Dentistry, Faculty of
DSpace
UBCV
Senini, Vincent
Master of Science - MSc
University of British Columbia
Background: Chronic periodontitis confers an increased risk for cardiovascular diseases, including thrombosis. However, the molecular mechanisms that potentially link periodontitis with thrombosis are undefined. Here I tested the hypothesis that gram-negative periodontal infection promotes pathological platelet activation and shape change. I focused specifically on lipopolysaccharide (LPS) signaling through Cdc42. Methods: Platelets were isolated from blood samples and allowed to spread on coverslips in the presence or absence of LPS purified from the periodontal pathogen Porphyromonas gingivalis. Platelets were fixed and stained with Alexa- 488-phalloidin to label the actin cytoskeleton. The degree of platelet spreading and shape change were quantified by confocal microscopy. In a translational pilot study, blood samples were obtained from human subjects exhibiting generalized severe chronic periodontitis (SP) or healthy periodontium (HP). Rotational thromboelastometry (ROTEM) was used to quantify the rate of clot formation via the intrinsic coagulation pathway. Results: LPS-treated platelets exhibited significantly (p<0.05) greater spreading and higher numbers of actin-rich filopodia (cell extensions) than controls. I also found that LPS stimulation directly activated Cdc42, the small GTPase responsible for filopodial formation. Exposure of whole blood samples to LPS significantly (p<0.05) reduced clotting times. Blood from SP patients clotted significantly (p<0.05) more rapidly and exhibited reduced partial thromboplastin times (aPTT) relative to HP controls. Conclusion: This is the first study to suggest a mechanism by which P. gingivalis LPS drives Cdc42 activation and platelet spreading. These data are consistent with the notion that periodontitis promotes accelerated clot formation and an increased risk of thrombosis.
Porphyromonas gingivalis lipopolysaccharide promotes platelet spreading and thrombosis via Cdc42 activation by Vincent Senini B.Sc., The University of British Columbia, 2009 M.Sc., The University of British Columbia, 2011 D.D.S., University of Toronto, 2015 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE AND POSTDOCTORAL STUDIES (Craniofacial Science) THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver) August 2018 © Vincent Senini, 2018 ii The following individuals certify that they have read, and recommend to the Faculty of Graduate and Postdoctoral Studies for acceptance, a thesis/dissertation entitled: Porphyromonas gingivalis lipopolysaccharide promotes platelet spreading and thrombosis via Cdc42 activation submitted by Vincent Senini in partial fulfillment of the requirements for the degree of Master of Science in The Faculty of Graduate and Postdoctoral Studies Examining Committee: Hugh Kim Supervisor Edward Putnins Supervisory Committee Member Flavia Lakschevitz Supervisory Committee Member Babak Chehroudi External Examiner iii Abstract Background: Chronic periodontitis confers an increased risk for cardiovascular diseases, including thrombosis. However, the molecular mechanisms that potentially link periodontitis with thrombosis are undefined. Here I tested the hypothesis that gram-negative periodontal infection promotes pathological platelet activation and shape change. I focused specifically on lipopolysaccharide (LPS) signaling through Cdc42. Methods: Platelets were isolated from blood samples and allowed to spread on coverslips in the presence or absence of LPS purified from the periodontal pathogen Porphyromonas gingivalis. Platelets were fixed and stained with Alexa-488-phalloidin to label the actin cytoskeleton. The degree of platelet spreading and shape change were quantified by confocal microscopy. In a translational pilot study, blood samples were obtained from human subjects exhibiting generalized severe chronic periodontitis (SP) or healthy periodontium (HP). Rotational thromboelastometry (ROTEM) was used to quantify the rate of clot formation via the intrinsic coagulation pathway. Results: LPS-treated platelets exhibited significantly (p<0.05) greater spreading and higher numbers of actin-rich filopodia (cell extensions) than controls. I also found that LPS stimulation directly activated Cdc42, the small GTPase responsible for filopodial formation. Exposure of whole blood samples to LPS significantly (p<0.05) reduced clotting times. Blood from SP patients clotted significantly (p<0.05) more rapidly and exhibited reduced partial thromboplastin times (aPTT) relative to HP controls. Conclusion: This is the first study to suggest a mechanism by which P. gingivalis LPS drives Cdc42 activation and platelet spreading. These data are consistent with the notion that periodontitis promotes accelerated clot formation and an increased risk of thrombosis. iv Lay summary Periodontitis (gum disease) is a common oral infection caused by harmful bacteria in dental plaque. This disease affects nearly half of the adult North American population, leads to dental problems such as tooth loss, and also increases the risk for systemic (general) metabolic diseases such as diabetes, and cardiovascular diseases such as heart attack and stroke. However, the exact mechanisms that connect gum disease with cardiovascular disease are unknown. Platelets are tiny blood cells that spread out and stick together during hemostasis (blood clotting). Blood clotting is essential for healing after injury but inappropriate blood clots that form in the body obstruct blood flow to critical organs leading to heart attacks and strokes. In my research, I discovered that the dental bacteria that cause gum disease can also accelerate the process in which platelets spread and change shape, as they do during blood clot formation. Using a specialized instrument that measures blood clot formation, I also found that blood samples from patients with severe gum disease clot more rapidly than blood samples taken from patients with healthy gums. This work therefore provides additional evidence to support the link between oral health and cardiovascular health. v Preface This thesis is based on work that I conducted in Dr. Hugh Kim’s laboratory at the UBC Centre for Blood Research. I performed the platelet isolation, spreading assays and confocal microscopy. I performed the blood coagulation assays using rotational thromboelastometry (ROTEM). I was also responsible for the statistical analysis. The p21-activated kinase (PAK) pull-down assays for active Cdc42, and immunoblotting for active and total Cdc42, and assays for PT and aPTT were performed by Dr. Umme Amara (postdoctoral fellow). Blood samples were collected by Brana Culibrk and Iren Constantinescu. All research involving human subjects was performed with approval from the UBC Clinical Research Ethics Board (CREB) under the protocols #H13-0185 and #H14-00100. A manuscript based on this work will be submitted for publication. Figure 8 of this thesis was reproduced with written permission from Instrumentation Laboratory, Ltd. vi Table of contents Abstract………………………………………………………………………………………..iii Lay summary…………………………………………………………………………………. iv Preface…………………………………………………………………………………………. v Table of contents……………………………………………………………………………. vi List of tables……………………………………………………………..………………….. viii List of figures…………………………………………………………………………………. ix List of abbreviations…………………………………………………………………………… x Acknowledgements……………………………………………………………………............xi Dedication…………………………………………………………………………………….xii Chapter One: Literature review……………….…………………………….…...…..……….. 1 The periodontium…………………………………………...……………………………….... 1 Pathogenesis of periodontal disease……...…………………………………………………… 1 Bacteremia as a link between periodontal disease and cardiovascular disease………………...3 Evidence of associations between periodontitis and cardiovascular disease……………………4 Lipopolysaccharide (LPS) and toll-like receptors (TLRs) ……………………………………. 6 Platelets………………………………………………………………………………………... 8 Platelet activation, aggregation and clot formation…………………………………………...10 Platelet adhesion and spreading dynamics………………………………………………….....12 Actin assembly………………………………………………………….………………….... 13 GTPases……………………………………………………………………………….……... 14 Modulation of cell spreading by Rho GTPases………………………….………..…………..15 The coagulation cascade: intrinsic and extrinsic pathways……...………………..…………..16 Conventional measurements of coagulation…………………………..……………………....18 Rotational thromboelastometry (ROTEM)…………………..…..…………………………....19 Statement of the problem, aims and hypothesis………………………………………………21 Chapter Two: Porphyromonas gingivalis lipopolysaccharide promotes platelet spreading and thrombosis by signaling through Cdc42…………...…………………………………….. 22 Materials and methods…..…………..…………..…………..…………..…………………… 22 Platelet purification and spreading assays…………………………………………………… 22 Active Cdc42 pull-down assay and immunoblotting………………………………………… 23 Periodontal patient recruitment and blood collection………………………………………... 23 Measurements of the intrinsic (aPTT) and extrinsic (PT) clotting cascades………………… 24 Statistical analysis………………..…………..…………..…………..………………………. 25 Results….…………..…………..…………..…………..…………..…………..…………….. 25 Lipopolysaccharide (LPS) from Porphyromonas gingivalis enhances platelet spreading…... 25 P. gingivalis LPS promotes filopodial formation in platelets by activating Cdc42………….. 25 Lipopolysaccharide (LPS) from P. gingivalis shortens clotting times in whole blood……… 26 vii Patients with severe chronic periodontitis exhibit accelerated clotting and aPTT times…….. 27 Discussion…..…………..…………..…………..…………..…………..……………………. 33 P. gingivalis LPS mediates platelet activation & spreading in a Cdc42-dependent manner… 33 P. gingivalis LPS reduces clotting time………………..……………………...……………... 34 Severe periodontal infection may promote thrombosis…..…………..……………………… 34 Chapter Three: Conclusions and future directions…………..…………..…………..……… 37 Bibliography………..…………..…………..…………..…………..…………..…………….. 38 viii List of tables Table 1: Platelet receptors and corresponding agonists……………………………..………….. 11 Table 2: Periodontal characteristics of blood donors……………………………….…………... 27 ix List of figures Figure 1: Structure of the periodontium…………………………………………………………. 1 Figure 2: Pathogenesis of periodontitis………………………………………………………….. 3 Figure 3: Illustration of the oral-systemic connection…………..……………………………….. 4 Figure 4: Platelet spreading and clot formation………………..………………………….……. 12 Figure 5: Actin polymerization and cell extension formation..................................................... 14 Figure 6: Cdc42 activation and filopodial formation……………………………………..……. 16 Figure 7: Illustration of the extrinsic, intrinsic and common coagulation pathways………....... 18 Figure 8: Illustration of rotational thromboelastometry (ROTEM)……………………………. 21 Figure 9: Platelet spreading is enhanced by P. gingivalis LPS…………………………….….. 29 Figure 10: P. gingivalis LPS promotes filopodial formation via Cdc42 activation……………. 30 Figure 11: P. gingivalis LPS accelerates blood clotting………………………………….……. 31 Figure 12: Clotting time and aPTT are shortened in patients with severe periodontitis……….. 32 Figure 13: Proposed model for pro-thrombotic mechanisms of P. gingivalis………...………... 36 List of abbreviations ADP adenoside diphosphate ANOVA analysis of variance aPTT activated partial thromboplastin time BOP bleeding on probing CaCl2 calcium chloride Cdc42 cell division control protein 42 homolog CAL clinical attachment loss CEJ cemento-enamel junction CI confidence interval CM cementum CRP C-reactive protein GAP GTPase activating protein GDP guanosine diphosphate GTP guanosine triphosphate GE gingival epithelium GEF guanine nucleotide exchange factor GP glycoprotein H2O water HP healthy periodontium IB immunoblot INR International Normalized Ratio LPS lipopolysaccharide MGI Modified Gingival Index MMP matrix metalloproteinase NHANES National Health and Nutrition Evaluation Survey OCS open canalicular system P. gingivalis Porphyromonas gingivalis PAK p21-activated kinase PAR protease activated receptor PBD p21-activated kinase binding domain PD probing depth PDL periodontal ligament PI plaque index PMN polymorphonuclear cells PT prothrombin time ROTEM rotational thromboelastometry SD standard deviation SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis SP severe periodontitis TF tissue factor TLR toll-like receptor vWF von Willebrand factor WB whole blood xi Acknowledgements I would like to send special thanks to Dr. Hugh Kim and Dr. Umme Amara for their indispensable insights and aid with the direction of this project. I thank Brana Culibrk and Iren Constantinescu for their help with the collection of blood samples. I also thank Angela Tether for her assistance with the EndNote software and organization of the bibliography. xii Dedication To my wife Caroline. 1 Chapter One: Literature Review The periodontium Periodontitis (periodontal disease, or “gum disease”) is a chronic disease affecting the supporting tissues of the teeth, termed the periodontium (Fig. 1). The periodontium functions principally to provide support needed to sustain teeth in function. This support is mediated in part by the four major components that compose the periodontium including the gingiva, periodontal ligament, cementum, and alveolar bone (Fig. 1). FIGURE 1. Structure of the periodontium. Left: Photographs of teeth affected by periodontal disease (upper left image) and a patient with periodontal health after periodontal therapy (lower left image). Right: Diagram illustrating the major anatomic landmarks of the periodontium. Inset: focus on the dento-gingival (tooth-gum) interface. CEJ = cementoenamel junction, PDL=periodontal ligament, GE=gingival epithelium; CM=cementum. Pathogenesis of periodontal disease The onset of periodontal disease is characterized by a change in the microbial flora from a predominately Gram-positive to a predominately Gram-negative flora (Socransky et al., 1998). A number of specific Gram-negative bacteria have been identified as periodontal pathogens, 2 including the highly virulent and proteolytic species Porphyromonas gingivalis (van Winkelhoff et al., 2005). Notably, Porphyromonas gingivalis and associated virulence factors such as gingipains and lipopolysaccharide (LPS) are well-documented in the literature as important mediators of periodontal disease pathogenesis. For example, Porphyromonas gingivalis has been identified as one of a small group of pathogenic microbes that are associated with periodontal disease states at the site of the periodontal pocket (Socransky et al., 1998). While the presence of Gram-negative bacteria is the precipitating event, the actual tissue destruction (Fig. 1) results from complex interactions between the bacteria and the host immune cells (Silva et al., 2015). For example, the local influx of neutrophils (PMNs) in response to the bacterial infection results in the release of tissue-degrading matrix metalloproteases (MMPs) and associated tissue destruction will ensue (Johnstone et al., 2007). Also interceding between this host response and the resulting tissue degradation is a network of cytokines, which are soluble proteins used in cellular communication, and destructive enzymes released from host cells (Seymour and Taylor, 2004). Outcomes of disease include degradation of tooth supporting soft tissue integrity and resorption of the hard tissues supporting teeth, which ultimately lead to increasing non-physiologic tooth mobility and ultimately tooth loss (Page and Kornman, 1997). A simplified summary of the major elements of periodontal disease pathogenesis is depicted in Fig. 2. Periodontal disease affects up to 50% of adults in North America to various levels of severity (Eke et al., 2012). Periodontal disease is also associated with several systemic diseases including diabetes (Taylor et al., 1996), atherosclerosis and thrombosis (Schenkein and Loos, 2013). 3 FIGURE 2. Pathogenesis of periodontitis. Partial illustration of the elements involved in the pathogenesis of periodontitis. The initial infection (dental plaque) triggers a host response where pro-inflammatory cytokine signaling and MMP release result in tissue degradation and tooth loss. MMP=matrix metalloproteinase; PPD=probing pocket depth; CAL=clinical attachment loss. Bacteremia as a potential link between periodontitis and cardiovascular disease The circulatory system is normally a sterile environment (Horliana et al., 2014). Even so, transient bacteremia can result after non-surgical and surgical dental procedures or simply by daily brushing or flossing of teeth (Waghmare et al., 2013). Bacteremia may confer negative health consequences. For example, immune responses to the bacteria within the circulatory system can cause septic shock, which has risk of mortality (Singer et al., 2016). Bacteria can also spread through the circulatory system to numerous body regions and may elicit infections away from the original nidus of infection (Singer et al., 2016). A notable example of such precipitating infection is infective endocarditis and in such high-risk cases, treatment for bacteremia involves antibiotics and preventive efforts during high-risk scenarios using antibiotic prophylaxis (Yang et al., 2016). Accordingly, the dissemination of oral bacteria into the bloodstream represents a plausible mechanism by which periodontal pathogens could modulate cardiovascular diseases such as atherosclerosis. Dental Plaque Bacteria &Virulence factorsHost CellsCytokinesMMPsProstaglandinsConnective Tissue Degradation& Bone ResorptionDeep PPDsCALTooth MobilityTooth Loss 4 FIGURE 3. Schematic illustration of the potential relationships between periodontal infection and systemic disease. Oral bacteria may enter the systemic circulation; in addition, an elevated inflammatory response from periodontitis may also contribute to vascular lesions. Evidence of associations between periodontal disease and cardiovascular disease Atherosclerosis is a chronic condition associated with hyper-inflammation resulting in the accumulation of lipid-rich arterial plaques inside arteries. As these lesions mature, they harden and progressively restrict blood flow to various vital organs (such as the heart and brain) (Libby, 2001; Woollard, 2013). There are several lines of evidence suggesting an association between periodontitis and cardiovascular diseases and they are outlined as follows: Evidence from cross-sectional epidemiological research: Poor dental health has a documented association with an increased risk for cerebrovascular ischemia and stroke (Syrjanen et al., 1989). In men younger than 50 years, 25% of patients who experienced a stroke had significant dental disease, compared with only 2.5% of control patients without cerebrovascular disease (Syrjanen et al., 1989). The dental status, including dental caries, periodontitis, and endodontic lesions, was examined of more than 300 patients with strokes, brain infarction, and transient cerebral ischemia (Grau et al., 1997). This study showed an increased risk for stroke Chronic Periodontitis (Gum Infection)LiverPlatelet aggregation, Endothelium invasionVascular LesionC-reactive protein, FibrinogenAntibodies to bacteria, Cross-reactive antigens, Sensitization of T-cells 5 (odds ratio 2.60 [95% CI; 1.18 to 5.70]) was associated with periodontitis and periapical lesions. Also, analysis of the National Health and Nutrition Examination Survey (NHANES) I data showed that periodontal disease was a significant risk factor for stroke (Wu et al., 2000) with the relative risk at 1.41 (95% CI; 1.30 to 3.42) for periodontitis, indicating a significant increased stroke risk for patients with chronic periodontitis. In another study, periodontitis was associated with stroke in the with similar odds ratios (1.33; 95% CI; 1.03 to 1.70) (Joshipura et al., 2003). Evidence from blood-based markers: Periodontal disease is associated with elevations of circulating levels of cardiovascular risk factors such as C-reactive protein (CRP) (Loos et al., 2000), plasma fibrinogen (Chandy et al., 2017), matrix metalloproteases (Franco et al., 2017) and various coagulation factors (Tatakis, 1992). Patients with chronic periodontitis reportedly have increased systemic levels of CRP and fibrinogen after adjustment for various contributing disease factors dental calculus, education, sex, age, poverty index, body mass index, family history of myocardial infarction, diabetes, and tobacco and alcohol use (Slade et al., 2000). Colonization of atheromas by periodontal pathogens: Perhaps most importantly, notable periodontal pathogens including Porphyromonas gingivalis and Tannerella forsythia have been identified within human carotid atheromas (Haraszthy et al., 2000), suggesting that periodontal pathogens may participate in the initiation and progression of atherosclerosis. The rupture of atherosclerotic plaques causes local platelet aggregation, thrombus formation and the occlusion of the blood vessels, leading to myocardial infarction, or stroke. Evidence of association between periodontal infection and thrombosis: Chronic periodontitis is associated with increased prevalence of thrombosis (Wu et al., 2000) although the mechanisms linking the two conditions are undetermined (Schenkein and Loos, 2013). Periodontal pathogens can enter the systemic circulation from the oral environment (Iwai, 2009). 6 This concept fuels the premise that P. gingivalis is potentially thrombogenic since seropositivity for P. gingivalis is associated with an increased risk for stroke (Pussinen et al., 2007). Previous research has provided partial insights into the mechanisms by which P. gingivalis may promote thrombosis. For example, P. gingivalis reportedly increases the expression of the procoagulant tissue factor in cultured human aortic endothelial cells (Roth et al., 2006). Moreover, P. gingivalis produces cysteine proteases termed gingipains (Potempa et al., 2003) that are capable of activating prothrombin (Imamura et al., 2001a) as well as blood coagulation factors IX and X (Imamura et al., 1997; Imamura et al., 2001b). Lipopolysaccharide (LPS) and toll-like receptors (TLRs) In addition to gingipains, another virulence factor of P. gingivalis is an endotoxin termed lipopolysaccharide (LPS) that are structural components of the outer membrane of Gram-negative bacteria (Bosshardt and Lang, 2005). LPS consists of 3 domains: (1) the lipid anchor domain (lipid A), which is the primary pathogen-associated molecular pattern for LPS, (2) a short core oligosaccharide and an (3) O side chain that may be a long polysaccharide (Raetz and Whitfield, 2002). The innate immune system recognizes LPS via a family of pathogen recognition receptors called toll-like receptors (TLRs) (Miller et al., 2005). A number of TLRs have been identified and are classified into two categories based on their cellular location and their interacting ligand. Specifically, TLR-associated ligands are referred to as pathogen-associated molecular patterns and may include proteins, nucleic acids or lipid components of a number of pathogens such as fungi, Gram–negative bacteria lipopolysaccharide and viruses or fungi (Akira et al., 2006). One category of TLRs are those that are cell surface-expressed, which include 7 TLR1, -2, -4, -5, -6 and -11, which typically interact with membrane structures of pathogens (ex. lipids, proteins) (Akira et al., 2006). The other group of TLRs includes TLR3, -7, -8 and -9, that recognize cellular elements such as nucleic acids and, which are expressed as receptors in intracellular vesicles, including for example, vesicles associated with lysosomes and endoplasmic reticulum (Akira et al., 2006). Importantly, research using mice deficient in various TLRs show that these receptors individually provide distinct functions with regards to the resulting immune response (Akira et al., 2006). Engagement of TLRs with lipopolysaccharide induces dimerization by bringing together two signaling domains, which do provide platform for recruitment of a number of intercellular signaling components. Such dimerization will encourage cellular signaling that will culminate for example in alterations in gene expression that promotes a pro-inflammatory state (van Vliet et al., 2007). Indeed, LPS binding to TLRs does activate transcription factors for the synthesis of pro-inflammatory cytokines (Rosadini and Kagan, 2015). TLRs are found on various immune cells, including neutrophils (Subramanian et al., 2016), monocytes (Seneviratne et al., 2012), lymphotcytes (Buchta and Bishop, 2014; Reynolds and Dong, 2013) and platelets (Hamzeh-Cognasse et al., 2018). Regarding platelets, it is understood that four types of TLRs are expressed by platelets, namely TLR1, -2, -4 and -6 (Clark et al., 2007). Research does indicate an important role for TLR4 in LPS-induced actin assembly and associated platelet shape alterations that drive critical platelet functions. Specifically, TLR4 activation by its ligands upregulates phosphatidylinositol (3, 4, 5) triphosphate (PIP3) through PI3-kinases, and activates small GTPases such as Rac and Cdc42, which promote actin polymerization and kinase activation at the leading edge of cells that influence alterations in cellular shape (Kuiper et al., 2011). Such actin-associated alterations in cellular shape are critical for platelet activation, adhesion and spreading events associated with clot formation (Hartwig et 8 al., 1999). For example, such activated platelets show rapid actin polymerization giving rise to cell extensions such as filopodia (Hartwig et al., 1999) and these formations are mediated by Cdc42, a small GTPase that cycles between an inactive, GDP-bound form and an active, GTP-bound form (Kong and Ge, 2008). In the context of periodontal disease pathogenesis, it is currently undetermined whether GTPases such as Cdc42 are activated following the ligation of platelet TLRs by LPS from periodontal pathogens such as P. gingivalis. Platelets Platelets are the anucleate products of bone megakaryocytes and the smallest (0.5-3 m diameter) of the blood cells. Being anucleate, platelets cannot transcribe new mRNA (Shashkin et al., 2008). Platelets nonetheless contain a number of transcript elements such as spliceosomes that transform important biochemical transcripts into translatable mRNAs (Schwertz et al., 2006) such as the transcript for production of the pro-inflammatory cytokine interleukin-1β (Denis et al., 2005). The development of platelets from the megakaryocytes has been studied in mouse models where the system has shown platelets forming from megakaryocytes through the intermediary stage as pro-platelets (Lecine et al., 1998). Platelets are released from the pro-platelet membrane via formation of extensions of cytoplasm termed megapodia (Lind et al., 1987). These released platelets exhibit characteristics of the typical circulating platelet such as a discoid cellular structure that is maintained by a scaffolding of actin filaments within the cytoplasm and an innate capability to form actin-associated shape rearrangements associated with their activation in effort to seal sites of vascular injuries (Hartwig et al., 1999). In their quiescent forms, platelets circulate as round discs that become activated upon binding of their surface receptors with soluble extracellular signals (Stalker et al., 2012). Upon 9 activation, platelets quickly alter their shape in large part due to actin-based cytoskeleton arrangements of new actin filaments into cellular extensions of filopodia and lamelopodia (Hartwig et al., 1999). Notably, such actin components constitute the most prevalent protein within platelets. They also play critical roles in establishing cellular form and for establishing strength for the platelet plugs used by the host to stop extravasation of fluid from injured blood vessels (Hartwig and DeSisto, 1991) These activated platelets also aggregate and secrete bioactive molecules stored in granules within their cytoplasm (White and Krumwiede, 1987). It is interesting to note that several hundred different molecules have been discovered within these secretions. These secreted molecules play critical roles in mediating responses to vascular injury that include promoting repair responses with hemostatic and angiogenic factors such as fibrinogen (Harrison, 1992) and vascular endothelial growth factor (Italiano et al., 2008). Granules also contain tissue-degrading proteases such as MMP-2 and MMP-9 (Mastenbroek et al., 2015) and pro-inflammatory cytokines such as (TNF-α) (Gawaz and Vogel, 2013). Importantly, activated platelets aggregate together to form a plug that is eventually converted into a fibrin clot following a well-defined coagulation cascade (Mackman et al., 2007). The plasma membrane of platelets contains numerous surface receptors that recognize extracellular signaling molecules (Table 1). Several lines of published evidence also indicate that P. gingivalis can promote thrombosis by signaling to blood platelets (Kerrigan and Cox, 2009; McNicol, 2015; Nakayama, 2010). For example, P. gingivalis gingipains have been found to promote platelet aggregation (Klarstrom Engstrom et al., 2015) by signaling through the platelets’ protease-activated receptors (PARs) (Lourbakos et al., 2001), the major thrombin receptor on the platelet surface (Coughlin, 1999). In addition to PARs, platelets also express toll-like receptor 4 (TLR4), which recognizes 10 lipopolysaccharide (LPS), a significant virulence factor of P. gingivalis (Olsen and Singhrao, 2018). However, it is currently undetermined how LPS from periodontal pathogens such as P. gingivalis might influence platelet activation and clot formation, specifically in terms of cytoskeleton-mediated platelet shape change. Platelet activation, aggregation and clot formation Platelet behavior during clot formation can be roughly separated into three sequential stages namely: (1) adhesion, (2) activation, and (3) aggregation (Tomaiuolo et al., 2017). The adhesion stage begins with endothelial cell layer disruption that promotes collagen and von Willebrand factor to encourage anchorage of the platelets to the subepithelium (Coenen et al., 2017). Specifically, platelet-associated GP1b-9-5 receptor interacts with newly available von Willebrand factor whereas the glygocprotein-6 receptor and integrin α2β1 interact with the newly exposed collagen (Dubois et al., 2006). Platelet activation processes are initiated when exposed collagen from the subendothelium interacts with putative platelet surface receptors. This activation promotes platelet-degranulation and releases of mediators of the coagulation system such as fibrinogen and thromboxane A2 (Heemskerk et al., 2002). Enhanced production of thromboxane A2, for example, promotes positive feedback signaling of platelet-associated thromboxane receptors via paracrine and autocrine mechanisms and these thromboxane receptors subsequently activate the GPIIb/IIIa receptors to promote platelet aggregation (Yip et al., 2005). 11 TABLE 1. Known platelet receptors and their corresponding agonists. Platelet receptor Corresponding agonist(s) Reference(s) α2β1 Integrin Collagen (Clemetson and Clemetson, 2008) α5β1 Integrin Fibronectin (Kasirer-Friede et al., 2007) α6β1 Integrin Laminin (Chang et al., 2005) αLβ2 Integrin Neutrophil adhesion, platelet-leukocyte interaction (Diacovo et al., 1994) αIIbβ3 Integrin Fibrinogen, fibrin, vWf, fibronectin, vitronectin, thrombospondin (Bennett, 1990) αVβ3 Integrin Vitronectin, fibrinogen, vWF, prothrombin, thrombospondin, osteopontin (Bennett, 1996) TLRs (1, 2, 4, 6) Products of bacteria (ex. LPS), viruses, protozoa, fungi (Beutler, 2004; Clark et al., 2007) Protease activated receptor-1 Thrombin (da Silva et al., 2014) Α2 adrenergic receptor Epinephrine (Spalding et al., 1998) P2Y12 receptor ADP (Dorsam and Kunapuli, 2004) P-selectin Neutrophils, monocytes, endothelial cells, pro-coagulant microparticles (P-selectin glycoprotein ligand-1, tissue factor) (Falati et al., 2003) C-type lectin-like receptor-2 Podoplanin, rhodocytin (Ozaki et al., 2009) Adenosine diphosphate receptor Adenosine diphosphate (Gachet, 2001) Prostacycline receptor PGI2 Prostacyclin, G proteins (Katsuyama et al., 1994) Prostaglandin PGE2 ADP, collagen (Fabre et al., 2001) Glycoprotein VI Collagen (Moroi and Jung, 2004) Thrombopoietin receptors c-mpl Thrombopoietin (Kaushansky, 2008) Platelet insulin receptors Insulin (Udvardy et al., 1993) Platelet-derived growth factor receptor Platelet-derived growth factor (Raica and Cimpean, 2010) 5-hydroxytryptamin 2A Serotonin (Li et al., 1997) Glycoprotein 3b Microparticles (Su and Abumrad, 2009) Glycoprotein Ibα vWF (Bergmeier et al., 2006) Glycoprotein Ib-IX-V vWF, thrombin, P-selectin, factor IX, factor XII (Li and Emsley, 2013; Solum et al., 1980) C1q receptor Collagen, immune complexes (Peerschke and Ghebrehiwet, 1998) Thromboxane A2 receptor Thromboxane A2 (Nakahata, 2008) Tyrosine kinase Syk Collagen (Poole et al., 1997) G-protein-coupled receptor ADP, thromboxane, thrombin (Gurbel et al., 2015) 12 Platelet adhesion and spreading dynamics The formation of the platelet plug is an intricate process involving a multitude of protein interactions (Tomaiuolo et al., 2017). An adhesion stage begins the process and involves endothelial cell layer injury promoting newly exposed components such as collagen and von Willebrand factor, to jointly function to anchor platelets to the subepithelium (Dubois et al., 2006). Specifically, fundamental interactions in this process involves the platelet-associated GP1b-9-5 receptor bonding with von Willebrand factor and the glycoprotein-6 (GP6) receptor in conjunction with integrin α2β1 bonding with the newly exposed collagen in effort to encourage platelet adhesion (Dubois et al., 2006). FIGURE 4. Platelet spreading and clot formation. Illustration of platelet activation and shape change (spreading) as an integral component of clot formation. Following their adhesion to an appropriate substratum, the platelets’ ability to shape change and spread plays an important role in clot formation (Aslan et al., 2013) (Fig. 4). This process is driven in large part by changes in the structural framework of the platelets, known as the actin cytoskeleton, and is mediated by increases in intracellular calcium, as well as the activity of actin-accessory proteins and associated interactions with components of the platelet cell TIME(1) Resting platelet (2) Activated & spreading platelet (3) Clot: platelet aggregation & fibrin maturation(1) Resting platelet (2) Generation of filopodial protrusions (3) Spread platelet: lamellipodiafill gaps between filopodiafilopodialamellipodiumTIME 13 membrane (Stossel et al., 2006). This spreading process includes the following sequential events, outlined as follows in a simplistic fashion: (1) early rounding, (2) early spread, and (3) complete spread (Aslan and McCarty, 2013; Behnke, 1970). ‘Early rounding’ is characterized by a well-circumscribed platelet with a highly grooved cell surface that functions to enhance available platelet surface area to enable adhesions functionality (Aslan et al., 2013; Behnke, 1970). Development into the subsequent ‘early spread platelet’ resembles a well-circumscribed cell body with multiple cellular extensions called filopodia (Aslan et al., 2013; Behnke, 1970). Lastly, the ‘completely spread’ platelet is characterized by broader cellular extensions termed lamellopodia that form between the spike-like fillopodia extensions and there is a characteristically dense central cell body (Aslan et al., 2013; Behnke, 1970). Actin assembly Platelet shape change is contingent on the rapid assembly and rearrangement of the platelet’s actin cytoskeleton (Hartwig et al., 1999). The actin cytoskeleton is comprised of globular monomeric subunits of (termed G-actin) that rapidly assemble into filamentous actin polymers (termed F-actin) at the cell membrane (Stossel et al., 2006), thus creating cellular protrusions and extensions that are characteristic of activated, spreading platelets (Aslan and McCarty, 2013). The assembly of actin filaments occurs in a number of stages and these include activation, nucleation and elongation (Lee and Dominguez, 2010). Activation occurs with the bonding and exchange of divalent cations occurs in various locations on G-actin (Knowles, 1980). During the subsequent nucleation phase, small unstable fragments of F-actin that are able to polymerize and form short filament structures. The ensuring elongation phase begins when there are a sufficiently large number of these short polymers. In this elongation phase, the actin filament 14 rapidly forms and grows in size through the reversible addition of new actin monomers at both ends of the filament structure (Korn et al., 1987). Lastly, a stationary equilibrium phase is achieved where the G-actin monomers are exchanged at both ends of the microfilament without any alteration to its length (Korn et al., 1987). In this final phase, the dynamics involving addition and elimination of actin components does not promote any alteration in the microfilament's length. As is observed in most cell types, activated platelets exhibit rapid shape change characterized by spike-like cellular extensions known as filopodia and sheet-like extensions known as lamellipodia (Aslan and McCarty, 2013; Hartwig et al., 1999). Formation of these F-actin-rich cellular extensions (Fig. 5) is regulated by low-molecular weight proteins known as Rho GTPases (Kong, 2008). FIGURE 5. Actin polymerization and cell extensions. Illustration of the formation of F-actin-rich cell extensions during platelet spreading, including the spike-like filopodia (2) and sheet-like lamellipodia (3). GTPases The GTPases are a family of hydrolase enzymes that bind and hydrolyze guanosine triphosphate (GTP). As a result, GTPases play important regulatory roles in biochemical signaling systems acting as a ‘switch’ to activate and inactivate cellular signaling cascades (Scheffzek and Ahmadian, 2005). This switch involves, in a simplistic sense, hydrolysis-induced structural TIME(1) Resting platelet (2) Activated & spreading platelet (3) Clot: platelet aggregation & fibrin maturation(1) Resting platelet (2) Generation of filopodial protrusions (3) Spread platelet: lamellipodiafill gaps between filopodiafilopodialamellipodiumTIME 15 alteration of the GTPase that occurs through (1) intrinsic enzyme activity and (2) GTPase-activating proteins (GAPs), that switch the GTPase from the active “On” structure (GTP-bound GTPase) to the inactive “Off” structure (GDP-bound GTPase) (Scheffzek and Ahmadian, 2005). The “On” switch involves, guanine nucleotide exchange factors (GEFs) that encourage dissociation of the GDP from the GTPase, enabling it to functionally interact with a new GTP molecule (Scheffzek and Ahmadian, 2005). GTPases are classified as part of a “superfamily” of monomeric proteins homologous to Ras (Goitre et al., 2014), which characteristically possess kilodalton weights of ~ 21 and function as molecular switches for various of biochemical signaling processes (Goitre et al., 2014). This superfamily is divided into a number of categories that include Ras, Rab, Rho, Arf and Ran (Goitre et al., 2014). Modulation of cell spreading by the Rho GTPases The Rho subfamily includes the proteins RhoA, Rac1, and Cdc42, which have critical roles in actin-associated cellular formations, specifically, stress fiber formation, lamellopodia and filopodia formation, respectively (Tapon, 1997). A critical enzyme that is implicated in mediating biologic activity of Rac1 and Cdc42 is p21 activated kinase (PAK). Specifically, p21 activated kinase is a downstream target of Rac1 and Cdc42 and thus regulates actin assembly and the formation of spike-like filopodia and sheet-like lamellipodia (Caron and Hall, 1998; Rane and Minden, 2018). This is also observed in platelets, where actin assembly and cell spreading are essential for clot formation (Fig. 6). 16 FIGURE 6. Cdc42 activation and filopodial formation. Illustration of Cdc42 activation and filopodial formation in a platelet activated by ligation of TLRs (ex. TLR4) by P. gingivalis LPS. Inset: Activation of Cdc42 by substitution of GDP for GTP results in actin assembly thus producing a spike-like filopodium. The coagulation cascade: intrinsic and extrinsic pathways The coagulation cascade includes 3 biochemical pathways: an extrinsic and an intrinsic pathway and one final common pathway (Chaudhry and Babiker, 2018). These pathways ultimately lead to the same reactions that elicit cross-linked fibrin formation associated with a maturing blood clot. Initial pathways are the intrinsic contact activation pathway, and the extrinsic tissue factor pathway (Chaudhry and Babiker, 2018). The pathways consist of a series of biochemical reactions, in which an inactive zymogen enzyme precursor of a serine protease and its glycoprotein co-factor are activated to become active components that catalyze the subsequent reactions in the series (Furie and Furie, 2008). The intrinsic contact activation pathway starts with formation of the primary complex on collagen by high-molecular-weight kininogen, prekallikrein, and factor 12. Prekallikrein converts to kallikrein and factor 12 becomes factor 12a. Factor12a then promotes factor 11 to transform into factor 11a. Factor 11a subsequently activates factor 9, which with its co-factor factor 8a form the tenase complex. The tenase complex then transforms factor 10 to factor 10a. Notably, this intrinsic contact activation system appears to respond and play a significant role in inflammation and innate immunity TLR4P.g. LPSCdc42‘off’’ GDPCdc42‘on”GTPCell membraneTLR4GTPbindingGTPhydrolysisActin filament assemblyLPS 17 processes (Long et al., 2016) as putative inflammatory pathogens do activate contact system proteins. For example, the contact system protein factor 12 has been shown to be activated via the inflammatory disease-inducing bacterial cell surface protein called polyphosphate (Smith et al., 2006). Moreover, inflammatory mediators influence blood clotting cascades by inducing thrombin formation in mammalian species (van der Poll et al., 2001). Such a role has been documented for the cytokines IL-1α (Jansen et al., 1995), IL-6 (Stouthard et al., 1996) and TNF (van der Poll et al., 1990). The extrinsic tissue factor pathway has a primary role to generate a thrombin burst, which is critical for achieving hemostasis (Salvagno and Berntorp, 2010). The process includes the following steps: (1) Immediately after the integrity of a blood vessel is degraded by injury to the blood vessel, factor 7 leaves the circulation and contacts tissue factor (TF) expressed on fibroblasts and leukocytes in the region, forming an activated complex TF-factor 7a complex., (2) the TF-factor 7a complex subsequently activates factor 9 and factor 10, (3) Factor 7 is activated by and number of factors including thrombin, factor 11a, factor 12 and factor 10a. (4) factor 10a and its co-factor factor 5a then form the prothrombinase complex, which transforms prothrombin to thrombin (Krishnaswamy, 2013). The coagulation factors typically circulate as inactive zymogens and structurally are mostly serine proteases enzymes that function to cleave and activate downstream proteins in the signaling cascade. The exceptions are factors 3, 5, 8 and 13 where 3, 5 and 8 are glycoproteins, and factor 13 is a transglutaminase (Furie and Furie, 2005). In essence, the three pathways of the coagulation cascade involve extrinsic tissue factor and intrinsic contact activation pathways both activating the final common pathway of factor 10, thrombin and fibrin (Furie and Furie, 2005) (Fig. 7). 18 FIGURE 7. Illustration of the extrinsic, intrinsic and common coagulation pathways. The intrinsic coagulation cascade is comprised of the following factors: XII, prekallikrien (PK), XI, IX, and VIII and high-molecular-weight kininogen (HK). Extrinsic coagulation cascade comprises tissue factor and factor VII. The common coagulation pathway consists of the following factors: X, V, II and fibrinogen. Activated partial thromboplastin time relies on the presence of most of the above listed factors with the exception of tissue factor and factor VII. In contrast, prothrombin time (PT) relies on the following: tissue factor; factors VII, X, V, II and fibrinogen. Conventional measurements of coagulation There are a number of commonly used laboratory tests to evaluate hemostatic function. These include the activated partial thromboplastin time (aPTT), prothrombin time (PT), bleeding time, fibrinogen testing, assessments of platelet count, and platelet function testing (Lassila, 2016; Paniccia et al., 2015). The intrinsic contact activation pathway is initiated by activation of the "contact factors" of plasma, and can be measured for example, by the activated partial thromboplastin time (aPTT) test. Notably, this intrinsic cascade, although integral to the coagulation, has a relatively small role in clot development and this is in contrast to the extrinsic pathway, which has a fundamental role in producing the integral thrombin seal involved in initial clot formation (Long et al., 2016). Specifically, this has been shown in patients with deficiencies of various intrinsic coagulation factors, such as factor XII, whom do not demonstrate a severe bleeding disorder (Renne and Gailani, 2007). Rather, intrinsic coagulation cascades appear to be relatively more associated with innate immunity and inflammatory events (Long et al., 2016; Nickel and Renne, 2012). An example of this interaction involves the role of the intrinsic 19 coagulation factor XII in binding to the surfaces of gram-negative and –positive bacteria, viruses, fungi and human simplex virus-1 (Nickel and Renne, 2012). In contrast, the extrinsic tissue factor pathway is initiated by release of tissue factor, and can be assessed by the prothrombin time (PT) test. Notably, PT results are often reported as International Normalized Ratio (INR), which is used to monitor anticoagulant effectiveness of warfarin in the individual patient (Hayward et al., 2012; Tripodi et al., 2016). Bleeding time is a coagulant test that examines platelets function and involves inducing a patient to bleed and then determining the time required for bleeding to arrest. The bleeding time assessment is indicated when other more reliable and less invasive tests for coagulation are unavailable (Lehman et al., 2001). Rotational thromboelastometry (ROTEM) Hemostasis is controlled by both endothelium and various mediators that include but are not limited to cellular-derived components, coagulant proteins and anti-coagulant proteins. Common assessments of hemostasis involve plasma-based assays that rely on plasma-based clotting proteins and not whole blood samples. In contrast, global hemostasis assays such as rotational thromboelastometry (ROTEM) use whole blood samples to assess platelet function, clot strength, and analyses can be performed in a relatively short amount of time (Da Luz et al., 2014). Specifically, ROTEM relies on whole venous blood samples in effort to quantify viscoelastic alterations of clotting parameters that occur during clot formation (Whiting and DiNardo, 2014). In doing so, ROTEM analysis enables quantification of clot formation kinetics that can be tested, for example, in the presence of various blood components or other factors of interest that may modulate clotting parameters (Da Luz et al., 2014). 20 The fundamental components of the ROTEM system include a heated cup (body temperature 370C) for containing blood samples and a pin transduction system that is placed into this cup and blood sample of interest. During the analysis, the blood samples in the cup remain motionless while the pin oscillates at a fixed rate (Fig. 8). As coagulation progresses, the forming clot along with its associated meshwork of fibrin, produce a physical impedance to pin oscillation, which transfers a measure of torque of the motionless cup relative to the oscillating pin. ROTEM relies on an optical detection and motion sensor system that generates the electrical signal that a computer documents (Whiting and DiNardo, 2014). Computer software produces quantitative parameters and a graphical representation, which allows evaluation of the different phases of clotting. Coagulation may be initiated purely by contact activation with the cup (called “native”), or with specific activators targeting either extrinsic pathway or intrinsic pathway. Clotting factor reagents are available for both activation of the intrinsic coagulation cascade (ex. INTEMTM, partial thromboplastin phospholipid), and EXTEMTM (ex. recombinant tissue factor) for stimulation of the extrinsic coagulation cascade. Like any other hemostasis evaluating method, ROTEM has limitations. For example, ROTEM is not sensitive to the effect of von Willebrand factor or platelet antagonists such as acetylsalicylic acid (Da Luz et al. 2014). Moreover, ROTEM’s sensitivity for coagulation factor deficiencies is less pronounced compared to traditional clotting assays such as activated partial thromboplastin time or prothrombin time. 21 FIGURE 8. Illustration of rotational thromboelastometry (ROTEM). A cuvette (cup) holds a whole blood (WB) sample with an immersed cylindrical pin. A spring rotates the pin to the right and to the left. When blood starts clotting, the clot increasingly restricts the rotation of the pin with rising fibrin maturation, enhanced platelet aggregation, which leads to an associated increase in clot firmness. This mechanical kinetic is detected with an optical device and digitally quantified by a computer. Image reproduced with permission. AIMS AND HYPOTHESES OF THIS RESEARCH: AIM 1: Quantify platelet spreading in the presence of lipopolysaccharide (LPS) from Porphyromonas gingivalis, a pathogen central to the pathogenesis of periodontitis. Hypothesis: Platelet spreading is enhanced following stimulation by P. gingivalis LPS. AIM 2: Compare clot formation (mediated by the intrinsic pathway) in the presence or absence of LPS, and in blood samples collected from patients with severe periodontitis and controls. Hypothesis: Following stimulation of the intrinsic pathway, clotting time is reduced in patients with severe periodontitis. STATEMENT OF THE PROBLEM: Chronic periodontitis is reportedly associated with cardiovascular disease including thrombosis but the cellular and molecular mechanisms linking periodontitis to thrombosis are undefined. 22 Chapter Two: Porphyromonas gingivalis lipopolysaccharide promotes platelet spreading and thrombosis via Cdc42 activation Materials and Methods Platelet purification and spreading assays To study the in vitro platelet response to Porphyromonas gingivalis lipopolysaccharide (LPS), human blood samples were obtained from healthy volunteers with approval obtained from the University of British Columbia Clinical Research Ethics Board (CREB) and informed consent in accordance with the Declaration of Helsinki. Platelets were isolated by sequential centrifugation of whole blood and the resultant platelet-rich plasma as previously described (Kim et al., 2013). Resting platelets were incubated either with 1 g/mL of lipopolysaccharide (LPS) from P. gingivalis (Invivoven, San Diego, CA) or vehicle control (ultrapure H2O) immediately prior to plating on to glass coverslips. Platelets were allowed to spread on the coverslips over a time course ranging from 1 to 15 minutes. Platelets were then fixed with 4% paraformaldehyde, permeabilized with 0.1% (v/v) Triton-X and labeled with Alexa-488-phalloidin (Thermo Fisher Scientific, Grand Island, NY) to allow visualization of the actin cytoskeleton by confocal microscopy. Confocal images were captured with a Zeiss spinning disk confocal microscope and processed by SlideBook software (Intelligent Imaging Innovations, Denver, CO). Cell spreading was quantified by visual assessment of cell morphology. Cells were classified as either (1) spread or (2) rounded; the percentage of spread vs. total cells was calculated per field of view. Cell spreading was also quantified by counting the average number of filopodial projections at the platelet surface. A minimum of 3 fields of view were analyzed per experiment; moreover, all quantifications were based on a minimum of 3 independent experiments. 23 Active Cdc42 pull-down assay and immuoblotting The Rho GTPase Cdc42 is directly responsible for filopodial formation in platelets(Akbar et al., 2011). To quantify the active, GTP-bound form of Cdc42 in platelets, lysates of vehicle- and LPS-treated platelets were incubated with p21-activated kinase binding domain (PAK-PBD) beads (Thermo Fisher Scientific, Grand Island, NY) to precipitate the active, GTP-bound form of Cdc42. Bead-associated proteins were eluted, resolved by SDS-PAGE and immunoblotted with an anti-Cdc42 antibody. Prior to the PAK pull-down procedure, a portion of the whole platelet lysates were retained and blotted for Cdc42 to verify the equivalence of total Cdc42 in all samples. Rotational thromboelastometry (ROTEM) We employed rotational thromboelastometry (ROTEM) to simultaneously analyze the coagulation time of the clots formed from human blood samples (Whiting and DiNardo, 2014). Clotting time is defined as the time elapsed from the addition of the clotting agonist until the start of blood clot formation (Whiting and DiNardo, 2014). The ROTEM® delta (Tem International GmbH, Munich, Germany) apparatus consists of an oscillating sensor pin immersed in a sample cup containing 330 µl of whole blood. To trigger the intrinsic coagulation pathway, 4 µl of ellagic acid (INTEMTM agonist) is added to the blood sample in the presence of 30 µl of 0.2M CaCl2. The nascent clot then forms a physical connection thus creating resistance between the cup and the pin. The degree of pin motion allowed by the clot is proportional to the clot’s elastic strength and is quantified as the maximal clot strength in millimeters (mm). Periodontal patient recruitment and blood collection Peripheral venous blood was obtained from 16 systemically healthy, non-smoking and completely medication-free dental patients recruited from the University of British Columbia 24 dental clinic. Patients had no history of any form of periodontal treatment within the past 6 months. Full-mouth periodontal probing was performed for each patient. The presence and extent of bone loss was determined from dental radiographs. The subjects were age- and sex-matched and classified into the following 2 groups: 1. Healthy periodontium (n=8): patients showed minimal signs of gingival inflammation and no radiographic evidence of alveolar bone loss (2015; Armitage, 1999). 2. Generalized severe periodontitis (n=8): patients with clear radiographic evidence of alveolar bone loss and periodontal pocketing (probing depths ≥5 mm) at a minimum of 30% of sites (2015; Armitage, 1999). The detailed periodontal characteristics of the blood donors are summarized in Table 2. Measurements of the intrinsic (aPTT) and extrinsic (PT) clotting cascades In addition to ROTEM analysis of whole blood samples, separate aliquots of plasma were retained for determination of the activated partial thromboplastin time (aPTT) and prothrombin time (PT) for each periodontitis and healthy control patient. The aPTT and PT were measured using the Stago ST4 Coagulation Analyzer semi-automated benchtop system. To determine the aPTT, 120 µl of plasma were incubated at 37°C for 180 seconds and mixed with an equal amount of aPTT reagent (Dade Actin FS Activated PTT, Siemens/Dade-Behring). Of this mixture, 100 µl was then transferred into a cuvette where clotting was initiated by adding CaCl2 to a final concentration of 8.33 mM. To measure PT as a determinant of the extrinsic clotting cascade, 50 µl of plasma was incubated in a cuvette at 37°C for 180 sec prior to the addition of 100 µl of clotting reagent, also known as PT reagent (recombinant tissue factor, Siemens/Dade-Behring). All assays were performed three times in duplicate. 25 Statistical analysis Using GraphPad Prism software (La Jolla, CA), a two-way analysis of variance (ANOVA) and Bonferroni post-hoc multiple comparison tests were used to assess the effects of (1) treatment (LPS vs. vehicle control) and (2) time on platelet spreading. The Student’s t-test was used to determine the effect of periodontal status (severe periodontitis vs. healthy control) on clotting time, activated partial thromboplastin time (aPTT), prothrombin time (PT) and maximum clot thickness. Statistical significance was set at p<0.05. Results Lipopolysacharide (LPS) from Porphyromonas gingivalis enhances platelet spreading To improve our understanding of how periodontal infection may increase the risk of thrombosis, we evaluated the effect of P. gingivalis LPS on cell spreading since platelet shape change is a pivotal event in clot formation. Vehicle-treated (control) platelets exhibited a spread morphology characterized by a gradual increase in cell area and also exhibited spike-like filopodial projections during the 15 minutes of spreading on glass coverslips (Fig. 9A). However, LPS-treated platelets harbored a significantly (p<0.05) greater proportion of spread cells than did control platelets (Fig. 9B). These data indicate that cell surface actin assembly and platelet shape change are directly catalyzed by P. gingivalis LPS. P. gingivalis LPS promotes filopoidal formation in platelets by activating Cdc42 In addition to exhibiting greater proportions of spread cells, platelets pre-treated with P. gingivalis LPS also exhibited significantly (p<0.05) greater numbers of filopodial extensions within the first 2 minutes of spreading than did control cells (Figs. 9A-9B, 10A). Therefore, to further dissect the signaling events in platelets that occur downstream of LPS challenge, we 26 evaluated the LPS-induced activation of Cdc42, a small GTPase with a clearly documented role in filopodial formation (Akbar et al., 2011). We used p21-activated kinase (PAK-PBD) beads to precipitate active, GTP-bound Cdc42 from lysates of platelets treated with either LPS or vehicle. The PAK-PBD pulldown assay clearly shows higher levels of active Cdc42 in LPS-treated platelets relative to controls (Figs. 10B-10C). Remarkably, peak Cdc42 activity occurs after 2 minutes of LPS treatment; this is consistent with the finding that greatest increase in filopodial formation occurs within the first 2 minutes of platelet spreading (Fig. 10B). Levels of Cdc42 activity are attenuated by the 15-minute time point (Fig. 10B); this is also consistent with our observation that filopodial numbers are slightly decreased in LPS-treated platelets between the 5- and 15-minute time points (Fig. 10A). Collectively, these data clearly indicate that gram-negative infection amplifies platelet activation and shape change. Lipopolysacharide (LPS) from P. gingivalis shortens clotting times in whole blood The potential for periodontal pathogens to enter the systemic circulation is well-documented (Iwai, 2009) and P. gingivalis seropositivity is associated with thrombosis risk (Pussinen et al., 2007). To determine how LPS signaling in platelets may contribute to thrombosis, we pre-incubated whole blood samples with P. gingivalis LPS for 6 hours prior to the addition of the ellagic acid (INTEMTM) reagent to trigger the intrinsic clotting cascade. LPS-treated blood samples clotted significantly (p<0.05) more rapidly than control blood samples, as measured by the rotational thromboelastometer (ROTEM) (Fig. 11). These data, obtained from whole blood samples, validate our initial observations in purified platelets in terms of the pro-thrombotic effects of P. gingivalis LPS. 27 Patients with generalized severe chronic periodontitis exhibit accelerated clotting and activated partial thromboplastin (aPTT) times Since the addition of exogenous LPS reduces clotting time (Fig. 11), we then wished to assess the effects of long-standing severe periodontal infection on blood coagulability. We obtained blood samples from 8 systemically healthy, medication-free subjects diagnosed with generalized severe chronic periodontitis (SP) as well as 8 age- and sex-matched periodontally healthy controls (HP) (Table 2). TABLE 2. Periodontal characteristics of blood donors. Healthy (n=8) Periodontitis (n=8) Age (mean  SD) 56.5  14.1 58.3  12.6 Sex distribution 6 M; 2 F 6 M; 2 F Mean PD (mm) 2.7  0.5 5.5  0.3 PD range (mm) 1-4 1-13 PD4 mm (% sites) 100 43 5mm<PD<7 mm (% sites) 0 39 PD7 mm (% sites) 0 15 BOP (%  SD) 18.0  2.4 73.0  18.7 PI (%  SD) 14.0  1.8 82.0  19.1 Legend: SD: Standard deviation PD: Probing depth BOP: Bleeding on probing PI: Plaque index 28 The intrinsic coagulability of the blood samples (without the addition of LPS) was assessed by ROTEM as described in Section 2.4 above. The clotting time of the blood samples obtained from the SP patients were significantly (p<0.05) reduced relative to the HP control blood samples (Fig. 12A). Moreover, relative to healthy controls, SP patients exhibited significantly (p<0.05) faster active partial thromboplastin times (aPTT) (Fig. 12B) but there was no significant (p>0.05) difference in prothrombin times (PT) (Fig. 12C). In addition, there was no significant difference (p>0.05) in maximal clot firmness in SP patients and controls (Fig. 12D). These data indicate that severe periodontal infection affects coagulation via the intrinsic pathway but does not appear to influence the extrinsic pathway or the physical properties of the formed blood clot. 29 FIGURE 9. Platelet spreading is enhanced by P. gingivalis LPS. A. Confocal micrographs depict purified platelets spreading on glass coverslips and stained with Alexa-488-phalloidin to label F-actin. Platelets were pre-treated with either ultrapure H20 vehicle (top panels) or with lipopolysaccharide (LPS) from Porphyromonas gingivalis (bottom panels) and allowed to spread for the indicated times. Bar=10m. B. Histogram depicts the relative percentage of spread cells in control platelets (white bars) and LPS-treated platelets (black bars). Data are mean  SD and represent three (3) independent experiments using blood from different donors. *, p<0.05, based on two-way ANOVA and Bonferroni multiple comparison tests. Figure 1Control1 minControl2 minControl5 minControl15 minLPS1 minLPS2 minLPS5 minLPS15 minA.B.Figure 1Control1 minControl2 minControl5 minControl15 minLPS1 minLPS2 minLPS5 minLPS15 minA.B. 30 FIGURE 10. P. gingivalis LPS promotes filopodial formation via Cdc42 activation. A. Histogram depicts the relative numbers of filopodial extensions in control platelets (white bars) and LPS-treated platelets (black bars). Data are mean  SD and represent three (3) independent experiments using blood from different donors. *, p<0.05, based on two-way ANOVA and Bonferroni multiple comparison tests. B. Whole cell lysates were prepared from platelets incubated with either ultrapure H20 vehicle (-) or P. gingivalis LPS (+) for the indicated time periods. Protein was resolved by SDS-PAGE and blotted (IB, immunoblot) with an antibody against the small GTPase Cdc42. C. Platelet lysates prepared as described in (B) were incubated with p21-activated kinase (PAK) beads to selectively bind and precipitate the active, GTP-bound form of Cdc42. Bead-associated proteins were eluted, resolved by SDS-PAGE and blotted (IB, immunoblot) for Cdc42. Figure 2A.C. PAK pulldownIB: Cdc420 1 2 5 15Time (mins)P. gingivalis LPS - + + + + B. Whole cell lysatesIB: Cdc420 1 2 5 15Time (mins)P. gingivalis LPS - + + + + 31 FIGURE 11. P. gingivalis LPS accelerates blood clotting. Dot plot depicts the clotting times of whole blood samples pre-incubated with either ultrapure H20 vehicle (Control, black circles) or P. gingivalis LPS (LPS, black squares) as measured by rotational thromboelastometry (ROTEM). Error bars are SD and represent three (3) independent experiments using blood from different donors. *, p<0.05, based on t-test. Figure 305 01 0 01 5 02 0 02 5 03 0 03 5 0Clotting time (sec)*C o n tro lL P S 32 FIGURE 12. Clotting time and partial thromboplastin time are shortened in patients with severe chronic periodontitis. A. Dot plot depicts the clotting times of whole blood samples (without pre-incubation) obtained from periodontally healthy control patients (Health, black circles, n=8) and age- and sex-matched patients with generalized severe chronic periodontitits (Periodontitis, black squares, n=8) as measured by rotational thromboelastometry (ROTEM). Error bars are SD. *, p<0.05, based on t-test. B-C. Dot plots depict the activated partial thromoboplastin times (B) or prothrombin times (C) of blood samples (without pre-incubation) obtained from periodontally healthy control patients (Health, black circles, n=8) and age- and sex-matched patients with generalized severe chronic periodontitits (Periodontitis, black squares, n=8). Error bars are SD. *, p<0.05, based on t-test. D. Dot plot depicts the maximum clot firmness of whole blood samples (without pre-incubation) obtained from periodontally healthy control patients (Health, black circles, n=8) and age- and sex-matched patients with generalized severe chronic periodontitits (Periodontitis, black squares, n=8) as measured by rotational thromboelastometry (ROTEM). Error bars are SD. *, p<0.05, based on t-test. Figure 405 01 0 01 5 02 0 02 5 03 0 03 5 04 0 0Clotting time (sec)*H e a lthP e r io d o n t it isA.C.051 01 52 02 53 03 54 0aPTT (sec)*H ea lthP e rio d o n tit is051 01 52 02 53 03 54 0PT (sec) H e a lthP e r io d o n tit isD.01 02 03 04 05 06 07 08 0Maximum clot firmness (mm)H ea lthP e r io d o n tit isB. 33 Discussion Published cross-sectional epidemiological studies provide considerable indirect evidence to support an association between periodontal and cardiovascular diseases (Dietrich et al., 2013; Persson and Persson, 2008; Teeuw et al., 2014; Tonetti et al., 2013). However, there is relatively little direct evidence regarding the cellular and molecular mechanisms linking chronic periodontitis and thrombosis. Here we report the novel finding that LPS from P. gingivalis directly amplifies Cdc42 activation, platelet shape change and spreading, a critical step for clot formation and thrombosis. We further translate this finding to human samples using advanced ROTEM technology to measure the formation rate and physical properties of blood clots. Notably, data from our human pilot study indicate that generalized severe periodontitis confers an increased intrinsic propensity for clot formation. P. gingivalis lipopolysaccharide (LPS) mediates platelet activation and spreading in a Cdc42-dependent manner Our study is the first to report a specific role for P. gingivalis LPS and Cdc42 signaling with regards to the platelet cytoskeleton and GTPase activation. While previously published data also indicate increased platelet activation in response to P. gingivalis, it should be noted that those studies focused on different aspects of P. gingivalis-platelet interactions. For example, Engström and coworkers incubated wild-type and gingipain-mutant P. gingivalis with platelets and discovered that bacteria-induced platelet aggregation is gingipain-dependent (Klarstrom Engstrom et al., 2015). Using purified gingipains, Lourbakos and colleagues determined that P. gingivalis promotes platelet aggregation by gingipain-driven activation of protease-activated receptors (PARs) (Lourbakos et al., 2001). Our finding of a novel pro-coagulant pathway 34 involving Cdc42 activation in response to LPS challenge thus represents an advance in our understanding of the interplay between P. gingivalis and platelets. P. gingivalis LPS reduces clotting time Our initial findings of the effects of P. gingivalis LPS on purified platelets were validated by the accelerated clotting times observed in whole blood samples by ROTEM. Other groups have reported the activation of coagulation factors by P. gingivalis gingipains (Imamura et al., 2001a; Imamura et al., 1997; Imamura et al., 2001b) but their studies employed purely biochemical assays and were not conducted on blood samples. Yu and coworkers did assess the effect of P. gingivalis on whole blood samples from rats (Yu et al., 2011) although this was done by incubating the blood samples with the P. gingivalis bacteria rather than specific molecular components of the bacteria (e.g. LPS, gingipains). Consequently, our study provides, to our knowledge, the first direct evidence that LPS from P. gingivalis promotes the intrinsic coagulability of whole blood as determined by clotting time. Severe periodontal infection may promote thrombosis Recently published reports associate periodontitis with elevated platelet counts (Al-Rasheed, 2012), elevated platelet activity (Brousseau-Nault et al., 2017; Laky et al., 2018; Zhan et al., 2016), increases in circulating levels of platelet-derived cytokines (Brousseau-Nault et al., 2017; Papapanagiotou et al., 2009) and greater responsiveness of platelets to oral bacteria (Nicu et al., 2009). Collectively, these data suggest that platelet activity is heightened in periodontitis patients who would therefore be at increased risk for thrombosis. Again, to our knowledge, we are the first to report shorter intrinsic, non-LPS stimulated clotting times in ROTEM-analyzed whole blood samples obtained from periodontitis patients and healthy controls. Based on the collective 35 existing knowledge, we propose a model illustrating the different P. gingivalis-driven pro-thrombotic pathways (Fig. 13). A limitation of this work was the relatively small numbers of periodontitis patients recruited for the clinical portion of this study. We sought patients with generalized and severe periodontal destruction but who also met the highly stringent exclusion criteria, including the total absence of any medication or systemic condition. This was a significant impediment given the high prevalence of adults using prophylactic aspirin (Stuntz and Bernstein, 2017) and/or non-steroidal anti-inflammatory drugs (NSAIDs) (Zhou et al., 2014). In summary, our study elucidates a new signaling pathway by which P. gingivalis lipopolysaccharide promotes thrombosis by activating platelet Cdc42, thus stimulating actin assembly and spreading of platelets. Our data also provide direct evidence that chronic periodontal infection may amplify the intrinsic blood coagulability, thus increasing the risk for thrombosis. It would be interesting to evaluate platelet Cdc42 as a potential therapeutic target to mitigate the pro-thrombotic effects of periodontal infection. 36 FIGURE 13. Proposed model illustrating different pro-thrombotic mechanisms of Porphyromonas gingivalis infection. A. Gingipain-mediated signaling. Cysteine proteases (gingipains) activate protease-activated receptor (PARs) on platelets (A1) thus promoting platelet aggregation (A2). Gingipains also promote thrombosis by activating coagulation factors and prothrombin (A3). B. LPS-mediated signaling. Lipopolysaccharide from P. gingivalis recognizes toll-like receptor 4 (TLR4) on the platelet surface (B1) resulting in an increased activation of Cdc42, cell surface actin assembly, filopodial formation and platelet spreading (B2). The combined effects of these 3 P. gingivalis-mediated pathways presumably increase the risk for thrombosis (C). Figure 5A. B.GingipainsLPSPAR1TLR4THROMBOSISACTIVATION OF:- Coagulation factor XI- Coagulation factor X- ProthrombinCdc42GTPActin-rich filopodiaPlatelet spreadingC.Platelet aggregationActin assemblyP. gingivalis P. gingivalisA1.A2.A3.B1.B2. 37 Chapter Three: Conclusions and future directions Conclusions The connection between oral and systemic health is a broadly examined topic of research (Dietrich et al., 2013; Persson and Persson, 2008; Teeuw et al., 2014; Tonetti et al., 2013) although the available evidence is largely based on correlational studies that do not provide evidence of causation. The work that I have performed for this thesis addresses an important general health problem (thrombosis) by focusing on the specific cellular elements of this problem (platelets). In addition, I use a highly specific molecular aspect (LPS) of P. gingivalis, a dental pathogen with a widely-recognized role in the etiology of periodontitis, a very common condition (Eke et al., 2012). My work, which combines both basic and translational research, makes an original contribution by suggesting a specific molecular mechanism by which chronic periodontal infection can promote platelet spreading which is a critical aspect of thrombosis. These data are corroborated by evidence obtained from human blood samples and provide a plausible mechanism by which chronic periodontitis can increase the risk of thrombosis. Future Directions: 1. For the human subjects (blood donors), I sought to include patients with generalized severe chronic periodontitis. However, I also applied very strict inclusion/exclusion criteria that excluded any subject taking any medication (including commonly-used aspirin and NSAIDs). This significantly restricted the numbers of subjects. Future studies should seek to analyze blood samples from greater numbers of periodontal patients. 2. 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Thesis/Dissertation
eng
Craniofacial Science
Vancouver : University of British Columbia Library
Attribution-NonCommercial-NoDerivatives 4.0 International
Graduate
Porphyromonas gingivalis lipopolysaccharide promotes platelet spreading and thrombosis via Cdc42 activation
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