1993). However, there has been no direct evidence to support this hypothesis. The first study reported here demonstrates that the addition of a specific complement inhibitor, N A A G A , to PCs improves the quality of the platelets in 60  comparison to the absence of this complement inhibitor. N A A G A is thought to function by inhibiting the C3/C5 converting enzyme complexes of the complement cascade; however, its precise mechanism of action remains to be clarified. In this study, changes in a number of platelet parameters were detected at 2 hours after the second centrifugation (day 0) step as well as during the storage period. Consistent with the reports in literature, the complement activation in the platelet storage container was time dependent and proceeded to the formation of membrane attack complex (Bode, A. et al 1989, Schleuning, M . et al 1992). Significant complement activation, as measured by C3a and SC5b-9 levels, was detected at day 0 indicating that activation of complement is not restricted to the storage period, rather it starts during the collection and processing of WB. The platelet activation was demonstrated on day 0 and during storage as well. During storage the overall platelet count was reduced, probably due to activation, lysis or binding of platelets to the polymer surface of the storage container. Storage induced lesion was evident in a number of platelet parameters; signs of platelet activation, decline in platelet morphology scores and the reduced recovery of platelets from HSR, all represent the characteristics of storage lesion. The inclusion of N A A G A to PCs improved the overall quality of platelets by inhibiting the activation of complement. N A A G A inhibited the complement activation throughout the storage period; however, it was not effective enough to block the complement activation completely. E D T A treatment was a very effective way to inhibit complement activation; however, this is not ideal for the maintenance of normal discoid shape of platelets and could not be used for transfusion. The molar concentration of  61  NAAGA used was 5 mM. It remains to be determined whether higher concentrations of NAAGA would be more efficacious in inhibiting complement activation. The inhibition of complement activation by pharmacological intervention is an area of active investigation. The widespread destructive potential of complement activation has been targeted by a number of pharmacological agents, which have been developed as a result of the increased understanding of the biochemistry of the complement system. NAAGA was chosen for this study in part because this low molecular weight dipeptide is very similar to a natural peptide originally isolated from mammalian brains and has been used at similar concentrations without reported side effects in humans. In this study, the platelet morphology score was significantly improved by treatment with NAAGA. HSR, which has been shown to correlate with clinical outcome, was improved in the PCs treated with NAAGA throughout the storage period compared to paired controls treated with PBS alone. The recovery of platelets on day 0 was lower than on day 1 of storage, presumably because of the shape change due to partial activation during processing of WB; as they recovered their discoid shape by day 1 they showed better recovery. Platelet aggregations were not performed in this study, as platelets in 5-days stored PCs are unable to aggregate when stimulated with low doses of agonists, so this parameter is perhaps not a reliable indicator of platelet quality (Murphy, S.ef a/ 1994). Consistent with previous studies from our laboratory (Gyongyossy-Issa, M. et al 1994) and the studies of other investigators (Fijnheer, R. et al 1990, Metcalfe, P. et al 1997), a continuous increase in the activation marker expression, CD62 and CD63, was  62  detected over time in storage. A significant number of platelets were positive for these markers at day 0, indicating that full platelet activation with its associated granule release occurs before storage probably during collection and processing of PCs. However, N A A G A was unable to decrease the expression of this marker of the storage lesion. In addition to activation marker expression, changes in the expression of GPIb-IX (a receptor for vWF) were also studied. Inconsistent studies on the effects of storage on GPIb have been reported; GPIb has been shown to either increase, decrease or remain constant during the storage period (George, J. et al 1988, Michelson, A. et al 1994, Bode, A. et al 1990). Also, the activation-induced decrease in the platelet surface expression of GPIb-IX has been reported to be reversible (Michelson, A . et al 1994) due to relocation of GPIb-IX to platelet surface from an intracellular pool (Michelson, A . et al 1988). However, in this study an antibody to GPIb-IX complex did not report any loss of this membrane glycoprotein over storage. This may be explained in terms of the dependency of the flow cytometric detection of this complex on the surface of platelets, on the type of antibody conjugate used (Goodall, A. et al 1993). It may be possible that the monoclonal antibody, directly conjugated to FITC, used in this study was also able to detect the internalized GPIb-IX complexes, which might have been lost from the platelet surface over the storage period and did not report any loss of this complex over storage. The differences seen in this study may reflect the relative utility of these assays for the determination of the quality of stored platelets. However, there is no established simple in vitro assay that can be used to translate accurately the measured loss of platelet function into an appropriate scale of PC quality. An extensive review of methods (Murphy, S. et al 199'4) concerning the relationship between in vitro evaluation of stored  63  platelet quality and their in vivo effectiveness indicated that some assays change little over the duration of storage, e.g., mean platelet volume, serotonin uptake and lactate dehydrogenase (LDH) release, and lack sufficient sensitivity to be of use for the evaluation of PC quality. Other assays are too sensitive. Aggregation and release of platelets in response to widely used agonists ADP, collagen and epinephrine markedly deteriorate in PCs, which, in spite of this, still possess the capability to aggregate after incubation in fresh plasma and provide adequate hemostasis. So, these in vitro assays are often misleading and show less correlation with in vivo results. As determined by this review, the best practical markers of the overall platelet storage lesion which correlate best with in vivo platelet function are the traditional platelet morphology score and the HSR; two parameters that were improved by N A A G A in this study. This review (Murphy, S. et al. 1994) also indicated that the lack of understanding of the detailed nature of the platelet storage lesion makes it difficult to identify parameters that predict in vivo hemostasis. As the time a complement inhibitor remains functional in the platelet storage container could influence the degree of complement inhibition, the second study described here was specifically designed to assess the length of time that the complement inhibitor remains active in the PC over the storage period. Ideally, an inhibitor of complement should remain active for the duration of the 5-day storage period. N A A G A Na was used in this study. In order to assess the activity of this inhibitor in the PC, its complement inhibiting ability was monitored over time of storage using a modified hemolytic assay. To achieve this goal, the idea of heat inactivation of serum/plasma containing N A A G A - N a was employed which was later used as a source of complement  64  inhibitor to measure its residual activity to inhibit the lysis of E A cells by fresh serum. When the N A A G A - N a was compared to N A A G A , they showed different effects; at low concentrations N A A G A appeared to be more effective inhibitor than the salt but at higher concentrations, > 10 mM, they showed similar effects. When this assay using HIS was employed to determine the stability of N A A G A Na in PC over storage, by making serum from the aliquots of PC in the presence of 20 m M final concentration CaCb which was later heat inactivated, the assay did not work. The reason was an excessive amount of CaCl2 in the system that was inhibitory to the activation of complement. In order to avoid the conversion of PC samples to serum and the involvement of CaCh, HDP was used. But, again the anticoagulant, CP2D, present in PC samples which were heat defibrinogenated was able to chelate C a buffer and did not leave enough C a  2+  2+  present in VBS"^  required for the complement activation. Two  millimolar final concentration CaCb in HDP was able to enhance complement activation. PC samples from the bag could not be used directly as the hemolytic assay requires an incubation step at 37 °C for one hour, which could clot the plasma. So, the approach to defibrinogenate the plasma, prepared from the PC samples containing N A A G A - N a , was adopted. It had dual beneficial effects, on one hand, it removed fibrinogen from the plasma thereby rendered it unclottable during the assay and on the other hand, it inactivated complement present in those plasma samples. So using this approach the residual complement inhibitory activity of N A A G A - N a was monitored to determine its stability in the bag over the storage period. This study clearly demonstrated that this complement inhibitor remained active in the PC throughout the 5-day storage period.  65  Other parameters, including the measurement of complement activation as well as parameters to assess the quality of platelets in stored PCs, were also studied. Time dependent complement activation up to the formation of M A C , C5b-9 was detected. However, very low level of complement activation was detected at day 0 in this study. This may be attributed to the difference in the method of preparation of PCs in the two different studies reported here. In this study, platelets prepared were leukoreduced and also the adsorption of anaphylatoxins through electrostatic interactions with negatively charge filters has been reported previously (Shimizu, T. et al 1994, Geiger, L. et al 1997). Therefore, it could be possible that the complement activation products present in PRP at the time of filtration got adsorbed on to the filters leaving behind very low concentrations, which were detected on day 0. But this is inconsistent with studies done in our laboratory (Devine, D. et al 1999). This issue needs further investigation. The parameters measured to assess the quality of platelets in this study showed different response to the presence of N A A G A - N a as compared to the last study. There was no difference in the morphology scores and HSR. Neither N A A G A in this first study nor N A A G A - N a in this study was able to lessen the activation marker expression. Rather the expression of all markers, CD62, CD63 and GPIIb-IIIa, in the presence of N A A G A Na was significantly higher in this study. Also, in contrast to previous studies from our laboratory (Gyongyossy-Issa, M . et al 1994) and from other groups (Fijnheer, R. et al 1990, Metcalfe, P. et al 1997), we here did not detect a continuous increase in CD62 and CD63 expression over the storage period. The addition of N A A G A - N a had no significant effect on microparticle generation in PCs over storage. Annexin V binding to platelets, as an indicator of the loss of membrane asymmetry, showed only slight  66  increases until day 5; whereupon the rate of binding rapidly rose to 100% in all PCs by day 11 of storage, which is consistent with the studies described previously (Geffet, P. et al 1994, Matsubayashi, H. et al 1999). The differences seen in the response of platelets between two studies reported here might be attributed to the behaviour of platelets prepared with or without W B C reduction filters. The first study was performed on PCs containing contaminating WBCs. Activation or fragmentation of WBCs during storage of PCs release several substances that affect platelets. As discussed before (in Section 1.2.2.4), destructive lysosomal enzymes present in the neutrophils are known to digest various platelet proteins. Biologically active cytokines released by WBCs are able to cause platelet activation (Lumadue, J. et al 1996). In this study, the quality of the starting component was not as good as in the second study, which was performed on filtered PCs. N A A G A demonstrated an improvement in platelet quality in the first study. However, prestorage WBC-reduction filtration used in the second study removed WBCs before the cells had a chance to effect platelets negatively, thereby produced a starting component of better quality. The platelets in the second study were not of poor quality for N A A G A - N a to exert its effect. The development of the platelet storage lesion is unlikely to be due only to complement activation. Studies have shown that the functional defect can arise by many different mechanisms. As mentioned before, collection of WB and processing and storage of PC expose platelets to a variety of influences that could lead to platelet activation. The storage of platelets although providing clinically acceptable products, is associated with a reduction in viability and functional characteristics.  67  Although the current storage period for PCs is limited to 5 days, it has been previously shown that platelet function can be preserved for much longer periods (Bode, A. and Miller, D. 1988, Holme, S. et al 1992). Various attempts to improve platelet quality have been made by manipulating the platelet microenviroment. Platelets stored in synthetic media have less evidence of storage-induced activation and a reduced metabolic activity (Rock, G. et al 1991, Murphy, S. et al 1991, Holme, S. et al 1987), which has been attributed to the decreased levels of plasma enzyme and proteins that cause platelet activation. Another approach toward the improvement of platelet quality in stored PCs has been the addition of inhibitors of platelet activation to plasma concentrates or to the synthetic media (Holme, S. et al 1992, Bode, A . and Miller, D. 1988, Bode, A . and Miller, D. 1989). For the most part, these approaches address the physiological response of platelets to exposure to activators during storage rather than targeting the activation mechanisms. To inhibit the generation of the activators, it is necessary to understand the processes that may have an impact on long-term storage of platelets, which in turn will help to extend the shelf life of PCs. The results from the first study provide direct evidence that complement activation is one mechanism by which platelet activation occurs in stored PCs and that the inhibition of complement activation improves the quality of stored platelets. Although we did not find an improvement of platelet quality in the second study, we can not exclude the possibility that complement is one of the likely candidates that may have an impact on the extended storage of PCs. As mentioned above, this could be due to the behaviour of platelets in the unfiltered and filtered units.  68  For a real comparison, P C s prepared by similar method and same unit treated with similar dose o f N A A G A or N A A G A - N a should be compared. If complement inhibition results in the improvement of platelet quality, then the studies should be done on extended storage periods and platelet function and integrity o f platelets stored for periods longer than 5 days should be assessed. The post transfusion platelet recovery should then be determined by radiolabeled platelet survival studies in normal volunteer donors. The identification o f complement as one o f the mediators o f the storage lesion provides a defined target for practical strategies to control platelet activation during storage.  69  Bibliography Ando B, Wiedmer T, Hamilton K , Sims P, 1988. Complement proteins C5b-9 initiate secretion of platelet storage granules without increased binding of fibrinogen or von Willebrand factor to newly expressed cell surface GPIIb-IIIa. J Biol Chem 263:1190711914.  Andrade J, 1985. Surface and Interfacial Aspects of Biomedical Polymers. Vol 2, Protein Adsorption. New York: Plenum Press.  Aye M , Palmer D, Hashemi S, 1995. Effect of filtration of platelet concentrates on the accumulation of cytokines and platelet release factors during storage. Transfusion 35:117-124.  Aziz K , Cawley J, Kamiguti A , Zuzel M , 1995. Degradation of platelet glycoprotein lb by elastase released from primed neutrophils. Br J Haematol 91:46-54.  Blamoutier J, Luyckx J, 1988. A double-bind crossover study comparing N-acetyl aspartyl glytamic acid ( N A A G A ) with disodium cromoglycate (SDCG) in the treatment of perennial rhinitis. Acta Therapeut 14:145-154.  Blomback M , Chmielewska J, Netre C, Akerblom O, 1984. Activation of blood coagulation, fibrinolytic and kallikrein systems during storage of plasma. Vox Sang 47:335-342. 70  Bode A , 1990. Platelet activation may explain the storage lesion in platelet concentrates. Blood Cells 109-124.  Bode A , Knupp C, Miller D, 1990. The effect of platelet activation inhibitors on the loss of glycoprotein lb during storage of platelet concentrates. J Lab Clin Med 115:669-679.  Bode A , Miller D, 1988. Preservation of in vitro function of platelets stored in the presence of inhibitors of platelet activation and a specific inhibitor of thrombin. J Lab Clin Med 111:118-124.  Bode A , Miller D, 1989. Metabolic status of platelet concentrates during extended storage. Improvement with pharmacological inhibitors and reduced surface to volume ratio. Vox Sang 57:19-24.  Bode A , Orton S, Frye M , Udis BJ, 1991. Vesiculation of platelets during in vitro aging. Blood 77:887-895.  Bode AP, Miller D, Newman S, Castellani W, Norris H , 1989. Plasmin activity and complement activation during storage of citrated platelet concentrates. J Lab Clin Med 113:94-102.  71  Connor J, Currie L , Allan H and Livesey S, 1996. Recovery of in vitro functional activity of platelet concentrates at 4° C and treated with second-messenger effectors. Transfusion 36:691-698.  Currie L, Harper J, Allan H and Connor J, 1997. Inhibition of cytokine accumulation and bacterial growth during storage of platelet concentrates at 4° C with retention of in vitro functional activity. Transfusion 37:18-23.  Devine D, 1992. The effects of complement activation on platelets. Curr Topics in Microb and Immunol 178:101-113.  Devine D, 1994. "Complement". In: Anderson K , and Ness P M , eds. The Scientific Basis of Transfusion Medicine. W B Saunders Co. 147-163.  Devine D, Rosse W, 1987. Regulation of activation of platelet-bound C3 convertase of the alternative pathway of complement by platelet factor H . Proc Nat Acad Sci USA 84:5873-5877.  Etievant M , Leluc B and David B, 1988. In vitro inhibition of the classical and alternate pathways of activation of human complement by N acetyl aspartyl glutamic acid ( N A A G A ) . Agents and Actions 24:137-144.  72  Federowicz I, Barrett B, Anderson J, et al 1996. Characterization of reactions after transfusion of cellular blood components that are white cell reduced before storage. Transfusion 36:21-28.  Feuillard J, Maillet F, Goldschmidt P, Weiss L and Kazatchkine M , 1991. Comparative study of in vitro inhibition of the classical and alternative pathways of human complement by the magnesium and sodium salts of anti-inflammatory peptide N-acetylaspartyl-glutamic acid. Agents and Actions 32:343-346.  Fijhneer R, Modderman P, Veldman H, Ouwehand W, Nieuwenhuis H , Ross D, de Korte D, 1990. Detection of platelet activation with monoclonal antibodies and flow cytometry. Changes during platelet activation. Transfusion 30:20-25.  Fijnheer R, Pietersz R, de Korte D, et al 1990. Platelet activation during preparation of concentrates: a comparison of the platelet-rich plasma and the buffy coat methods. Transfusion 30:634-638.  Filip D and Aster R, 1978. Relative hemostatic effectiveness of human platelets stored at 4°C and 22°C. J Lab Clin Med 91: 618-624.  Fukuoka Y , Hugh T, 1988. Demonstration of a specific C3a receptor on guinea pig platelets. J Immunol 140:3496-3501.  73  Geffet P, Basse F, Bienvenue A , 1994. Loss of phospholipid asymmetry in human platelet plasma membrane after 1-12 days of storage. A n ESR study. Eur JBiochem 222:1033-1040.  Geiger L, Perotta PL, Davenport R, Baril L and Snyder E, 1997. Removal of anaphylatoxins C3a and C5a and chemokines interleukin 8 and R A N T E S by polyester white cell-reduction and plasma filters. Transfusion 37:1156-1162.  George J, Pickett E, Heinz R, 1988. Platelet membrane glycoprotein changes during the preparation and storage of platelet concentrates. Transfusion 28:123-126.  Goodall A , de Oliveira Domingos M , Chronos N , Janes S, Wilson D, 1993. Flow cytometric detection of the redistribution of the glycoprotein Ib-IX complex on the thrombin-stimulated platelets is dependent on the type of antibody conjugate used. Blood 81:1407-1409.  Gottschall J, Rzad L and Aster R, 1986. Studies of the minimum temperatures at which human platelets can be stored with full maintenance of viability. Transfusion 26:460-462.  Greenberg J, et al 1979. Survival of rabbit platelets treated in vitro with chymotrypsin, plasmin, trypsin or neuraminidase. Blood 53:916-927.  74  Gyongyossy-Issa M , McLeod E, Devine D, 1994. Complement activation in platelet concentrates is surface-dependent and modulated by the platelets. J Lab Clin Med 123:859-868.  Hogman C, 1993. Components preparation and quality assurance, in Rock G, Seghatchian M , (eds): Quality assurance in Blood Transfusion Medicine, vol II, CRC Press, Boca Raton, Florida, 59-98.  Holme S, Bode A , Heaton W, Sawyer S, 1992. Improved maintenance of platelet in vivo viability during storage when using a synthetic medium with inhibitors. J Lab Clin Med 119:144-.  Holme S, Heaton W, Coutright M , 1987. Improvement in vivo and in vitro viability of platelet concentrates stored for 7 days in a platelet additive solution. Br J Haematol 66:233-238.  Holme S, Sawver S, Heaton A and Sweeney J, 1997. Studies on platelets exposed to or stored at temperatures below 20° C or above 24° C. Transfusion 37:5-11.  Kilkson H, Holme S, Murphy S, 1984: Platelet metabolism during storage of platelet concentrates at 22 °C. Blood 64:406-414.  75  Kunicki T, Tuccelli M , Becker G, Aster R, 1975. A study of variables affecting the quality of platelets stored at "room temperature". Transfusion 15:414-421.  Kunicki T, 1996. Role of platelets in hemostasis. In: Rossi, Simon, Moss and Gould eds. Principles of Transfusion Medicine. Williams and Wilkins Co. 221-230.  Kuter D, 1991. Hemorrhagic Disorders II. Platelets. In: Beck WS ed. Hematology. The MIT press. 543-575.  Lazarus H, Herzig R, Warm S and Fishman D, 1982. Transfusion experience with platelet concentrates stored for 24 to 72 hours at 22°C. Transfusion 22:39-43.  Lumadue J, Lanzkron S, Kennedy S, Kuhl D, Kickler T, 1996. Cytokine induction of platelet activation. Am J Clin Pathol 106:795-798.  Matsubayashi H , Weidner J, Miraglia C and Mclntyre J, 1999: Platelet membrane early activation markers during prolonged storage. Thromb Res 93:151-160. Med 280: 1094-1098.  Metcalfe P, Williamson L, Reutelingsperger C, et al 1997. Activation during preparation of therapeutic platelets affects deterioration during storage: a comparative flow cytometeric study of different production methods. Br J Haematol 98:86-95.  76  Meuer S, Hugli T, Andreatta R, Hadding U , Bitter-Seurmann D, 1981. Comparative study on biological activities of various anaphylatoxins (C4a, C3a, C5a). Inflammation 5:263-273.  Michelson A , Adelman B, Barnad M , Carrol E and Handin R, 1988. Platelet storage results in a redistribution of glycoprotein lb molecules. J Clin Invest 81:1734-1740.  Michelson A , Benoit S, Kroll M , L i J-M, Rohrer M , Kestin A , Barnard M , 1994. The activation-induced decrease in the platelet surface expression of the glycoprotein Ib-IX complex is reversible. Blood 83:3562-73.  Miletic V , Popovic O, 1993. Complement activation in stored platelet concentrates. Transfusion 33:150-154.  Miller T and Bode A , 1988. Changes in the activation state of stored platelets. Plasma Ther Transfus Technol 9:283-294.  Minno G, Capitanio A , Thiagarajan P, Martinez J and Murphy S, 1983. Exposure of fibrinogen receptors on fresh and stored platelets by A D P and epinephrine as single agents as a pair. Blood 61:1054-1059.  Moroff G and Holme S, 1991. Concepts about current conditions for the preparation and storage of platelets. Trans Med Reveiws V:48-59.  77  Murphy S and Gardner F, 1969. Platelet preservation-Effect of storage temperature on maintenance of platelet viability-deleterious effect of refrigerated storage. New EngJ Med 280: 1094-1098.  Murphy S, Kagen L, Holme S, Gottlieb B, Heaton W, Grode G, Davisson W, Buchholz D, 1991. Platelet storage in synthetic media lacking glucose and bicarbonate. Transfusion 31:16-20.  Murphy S, Rebulla P, Bertolini F, Holme S, Moroff G, Snyder E, Stromberg R, 1994. In vitro assessment of the quality of stored platelet concentrates. Trans Med Rev 8:29-36.  Murphy S, Seyed S and Frank G, 1970: Storage of platelet concentrates at 22 °C. Blood 35:549-557.  Murphy S. and Frank G, 1975. Platelet storage at 22 °C: Role of gas transport across plastic containers in maintenance of viability. Blood 46:209-218.  Muylle L , Joos M , Wouters E, De Bock R, Peetermans M , 1993. Increased tumour necrosis factor a (TNF-a), interleukin-1 (IL-1) and interleukin-6 (IL-6) levels in the plasma of stored platelet concentrates: relationship between T N F - a and IL-6 levels and febrile transfusion reactions. Transfusion 33:195-199.  78  Nieuwenhuis A , van Oosterhout J, Rozenmuller E, van Iwaaden F, Sixma J, 1987. Studies with a monoclonal antibody against activated platelets: Evidence that a secreted 53,000 molecular weight lysosome-like protein is exposed on the surface of platelets in the circulation. Blood 70:838-845.  Packham M , Kinlough-Rathbone R and Mustard J, 1987. Thromboxane A2 causes feedback amplification involving thromboxane A2 formation on close contact of human platelets in media with a low concentration of ionized calcium. Blood 70:647-651.  Peerschke E, Ghebrehiwet B, 1987. Human blood platelets possess specific binding sites for C l q . J Immunol 138:1537-1541.  Peerschke E, Ghebrehiwet B, 1992. Platelet interactions with C l q in whole blood and in the presence of immune complexes or aggregated IgG. Clin Immunol Immunopathol 63:45-50.  Peerschke E, Reid K , Ghebrehiwet B, 1993. Platelet activation by C l q results in platelet activation. Am J Clin Pathol 106:795-798.  Peerschke E, Reid K , Ghebrehiwet B, 1993. Platelet activation by C l q results in the induction of anb/fc integrins (GPIIb-IIIa) and the expression of P-selectin and procoagulant activity. J Exp Med 178:579-87.  79  Polley M , Nachman R, 1983. Human platelet activation by C3a and C3a des arg. JExp Med 158: 603-615.  Rinder H and Snyder E, 1992. Activation of platelet concentrate during preparation and storage. Blood Cells 445-455.  Rinder H, Murphy M , Mitchell J, Sticks J, Ault K, Hillman R, 1991. Progressive platelet activation with storage: evidence for shortened survival of activated platelets after transfusion. Transfusion 31:409-414.  Rock G, White J, Lebow R, 1991. Storage of platelets in balanced salt solution: a simple platelet storage medium. Transfusion 31:21-25.  Schleuning M , Bock M and Mempel W, 1994. Complement activation during storage of single-donor platelet concentrates. Vox Sang 67:144-148.  Schleuning M , Schmid-Haslbeck M , Utz H , Jochum M , Mempel W, Wilmanns W, 1992. Complement activation during storage of blood under normal blood bank conditions. Effect of proteinase inhibitors and leukocyte depletion. Blood 79:3071-3075.  Seghatchian J, Krailadsiri P, 1997. The platelet storage lesion. Tranfus Med Rev 11:130-144.  80  Sevastinov V , Tseytlina E, 1985. The activation of the complement system by polymer materials and their blood compatibility. Biomed Mater Res 18:969.  Shiba M , Tadkoro K , Sawanobori M , et al 1997. Activation of the contact system by filtration of platelet concentrates with a negatively charged white cell-removal filter and measurement of venous blood bradykinin level in patients who received filtered platelets. Transfusion 37:457-462.  Shimizu T, Ishikawa Y , Morishima Y , Fukuda T and Kato K, 1985. Platelet factor 4 release from the platelets stored in platelet concentrates. Transfusion 25:420-423.  Shimizu T,Uchigiri C, Mizuno S, Kamita T and Kokubo Y , 1994. Adsorption of anaphylatoxins and platelet-specific proteins by filtration of platelet concentrates with a polyester leukocyte reduction filter. Vox Sang 66:161-165.  Sims P, Faioni E, Wiedmer T, Shattil S, 1988. Complement proteins C5b-9 cause release of membrane vesicles from the platelet surface that are enriched in the membrane receptor for coagulation factor Va and express prothrombinase activity. JBio Chem 263:18205-18212.  Skojonsberg O, Kierulf P, Fagerhol M , Godol H, 1986. Thrombin generation during collection and storage of blood. Vox Sang 50:33-37.  81  Slichter S and Harker L, 1976. Preparation and storage of platelet concentrates. II Storage variables influencing platelet viability and function. Br J Haematol 34:403-419.  Snyder E, 1992. Activation during preparation and storage of platelet concentrates. Transfusion 32:500-502.  Snyder E, Hezzey A , Katz A , Bock I, 1981. Occurrence of the release reaction during preparation and storage of platelet concentrates. Vox Sang 41:172-77.  Stack G, Snyder E, 1994. Cytokine generation in stored platelet concentrates. Transfusion 34:20-25.  Tallan H , Moore S, Stein W, 1956. N-acetyl-L-aspartic acid in brain. J Biol Chem 219:257-264.  Valeri C, Feingold H and Marchionni L, 1974. The relation between response to hypotonic stress and the 51Cr recovery in vivo of preserved platelets. Transfusion 14:331-336.  Wadhwa M , Seghatchian M , Lubenko A , et al 1996. Cytokine levels in platelet concentrates: quantitation by bioassays and immunoassays. Br J Haematol 93:225-234.  82  Washitan Y , Irita Y , Yamamoto Y , et al 1988. Prevention of acquired defects in platelet function during blood processing. Transfusion 28:571-75.  White JG, 1981. Ultrastructural lesions of stored platelets. Vox Sang 40:62-8.  Zwaal R, Schroit A , 1997. Pathophysiologic implications of membrane phospholipid asymmetry in blood cells. Blood 89:1121-1132.  83  

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