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Enhancement of the epitopic activity of anti-thrombotic peptidomimetics by conjucation to a macromolecular… Zhang, Jerry Gongdu 2008

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Enchancement of the Epitopic Activity of Anti-Thrombotic Peptidomimetics by Conjugation to a Macromolecular Carrier by JERRY GONGDU ZHANG B.Sc. (Honours.), The University of British Columbia, 2007 A THESIS SUBMITTED iN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE In THE FACULTY OF GRADUATE STUDIES (Pathology and Laboratory Medicine) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) July 2008 ©Jerry Gongdu Zhang, 2008 Abstract Background: Novel peptides with epitopic activity can be discovered by a combination of peptide arrays and computational interpolation. These peptides can mimic the activity of cellular receptors or their ligands, and are thus called peptidomimetics. The activity of peptidomimetics evidently varies in accordance with the attributes of the peptide. The fibrin-mimetic, Arginine-Glycine-Aspartate (RGD), is a well known anti-thrombotic peptide which inhibits the interaction between the platelet integrin GPITbIIIa and fibrinogenlfibrin. Short peptides often suffer from high inhibitory concentrations in vitro and short clearance times in vivo. To increase vascular residence times of such antithrombotic peptides, they were coupled to macromolecular carriers. Hyperbranched polyglycerols (I{PG), macromolecules designed as a dendritic carrier species, at a range of HPG molecular weights (MW) were tested as the carriers for antithrombotic peptidomimetics. Methods: HPG of MW 3 to 500 kDa were conjugated with RGD at a range of substitution ratios. The optimum molecular weight and substitution ratio were then applied to the peptide SHAYIGLKDR, a vWf mimic peptide discovered through the use of bioinformatics. For peptidomimetics (RGD and SI{AYJGLKDR), enzymatic proteolysis was employed to distinguish the specificity of the inhibitory activity among HPG, peptide, and the HPG-peptide conjugate. Flow cytometry, UV spectroscopy, compound light microscopy, and lummiaggregometry were used to characterize the function of the conjugates in vitro. 11 Results: Conjugation of RGD to HPG resulted in a decrease of the inhibitory concentration required to interrupt platelet-fibrinogen interactions by up to three orders of magnitude. Inhibitory activity was directly related to the number of peptides attached per HPG. Similar results were found when high molecular weight HPG, selected from the RGD-related experiments, was used to carry the peptide SHAYIGLKDR. None of HPG, RGD, SHAYIGLKDR, or their conjugates caused spontaneous platelet activation, or inhibited thrombin-mediated platelet activation, showing that the peptides’ activity is directed specifically toward their targets: GPllblllalfibrinogen (RGD) and GPIb/vWf (SHAYIGLKDR) interactions. Tryptic digestion of conjugates confirmed that the inhibitory activity of HPG conjugates was dependent on the presence of the intact peptides. Conclusions: Conjugation of peptidomimetics or other molecules to macromolecular platforms such as HPG is a viable method to enhance the peptidomimetics’ activity. The degree of enhancement is dependent upon the level of peptide substitution as well as the size of the carrier. 111 Table of Contents Abstract ii Table of Contents iv List of Tables Vi List of Figures vii List of Abbreviations ix Acknowledgements X CHAPTER 1. Introduction 1 1.1 General Platelet Characteristics and Function 1 1.2 Platelet Integrins and Mechanism of Action 3 1.3 Anti-thrombotics 7 1.4 Peptidomimetics 10 1.5 Macromolecular Carriers and Conjugation 14 1.6 Peptide Polarity and Chirality 18 1.7 Hypothesis 21 CHAPTER 2. Materials and Methods 23 2.1 Peptides and Proteins 23 2.2 Preparation of Human Platelets 23 2.3 HPG-RGD Conjugates 24 2.4 HPG-SHAYIGLKDR Conjugates 28 CHAPTER 3. Results 35 3.1 HPG-RGD Conjugates 35 3.2 HPG-SHAYIGLKDR Conjugates 45 CHAPTER 4. Discussion 52 4.1 HPG-RGD Conjugates 52 4.2 HPG-SHAYIGLKDR Conjugates 57 4.3 Summary 61 CHAPTER 5. Conclusion 63 iv Bibliography 65 Appendices 72 Appendix A 72 V List of Tables Table 3.11 Synthesis, characterization, and evaluation of RGD and RGD-HPG conjugates 36 Table 3.12 Light microscopy of RGD-HPG conjugates and aggregation of resting and Mn2 activated platelets in the presence of free RGD peptides and RGD-HPG conjugates at their respective 1c5°values 41 Table 3.21 Functionalization and Characterization of HPG and HPG Conjugates 46 Table 3.22 Light microscopy, aggregometry, and flow cytometry of HPG-conjugates with resting andlor activated platelets 46 vi List of Figures Figure 1.11 Platelet-Platelet and Platelet-Subendothemlium Interactions During Thrombosis 2 Figure 1.21 Platelet Activation Signalling Pathways 6 Figure 1.31 Conventional Antithrombotics and Their Targets in Platelet Signalling Pathways 9 Figure 1.41 MIAX Interpolated vWfPeptidomimetic (SHAYIGLKDR) Inhibition of GPTb-vWf Interaction 12 Figure 1.51 Carrier Valency 15 Figure 1.52 Relationship Between Linker Length and Degrees of Freedom in High Density Brushes 16 Figure 1.61 Nomenclature of Peptide Polarity 19 Figure 1.62 The SHAYIGLKDR Peptide and its D-Enantiomer 20 Figure 2.31 Functionalization of ITPG and Conjugation of RGD Peptides 25 Figure 2.41 Functionalization of HPG and Conjugation of SHAYIGLKDR Peptides.... 30 Figure 3.12 Evaluation of the Effect of Unconjugated HPGs on Resting and Activated Platelets 37 Figure 3.13.1 Effect of HPG-RGD Conjugates on Fibrinogen Binding by Resting Platelets 38 Figure 3.13.2 Trypsin Proteolysis Assay of RGD and HPG-RGD Conjugates 39 Figure 3.14 Inhibitory Effects of HPG-RGD Conjugates on Platelet-Fibrinogen Binding 42 Figure 3.15 Relationships Among k50, HPG Polymer Molecular Weight and Amount of RGD Peptides Conjugated to HPG 43 Figure 3.16 Light Microscopy and Aggregometry 44 Figure 3.23.1 Inhibition of Platelet-vWf Binding by Peptide-conjugated HPG 49 Figure 3.23.2 Trypsin Proteolysis Assay of SIIAYIGLKDR and HPG SHAYIGLKDR Conjugates 50 vii Figure 3.23.3 Effect of Peptide on GPIIbIIIa Activation Through GPIb Signalling 51 Figure 4.11 Proposed Mechanism A: Polyvalency of HPG-RGD Conjugates 55 Figure 4.12 Proposed Mechanism B: Higher Local Concentration 56 viii List of Abbreviations DVS = Divinyl sulfone GPIb-IX-V = Glycoprotein Tb/TX/V integrin complex GPllbllIa = Glycoprotein TIb/ifia integrin complex HPG Hyperbranched Polyglycerols = Inhibitory Concentration, a concentration at which 50% inhibition is achieved M = Number Average Molecular Weight M = Weight Average Molecular Weight MW = Molecular weight PAR = Platelet activated receptor PDI = Polydispersity PEG = Polyethylene Glycol P1= Phosphoinositol RGD = Arginine-Glycine-Aspartic Acid SBTI = Soybean Trypsin Inhibitor SHAYIGLKDR polypeptide Serine-Histidine-Alanine-Tyrosine-Isoleucine-Glycine Leucine-Lysine-Aspartic Acid-Arginine TP Thromboxane/prostanoid receptor VS = Vinyl sulfone vWf= von Willebrand Factor ix Acknowledgements I would like to thank Canadian Blood Services, Canadian Institutes of Health Research, NSERC, the Canadian Foundation for ftnovation, the Heart and Stroke Foundation of Canada, and the UBC Centre for Blood Research for their funding support of this project and its personnel. People with experimental contributions are gratefully acknowledged: Dr. Rajesh Kainthan produced all HPG molecules of varying molecular weights, and all HPG-RGD conjugates. Dr. William Campbell produced the vWf sequence peptide array from which we derived the preliminary vWf peptidomimetics and Dr. Carlos Del Carpio used MIAX bioinformatics software to narrow down our selections of those peptides. Mr. Oren B. Krajden performed the preliminary tests on the HPG-RGD conjugates and Ms. Wendy W.Y. Lin contributed to the establishment of the ristocetin-vWf mediated platelet activation assays in flow cytometry, microscopy, and aggregometry. I would like to further acknowledge the people of Canadian Blood Services Devine-Issa Lab: Peter Shubert, fren Constantinescu, Katherine Serrano, Elena Levin, and Brankica Culibrk for their assistance with my experiments. Finally, I would like to thank my research supervisor, Dr. Maria Gyongyossy-Issa, without whose source of crazy ideas, awesome support, and endless cat-sitting chores, these projects and this thesis could never have materialized. No graduate students were badly harmed in the production of this thesis. x Chapter 1 — Introduction 1.1 General Platelet Characteristics and Function Platelets are anucleate cell fragments of megakaryocytes, 1-2 im in diameter (Kaushansky, 2005). Platelets enclose a complement of secretory granules: ct-granules contain thrombospodin, von Willebrand Factor (vWf), coagulation factors, fibrinogen, ADP, P-selectin; and dense granules contain ADP/ATP, Ca2 histamines, serotonin, and epinephrine; though the majority of compounds/proteins in these granules have not been identified (Coppinger & Maguire, 2007). The contents of these granules mediate thrombosis and vascular repair through extensive signalling mechanisms involving endothelial cells, leukocytes, plasma proteins, and the platelets themselves. The primary function of platelets is to participate in thrombus formation by anchoring through Glycoprotein Ib-vWf (Varga-Szabo et al., 2008) to collagen exposed on the damaged subendothelium and cross-linking each other through Glycoprotein IIbIIIa-fibrin(ogen) (Varga-Szabo et al., 2008). The consequent release of cytokines and chemokines signal other platelets (Coppinger & Maguire, 2007), and interact with polymerized fibrin fibrils to perform clot retraction (Mosesson, 2007; Weyrich et a!., 2007). To realize these functions, platelets are also highly amoeboid: cytoskeletal remodelling of actin allows platelets to assume a variety of shapes both for motility and during activation (Bearer et a!., 2002). Although platelets do not contain nuclei, regulation and control of cytoplasmic proteins is thought to be controlled by translation of innate mRNA, inherited from megakaryocytes (Weyrich et al, 2007). Platelets have a maximum in vivo lifespan of 9-11 days before apoptosis mechanisms are triggered, but they may be cleared by ( Figure 1.11 Platelet-platelet and Platelet-subendothelium Interactions During Thrombosis. Platelets are cross-linked by interaction of GPIIbIIIa with polymerized fibrinogenlfibrin fibrils and anchored to the site of damage by interaction of GPIb and vWf to exposed collagen. Endothelial Cell = Fibrinogen b Von Willebrand Factor < =Site ofEndothel ial bamoge Collagen 2 macrophages before this time due to a steady basal level of activation stimulus experienced during circulation (Mason et al., 2007). 1.2 Platelet Integrins and Mechanism of Activation Platelet activation and thrombus formation are initiated by signalling pathways rooted in the cell-surface integrin receptors and the receptors’ binding of their ligands (Gibbins, 2004; Calvete, 2004; Du 2006; Figure 1.2). Several such receptors and their respective ligands are particularly important: GPIb with vWf PAR1/PAR4,P2Y1/P2 TP receptors with soluble antagonists thrombin, ADP, and thromboxane A2; GPIIbIIIa - fibrinogen/fibrin. Platelet activation can be triggered through mechanisms related to these integrins (Shattil & Newman, 2004). Probably the initial activation mechanism is through von Willebrand factor. Conditions of high hydrodynamic shear, often induced by endothelial damage, activate vWf (250 kDa) in the plasma (Ruggeri, 2007). vWf will spontaneously bind to collagen, which is only exposed where there is endothelial damage (Andrews et at., 1997; Lopez & Dong, 2005; Ruggeri, 2007). Activation of vWf by conformational change (Andrews et at., 1997; Ruggeri, 2007) exposes the Al-domain, which will cause vWf to bind to the platelet surface integrin GPIb-IX-V complex (Lopez & Dong, 2005; Varga-Szabo et al., 2008). This binding induces conformational changes of the integrin complex that trigger tyrosine kinases attached to the cytoplasmic portion of GPIb. In turn, these kinases trigger PKG and phospholipase C activation (Yin et at., 2008), resulting in platelet activation by degranulation of the cytokine/chemokine containing u-granules and dense 3 granules, and result in the collateral activation of GPIIbIIIa to the active conformation (Gibbins, 2004; Beer eta!., 1992). A subsequent mechanism of platelet activation is also thought to function during the amplification of the platelet activation cascade and is triggered by soluble agonists (Dawood et a!., 2007). These agonists are stored in platelet ct-granules and include ADP, thromboxane A2 and platelet-derived thrombin and they are used in an autocrine/paracrine manner by platelets to stimulate the surface integrins (Dawood et a!., 2007; Vilahur et a!., 2007). Integrins activated by these cytokines include PAR1, PAR4, P2Y1,P2Y12, and TP. These receptors are involved in platelet aggregation, P1-3 kinase activation, Ca2 influx, and phospholipase C activation, which all lead to iterative degranulation and activation of GPIIbIIIa to the active conformation (Dubois et a!., 2004; Du, 2007). Another of these activation mechanisms is dependent upon thrombin (Factor ha), which is the end target for activation of the coagulation cascade (Green, 2006). Thrombin cleaves fibrinogen into fibrin, which polymerizes and binds to platelet GPIIbIIIa in its open conformation (Kasirer-Friede et a!., 2001). This interaction causes Ca2 influx and phospholipase C activation, which again leads to platelet activation by degranulation. Fibrinogen binding to platelet GPIIb/IIIa induces a conformation change of the integrin such that signal transduction leads to Ca2 influx into the platelet, which triggers further degranulation (Watson eta!., 2005; Du, 2007). It should be noted that thrombin interacts with a variety of platelet integrins including GPIb, GPIIbIIIa, PAR1/PAR4, etc. Thrombin binding may enhance ligand binding or help localize fibrin generation to platelet surfaces (GPIb), or may serve to 4 induce platelet activation by direct interaction with those integrins (PAR receptors) (Dubois et al., 2004; Du, 2007). MI of these pathways result in the release of ct-granule contents and the exposure of P-selectin (CD62) on platelet surfaces, which allow platelets to attach to endothelial cells (Jurk & Kehrel, 2005) and leukocytes (McGregor et a!., 2006) and initiate a rolling mechanic also observed for leukocytes. Hence CD62 surface expression is often considered a global sign of platelet activation — it is a sign that one or more of the above mentioned mechanisms has been triggered. Other ways to evaluate platelet activation also include platelet-fibrinogen binding, and platelet-vWf binding. It is useful to note that independent of the coagulation cascade, which produces thrombin from plasma-derived prothrombin, platelet activation is a step-wise process. Platelet GPIb binds activated vWf the platelet degranulates and rolls along the endothelium, the cytokines and chemokines released by platelets during degranulation trigger further degranulation and activate GPIIbIIIa. GPIIbIIIa binds fibrinogenlfibrin and further facilitates platelet activation. Eventually platelets will roll into the damaged site and be anchored to collagen by the other end of vWf multimers, and as more and more thrombin as well as platelets accumulate, platelets become cross-linked by fibrin fibrils through their activated GPIIbIIIa receptors, and a thrombus is formed. Further thrombus refinement such as clot retraction occurs within hours of initial thrombus formation (Zimmerman & Weyrich, 2008). Hence, GPIb-vWf interaction is often considered to be upstream of most platelet activation mechanisms, a potentially useful target for clinical applications (Clementson & Clementson 2008). 5 1 I 1 1 — PA1/PAR4 P13 K 3zta 411 -)Akt PhosphoHpaeC411 eNQS J,NO PKG’ •— ERK I (I GPIb-IX-V vW rnnn Co2+ ,IF? GPIIb/tIIa I ill ii I_j,1l1u II I 1’ Thromboxane A2 I TP receptor frK1 Rap 1 + Unknown Kfriase AtP P2Y1/P2Y12 Figure 1.21 Platelet Activation Signalling Pathways This is a generalized graphic representation of platelet signalling during activation. Items in red denote cytokines and chemokines that act as agonists for various platelet integrins. SFK: Src family kinases; P13K: P13 kinase; PKC: protein kinase C; PKG: Protein kinase G; eNOS: endothelial nitric oxide synthase; NO: Nitric Oxide; ERK: extracellular responsive kinase; TP: 6 1.3 Antithrombotics Antithrombotics are a series of inhibitors aimed at mitigating the coagulation cascade, platelet activation, platelet attachment to the endothelium, and subsequent thrombus formation (Vanhoorelbeke et al., 2003; Clementson & Clementson 2008). Due to the complexity of coagulation and thrombosis, many drugs exist which target different aspects of thrombus formation. These drugs can be categorized with respect to their effect on the coagulation cascade or platelet activation: vitamin K antagonists, anti thrombin III enhancers, GPIIb/IIIa inhibitors, platelet aggregation inhibitors, plasminogen activators, direct thrombin inhibitors, and non-specific chemical compounds (Figure 1.31), inhibitors of the thromboxane/prostanoid receptor; P2Y1/P2Y12: G coupled receptors of the P2Y nucleotide-sensing family (Du, 2006). Vitamin K antagonists interfere with vitamin K dependent synthesis of clotting factors II (thrombin), VII, IX, X, and in turn disrupt the coagulation cascade, preventing fibrin generation and platelet cross-linking (Merli & Fink, 2008); classic examples include warfarin and FluindioneTM (Bossavy et aL, 1999). Anti-thrombin enhancers bind to anti-thrombin and increase its affinity to thrombin by inducing slight conformational changes, resulting in reduced active thrombin and lowered platelet activation as well as reduced fibrin generation (Selwyn, 2003); heparin is a classical example of such a compound (Bjork & Lindahi, 1982; McRae & Ginsberg, 2005; Prandoni et al. 2008). GPIIbIIIa inhibitors block fibrinogen/fibrin attachment to GPIIbIIIa, thus preventing platelet-platelet cross-linking and GPIIbIIIa-mediated platelet activation via phospholipase C: examples include AbciximabTM (Gabriel & Oliveira 2006), EpitifibatideTM (Zeymer & Wienbergen 2007), and TirofibanTM (Bukow et al., 2006). 7 Platelet aggregation inhibitors are a large family of platelet inhibitors on their own; they act on aggregation-associated platelet integrins PAR1/PAR4, P2Y1/P2 TP, and their associated binding targets. For example, the popular AspirinTM (Watson & Chee YL, 2008; Yasuda et al., 2008) blocks thromboxane A2 generation (which triggers TP activation), and PrasugrelTM (Niitsu et al., 2005) inhibits ADP receptors P2Y1/P2 to prevent amplification of platelet activation. Plasminogen activators such as streptokinase convert plasma derived plasminogen into plasmin, which is an enzyme that breaks down fibrin fibrils, and represent the so-called clot-busting drugs rather than clot preventing drugs (Juttler et al., 2006). Direct thrombin inhibitors such as ArgatrobanTM (Bauer, 2006) are almost self-explanatory, they inhibit thrombin and hence thrombin generation of fibrin, which drastically reduces platelet-platelet cross-linking. Lastly, there are non specific chemical compounds such as citrate, EDTA, and oxalates, which chelate plasma Ca2 (Chappell, 2007) and in turn reduce Ca2 influx into platelets thus inducing platelet activation; these compounds are not used in vivo due to their non-specific nature. Antithrombotics are primarily employed in clinical scenarios where thrombus formation could lead to life-threatening ischemic events such as stroke or pulmonary embolism (Agnelli & Sonaglia, 2002; Katira et aL, 2005). Hence antithrombotics are widely used during and post surgery and their popularity is expected to rise with the increase in the prevalence of cardiovascular disease. However, it should be noted that many current drugs (warfarin, heparin, and their analogues) are mostly focused on the coagulation cascade and not necessarily on the platelets themselves. This could be problematic as platelets will still attach to the damaged endothelium and cross-link through fibrinogen even in the absence of thrombin and clotting factors; platelet-rich 8 GPIb-]x-v Antc-th’-Drnbin ( [arin _frbT4, Cftr’Gt vWf \ Ca2 Fibrinogen Thrombin —IArqatorbGn I GPrIb/IIIa PAR1/PA4 TQlin 14-3-3zta SFK P13K Abcxiiuib, Eptifb&id€, TrofibGn Ca2+ Akt Phospholipase C PKC Thrombo<are A2 eNO5 Rap 1 NO IIAspirin I TP receptor PKG ERK + Thromboxare A2 Unknown Fibrinogen thrombiri Kinase vWf ADP Co2+ Granule Secretion P13K AbP Prosure{ i— P2Y1/P2Y12 Figure 1.31 Conventional Antithrombotics and Their Targets in Platelet Signalling Pathways. The platelet signalling pathways are shown in gray. Platelet integrin antagonists are denoted in red. Antithrombotics are represented by black boxes proximal to their targets. 9 thrombi could still be formed under these conditions and could cause life threatening events. For example, heparin induced thrombocytopenia (HIT) is a disease resulting from the use of heparin that may lead to life-threatening thrombotic events (Castelli et al., 2007; Joost et a!., 2008; Linkins & Warkentin 2008). Hence understanding the function of platelet integrins and their associated signalling mechanisms may help identify therapeutic targets for the generation of future antithrombotics (Vanhoorelbeke et al., 2003; Clementson & Clementson 2008) In particular, the GPIb-IX-V receptor and its interaction with vWf were mentioned previously as being upstream of most platelet activation mechanisms and are a useful avenue to explore for potential therapeutics (Clementson & Clementson, 2008). In this study, I will verify the anti-thrombotic activity of RGD and, more importantly, investigate the efficacy of a peptidomimetic inhibitor of the GPIb-vWf interaction. 1.4 Peptidomimetics Peptidomimetics are short amino acid sequences that have shape and hydropathic profiles that provide epitopic behaviour. Based on their attributes, they can be screened selectively for ligand- or receptor-like activity to target specific binding sites. The prospect of deploying such polypeptides in a therapeutic setting presents a promising lead in rational drug-design. Novel peptidomimetics can be derived from peptide arrays of random amino acid sequences, or overlapping sequences from a ligand or receptor (Grainger et a!., 2007). In either case, the sequences with binding potential and possible inhibitory activity are probed with a target protein that may or may not bind sequences from the array. The materials that bind to the array can in turn be detected by a variety of detection systems, often based on antibodies in a manner similar to a western blot. 10 Positive sequences from peptide arrays may be tested for binding activity in two ways: by laboratory testing of compound libraries (Wiley & Rich, 1993), or by the application of bioinformatic models (Del Carpio Munoz et al., 2006; Urbanj et a!., 2006). One approach involves manufacturing all the candidate peptides and threading them through functional assays, and the other, perhaps more cost-effective method, involves computational interpolations of the free-energy release associated with peptide binding to the active sites of target proteins (Del Carpio Munoz et al., 1999; Yoshimori et a!., 2001). Applications of peptidomimetics are numerous; for example, a peptidomimetic was used to induce apoptosis of malignant leukemic cells without affecting healthy tissue Walensky et al. (Walensky et al., 2004); peptidomimetics have been used to inhibit thrombin (Hauptinann, 2002). Pertinent to this study, one of the best-known peptidomimetics, RGD was coupled to liposomes and used to target activated platelets as a method of vascular drug delivery (Gupta et a!., 2005). The RGD peptide sequence is a motif found in fibronectin, vitronectin, osteopontin, collagens, thrombospondin, and fibrinogen, and can mediate integrin adhesion and cellular communication (Rouslahti & Pierschbacher, 1987). The RGD motif disrupts platelet aggregation by binding to the platelet glycoprotein Jib/lila integrin receptor (GPIIbIIIa) to cause competitive exclusion of fibrinogen from its binding site (Beer et a!., 1992). RGD and RGD-containing conjugates are effective antithrombotics (Gould, 1994) because fibrinogen-mediated bridging and platelet aggregation are essential steps in primary haemostasis. The GPIIbIIIa - fibrinogen interaction is clinically relevant and a new generation of antithrombotics specifically targets this interaction (Gabriel & Oliveira, 2006). To this end, a number of laboratories have defined the pharmacophore of RGD (Ojima et a!., 11 Figure 1.41. MIAX Interpolated vWf Peptidomimetic (SHAYIGLKDR) Inhibition of GPIb-vWf interaction. The bottom protein domain in red is a truncated vWf-binding active site of GPJb. The top protein in blue is von Willebrand factor. The peptide in grey binds GPIb at its vWf-binding active site, and prevents vWf attachment to GPIb. 12 1995), and explored a wide variety of chemical modifications on simple peptidomimetic structures designed to compete with fibrinogen, in order to improve either their binding affinity for GPIIbIIIa or their pharmacokinetic profile (Walensky et al., 2004; Gould, 1994; Gabriel & Oliveira, 2006; Ojima et a!., 1995; Lee & Marchant, 2003; Mousa et a!., 2001). RGD, RGD variants, and RGD mimics have proven to be successful antithrombotics both in vitro and in vivo (Nicholson et a!., 1991; Sheu & Huang, 1994). Although RGD seems somewhat promising, it interferes with GPIIbIIIa’s interaction with fibrinogen, which is a step of platelet activation that occurs late in the activation cascade, especially compared to the vWfs ineraction with the GPIb-IX-V complex and its consequent effects. Relying on RGD-based antithrombotics still leaves activated and degranulated platelets. Since platelet granules are not renewed (Harrison & Cramer 1993), patients receiving RGD-based drugs would inevitably lose those activated platelets to macrophages, thereby lowering their platelet count. Hence, it is more beneficial to look for upstream targets in the platelet activation cascade that prevent platelets from becoming activated in the first place. The interaction of vWf with the GPIb-IX-V complex is an excellent upstream target for drug interference. For this purpose, we had identified SHAYIGLKDR as a high affinity peptide binding the platelet glycoprotein GPIb. This peptide was derived by synthesizing overlapping peptides from the native sequence of vWf on an L-peptide array and identifying active peptides by binding to GPIb. Using the MIAX bioinformatics program (Del Carpio Munoz et a!., 1999; Yoshimori et a!., 2001; Del Carpio Munoz et a!., 2003) SHAYIGLKDR was mapped to the Al domain of the vWf sequence and was projected by MIAX to bind GPIb in the 13 GPTb-vWf interactive domain. It was hoped that by locating to the GPIb interactive surface, this peptide would inhibit vWf binding to the same location. 1.5 Macromolecular Carriers and Conjugation In order to be clinically useful, a mimetic compound must evade rapid elimination from the host and demonstrate high efficacy at comparatively low doses. RGD, RGD variants, and RGD mimics suffer from limited in vivo retention due to their small size. A well known solution to this problem is to couple such peptides to long-circulating materials. Studies have described the conjugation of RGD to albumins (Temming et al., 2006), to polyethylenimine — polyethylene glycol (Kim et al., 2003) and to liposomes (Janessa et aL, 2003). Other macromolecules, such as hyperbranched polyglycerols are also potentially viable choices as carriers. HPGs are water soluble, biocompatible polyether polyois (Sunder et al., 2000; Sunder et al., 2000) that can be synthesized in a controlled manner with predetermined molecular weights and narrow molecular weight distributions (Kainthain et aL, 2006). There are two general choices when selecting a macromolecular carrier: monovalent/bivalent carriers, and polyvalent dendrimers (Figure 1.51). The first encompasses carriers such as linear PEG molecules to which peptides may be attached at one or both ends; the latter includes carriers with many “dendrites” that consequently can host a high number of peptides. While both serve to increase the MW of the peptide they carry, the latter carriers have the potential of also altering the equilibrium dynamics by presenting their target as a higher local concentration of peptides. Thus macromolecules with dendrites were chosen as the carriers for this study. Recently Kainthan et a!. reported the synthesis of very high molecular weight HPGs (MW values up to 1.48 x 106) with low PDI: where PDI M/M=1.1-1.4 where 14 A) B) C) Figure 1.51. Carrier Valeucy. Diagramatic representation of (A) monovalent, (B) bivalent, and (C) polyvalent carriers. Red ends on panel A, B, and C denote peptides. 15 Figure 1.52. Relationship Between Linker Length and Degrees of Freedom in High Density Brushes. The HPG (10 nm) is not to scale relative to the length of the peptides (2 nm). The left side of the figure shows the differences in the available space (red double tipped arrows) for the tips of the peptides conjugated to HPG at different heights. The right side of the figure demonstrates that some level of flexibility may be required for binding to receptors (GPIIbIIIa). Rotationa’ Hex i b ii it> / / HPG 16 M and M are the weight and number molecular weights respectively (Kainthain et a!., 2006). These polymers show excellent biocompatibility properties both in vitro (Kainthan et al., 2007) and in vivo (Kainthan & Brooks. 2007). The plasma circulation half lives were found to depend on the molecular weight and half lives as high as 60 hours were achieved for high molecular weight polymers (Kainthan & Brooks, 2007). As the number of hydroxyl groups approximately equals the degree of polymerization, synthesis of higher molecular weight HPGs is a major development given the difficulty of synthesizing high molecular weight dendrimers. These materials have numerous applications in nano-medicine and one such application is the use of HPGs as carriers of peptides for biomedical applications where multivalency/polyvalency can be exploited. The hydroxyl groups on HPG can be coupled to sulfhydryl groups on cysteines in a peptide through divinyl sulfone (DVS). First the HPG is functionalized by coupling to DVS, the resulting HPG molecule with DVS (HPG-VS) acquires an acryl group that is thiol-selective, which is excellent for coupling to the sulthydryl group on a cysteine (Bulmus et a!., 2000). A terminal cysteine residue can be added to the peptidomimetic sequence for this purpose. In addition, a poly-glycine linker is used as a spacer, which could grant the peptidomimetic more flexibility as well as more space to maneuver (Figure 1.52). Due to the novelty of high molecular weight HPG, no information was available about its potential to function as a carrier for peptidometics. To test the potential of this molecule as a carrier for peptidomimetics, the well-known peptidomimetic, RGD was tested instead of directly proceeding to our novel peptide, SHAYIGLKDR. Therefore, our theories of polyvalency were tested initially with the 17 RGD molecule and then the optimized parameters from the RGD study were applied to the novel peptide. 1.6 Peptide Polarity and Chirality Conjugation of peptides to a surface such as a macromolecular carrier automatically confers polarity to that peptide. This directionality may, or may not affect the peptide’s activity by keeping the pharmacophore at a distance from the target site and closer to the surface of the macromolecular carrier; or by orienting it in a way that would prevent the peptide from fitting to its target. Hence it is always prudent to consider both forward and reverse (reverso-) forms of a peptide during conjugation (Figure 1.61). Short peptides sequences such as RGD are less susceptible to this problem compared to longer peptides such as SHAYIGLKDR, especially when the actual pharmacophore is not known in detail. In addition to the directional orientation imparted by conjugation to a carrier, peptide specificity is also controlled by charge and side-chain orientation. Although these two factors are often considered hand-in-hand, charge interactions are generally less specific than side-chain interactions, as side-chain orientation is important for fitting the peptide into a specific binding pocket that exists in a relatively fixed orientation. However, it is prudent to evaluate which of these factors dominates experimentally because these principles are general but may not be applicable to selected situations. Hence, for this study we also created the peptidomimetics as D-enantiomers (Figure 1.62), those peptides with opposite backbone/side-chain orientations, as well as their D reverso counterparts. This design was intended to evaluate whether the specificity of the SHAYIGLKDR peptide is primarily charge driven or orientation driven. Hence, the D 18 Forward N2 CGGGGGG 5AYIGLKb__>coo fteverso N- CGGGGGG RDKLGIYAHS FC00H Figure 1.61. Nomenclature of Peptide Polarity. All amino acids represented by their single letter amino acid names are written from the amino terminus to the carboxy terminus (left to right). The linker peptide is denoted in blue, and the peptidomimetic peptide is shown in red as either “forward” or “reverse.” This convention applies to the amino sequence only; a normal peptide bond (in green) indicates that the amino- and carboxy- termini were not altered/reversed. Large arrows housing the peptide sequences indicate arbitrary labelling convention for “forward” and “reverse.” 19 O’f’NHNH O:C3 NI-I Cl-I3 0 CH3 ICOH L _ H L. isomer Figure 1.62. The SHAYIGLKDR Peptide and its D-enantiomer. The SHAYIGLKDR peptide is represented as both L- and D- enantiomers, which are mirror images of one another. Coloured models at the bottom of the figure are guides to the L & D naming convention for amino acids. D isomer 20 enantiomers serve as an important control for SHAYIGLKDR as the peptide was originally derived as an L-enantiomer. As well, ideally for drug design, a peptide that resists in vivo enzymic degradation would be an asset and this was a further rationale for the assessment of D-enantiomer function. 1.7 Hypothesis We hypothesize that it is possible to create a new class of antithrombotic materials based on the combination of a peptidomimetic and a macromolecular carrier such that the peptidomimetic’s anti-platelet potential is enhanced by conjugation. Hence, we also hypothesize that: 1) As RGD is an inhibitor of the platelet-fibrinogen interaction, L-SHAYIGLKDR is an inhibitor of the platelet-vWf interaction. 2) RGD and SHAYIGLKDR do not trigger spontaneous platelet activation by degranulationlCD62 surface expression, fibrinogen binding, vWf binding, and/or GPIIbIIIa activation. 3) HPG of molecular weights 3 kDa, 100k Da, and/or 500 kDa do not trigger spontaneous platelet activation by degranulation/CD62 surface expression, fibrinogen binding, vWf binding, and/or GPIIbIIIa activation. 4) HPG of molecular weights 3 kDa, 100k Da, and/or 500 kDa do not inhibit platelet-fibrinogen and/or platelet-vWf interaction. 5) Conjugates resulting from combinations of HPG and RGD do not trigger spontaneous platelet activation by degranulationlCD62 surface expression, fibrinogen binding, vWf binding, and/or GPIIbIIIa activation. 21 6) Conjugates resulting from combinations of HPG and RGD are inhibitors of platelet-fibrinogen interactions: that is, the activity of RGD is conserved after conjugation. 7) The resulting activity of HPG-RGD conjugates is solely dependent and specific to the RGD moiety. 8) Conjugates resulting from combinations of HPG and SHAYIGLKDR do not trigger spontaneous platelet activation by degranulationlCD62 surface expression, fibrinogen binding, vWf binding, and/or GPIIbIIIa activation. 9) Conjugates resulting from combinations of HPG and SHAYIGLKDR are inhibitors of platelet-vWf interactions: that is, the activity of SHAYIGLKDR is conserved after conjugation. 10) The amplification of activity of either RGD or SHAYIGLKDR peptide would be dependent on the size of HPG and/or the amount of peptide conjugated per HPG molecule. 11) The resulting activity of HPG-SHAYIGLKDR is solely dependent upon and specific to the SHAYIGLKDR moiety. 12) Forward and reverso versions of the SHAYIGLKDR peptide would have different activities after conjugation to HPG as a result of polarity considerations. 13) L- and D- enantiomers of SHAYIGLKDR peptide have different activities in the inhibition of the interaction between GPIb and vWf. 22 Chapter 2— Methods 2.1 Peptides and Proteins The peptidomimetic RGDF was selected for its high 1c50, and a 6-mer cysteine polyglycine linker was added to create CGGGGGGRGDF, (MW = 882) and was synthesized by the University of BC’s Nucleic Acid and Peptide Synthesis unit to >90% purity as tested by HPLC. A 7-mer poly-glycine linker sequence waS defined such that with the active peptide SHAYIGLKDR the peptidomimetic sequences (MW = 1605) became: L CGGGGGGSHAYIGLKDR (L-peptide), L-CGGGGGGRDKLGIYAHS (L-reverso peptide), D-CGGGGGGSHAYIGLKDR (D-peptide), D-CGGGGGGRDKLGIYAHS (D reverso peptide). These were synthesized by the University of BC’s Nucleic Acid and Peptide Synthesis unit at the Brain Research Centre to >93% purity as tested by HPLC. The peptides are dissolved in HEPES buffered saline (HBS, 10 mM HEPES, 150 mM NaC1, pH 7.40) for spectroscopic studies and in vitro assays. 2.2 Preparation of Human Platelets As approved by CBS’ and UBC Ethics Board, samples of whole blood were drawn from healthy, consenting donors into 2.7 - 4.0 mL citrate tubes (BD Biosciences, USA). Platelet rich plasma (PRP) was prepared from whole blood by centrifugation in a Beckman (CS-6R) bench-top centrifuge (r = 203.57mm, Beckman Coulter, USA) 164xg for 15 minutes. PRP from a number of unrelated donors was evaluated on an Adiva 120 Hematology Analyzer (Bayer, Canada) and standardized to a concentration of 300 x 1 0 platelets/L in HEPES buffered saline (HBS, 10 mM HEPES, 150 mM NaC1, pH 7.4). 23 2.3 HPG-RGD Conjugates 2.31 Polyglycerols and Conjugation Hyperbranched polyglycerols of molecular weights of 3 kDa, 1 OOkDa, and 515 kDa were synthesized as described previously (Kainthan et al., 2006). The conjugation of the peptide to the HPG was achieved through the sulfhydryl group of cysteine coupling to vinyl sulfone functionalized polymers. A typical reaction procedure (HPG-RGD conjgate C-7) was as follows. Initially, the HPG-515 kDa polymer (100 mg) was dissolved in 3 mL dimethyl sulfoxide (DMSO) and up to 20% of the n 7000 theoretical hydroxyl groups were deprotonated with 20 mg potassium hydride (30 wt % dispersion in mineral oil, Sigma-Aldrich, Canada). Subsequently, 20 jiL of divinyl sulfone (corresponding to a polymer-peptide ratio of 1:1000) was added and stirred at 22 °C for 12 hours. After the reaction, methanol was added and the reaction mixture was passed through an Amberlite IRC-150 cation exchange column (Sigma-Aldrich, Canada) to remove the potassium ions. The polymer was then precipitated in acetone and dried. For the peptide coupling, 5 mg vinyl sulfone functionalized polymer was dissolved in 2 mL DMSO (Sigma-Aldrich, Canada) and stirred with an excess amount of peptide (15 mg) for 2 days at 22 °C. The excess peptide was removed by dialysis (cellulose acetate membrane, MW cut-off 1000, Spectrum Laboratories Inc., USA) against HEPES buffered saline and the conjugate was collected by lyophilization (Figure 2.31). The other conjugates were synthesized using appropriate amounts (Table 3.11) of divinyl sulfone and peptide. 24 a) b) 0 Cys-&Iy-&Iy-GIy-.GIy-GIy-Arg-Gly-Asp-Phe I-WG-VS Figure 2.31. Functionalization of HPG and Conjugation of RGD Peptides. a) functionalization of HPG hydroxyl groups with divinyl sulfone (DVS); b) conjugation of peptides to functionalized HPG via cysteine side chain. OH ,OH oJ /OH OH OH ‘OH (HPG) DVS 25 2.32 Characterization of the HPG-RGD Conjugates The HPG-RGD conjugates were characterized for peptide substitution levels by UV absorption at 260 nm ( = 144 cm1M’) on an ND-bOO Spectrophotometer, (Nanodrop Technologies, USA). A calibration curve was made using standards made from the free peptide molecules at a range of concentrations (1.0 x l0 M to 6.0 x M). 2.33 Flow Cytometry Effects of the Native HPG on Resting and Activated Platelets First we examined the effect of the various molecular weights of unconjugated HPG on resting and activated platelets. For resting platelets, 45 1iL aliquots of HBS were mixed with 5 j.tL PRP and 5 tL of 3 kDa, 100 kDa, and 500 kDa HPG polymers at each of 1.0 x M, 1.3 x M, and 2.9 x 108 M concentrations and incubated for 40 minutes, then 5 jiL of human fibrinogen (Sigma-Aldrich, Canada), at 5 mglmL dissolved in bicarbonate buffer (100 mM NaHCO3, pH 8.30) was added and the incubation continued for 30 mm before the addition of 5 tL 1.5 mglmL FITC-conjugated monoclonal mouse anti-human fibrinogen IgG (Biocytex, France) and continued incubation in the dark for 30 mm. Similarly, to assay the effect of unconjugated HPG on activated platelets, 5 jiL 10 mM MnC12 to activate GPIIbIIIa (Walsh et al., 2004) was included in the above assay. Effects of the Conjugated HPG on Resting Platelets Thereafter, I determined whether the conjugated HPGs were capable of activating resting platelets. For this, the fibrinogen-anti-fibrinogen detection system of the above 26 assay was replaced by 5 tL 2 mg/mL of PE-conjugated mouse anti-human CD62 IgGi (Beckman-Coulter, Canada) to detect P-selectin expression on the platelet surface. Effects of the Conjugated HPG on Activated Platelets Finally, I examined the effect of the RGD-conjugated HPGs first on the resting platelets’ ability to bind fibrinogen, then on activated platelets using appropriate modifications of the above assay systems. Negative controls included incubating with mouse IgG-FITC of the same isotype, and/or omitting the conjugate, and/or omitting the addition of MnCl2 to activate platelet GPIIbIIIa which allows for the detection of baseline binding to fibrinogen. Positive controls for platelet fibrinogen binding did not contain conjugates. After the incubations, the samples were diluted and fixed with 0.2% formolsaline (0.2% formaldehyde, 150 mM NaC1, pH 7.20) before being submitted for analysis on the FACS Canto II flow cytometer (BD Biosciences, USA). Enzyme Cleavage RGD conjugated to the 3 kDa HPG was dissolved in HBS to a final concentration of 1 mM and treated with 1 mM trypsin-TPCK (Sigma-Aldrich, Canada) for 15 minutes at 37 °C. Enzyme cleavage was stopped with the addition 2 mM soybean trypsin inhibitor (SBTI, Sigma-Aldrich, Canada) for another 15 minutes. Controls included pre mixed and incubated trypsin-TPCK with SBTI added to platelets only, to peptides only, and to the RGD conjugated HPG. Enzyme treatment was followed by flow cytometry as already described. 27 2.34 Microscopy In a standard ELISA plate, PRP was incubated at 37°C for 100 minutes with 10 jiL 100 mM MnC12, and an amount of conjugate with a final conjugate concentration equal to the conjugate’s 1c50 and a final platelet concentration of 300 x cells/L in a 200 tL volume. Negative controls consisted of platelets incubated alone, while positive controls included 10 1iL 100 mM MnCl2 as a platelet agonist. These samples were examined at 400x magnification on a Leica DMIL inverted microscope (Leica Microsystems, Germany) with 1-S55 filter. Pictures were taken with Micropublisher 3.3 Cooled RTV and the QCapture program (Q-Imaging, Canada). 2.35 Aggregometry PRP (300 x i0 cells/L, final concentration) was incubated at 37°C for 100 minutes with conjugates or unsubstituted HPG at a final concentration equal to the 1c50 of that conjugate, in a total volume of 315 jiL. The samples were loaded into the aggregometer (Chrono-Log, USA) and compared to a platelet poor plasma derived from the same blood source, produced by high speed centrifugation. To initiate aggregation 25 tL of 100 mM MnC12was added and the aggregation curve was recorded for 12 minutes. 2.4 HPG-SHAYIGLKDR Conjugates 2.41 Polyglycerols and Conjugation Synthesis and conjugation of 500 kDa hyperbranched polyglycerols were conducted as described previously. (Kainthan, Zhang) In a typical reaction procedure, the HPG-500 kDa polymer (100 mg) was dissolved in 3.0 mL dimethyl sulfoxide (DMSO, Sigma-Aldrich, Canada) and up to 20 % of the n - 7000 theoretical hydroxyl groups were 28 deprotonated with 20 mg potassium hydride (30 wt % dispersion in mineral oil, Sigma- Aldrich, Canada). Divinyl sulfone, 2 tL, (DVS, corresponding to a polymer-peptide ratio of 1:100) was added and stirred at 22 °C for 12 hours. After the reaction, 2.0 mL 5.0 M HC1 was added to quench the remaining KH and the reaction mixture was adjusted to netural pH, then dialyzed through a 1000 kDa MW cut-off membrane (Spectrum Laboratories Inc., USA) to remove the potassium ions, and the VS-HPG (HPG-vinyl sulfone, bifunctionality of DVS reduced to VS after attachment to HPG, Figure 2.41) was recovered by lyophilization. For peptide coupling, 5 mg vinyl sulfone functionalized polymer was dissolved in 2 mL DMSO and stirred with an excess amount of peptide (6 mg) for 4 days at 22 °C. Excess peptide was removed by dialysis through a 3000 kDa MW membrane, (Spectrum Laboratories Inc., USA) against deionized water and the conjugate was collected by lyophilization (Figure 2.41). All the conjugates were synthesized using similar amounts of divinyl sulfone and peptide. The conjugates are identified with abbreviated names: L10 through DR100 (Table 3.21), with the first letter denoting the chirality of the peptide, the second letter (if any,) denoting the polarity, and the subscript denoting the conjugation ratio of peptides to HPG. For example, LR10 represents an HPG conjugate with 10 L-Reverso peptides per HPG molecule. 2.42 Characterization of the VS-HPG Constructs The initial substituted VS-HPG were dissolved in DMSO and their substitution levels were quantified using a standard thiol-estimation assay (Grasetti et al., 1967; Reiner et al., 2002) by reaction with excess 2-mercaptoethanol for 2 hours and then titration of the excess 2-mercaptoethanol with 4,4-dithiodipyridine (DTDP). The molar 29 OH HO HO - 0 0 OH OH / 0- OH OH q 1’ OHHO HO-V -o. / OH OH —-0 o HO, OH q OH - ‘ P OH a) ,_O0 OH OH b bj HO o. OH HO ‘—OH f N ,OH OH HO / / OH - / HO OH (I HO” OH (HPG) bVS b) o \Hs-cys-GIy-GIy4y.&Iy-&Iy--GIy-Ser-His- Forward HPG-Q-\J17 o \Hs_cys_GIy-&Iy_6Iy&Iy-&Iy_&Iy-Arg-Asp_Lys-Leu_Gly-IIe_Tyr.AIn.H_ser HPG-V5 Reverso Figure 2.41. Functionalization of HPG and Conjugation of SHAYIGLKDR Peptides. a) functionalization of HPG hydroxyl groups with divinyl sulfone resulting in HPG-VS; b) conjugation of peptides to functionalized HPG via cysteine side chains. 30 absorption of the resulting 4-thiopyridone was measured at A324 nm (E = 19,800 M’ cm1) with a spectrophotometer (Farooqi et a!., 1984). 2.43 Characterization of the HPG-peptide Conjugates The HPG-peptide conjugates were assessed for substitution level by UV absorption at 278 nm (8 1440 cm1 M’) on an ND-bOO Spectrophotometer, (Nanodrop Technologies, USA). A calibration curve was made using standards made from the free peptide molecules at a range of concentrations (1.0 x 10 M to 1.2 x i0 M). 2.44 Flow Cytometry Effects of the Native and Peptide-conjugated HPG on Resting Platelets First I examined the effect of the native and peptide-conjugated HPG on resting platelets. In a flow cytometer tube, 40 1tL aliquots of HBS were mixed with 5 tL PRP and 5 jiL of the material to be tested: 500 kiJa HPG; peptidomimetic peptides; or peptide conjugated HPG at 1.0 x 0 M to 1.0 x 1 0 M. After 40 minutes of incubation at ambient temperature, 5 jiL 0.01 mg/mL FITC-conjugated polyclonal sheep anti-human vWf IgG (Abcam, USA) or 5 tL of 2 mglmL PE-conjugated monoclonal mouse anti- human CD62 IgG (Beckman-Coulter, Canada) was added and incubation was continued in the dark for 30 mm. Thereafter the samples were diluted and fixed with 1.0 mL 0.2 % formolsaline (0.2% formaldehyde, 150 mM NaCl, pH 7.20) before analysis on a FACS Canto II flow cytometer (BD, USA). Effects of the Native and Peptide-conjugated HPG on Activated Platelets Finally, we examined the effect of native HPG, peptidomimetic peptides, and peptide-conjugated HPG on activated platelets using modifications of the above assay 31 systems. Platelets were either activated with of 5 jiL 4.2 mg/mL ristocetin (Chrono-Log, USA) or 100U thrombin (T6884, Sigma-Aldrich, Canada) with 5 iL GPRP fibrin inhibitor (G5779, Sigma-Aldrich, Canada) dissolved in HBS to determine whether peptides and peptide-conjugates can mitigate vWf and thrombin mediated platelet activation. Controls included incubation with mouse IgG-FITC / IgG-PE (Beckman Coulter, Canada) of the same isotype, and/or omitting the conjugate. Enzyme Cleavage The LR-peptide and conjugate LR100 were dissolved in HBS to a final concentration of 0.1 mM and treated with 2 mg/mL Trypsin-TPCK (Sigma-Aldrich, Canada) for 4 hours at 37 °C. Enzyme cleavage was stopped with the addition 5 mg/mL soybean trypsin inhibitor (SBTI, Sigma-Aldrich, Canada) for another 30 minutes. Controls included pre-mixed and incubated trypsin with SBTI added to platelets only, to peptides only, and to the peptide conjugated HPG. Enzyme treatment was followed by flow cytometry as already described. GPIb Signaling Through GPIIbIIIa Activation Signal transduction through GPIb will eventually cause conformational changes of GPIIbIIIa resulting in its conversion to its active form which allows fibrinogenlfibrin binding (Figure 1.21), as detected by a FITC conjugated anti-PAC- 1 antibody. Anti-PAC 1 antibody detects only the active/open form of GPIIbIIIa. 35 tL resting platelets were incubated with 5 1iL L-peptide and L100 conjugate at their respective IcSO concentrations for 1 hour before activation via 5 jiL 4.2 mg/mL ristocetin and addition of 5 tL monoclonal FITC conjugated anti-PAC-1 antibody (BD Biosciences, USA). Negative 32 controls included incubation of resting platelets with mouse IgM-FITC (1/10 dilution in HEPES buffer, BD Biosciences, USA) of the same isotype, resting platelets with 5 iL anti-PAC-l FITC antibody, and resting platelets incubated with L-pep and L100 conjugate without treatment by ristocetin; the positive control involves activation with 5 jtL 4.2 mg/mL ristocetin and incubation with 5 tL anti-PAC-1 FITC antibody. 2.45 Microscopy In a standard ELISA plate, wells received samples corresponding to the same composition of fluids as the flow cytometry assays performed to determine the effect of conjugated HPG on activated and resting platelets. In the ELISA plates, the reactions were fixed with 150 jtL of formolsaline. The samples were examined at 400x magnification on a Leica DMIL inverted microscope (Leica Microsystems, Germany) with a l-S55 filter. Pictures were taken with Micropublisher 3.3 Cooled RTV and the QCapture program (Q-Imaging, Canada). 2.46 Aggregometry In a total volume of 500 iL, PRP (300 x i0 platelets/L) was incubated at 37°C for 100 minutes with peptide-conjugated HPG or native HPG at a final concentration calculated to give the Ic50 of that material. The samples were loaded into the aggregometer (Chrono-Log, USA) and agglutination was initiated by 15 jiL of 125 mg/mL ristocetin (which is equivalent to the amount used for flow cytometry) and the aggregation curve was recorded for 6 minutes. As reference, platelet poor plasma derived from the same blood source, produced by high speed centrifugation and extruded through 33 a 0.20 im filter was used. The results are represented by numerical/percentage values related to the decrease of optical diffraction (Table 3.22). 2.5 Statistical Analysis Statistical analysis of all data to include 95% confidence intervals, two-tailed Student’s t-tests, and graphics generation were performed on the Microsoft Office 2003 Excel software. Due to the volume of data, only relevant comparisons were made. 34 Chapter 3 — Results 3.1 HPG-RGD Conjugates The results of this section are published (Zhang et a!., 2008). 3.11 Synthesis and Characterization of HPG-peptide Conjugates Peptides carrying the RGD motif were conjugated to HPGs of different molecular weights (Table 1). Because the number of hydroxyl groups approximately equals the degree of polymerization, a large number of peptide moieties can be coupled to the high molecular weight HPG which are thus ideal for realizing polyvalency. It is to be noted that the hydrodynamic radii of these branched molecules increase only marginally with molecular weight and are in the range of 7-10 nm as described earlier (Kainthan et a!., 2006). The extent of functionalization was kept below 20 % of the total hydroxyl groups as highly deprotonated HPGs are insoluble in DMSO (Knischka et aL, 2000). The alkoxide groups were reacted with divinyl sulfone and the resulting vinyl sulfone end groups were utilized to couple the cysteine terminated peptides (Fig. 2.31). The presence of phenylalanine residues in the peptide allows for the quantitative determination of the degree of substitution of the conjugate by UV spectrophotometry. The degrees of substitution were determined to be 0.05-0.1 for the 3 kDa conjugates, 125-150 for 100 kDa conjugates, and 1000-1500 for 515 kDa conjugates. 35 Table 3.11. Synthesis, characterization, and evaluation of RGD and RGD-HPG conjugates. Name HPG UV Spec. Theoretical 1c50 (M) [RGD] used Platform RGD peptides MW to achieve MW per HPG 1c50 (M) HPG 3/100/5 15 n/a 3/100/5 15 kDa No activity n/a kDa RGDF n/a n/a 0.882 kDa 5.0 x i”t# 5.0 x iO c-i 3 kDa 0.11 3.1 kDa 5.0 x 55 X iO C-2 3 kDa 0.05 3.0 kDa 5.0 x 103* 2.5 x i0 C-3 100 kDa 125 210 kDa 3.3 x 4.1 x C-4 100 kDa 151 233 kDa 2,2 x jj 3.3 x C-5 515 kDa 1.01 x iO l4lOkDa i.2x 108# 1.2x iO C-6 515 kDa 1.42 x iO 1770 kDa 1.4 x 2.0 x iO C-7 515 kDa 1.51 x l85OkDa 1.2x 108# 1.8x iO ‘, I, and # denote significant differences (p<0.O5) between RGDF and its target of comparison 3.12 Effects of the Unconjugated HPG on Resting and Activated Platelets Naked, unconjugated HPGs do not induce fibrinogen binding to platelets above control levels (Fig. 3.12), nor do they inhibit fibrinogen binding to either resting or activated platelets (Fig. 3.12). Furthermore, the RGD peptides and conjugated HPGs did not cause any platelet activation above control levels as detected by degranulation and consequent CD62 surface expression. 3.13 Effects of the Peptide Conjugated HPG on Resting Platelets Peptide conjugated HPG increased fibrinogen biding to resting platelets in a size and substitution-dependent manner. Free RGD peptides and smaller (3 kDa) conjugates did not increase the levels of fibrinogen bound to resting platelets; however, the larger conjugates (100 kDa and 515 kDa) significantly enhanced (p < 0.05) fibrinogen binding. This trend also applies to large conjugates in a substitution-dependent manner: platelet fibrinogen binding is increased significantly (p < 0.05) with increasing degrees of 36 100 90 (1) - 0 80 70 60 50 z I Figure 3.12. Evaluation of the Effect of Unconjugated HPGs on Resting and Activated Platelets. NEG (1): resting platelets; 3 kDa, 100 kDa, and 515 kDa Rest (Dotted histograms): resting platelets incubated with 3, 100, and 515 kDa HPG, respectively; POS (Filled histogram): Mn2 activated platelets; 3 kDa, 100 kDa, and 515 kDa Act (Striped histograms): Mn2 activated platelets incubated with 3, 100, and 515 kDa HPG, respectively. ‘:No significant differences were found between resting platelets and resting platelets incubated with HPG. “: No significant differences were found between activated platelets and platelets incubated with HPG and then activated. 37 60 50 40 V 30 a, 0 .0 U- Figure 3.131. Effect of HPG-RGD Conjugates on Fibrinogen Binding by Resting Platelets. Resting platelets are incubated with ROD and various HPG-RGD conjugates. NEG: resting platelets; Dotted histograms: resting platelets incubated with ROD, C-i, C 2, C-3, C-4, C-5, C-6, and C-7 HPG-RGD conjugates at their respective 1c50 concentrations; POS: Mn2 activated platelets. ‘: No significant differences were found between resting platelets and resting platelets incubated with HPG-RGD or ROD. “: Significant differences (p < 0.05) were found between resting platelets and resting platelets incubated with some HPG conjugates (C-3, C-4, C-6, C-7). 0 0 F..-. 0 10 - b CD ( 38 80 - 70 - °‘ 6 - 50- 40 - g)30 C . 20 Ii- 10 0 Figure 3.132 Trypsin Proteolysis Assays of RGD and HPG-RGD Conjugates. Histogram 1 (POS): positive control, ADP activated platelets; Histogram 2 (RGD): platelets incubated with RGD at 1c50 and activated with ADP; Histogram 3 (RGD + Trypsin): platelets incubated with tryptic digest of RGD peptides and activated with ADP; Histogram 4 (RGD + TrypsinISBTI): platelets incubated with SBTI inhibited tryptic digest of RGD peptide and activated with ADP; Histogram 5 (C-5): platelets incubated with C-5 HPG-RGD conjugate at 1c50 and activated with ADP; Histogram 6 (C-5 + Trypsin): platelets incubated with tryptic digest of C-5 HPG-RGD conjugate and activated with ADP; Histogram 7 (C-5 + TrypsinISBTI): platelets incubated with SBTI inhibited tryptic digest of C-5 HPG-RGD conjugate peptide and activated with ADP. Histogram 8 (NEG): negative control, resting platelets (unactivated). Tryptic cleavage of RGD significantly (p < 0.05) decreased RGD and HPG-RGD conjugate’s activity. 0. F (3 (3 F +0 I- +0 f (3 LU z 39 substitution by RGD peptides (Fig. 3.131). Tryptic removal of the RGD conjugated to the HPG produced significant (p < 0.05) decreases in inhibitory activity of the HPG-RGD conjugates and confirmed that the inhibition of fibrinogen binding was RGD-dependent (Fig. 3.132). 3.14 Effects of the Conjugated HPG on Activated Platelets Conjugated HPGs’ inhibition of fibrinogen binding to platelets is concentration-, size- and substitution-dependent. This was true especially for the 100 kDa and 515 kDa conjugates (Fig. 3.14). That this inhibition was specific and required the presence of the ROD motif was confirmed by the loss of function after enzymatic removal of the RGD (Fig. 3.132). Based on the titration curves, I derived 1c50 values that are shown in Table 3.11. Significant differences (p <0.05) were observed between 3 kDa conjugates, 100 kDa conjugates, 5l5kDa conjugates, and RGD. No statistically significant differences were found within the groups. The mathematical relationship between HPG size, substitution, and resultant Ic50 is summarized in Figure 3.15: Ic50 can be reduced with an increase in the molecular weight of HPG as well as an increase of the RGD substitution ratio. The range of Figure 3.15 is exaggerated beyond experimental data in order to further extrapolate current findings. 3.15 Macroscopic Platelet Aggregation In general, both the free RGD peptides and the ROD-conjugated HPG inhibited agonist-induced, macroscopic platelet aggregation whether the aggregation was assessed visually via light microscopy or semi-quantitated by lumiaggregometry (Table 3.12, Figure 3.16). I also found that HPG-RGD conjugates do not cause spontaneous platelet 40 aggregation of resting platelets as could have been suggested by the increased fibrinogen binding by platelets incubated with conjugates of high substitution (C-7). Table 3.12. Light microscopy of RGD-HPG conjugates and aggregation of resting and Mn2 activated platelets in the presence of free RGD peptides and RGD-HPG conjugates at their respective I5O values. Assay Microscopy of Visible Aggregates Aggregometry Resting Platelets Mn2 Activated Platelets % Aggregation Negative Control Positive Control 70 RGDF 5 C-i 5 C-2 5 C-3 5 C-4 5 C-5 5 C-6 10 C-7 5 41 120 - 100 ) 80 C C 60 C G) 0 C I . LI 20 0 1.OE-03 1OE-04 1.OE-05 1.OE-06 1.OE-07 1.OE-08 1.OE-09 1.OE-10 1.OE-11 1.OE-12 Concentration (M) Figure 3.14. Inhibitory Effects of HPG-RGD Conjugates on Platelet-Fibrinogen Binding. Platelets are incubated with RGD or HPG conjugates and fibrinogen at varying concentrations and activated with Mn2. RGD: 3 kfla HPG conjugates are marked with triangles and dotted lines: C-I: ; C-2: ; 100 kDa conjugates are marked with squares and dashed lines with dots in between: C-3: C-4: 515 kDa conjugates are marked with diamonds and dashed lines: C-5: C-6: C-7: JA 47-. t... A II I / I 42 64 2 ( 0 - 0 -201 0 -4 -6 10 Figure 3.15. Relationships Among k50, HPG Polymer Molecular Weight and Amount of RGD Peptides Conjugated to HPG. Parameters are plotted on logarithmic scales, the graphed range covers areas that exceed experimental data and are meant to be there as interpolated representations. High RGD substitution ratios and high HPG molecular weight serve to reduce 1c50. 43 2 4 90 100 - -- ,r __ —. —. E P — — FQ 3.16. Light Microscopy and Aggregometry. Compound Light Microscopy 400x, Panel 1: Resting platelets, Panel 2: Mn2Activated 2+ . . 2+platelets, Panel 3: Mn activated platelets incubated in C-5 HPG-RGD, Panel 4: Mn activated platelets incubated with C-7 HPG-RGD. Aggregometry Graph, Negative control: resting platelets, Positive control: Mn2 activated platelets, C-5 HPG-RGD: platelets incubated with RGD conjugate and activated with Mn2,C-7 HPG-RGD: platelets incubated with RGD conjugate and activated with Mn2. *This figure is used only as a visual reference for data represented in tables, and will not be described in subsequent sections of the thesis. 0 10 20 30 40 50 60 70 100 90 80 70 60 50 30 10 1:UU 2:00 3:00 4:00 5:00 6:00 7:00 8:00 9:00 10:00 Time (min:sec) 44 3.2 HPG-SHAYIGLKDR Conjugates The Results of this section are being drafted for publication. 3.21 Synthesis and Characterization of Peptide-conjugated HPG HPG was first derivatized with divinyl sulfone (DVS) to allow for subsequent peptide attachment. For the targeted 10:1 DVS to HPG ratio, the actual HPG-bound vinyl sulfone (HPG-VS) was calculated to be 9.75:1 by the back-titration thiol-estimation assay. Similarly, a ratio of 99:1 was obtained for the targeted 100:1 DVS to HPG, suggesting that the vinyl sulfone groups were the limiting condition for conjugation as they were saturated by the peptides. Furthermore, there appeared to be no steric interference among the peptide molecules themselves that would interfere with their conjugation to the HPG carrier. Because excess peptide conjugation was at a level sufficient to saturate the vinyl sulfone groups, thiol-estimation was not used to quantify the peptides on the peptide HPG constructs. Instead, the peptide’s native tyrosine residue’s UV absorbance was exploited as a measure conjugation using the molar extinction coefficient of 1260 M’ cm1 at 278 nm. 10:1 conjugates (L10 to DR10) were found to have approximately 9.5 peptides per HPG, and 100:1 conjugates (L100 to DR100) were found to have approximately 96 peptides per HPG. This confirmed the near complete or complete reaction of the HPG-VS with excess peptide (Table 3.21). 3.22 Effects of the Native HPG and Peptide-conjugated HPG on Resting Platelets Degranulation, a part of the amplification pathway of platelet signalling during activation, exposes CD62/P-selectin on platelet surfaces such that CD62 quantitation is a useful biomarker for platelet activation. Native, unconjugated HPG, free 45 Table 3.21. Functionalization and Characterization of HPG and HPG conjugates. Name HPG DVS Peptides Theoretical 1c50 (M) [Peptidel used Platform per HPG per HPG MW to achieve 1c50 MW (M) HPG --- --- --- 500 kDa No activity 0 L-pep --- --- --- 1.605 kDa 4.8 ± 0.6 x 10-5*# 4.8 x LR-pep -— --- --- 1.605 kDa 3.4±0.3 x 105*ly 3.4 x D-pep --- --- --- 1.605 kDa No activity No activity DR-pep --- --- --- 1.605 kDa No activity No activity L10 500 kDa 9.8±0.1 10±1 516 kDa 6.7±0.3 x 10 6.7 x ER10 500 kDa 9.8±0.1 10±1 516 kDa 4.1±0.7 x 10l 4.1 x 1Q D10 500 kDa 9.8 ±0.1 10±1 516 kDa No activity No activity DR10 500 kDa 9.8 ± 0.1 11 ± 1 518 kDa No activity No activity L100 500kDa 98±2 113±10 68OkDa 1.1±0.5x 10 2.1 x ER100 500 kDa 98 ± 2 107 ± 10 671 kDa 4.9 ± 0.5 x 10y 2.2 x i05 D100 500 kDa 98 ± 2 93 ± 9 649 kDa No activity No activity DR100 500 kDa 98 ± 2 93 ± 9 649 kDa No activity No activity ‘, , #, , and ‘ denote p < 0.05 between the pairs. Table 3.22. Light Microscopy, Aggregoinetry, and Flow Cytometry of HPG conjugates with Resting and/or Activated platelets Assay Microscopy of Aggregometry CD62 Surface vWf-binding Visible Aggregates Expression Resting Activated % Aggregation Resting Activated Resting Platelets Platelets Platelets upon activation Platelets Platelets Negative +-H- 5±5 22±2* 30±2 20±3’ Control Positive +++ 80 ± 5 90 ± 2 78 ± 5” 99 ± I Control HPG +++ 76 ± 5 22 ± 1 * 74 ± 4” 26 ± 5’ L-pep 5±5 18±1* 72±8” 24±4’ ER-pep 5±5 19±2* 70±5” 25± 12’ D-pep +++ 80±5 18±2* 68± 10” 14± 8’ DR-pep +++ 78±5 27±18* 72±3” 28±3’ L10 5±5 17±1* 73±5” 22±5’ ER10 5±5 17±2K 72±4” 24±2’ D10 82±5 14±7* 70±6” 24±2’ DR10 +++ 80±5 9±1* 72 ±7” 30± 3’ L100 5±5 20±3* 71±5” 23±2’ ER100 5 ± 5 18 ± 1* 73 ± 2” 22 ± 2’ D100 +++ 73±5 18±1* 72±1” 26±3’ DR100 +++ 70±5 19±1* 75 ±3” 18±6’ “,“, ‘:no significant differences were found within these groups. 46 peptidomimetics, and peptide-conjugated HPG in contact with resting platelets do not cause platelet activation as detected by CD62 surface expression (Table 3.22). In addition, HPG do not increase vWf binding to resting platelets (Table 3.22), which could trigger vWf crosslinking, platelet activation, or both. 3.23 Effects of the Peptide-conjugated HPG on Activated Platelets Peptide-conjugated HPGs’ inhibition of vWf binding to platelets was concentration- and substitution-dependent (Fig. 3.231). The D-enantiomer based peptides and their conjugates showed no inhibitory activity while both the L and the L-Reverso peptides as well as their conjugates (L10, LR10, L100, LR100) proved to be effective inhibitors. The free L-Reverso peptide was a significantly better inhibitor (p < 0.05) of the GPIb-vWf interaction than the native L-peptide. However, this was not true for the conjugated forms (Table 3.21). Based on the titration curves (Figure 3a), and the 1c50 values determined from them (Table 3.21), the peptides’ inhibitory effectiveness was augmented by as much as two orders of magnitude (p < 0.05) by their conjugation to HPG. The materials did not inhibit platelet activation by thrombin as measured by CD62 surface expression. This confirms that. the HPG-conjugates interfere solely with platelet activation mechanisms through GPIb-vWf and that the inhibitory activities of the HPG peptide conjugates are specific. This is further underlined by the fact that neither the native HPG nor the free peptides, nor combinations thereof are sufficient for inhibition. The presence of the peptidomimetic motif on the HPG is a requirement, as the loss of this function after trypsin cleavage removes the function of the conjugates (Figure 3.232). 47 Further, neither the L-peptide nor its conjugate L100 were found to induce direct GPIb dependent platelet activation of resting platelets. This is demonstrated both by the lack of increase of CD62 expression/platelet degranulation shown in Table 3.22 as well as by GPIIbIIIa activation of resting platelets treated with these substances (Figure 3.233). Ristocetin treatment to induce platelet activation (CD62 expression!GPIIbIIIa activation) through the GPIb dependent signalling pathway via activation of vWf also failed to cause GPIIbIlIa activation of platelets incubated with L-peptide and L100 (Figure 3.233). 3.24 Macroscopic Platelet Aggregation The inhibition of platelet agglutination by vWf was observed both by light microscopy and lumiaggregometry. No clumping was seen when resting platelets were incubated with free peptides, native HPG, or peptide-conjugated HPG (Table 3.22). To show inhibition of the GPIb-vWf interaction in PRP, activation must target the plasma component, specifically vWf by ristocetin rather than by platelet activators such as thrombin (Table 3.22). Platelets did not agglutinate in the presence of ristocetin if they had been incubated with either the peptides or the peptide-conjugated HPG at their respective I5O concentrations. 48 100 80 0) II 60 ji S 40 20 0 I I 1.OE-03 1.OE-04 1OE-05 1.OE-06 1.OE-07 1.OE-08 1.OE-09 1.OE-10 Concentration (M) Figure 3.231. Inhibition of Platelet-vWf Binding by Peptide-conjugated HPG PRP platelets incubated with either peptides or conjugates are activated by ristocetin and assessed for vWf binding under flow cytometry (FACS Canto II). L-peptide: LR peptide:’EI”,D-peptide:3,DR-peptide:’Z, L10: E,LR10:EI,D DRo: A.—, L100: 4, LR100: Doo: DR100: I, I, I, I, 1/III +1 -I. 49 110 100 90 80 o 70 . c 60- co 30 20 10 0] Figure 3.232. Trypsin Proteolysis Assays of SHAYIGLKDR and HPG SHAYIGLKDR Conjugates. Histogram NEG (1): PRP platelets are checked for baseline vWf binding. Histogram 2: platelets incubated with LR-peptide at 1c50 and treated with ristocetin. Histogram 3: platelets incubated with trypsin digested LR-peptide and treated with ristocetin. Histogram 4: platelets incubated with SBTI inhibited trypsin digested LR-peptide and treated with ristocetin. Histogram POS (5): Platelets treated with ristocetin. Histogram 6: platelets incubated with LR100 conjugate at 1c50 and treated with ristocetin. Histogram 7: platelets incubated with trypsin digested LR100 conjugate at 1c50 and treated with ristocetin. Histogram 8: platelets incubated with SBTI inhibited trypsin and treated with ristocetin. 50 50 POS (2) 45 40 35 30 0 o 25 a, 4- NEG (1) 3’ 4’ 5” 6” C., <15 I Figure 3.233. Effect of Peptide on GPIIbIIIa Activation Through GPIb Signalling. Histogram NEG (1): resting platelets are checked for baseline GPIIbIIIa activation. Histogram POS (2): resting platelets treated with ristocetin. Histogram 3: resting platelets treated with L-peptide at I5O. Histogram 4: resting platelets treated with L100 conjugate at 1c50. Histogram 5: resting platelets treated with L-peptide at 1c50 and activated with ristocetin. Histogram 6: resting platelets treated with L100 at 1c50 and activated with ristocetin. “: No significant differences were found among resting platelets and ristocetin-activated platelets under peptide or conjugate suppression. 51 Chapter 4 — Discussion 4.1 HPG-RGD Conjugates The attachment of RGD peptides to HPG confers unique attributes to the resulting construct: the HPG gains specificity and thereby function while the peptidomimetic gains polyvalency and thereby binding avidity. 4.11 Characterization Peptide attachment increases the theoretical molecular weight of the HPG conjugates depending on the number of available sites that can be functionalized on the surface of the HPG polymer. The branch density of the I-fPG varies directly with the molecular weight of the polymer while the Stokes’ radius of the molecule remains relatively stable at about 7-10 nm for the 100 kDa and 515 kDa HPG molecules (Kainthan et al., 2006). The peptidomimetic is predicted to be <1 nm in length, thus based on the level of substitution, peptide conjugation can approximately triple the theoretical MW of the 515 kDa HPG polymer to up to 1850 kDa for the HPG-RGD conjugate. Since attachment of the peptides is through divinyl sulfone, a reaction with limiting yields and kinetics (compared to DVS attachment to HPG), it is not surprising that the 3 kDa HPG (C-l and C-2) carried few RGD peptides after conjugation. The achieved peptide density has steric implications for subsequent function due to its polyvalent binding to multiple GPIIbIIIa receptors on the platelet surface. The design of the peptide included an N-terminal cysteine for attachment and five glycine residues to facilitate the RGD motif’s access to the GPIIbIIIa binding pocket (Beer et a!., 1992). The RGDF sequence was considered by Foster et a! (Foster et a!., 52 2003) to be the most potent of the small RGD-peptidomimetics (1c50 = 8 x i0 M) and the phenylalanine was also useful for detection UV tracking of the peptide (Foster et a!., 2003). 4.12 Function In order to evaluate whether RGD-conjugated HPGs have specific interactions with platelets, the range of MW of naked, unconjugated HPG were examined first. Both the original studies (Kainthan et a!., 2007; Kainthan & Brooks, 2007) and the assays presented here involving either resting or activated platelets show no significant alterations of fibrinogen binding to platelets. Although plasma fibrinogen concentration, platelet surface GPIIbIIIa levels and degree of preparation-induced platelet activation vary with the individual, similar 1c50 values were obtained with the plasma and cells of all the blood donors tested. Furthermore neither the unconjugated HPG nor the RGD conjugated HPG induced platelet activation as defined by degranulation and CD62 expression. However, the RGD-conjugated HPG promoted some fibrinogen binding to resting platelets in a manner that could be related to both HPG molecular size and degree of RGD substitution: the dense RGD brushes on HPG increased fibrinogen binding to platelets more than the diffuse RGD brushes. This can be attributed to the cooperativity of GPIIbIIIa binding of RGD motifs such that RGD binding to a GPIIbIIIa receptor enhances the ability of other GPIIbIIIa molecules to bind fibrinogen (Bassler eta!., 2007; Dickfeld et a!., 2001). The most important finding was that fibrinogen binding to activated platelets, and subsequent platelet aggregate formation is inhibited by RGD-conjugated HPG in a manner related to HPG size (Fig. 3.14), and to its degree of substitution (Fig. 3.14). As 53 the 3 kDa conjugates carried few RGD peptides, higher concentrations were needed to achieve the same degree of inhibition as obtained for free RGD peptides. However, both the 100 kDa and 515 kDa HPG conjugates carry 100 - 1500 RGD peptides, and thus they are able to reduce the 1c50 of free RGD peptides by two to three orders of magnitude. Extrapolating from this data (Fig. 3.15), it is possible to derive the substitution/MW relationship to achieve optimal platelet inhibition. The large decrease of the 1c50 and the corresponding increase of the HPG conjugated RGD peptides’ inhibitory effect may be a matter of kinetics. As a result of the polyvalent presentation of the RGD conjugated to HPG (Figure 4.11), the binding of RGD to platelet GPIIbIIIa is enhanced due to both a higher local concentration of RGD peptides, and a slowed dissociation due to several concurrent dynamic associations of HPG-RGD with two or more GPIIbIIIa integrins (Figure 4.12). As well, there is cooperativity among GPIIbIIIa receptors, such that a single bound RGD changes the GPIIbIIIIa conformation to increase the likelihood of further ligand binding (Bassler et al., 2007; Dickfeld et al., 2001). It is known that free RGD peptides can activate the GPIIbIIIa receptors such that an increased number of the receptors remain in the “open” configuration that is capable of binding fibrinogen. (Bassler et al., 2007; Dickfeld et al., 2001) Thus it is not unlikely that the HPG-carried RGD generate a similar activating effect. Increased fibrinogen binding seen on flow cytometry of resting platelets is caused by high molecular weight, high substitution HPG-RGD. However, this fibrinogen binding does not seem to cause spontaneous platelet macroscopic aggregation, as observed via light microscopy and lumiaggregometry. The conjugates however, still inhibit the macroscopic aggregation of 54 HPG-RGb f33 alib GPIIbIIIa Complex Figure 4.11. Proposed Mechanism A: Polyvalency of HPG-RGD Conjugates. Simultaneous binding to multiple GPIIbIIIa receptors on platelet surfaces by a corresponding number of RGD peptides attached to HPG resulting in significantly reduced dissociation kinetics. Differential color label (blue and green) for the HPG-RGD conjugates denote the conjugates as a heterogeneous population. 55 Figure 4.12. Proposed Mechanism B: Higher Local Concentration. Increased availability of RGD molecules brought by HPG shifts the equilibrium towards binding association with platelet surface integrin GPIIbIIIa. Red, up-side-down flag-like objects denote RGD peptides. HPG inding bomarn Free Gb 56 Mn2 activated platelets as detected both macroscopically (Fig. 3.16, Table 3.12) and by flow cytometry (Fig. 3.14). Figure 3.15 demonstrates the mathematical relationship between 1c50, substitution ratio, and HPG molecular weight. We see that an increase in substitution and HPG molecular weight causes a decrease in 1c50. Base on Figure 3.15 it would seem that we should use 515 kDa HPG with 1000:1 substitution ratio. However, this construct causes platelets to spontaneously bind platelets as well as the same molecular weight with 500:1 substitution ratio (Figure 3.131). As a result, the 500 kDa HPG with lower conjugation ratios (10:1 and 100:1) were chosen and the results are discussed in the following section. 4.2 HPG-SHAYIGLKDR Conjugates 4.21 Peptide Selection Here, the peptidomimetic peptide is a native sequence of the vWf molecule selected by probing on a tandem peptide array. The peptide binds at or near the GPIb active site for vWf, but does not spontaneously trigger platelet activation either through GPIb. Neither are CD62 expressionldegranulation (Table 3.22) and GPIIbIIIa activation triggered (Figure 3.233), but GPIb-dependent platelet activation is inhibited (Figure 3.231, Figure 3.233): this is defined as a dominant-negative peptide (Kirchmaier & Sugden, 1997). 4.22 Choice of Platform and Linker We chose the 500 kDa HPG molecule as the platform because for higher molecular weight polymers molecular size increases only modestly with increasing 57 molecular weight (Kainthan eta?., 2006). Based on the data from Figure 3.15 and Table 3.11, we had determined that the number of peptides attached to HPG is vital to the level of enhancement of peptide activity. The 500 kDa HPG molecules have the highest density of branches and hence the most number of available hydroxyl groups for conjugation. Peptides were again attached to HPG through a terminal cysteine sulthydryl’s side chain in a thiol-selective manner using divinyl sulfone (Houen et a?., 1995; Bulmus eta!., 2000). The peptidomimetic was designed to contain a cysteine in the linker moiety (Zhang et a?., 2008) le the 7-mer peptide linker: CGGGGGG described in this study. In the design of the linker, considerations that become important include length, composition, and polarity, as well as the inclusion of reactive groups for conjugation. The linker length and composition may adversely affect the peptide’s activity if an inadequate length prevents access of the peptide to the GPIb receptor’s binding site. Based on previous studies (Zhang et a?., 2008), we somewhat arbitrarily decided to lengthen the linker to 7 glycines to allow for increased flexibility in order to be able to attach to the linear binding domain of GPIb. Conjugation to a macromolecule, whether with or without a linker, fixes the spatial orientation of the peptidomimetic. Both attachment and the addition of linkers bring peptide sequence and polarity into question as the direction of peptide binding may be an important component of that peptide’s activity. Therefore, forward and reverso variations of the L-amino acid sequence were made. As well, the peptides were synthesized using non-natural D-amino acids in order to determine the relevance to the GPIb-vWf binding activity of both the peptide bond and side chain orientation. 58 4.23 Characterization Peptide attachment increases the theoretical molecular weight of the HPG-peptide conjugates: this is relative to the number of available sites for attachment as well as the number of peptides attached. The conjugation of DVS to HPG is clearly characterized by the thiol-estimation assay, which indicates a near complete reaction, attaining greater than 95% of the targeted conjugation ratio (Table 3.21). It should be noted that side reactions that polymerize HPG result in large insoluble clumps but are filtered away. The Strokes Radius of the 500 kDa naked HPG is around 8 to 10 nm by dynamic light scattering experiments (Kainthan et a!., 2006), and with the 1 7-mer peptide (calculated to be -l-2 nm), brings the final conjugate size to 10 to 11 nm. The terminal cysteine residues and the six glycine linkers respectively, allow conjugation to HPG-VS (Figure 3.22) and freedom for the peptidomimetic end to bind the vWf binding domain of GPIb. The tyrosine within the peptidomimetic allows quantification of the final conjugate constructs. Although other wavelengths (274 nm) may be better for tyrosine-dependent absorbance measurements, the A278 molar extinction coefficient is certainly comparable and hence it is utilized here. 4.24 Function HPG was found to be biocompatible in past studies involving both in vitro and in vivo methods (Kainthan et al., 2006; Kainthan et al., 2007; Kainthan & Brooks, 2007), but its interactions specifically with platelet integrins remains unknown. A study by our group has indicated that naked HPG does not affect fibrinogen binding to either resting or activated platelets (Zhang et al., 2008). Here we showed that naked }-IPG does not 59 contribute to vWf binding in resting or activated platelets (Table 3.22), as it does not seem to activate vWf or bind to the GPIb-vWf interactive domain on its own. Neither the peptide-derivatized HPG conjugates nor the peptidomimetic peptides themselves promote vWf binding to resting platelets (Table 3.22); nor do they activate platelet GPIb, to contribute to platelet outside-in signalling as determined by CD62 surface expression (Table 3.22) and GPIIbIIIa activation (Figure 3.233). These conjugates do not interfere with thrombin stimulated platelet activation, and thus seem to block only the GPIb-vWf dependent signalling (Table 3.22, Figure 3.231, Figure 3.233). However, it is evident that these peptides and conjugates interfere with vWf binding to platelets (Figure 3.231, Table 3.22) in a concentration- and substitution-dependent manner. This inhibitory effect can be attributed directly to the peptidomimetic peptide itself as proteolytic digestion of the peptides with trypsin causes a loss of activity (Figure 3.232). Significant differences (p < 0.05) were observed between the inhibitory activities of the L- and L-Reverso peptides but not between the peptide conjugated to their carrier. The D-peptides, DR-peptides, and their conjugates showed no activity. However, it should be noted that the difference in 1c50 between the L and the LR-peptides are quite small and are on the borderline of significance and hence the loss of this difference is assumed to become negligible after conjugation. The lack of inhibitory function by D peptides is intuitive as the original peptide array is based on an L-enantiomer design while the targeted GPIb-vWf binding sequence is relatively linear. The inhibitory effects of both the peptides and the HPG-peptide conjugates in the presence of ristocetin activated platelets is substantiated by macroscopic evidence viewed 60 by the compound light microscope and by aggregometry, where an absence of the agglutination response is seen (Table 3.22). The attachment of peptidomimetic peptides to HPG confers unique attributes to the resulting construct: the HPG gains specificity and function; while the peptidomimetic gains polyvalency and binding avidity. It should be noted that although the 1c50 of the peptidomimetic peptide itself is not very high, it remains very clear that drug conjugation to macromolecular carriers is a valuable new method to increase the effectiveness and most probably increase the in vivo retention of small drug molecules. In this set of experiments, we observe the same inverse linear relationship between 1c50 and the substitution ratio of the peptidomimetic, that is, HPG-peptide conjugates carrying 100 peptides necessarily lower the 1c50 by approximately the same amount. We have also found no difference between the forward and reverso. versions of our peptidomimetic, which suggests that conjugation defined directional polarity does not affect function in this particular case where the interactive domain is linear. Since the effectiveness (Ic50) of the peptides attached to HPG do not differ significantly from that the peptides in their free form, we classify the interaction as polyvalency dependent. This may be a result of higher loading rates of the conjugate due to higher local concentrations and lower off- loading rates as a result of accumulated attachment to multiple GPIb receptors. 4.3 Summary We see that the HPG conjugates’ activities are modulated by the number of peptides attached to the carrier HPG. Higher molecular weight HPGs have more groups that can be functionalized on their surface and hence have the highest potential for drug enhancement. However, high conjugation ratios could increase the peptide density on the 61 HPG surfaces to a point where the peptides’ mobility is reduced and steric hindrance to binding their receptor target becomes significant, and activity could be lost. This is perhaps a more dominant issue when considering receptors with complex internal active sites such as GPIIbIIIa, although in this study we did not seem to have approached this limit. These possibilities may not affect the vWf mimic as significantly because the binding domains are relatively linear and highly exposed. At the same time, the linker/spacer composed of poly-glycines could be either lengthened or shortened and other biocompatible materials such as poly(ethylene)glycol (PEG) can be used. The longer the linker, the more space the tips of the peptides (the active end) have to maneuver and attach to targeted sites (Figure 1.5). An optimum linker length may be titrated for binding specific receptors. Conjugation of small molecular species to macromolcules increases the molecular weight of the resulting species, and could decrease chemical kinetics. However, an increased local concentration of peptides (Figure 4.12) as a result of high conjugation ratios and polyvalency of the HPG-peptide attachment to receptors (Figure 4.11) are able to enhance the binding kinetics and/or decrease the ligand off-loading from receptors. These enhancements appear to overcome the decrease induced by the increase in their molecular weights. Considering the above constraints, an increase in efficacy is directly related to the number of peptides attached to the carrier HPG molecule. 62 Chapter 5 — Conclusion Here we address the some of the issues raised in the introduction and hypotheses regarding peptidomimetics, their conjugates, and anti-platelet inhibitor design: The RGD peptide has biological activity in the inhibition of GPIIbIIIa/fibrinogen interaction. The L-SHAYIGLKDR peptide has biological activity in the inhibition of GPIb-vWf interaction; its forward and reverso versions show similar activity while its D enantiomers were found to have no inhibitory activity, which suggests that the inhibitory interaction is primarily orientation-, but not charge-dependent. These peptides do not cause spontaneous platelet activation as detected by CD62 surface expression/degranulation, GPIIbIIIa activation, compound light microscopy, and/or aggregometry. Conjugation to HPG through DVS functionalization of these peptides (RGD or SHAYIGLKDR) creates a biocompatible molecule/conjugate after the peptides are attached and the remaining VS groups are neutralized. HPG and HPG conjugated molecules do not cause spontaneous platelet activation macroscopically, or microscopically. Enhancement of peptide activity is observed to be linearly related to the number of molecules attached per HPG, within theoretical limits of peptide brush density, linker length, length of the pharmacophore, peptide chirality, peptide polarity, and the nature of the binding site interaction (linear vs internal). Peptide conjugation to HPG enhances peptide activity in a peptide-specific manner and increases the molecular weight of the peptide. This presents an excellent solution to the issues of low molecular weight, raised in the introduction with regards to peptidomimetics, and the same concepts can be widely applied to other molecules and are not limited to peptides. 63 In addition to the hypotheses, it must be reiterated that we have discovered a peptide that interferes with the GPIb-vWf interaction. 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SC V5Z 4i$ ETHICS CERTIFICATE OF EXPEDITED APPROVAL RENEWAL OdNVESTIGATOR(S): . ... BLnWfl Ith. Sraoo aLein moo m. Cm4oo p •oK mm. Soomth I’j’e, althHofl ooflo Thou .*jC C&iBm PONSOflNG AGSNC)SS: aneia B’ood Sooeo - “General lnotromo’t CabraSaq aod Referenne Matnoaf ROJECT TTTLE: sofa Blood “ft foerw l ll,t Tent Cabhralfo Reference Matenaf wld R0300r’l — io0Pafho4oBy ISfltOTlON(S WHERE RESEARCH WILL BE CARRiED OUT: fn.thfBlen -_ $ft ‘BC Vacoovcr leacluftos USC oor leestions where th research wIll be conducted “Oa0 an Il-nsf Sftrn,ftrf NotwOrO Co-f’s lot P00 lad Deoffopterft where et ares. onn0 It’s b-Odd sfI On d”on wool caas ltln-,tI wIll Ow too, of 100 LOl mnsnh BOO’” Colecl,on SuOe 72 SXPIRY Gilt OF THIS APPROVAL:i respect ci clinical trials: Thn owwhcrsriip ti Bin fdasfl Specs Baird ccwpuias with tile rnawcnrstno rn’cicnwarpe let iheaeascir tlidcflsain&darjend\ulQirhnjelnb pt thai Food nnir Urac rduiahhna. Tire Rerccrdn Ethics enard cardas out is torrcticnn in a macne. consistent rash Grad Cilnicial Praccicart mr Rmsaaicit Ethics Board has reirrorond and aporaved thc.pnaicnt Inal protocol and srOit.ccriilanj igmnt (cc it,. car attn in ip ire ccnthecsed Dy the qaahtnad. named shorn a? theDoiccdlaêhhaa$ blat ada Thin approval and tire vans at Pita ReBBidhthBiias Board trace been dccnmnooiBd Ce hltttrip time USC Clinical Rnnoancdm EliPca BcanrThao conned Pam itcmcoamcniadcam in: tin abate canned project The rasnarcir study, as praneciad to Pta was lewd rEt pa ojamptable mr critic cr nd Iqi neee Is avqrv gi’ r hjnds wit 4n appmooed (c Ottawa t die USC .cd itmmaeerctm rtpprcmo: ot tire thrice) Rnscarctr Ethics tirmwd dry- r’ -K-- 73


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