Dissertations / Theses on the topic 'Platelet activation'

In order to provide more information on platelet activation that would be of interest to all clinicians involved in the care of the patient with atherosclerosis, this thesis sets out to provide data to either support or refute the following hypotheses. (1). Platelet activation in patients with stable coronary heart disease increases with the angiographic severity of disease. (2). Patients presenting with the acute manifestations of atherosclerosis have a greater degree of platelet activation than patients with stable disease. (3). Peripheral artery intervention results in the release of the cytokine, sCD40L, by platelets. (4). Platelet microparticle levels are higher in patients with Type 2 Diabetes who develop symptomatic macrovascular disease.;Using both flow cytometric and Enzyme Linked Immunosorbent Assay (ELISA) based measurements of platelet activation, this thesis confirms that platelet activation can be related to the clinical severity of atherosclerotic disease. In addition, the development of symptomatic atherosclerotic disease is associated with increased platelet microparticle levels. Peripheral artery angioplasty has also been shown to increase sCD40L release. However, there is no association between platelet activation status and the angiographic severity of coronary heart disease.

Platelets play a pivotal role in coagulation and haemostasis. Their most prominent task is to seal damaged blood vessels by the formation of a platelet plug at the damaged area. Once the injury is covered, platelets retract the coagulum to close the wound and allow the blood to flow freely in the vessel. Platelets are strongly activated by the essential enzyme thrombin, formed in the coagulation cascade. Activation of the platelet thrombin receptors PAR1 and PAR4 leads to shape change, secretion of granule content, and aggregation, all of which can be accomplished by each receptor individually. However more and more findings indicate that there are differences between the receptors and that they have different physiological functions. This thesis presents studies performed to elucidate the relative role of PAR1 and PAR4 in platelet activation and coagulation. We have studied the effects on platelet activation and coagulation, and revealed a possible physiological role for PAR4 in the stabilisation of the coagulum. We also investigated the relative role of PAR1 and PAR4 in the cross-talk between thrombin and epinephrine with and without inhibition of COX-1. We demonstrated that PAR4 interacts with adrenergic receptors and causes an aggregation of platelets dependent on released ATP and its receptor P2X1, thereby circumventing the inhibition by aspirin. Not only is this an interesting specific role for PAR4, but it may also be of clinical importance considering that COX-1 inhibition is the most common treatment for patients with cardiovascular disease to prevent thrombosis. We show that the number of PAR1 receptors varied between donors and that this variation was correlated to the response on receptor activation. The number of PAR1 receptors on the platelet surface was decreased after PAR1 stimulation but increased after stimulation of other receptors. In a final attempt to elucidate the nature of PAR1 and PAR4 we used mathematics to evaluate the effect of co-stimulation of the receptors. We found a strong synergistic effect for both platelet activation and aggregation. This indicates that PAR1 and PAR4 interact in a yet unknown way to regulate or amplify the effect of each other rather than merely transmitting the incoming signal the same way.

Biomedical Sciences
Ph.D.
Platelets are anucleate cells that are crucial mediators of hemostasis and thrombosis. Under physiological conditions, platelets are maintained in a quiescent state within the vasculature. Upon vascular injury, an essential receptor that initiates platelet activation upon interaction with sub-endothelial collagen is Glycoprotein VI (GPVI). The activation of platelets leads to platelet shape change, granular secretion, thromboxane A2 (TXA2) synthesis, and integrin IIb3-mediated platelet aggregation and thrombus formation. In the past, a lot of effort has been placed in understanding GPVI and its signaling in platelets, however, much is still unknown. Therefore, the focus of this thesis is to identify novel regulators of GPVI-mediated platelet signaling. Phosphoinositide 3-kinase (PI3K) is an important signaling molecule that is activated downstream of various receptors including GPVI upon platelet activation. PI3K activation leads to the generation of phosphatidylinositol (3,4,5)-trisphosphate (PIP3) subsequently leading to the recruitment of pleckstrin homology (PH) domain-containing proteins to the plasma membrane. We performed a proteomic screen to identify proteins that interacted with PIP3 using PIP3 beads, and among these proteins, we found engulfment and cell motility-1 (ELMO1). ELMO1 is a scaffold protein with no catalytic activity that regulates the actin cytoskeleton during cell motility and cell spreading in nucleated cells. ELMO1 is expressed in platelets and interacts with active RhoG. However, the function of ELMO1 is not known. Therefore, we utilized ELMO1-/- mice to investigate the role of ELMO1 in platelets. Aggregation, granular secretion, and thromboxane generation was enhanced in ELMO1-/- platelets in response to glycoprotein VI (GPVI) agonists, collagen-related peptide (CRP) and collagen, but unaltered using protease-activated receptor 4 (PAR4) agonist (AYPGKF). This suggests that ELMO1 plays a specific role downstream of GPVI despite normal surface expression level of GPVI. Furthermore, whole blood from ELMO1-/- mice, perfused over collagen, under arterial shear conditions, exhibited enhanced thrombus formation compared to blood from WT littermate controls. In an in vivo pulmonary thromboembolism model, ELMO1-/- mice showed reduced survival compared to WT littermate control. ELMO1-/- mice also showed shorter time to occlusion using the ferric-chloride injury model and reduced tail bleeding times compared to WT littermate control. This indicates that ELMO1 plays an essential role in hemostasis and thrombosis in vivo. At the molecular level, RhoG activity was enhanced in ELMO1-/- murine platelets compared to the WT littermate control in response to CRP. Together, these data suggest that ELMO1 negatively regulates GPVI-mediated thrombus formation via RhoG. Protein kinase C delta (PKC) is a serine/threonine kinase that positively and negatively regulate dense granule secretion downstream of PAR and GPVI receptors, respectively. However, the mechanism of such differential regulation is not known. We hypothesize that this differential regulation occurs via the phosphorylation of specific tyrosine sites on PKC downstream of GPVI and PARs. We observed that many of the tyrosine residues in PKC were phosphorylated in response to both GPVI and PAR activation. Interestingly, PKCY155 phosphorylation only occurred following GPVI stimulation. Hence, we generated PKCY155F KI mice to characterize the function of PKCY155 phosphorylation in platelets. Aggregation and dense granule secretion were unaffected in PKCY155F platelets upon stimulation with a PAR agonist. However, these platelet functional responses were decreased upon stimulation of PKCY155F platelets with GPVI agonists, compared to WT littermates, despite normal surface GPVI expression. Whole blood from PKCY155F mice perfused over collagen under arterial shear conditions showed decreased thrombus formation. Similarly, we observed that PKCY155F mice survive longer than controls using a pulmonary thromboembolism model. PKCY155F mice also exhibited longer time to occlusion using the ferric-chloride injury model. At the molecular level, Syk and PLC2 phosphorylation was decreased in the PKCY155F platelets following GPVI stimulation. In conclusion, PKCY155 phosphorylation positively regulates GPVI-mediated platelet activation. Together, the studies proposed in this thesis provide insights into regulation of GPVI-mediated platelet function by ELMO1 and PKCY155. ELMO1 negatively regulates GPVI-mediated platelet activation via RhoG and may provide a suitable target for antihemorrhagic therapy. While PKCY155, being a positive regulator of GPVI-mediated platelet activation, could be a potential drug target for anti-thrombotic therapy.
Temple University--Theses

Physiology
Ph.D.
Platelets are involved in many processes ranging from fighting microbial infections and triggering inflammation to promoting tumor angiogenesis and metastasis. Nevertheless, the primary physiological function of platelets is to act as essential mediators in maintaining homeostasis of the circulatory system by forming hemostatic thrombi that prevent blood loss and maintain vascular integrity. CLEC-2 is a C-type lectin-like receptor that is highly expressed in platelets and lesser extent, in other cell types such as activated dendritic cells and B cells. Rhodocytin was the first ligand used to identify CLEC-2 receptor and it’s signaling on platelets. In the first chapter we identified a new agonist for CLEC-2 receptor. Fucoidan, a sulfated polysaccharide from fucus vesiculosus, decreases bleeding time and clotting time in hemophilia, possibly through inhibition of tissue factor pathway inhibitor. However, its effect on platelets and the receptor by which fucoidan induces cellular processes has not been elucidated. In this study, we demonstrate that fucoidan induces platelet activation in a concentration-dependent manner. Fucoidan-induced platelet activation was completely abolished by the pan-Src family kinase (SFK) inhibitor, PP2, or when Syk is inhibited. PP2 abolished phosphorylation of Syk and Phospholipase Cγ−2. Fucoidan-induced platelet activation had a lag phase, which is reminiscent of platelet activation by collagen and CLEC-2 receptor agonists. Platelet activation by fucoidan was only slightly inhibited in FcRγ chain null mice, indicating that fucoidan was not acting primarily through GPVI receptor. On the other hand, fucoidan-induced platelet activation was inhibited in platelet-specific CLEC-2 knock-out murine platelets revealing CLEC-2 as a physiological target of fucoidan. Thus, our data show fucoidan as a novel CLEC-2 receptor agonist that activates platelets through a SFK-dependent signaling pathway. Furthermore, the efficacy of fucoidan in hemophilia raises the possibility that decreased bleeding times could be achieved through activation of platelets. Lipid rafts are distinct areas of the plasma membrane implicated in the regulation of signaling in a variety of cells including platelets. A previous study C-type lectin like receptor 2 (CLEC-2) has been reported to activate platelets through a lipid raft-dependent manner. Secreted ADP potentiates CLEC-2-mediated platelet aggregation. We have investigated whether the decrease in CLEC-2-mediated platelet aggregation, previously reported in platelets with disrupted rafts, is a result of the loss of agonist potentiation by ADP. We disrupted platelet lipid rafts with methyl-β-cyclodextrin (MβCD) and measured signaling events downstream of CLEC-2 activation. Lipid raft disruption decreases platelet aggregation induced by CLEC-2 agonists. The inhibition of platelet aggregation by the disruption of lipid rafts was rescued by the exogenous addition of epinephrine but not 2-methylthioadenosine diphosphate (2MeSADP), which suggests that lipid raft disruption effects P2Y12-mediated Gi activation but not Gz. Phosphorylation of Syk (Y525/526) and PLCγ2 (Y759), were not affected by raft disruption in CLEC-2 agonist-stimulated platelets. Furthermore, tyrosine phosphorylation of the CLEC-2 hemi-ITAM was not effected when MβCD disrupts lipid rafts. Lipid rafts do not directly contribute to CLEC-2 receptor activation in platelets. The effects of disruption of lipid rafts in in vitro assays can be attributed to inhibition of ADP feedback that potentiates CLEC-2 signaling. Tyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an Immune Tyrosine Activation Motif (ITAM) and hemi-ITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In chapter 3, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation. CLEC-2-mediated, but not GPVI-mediated, platelet activation and Syk phosphorylation were abolished by inhibition of PI3-Kinase, which demonstrates that PI3-Kinase regulates Syk downstream of CLEC-2. Ibrutinib, a Tec family kinase inhibitor, also completely abolished CLEC-2-mediated aggregation and Syk phosphorylation in human and murine platelets. Furthermore, embryos lacking both Btk and Tec exhibited cutaneous edema associated with blood-filled vessels in a typical lymphatic pattern similar to CLEC-2 or Syk-deficient embryos. Thus our data show, for the first time, that PI3-Kinase and Tec family kinases play a crucial role in the regulation of platelet activation and Syk phosphorylation downstream of CLEC-2 receptor.
Temple University--Theses

Background: Clopidogrel is commonly prescribed to cats with perceived increased risk of thromboembolic events, but little information exists regarding its antiplatelet effects. ObjectiveTo determine effects of clopidogrel on platelet responsiveness in cats with or without the A31P mutation in the MYBPC3 gene. A secondary aim was to characterize variability in feline platelet responses to clopidogrel. AnimalsFourteen healthy cats from a Maine Coon/outbred mixed Domestic cat colony: 8 cats homozygous for A31P mutation in the MYPBC3 gene and 6 wild-type cats without the A31P mutation. MethodsEx vivo study. All cats received clopidogrel (18.75 mg PO q24h) for 14 days. Before and after clopidogrel treatment, adenosine diphosphate (ADP)-induced P-selectin expression was evaluated. ADP- and thrombin-induced platelet aggregation was measured by optical aggregometry (OA). Platelet pVASP and ADP receptor response index (ARRI) were measured by Western blot analysis. ResultsPlatelet activation from cats with the A31P mutation was significantly (P = .0095) increased [35.55% (18.58-48.55) to 58.90% (24.85-69.90)], in response to ADP. Clopidogrel treatment attenuated ADP-induced P-selectin expression and platelet aggregation. ADP- and PGE(1)-treated platelets had a similar level of pVASP as PGE(1)-treated platelets after clopidogrel treatment. Clopidogrel administration resulted in significantly lower ARRI [24.13% (12.46-35.50) to 11.30% (-7.383 to 23.27)] (P = .017). Two of 13 cats were nonresponders based on OA and flow cytometry. Conclusion and Clinical ImportanceClopidogrel is effective at attenuating platelet activation and aggregation in some cats. Cats with A31P mutation had increased platelet activation relative to the variable response seen in wild-type cats.

Platelets play critical roles in thrombosis, inflammation, and wound healing, which are essential in response to trauma. These processes are primarily driven through the immunoreceptor tyrosine-based activation motif (ITAM)-containing receptors, glycoprotein VI (GPVI) and C-type lectin-like receptor 2 (CLEC-2). This study aimed to investigate; (i) the effects of Alarmins released following trauma on platelet reactivity and the mechanisms involved; (ii) establish whether soluble GPVI (sGPVI), a platelet activation marker is elevated in trauma and other inflammatory conditions; (iii) determine whether the CLEC-2 ligand, podoplanin, is elevated in inflammatory conditions and (iv) establishing the role of GPVI and platelets in cutaneous wound healing. The nuclear-related Alarmin, histones, induced robust platelet activation both in vitro and in vivo. Histone-induced platelet activation was mediated through GPVI in vitro However, this pathway was found not to underlie histone-induced lowering of platelet count in vivo and is most likely to result from mediators released following vascular damage. GPVI shedding was shown to be induced following activation by thrombin, through a pathway dependent on fibrin generation. sGPVI was found to be a marker for platelet activation during a variety of inflammatory disorders, notably in association with sepsis. Furthermore, GPVI shedding reflects platelet activation by collagen and potentially thrombin-induced fibrin generation.

Physiology
Ph.D.
Platelets act as a fundamental component of the hemostatic process and their activation leads to the formation of a stable clot at the injured endothelium surface. Thrombin, as the important physiological agonist, activates platelets through protease-activated receptors (PARs). Protease-activated receptors are one of the major receptors in platelets and belong to the seven-transmembrane G-protein couple receptor family. Four protease-activated receptors are found, named as PAR1, PAR2, PAR3 and PAR4. Human platelets express PAR1 and PAR4 and murine platelets express PAR4 and PAR3 instead of PAR1. Thrombin activates PARs through a unique mechanism, involving the cleavage of N-terminus of PAR receptors and the newly exposed N-terminus acts as its own tethered ligand to bind and activate the receptor. In this study, we characterized a new PAR1 specific activating peptide (TFRRRLSRATR), generated from the c-terminus of human platelet P2Y1 receptor, and evaluated its biological function. This peptide activated platelets in a concentration-dependent manner, causing shape change, aggregation, secretion and calcium mobilization. Its activation is completely inhibited by using BMS200261, a PAR-1 specific antagonist. Its specificity to PAR1 receptor is further confirmed by using TFRRR-peptide-pretreated washed platelets and murine platelets. The shape change induced by 10 microM peptide was totally abolished by Y-27632, an inhibitor of p160ROCK which is the downstream signal of G12/13 pathways. The TFRRR-peptide, YFLLRNP, and the physiological agonist thrombin selectively activated G12/13 pathways at low concentrations and began to activate both Gq and G12/13 pathways with increased concentrations. Similar to SFLLRN, the TFRRR-peptide caused phosphorylation of Akt and Erk in a P2Y12 receptor-dependent manner, and p-38 MAP kinase activation in a P2Y12-independent manner. The effects of this peptide are elicited by the first six amino acids (TFRRRL) whereas the remaining peptide (LSRATR), TFERRN, or TFEERN had no effects on platelets. Beside thrombin, PARs also can be activated by other proteases. Previous studies in our lab show that plasmin, a major extracellular protease, activates both human and murine platelets through prototypical cleavage of PAR4 (Quinton et al., 2004). In this study, we continue our study and investigate the molecular basis for the differential activation of murine and human platelets by plasmin. Plasmin-induced full aggregation is achieved at lower concentrations (0.1 U/mL) in murine platelets as compared to human platelets (1 U/mL). In COS7 cells expressing the murine PAR4 (mPAR4) receptor, 1 U/mL plasmin caused a higher intracellular calcium mobilization than in cells expressing the human PAR4 (hPAR4) receptor. This difference was reversed when the tethered ligand sequences of mPAR4 and hPAR4 were interchanged through site-directed mutagenesis. This difference between human and murine PAR4 is not because of the cofactor effect of PAR3 in murine platelets by showing that in both transfected cell lines and platelet system, PAR3 inhibits plasmin-induced PAR4 stimulation. All of the data suggest that murine platelets are more sensitive to activation by plasmin than human platelets due to differences in the primary sequence of PAR4. In contrast to thrombin-dependent activation of platelets, wherein PAR3 acts as a co-receptor, mPAR3 inhibits plasmin-induced PAR4 activation. Abnormal platelet activation causes thrombus formation and induces pathological conditions including stroke and atherosclerosis. Antithrombotic therapy is a widely used therapeutic method for stroke. However, currently used agents based on the irreversible inhibition of the platelet cyclooxygenases 1 and 2 or inhibition of P2Y12 receptors can cause unexpected bleeding or resistant side effects. Antithrombotic therapy targeting thrombin signaling is one of the new treatments under investigation and PAR1 antagonists are now in clinical trials. In this study, we investigate the effect of one of thrombin receptors, protease-activated receptor 4 (PAR4) in mice transient middle cerebral artery occlusion/ reperfusion (tMCAO/R) model. Our data show that PAR4 -/- mice have more than 80% reduction in infarct volume and significant improved neurological and motor function after 1 h MCAO followed by 23 h reperfusion. Examination of cellular responses to tMCAO/R indicates that PAR4-/- mice have less cellular death. Platelet/endothelial and leukocyte/endothelial interactions have been shown to play a critical role in the inflammatory responses during cerebral ischemic/reperfusion injury. Comparing wild-type with PAR4-/- mice platelets/endothelial and leukocyte/endothelial interactions, deficiency of PAR4 causes a significant decrease in both platelet/endothelial and leukocyte/endothelial interactions. In addition, PAR4-/- mice attenuate blood-brain barrier (BBB) disruption during tMCAO/R. All the data suggest that deficiency of PAR4 will protect against brain ischemic injury though attenuation of cerebral inflammatory responses including inflammatory cells extravasation and BBB disruption. Protease-activated receptor 4 (PAR4) is the only thrombin receptor existing in both human and murine platelets. The data we get in this study also have a beneficial effect for human study and inhibition of PAR4 may provide a novel potential therapeutic strategy for ischemic injury.
Temple University--Theses

Diabetics suffer from a pro-thrombotic condition. It is established that platelets are activated in type 2 diabetes. However the mechanisms of platelet activation in this disease are not yet known. The purpose of these studies was to elucidate the mechanisms of platelet activation and the effect these mechanisms have on type 2 diabetic platelets. Apoptosis of cells is regulated by caspases, a group of cysteine proteases. When platelets become activated, they express phosphatidylserine (PS) on the outer leaflet of the plasma membrane as well as form platelet microparticles (PMPs). In addition, platelets aggregate when activated. We found that platelets from diabetic subjects contain activated caspases. These platelets also formed increased numbers of PMPs compared to platelets from non-diabetic subjects. We observed a 30-fold increase in thrombin activity in the plasma from diabetics. To determine if caspases were involved in platelet activation, we determined if caspase inhibition (using the pan-caspase inhibitor zVAD-fmk); (1) decreased PS expression and (2) decreased platelet aggregation following activation. We found that platelets treated with zVAD-fmk significantly decreased A23187-induced PS exposure as well as aggregation. There is limited information on the role of caspases on platelet adhesion proteins such as P-selectin or GPIIb/IIIa during platelet activation. Therefore, we tested if caspase inhibition attenuated P-selectin and GPIIb/IIIa expression. Using blood from non-diabetic volunteers, we found that treatment with zVAD-fmk caused a significant attenuation of P-selectin expression in stimulated platelets. Together these data suggest that caspases play a novel role in platelet activation. We also wanted to determine if treating type 2 diabetic rats (Zucker Diabetic Fatty rats, ZDF) with the caspase inhibitor zVAD-fmk in vivo ; (1) attenuated PS expression, (2) attenuated platelet microparticle (PMP) formation and (3) attenuated platelet aggregation. ZDF rats were treated in vivo with 40 μg zVAD-fmk for 4 days. We found attenuated PS exposure, PMP formation as well as decreased aggregation in ZDF rats treated with zVAD-fmk. Our overall results demonstrate a novel role of caspases in platelet activation. Together, these studies may lead to development of novel treatments for pathophysiologic states associated with platelet activation such as diabetes, myocardial infarction, and stroke.

Atherosclerosis is a major cause of mortality in industrialized nations. Atherosclerosis is characterized by plaque deposition which decreases the lumen diameter into a stenosis. The creation of a restriction increases shear rates pathologic levels exceeding 3,500/s. Following plaque cap rupture, thrombus may form from the accumulation of millions of platelets, occluding the vessel, leading to heart attack and stroke. Studies of high shear thrombosis show that platelet activation, GPIIb/IIIa and vWF are involved. However, some recent studies also suggest that high shear aggregation is not dependent on activation or GPIIb/IIIa. Several antiplatelet pharmaceuticals against activation and GPIIb/IIIa have been proposed, but their efficacy in patients remains mixed. The overall objective of this project is to determine the factors necessary for thrombosis to occlusion in very high shear regions seen in diseased arteries. Our central hypotheses are that platelet activation and the subsequent conformational change in GPIIb/IIIa are necessary for thrombosis, and that higher concentrations of vWF in the plasma will increase thrombosis. To this end, we developed a new high shear hemodynamic model utilizing 30mLs of whole blood and quantified thrombus thickness, volume accumulation and accumulation rates. We demonstrate that thrombosis to occlusion stems from a second phase of Rapid Platelet Accumulation (RPA). Thrombus accumulation is completely prevented by PGE1 inhibition of platelet activation. Similarly, GPIIb/IIIa blockade via abciximab prevented significant thrombus deposition and RPA. We also found that increasing plasma vWF levels in high shear regions increased thrombus thickness and suggestively increased RPA rates. The results clarify the need for activation of mural platelets for long term thrombus accumulation without the activation of circulating platelets.

Thesis (PhD)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: Introduction In the era of antiretroviral therapy (ART), people living with Human Immunodeficiency Virus (HIV) now have prolonged life spans. An emerging trend of non- acquired immune deficiency syndrome (AIDS) related complications now prevails in the aging HIV infected population. Increased levels of inflammation and chronic immune activation are associated with HIV infection. In the era of ART people living with HIV are at an increased risk of cardiovascular disease (CVD). Platelets play a pivotal role in both inflammation and immune activation and upon activation platelets degranulate and secrete various inflammatory, coagulatory and adhesion molecules. Activated platelets express surface P-selectin (CD62P) and are a key component of the coagulation pathway and serve as a link between inflammation and thrombosis. Activated platelets have been implicated in inflammatory and cardiovascular disease and have been identified as immune cells that play a crucial role in pathogen recognition and modulation of immune cells during infections. Several antiviral and antibacterial properties of platelet alpha granule contents have been established. Platelet aggregometry remains the most widely used technique to evaluate platelet function even though this technique is limited by many pre-analytical variables. Platelet flow cytometry on the other hand offers a rapid measurement of platelet function in their physiological environment with minimal artefactual activation. Few studies have however reported on standardized methods to evaluate platelet function in the context of HIV. Platelet function remains unclear and data on HIV infected treatment naïve individuals remains scarce. The aim of this project was to examine the relationship between platelet function and immune activation in patients with HIV Materials and methods This study consisted of five sub-studies, firstly platelet indices and levels of platelet activation were determined in a cohort of 330 participants (185 HIV infected ARV naïve and 145 uninfected healthy controls) using; flow cytometry and haemotology analyzers. The relationship between these indices and markers of platelet activation, disease progression and immune activation were assessed. Furthermore, levels of platelet activation and aggregation were evaluated in a cohort of 82 participants (41 HIV infected (ARV naïve) individuals and 41 uninfected healthy controls), using a novel whole blood flow cytometry based functional assay. These baseline levels were then correlated with markers of immune activation and disease progression in HIV. In a subsequent study, platelet function in a cohort consisting of 58 HIV infected (ARV naïve) and 38 uninfected controls was evaluated using flow cytometry. Platelet response was measured post stimulation with adenosine diphosphate (ADP) at concentrations known to induce reversible (0.04mM) and irreversible (0.2mM) platelet aggregation. In order to assess platelet function in HIV, platelet response was evaluated in a cohort consisting of 58 HIV infected (ARV naïve) and 38 uninfected controls. Platelets were activated using varying concentrations of ADP, arachidonic acid (AA) and collagen and platelet function was measured using flow cytometry. Levels of circulating platelet leukocyte aggregates (PLAs) were also measured using flow cytometry in a cohort consisting of 35 HIV-infected (ARV naïve) individuals and 32 uninfected healthy controls. Associations between PLAs, immune activation and disease progression in HIV infected individuals were determined. The final study evaluated platelet aggregates, platelet derived microparticles (PMPs) and microparticles (MPs) in a cohort consisting of 46 HIV infected (ARV-naïve) and 40 uninfected healthy controls. Associations between MPs, PMPs, platelet aggregates and markers of immune activation and disease progression were evaluated. Results HIV infected individuals showed decreased mean platelet volume levels (HIV mean 7.91 ± 0.85 vs. 8.52 ± 1.12, p<0.0001) that directly correlated with CD4 counts (r=-0.2898, p=0.0075) and viral load (r=0.2680, p=0.0177). Platelet distribution width (PDW) levels directly correlated (r=0.3455, p=0.0362) with active coagulation and inversely correlated (r=-0.3666, p=0.0463) with platelet aggregation. HIV infected individuals showed increased levels of platelet activation (%CD62P median 11.33[5.96-29.36] vs. control group 2.48[1.56-6.04], p=0.0001). In HIV, platelet function is retained and platelets showed increased response to submaximal concentrations of endogenous agonists. HIV infected individuals showed increased levels of circulating platelet monocyte aggregates (25.26[16.16-32.28] vs. control group 14.12[8.36- 18.83], p=0.0001) that directly correlated with markers of immune activation; %CD38/8 (r=0.54624, p=0.0155), viral load (r=0.633, p<0.009). Furthermore we report on increased levels of circulating MPs (median %MPs 1.7[0.95-2.83] vs. Control group 1.12[0.63-1.57], p=0.0160); PMPs (median %PMPs 26.64[11.33-36.62] vs. Control group 20.02[18.08-26.08], p=0.0133); activated PMPs (median CD62P MFI 3.81[3.46-4.54] vs. Control group 3.41[3.16-3.6], p=0.0037) and platelet aggregates (Median %CD62P 14.10[5.49-39.94] vs. Control group 0.17[0.10-10.99], p= 0.0097) in HIV infected asymptomatic individuals. Conclusion This study supports the potential use of the MPV and PDW as readily available markers of platelet activation and immune activation in HIV. We also showed elevated levels of activated platelets in HIV infected individuals that were hyper responsive to endogenous agonists in a concentration dependent manner. Platelet flow cytometry is a rapid and valuable technique in the evaluation of platelet function in HIV. The measurement of platelet function using flow cytometry allows the evaluation of platelet signalling pathways that may be modified in HIV infected individuals. Lastly we describe an optimized whole blood flow cytometry based assay for the evaluation of circulating microparticles (MPs), platelet derived microparticles (PMPs) and levels of activated platelets and aggregates which mimics the in vivo physiological environment of MPs. To the best of our knowledge, this study is the first to report on a novel approach in evaluating platelet function in HIV using a series of optimised whole blood flow cytometry based platelet assays. In addition, minimal work has been performed previously on platelet function in the context of HIV-infection; and particularly in a cohort of asymptomatic, untreated patients as defined for this study.
AFRIKAANSE OPSOMMING: Inleiding In die era van antiretrovirale terapie (ART), het mense wat met die menslike immuniteitsgebreksvirus (MIV) leef, het nou 'n verlengde lewensduur. 'N opkomende neiging van nie-verworwe immuniteitsgebreksindroom (vigs) heers nou in die verouderende MIV-besmette bevolking. Verhoogde vlakke van inflammasie en chroniese immuun aktivering word geassosieer met MIV-infeksie en in die era van ART loop mense wat met MIV leef, 'n verhoogde risiko van kardiovaskulêre siekte (KVS). Plaatjies speel 'n belangrike rol in beide inflammasie en immuun aktivering en met aktivering degranulate en skei plaatjies verskeie inflammatoriese, coagulatory en adhesie molecule af. Geaktiveerde plaatjies druk oppervlak P-selectin (CD62P) is 'n belangrike komponent van die stollings weg en dien as 'n skakel tussen inflammasie en trombose. Geaktiveerde plaatjies is in beide inflammasie en kardiovaskulêre siekte betrokke en is geïdentifiseer as immuun selle wat 'n deurslaggewende rol speel in die patogeen erkenning en modulasie van immuun selle tydens infeksies. Verskeie antivirale en antibakteriese eienskappe van plaatjie alpha korrel inhoud is vasgestel. Plaatjie aggregometry bly die mees gebruikte tegniek om plaatjie funksie te evalueer, alhoewel hierdie tegniek is beperk deur baie pre-analitiese veranderlikes. Plaatjie vloeisitometrie aan die ander kant bied 'n vinnige meting van plaatjie funksie in hul fisiologiese omgewing met 'n minimale artefactual aktivering. Min studies het egter berig op gestandaardiseerde metodes om plaatjie funksie in die konteks van MIV te evalueer. Plaatjie funksie is steeds onduidelik en data oor MIV besmet behandeling naïef individue bly skaars. Die doel van hierdie projek was om die verhouding tussen die plaatjie funksie en immuun aktivering in pasiënte met MIV te ondersoek. Materiaal en metodes Hierdie studie het bestaan uit vyf sub-studies. In die eerste plekis plaatjie indekse en vlakke van plaatjie aktivering bepaal in 'n groep van 330 deelnemers (185 MIV-besmette ARV naïef en 145 onbesmette gesonde kontrole) met behulp van vloeisitometrie en hematologie ontleders. Die verhouding tussen hierdie indekse en merkers van plaatjie aktivering, die siekte se progressive en immuun aktivering is beoordeel. Verder is die vlakke van plaatjie aktivering en samevoeging in 'n groep van 82 deelnemers (41 MIV-besmette (ARV naïef) individue en 41 onbesmette gesonde kontrole) geëvalueer, met behulp van 'n nuwe vol bloed vloeisitometrie gebaseerde funksionele toets. Hierdie basislyn vlakke is dan gekorreleer met merkers van immuun aktivering en die progreessie van die siekte in MIV. In 'n daaropvolgende studie, is plaatjie funksie in 'n groep wat bestaan uit 58 MIV besmet te (ARV naïef) en 38 onbesmette beheer geëvalueer met behulp van vloeisitometrie. Plaatjie reaksie is na stimulasie gemeet met adenosine diphophate (ADP) by konsentrasies bekend omkeer (0.04mM) te oorreed en onomkeerbaar (0.2mm) plaatjie aggregasie. Ten einde plaatjie funksie in MIV te evalueer, is plaatjie reaksie in 'n groep wat bestaan uit 58 MIV-besmette (ARV naïef) en 38 onbesmette kontrole geëvalueer. Die plaatjies is geaktiveer deur gebruik te maak van wisselende konsentrasies van ADP, is aragidoonsuur (AA) en kollageen en plaatjie funksie gemeet met behulp van vloeisitometrie. Vlakke van sirkulerende plaatjie leukosiet gemiddeldes is ook gemeet met behulp van vloeisitometrie in 'n groep wat bestaan uit 35 MIV-positiewe (ARV naïef) individue en 32 onbesmette gesonde kontrole. Assosiasies tussen leukosiet gemiddeldes, immuun aktivering en die progressive van ie siekte in MIV-besmette individue is ook bepaal. Die finale studie het plaatjie-gemiddeldes, plaatjie afgelei mikrodeeltjies en mikrodeeltjies geëvalueer in 'n groep wat bestaan uit 46 MIV besmet (ARV-naïewe) en 40 onbesmette gesonde kontrole. Assosiasies tussen mikrodeeltjies, plaatjie afgelei, plaatjie gemiddeldes en merkers van immuun aktivering en die siekte se progressie is geëvalueer. Resultate MIV-besmette individue het gedaalde gemiddelde plaatjie volume vlakke getoon (HIV gemiddelde 7,91 ± 0,85 8,52 ± 1,12 teen, p <0,0001) wat direk gekorreleer het met CD4-tellings (r = -0,2898, p = 0,0075) en virale (r = 0,2680, p = 0,0177). Plaatjie verspreiding breedte vlakke het direk gekorreleer met (r = 0,3455, p = 0,0362) met 'n aktiewe koagulasie en omgekeerd gekorreleer (r = -0,3666, p = 0,0463) met plaatjie aggregasie. MIV-besmette individue het verhoogde vlakke van plaatjie aktivering getoon (% CD62P mediaan 11,33 [5,96-29,36] teen kontrole groep 2,48 [1,56-6,04], p = 0,0001). In MIV, was plaatjie funksie behou en plaatjies het 'n verhoogde reaksie op submaksimale konsentrasies van endogene agoniste getoon. MIVbesmette individue het verhoogde vlakke van sirkuleer plaatjie monosiet-gemiddeldes gedemonstreer (25.26 [16,16-32,28] teen kontrole groep 14,12 [8,36-18,83], p = 0,0001) wat direk gekorreleer het met merkers van immuun aktivering; % CD38 / 8 (r = 0,54624, p = 0,0155), virale lading (r = 0,633, p <0,009). Verder rapporteer ons op verhoogde vlakke van sirkulerende mikrodeeltjies (mediaan% LP 1.7 [0,95-2,83] teen kontrole groep 1,12 [0,63-1,57], p = 0,0160); PMPs (mediaan% PMPs 26,64 [11,33-36,62] teen kontrole groep 20,02 [18,08-26,08], p = 0,0133); geaktiveer PMPs (mediaan CD62P MFI 3,81 [3,46-4,54] teen kontrole groep 3,41 [3,16- 3,6], p = 0,0037) en plaatjie gemiddeldes (Mediaan% CD62P 14,10 [5,49-39,94] teen 0.17 [0,10- 10,99], p= 0.0097) in MIV besmet asimptomatiese individue. Gevolgtrekking Hierdie studie ondersteun die potensiële gebruik van die MPV en PDW as waardevolle geredelik waardevolle merkers van plaatjie aktivering en immuun aktivering in MIV. Ons het ook getoon verhoogde vlakke van geaktiveer de plaatjies in MIV-besmette individue getoon wat hyper reageer op endogene agoniste was in 'n konsentrasie-afhanklike wyse. Plaatjie vloeisitometrie is 'n vinnige en waardevolle tegniek in die evaluering van plaatjie funksie in MIV. Die meting van plaatjie funksie gebruik vloei cytometry maak die evaluering van plaatjie sein paaie wat in MIVgeïnfekteerde individue verander moontlik. Laastens het ons beskryf 'n hele bloed vloeisitometrie gebaseer de toets vir die evaluering van sirkulerende mikrodeeltjies, plaatjie afgelei mikrodeeltjies en vlakke van geaktiveer plaatjies en gemiddeldes wat lyk soos die in vivo fisiologiese omgewing van MP's. Na die beste van ons kennis, is hierdie studie die eerste om te rapporteer oor 'n nuwe benadering in die evaluering van plaatjie funksie in MIV met behulp van 'n reeks van new hele bloed vloeisitometrie gebaseer de plaatjie toetse. Daarbenewens is minimale werk voorheen uitgevoer op die plaatjie funksie in die konteks van MIV-infeksie; en veral in 'n groep van asimptomatiese, onbehandelde pasiënte soos vir hierdie studie. Hierdie projek het bewyse bygevoeg tot die teorie dat plaatjies, in MIV, kan 'n skakel wees tussen die aktiewe inflammatoriese reaksie en die toename in die aantal trombotische en kardiovaskulêre siekte waargeneem in pasiënte wat met hierdie siekte saamleef.

There are many conflicting views about the blood-response to polymeric materials. In order to be satisfied that a material performs appropriately when used as a device in contact with blood it must be evaluated under relevant conditions. Central venous catheters suffer from problems related to thrombosis and embolism since they are implanted for very long periods of time within the vascular system. The aim of this study was to evaluate the most appropriate method for assessing catheter thrombogenicity, establish data for a number of relevant parameters and correlate these findings with various physico-chemica1 characteristics of the materials. Accordingly, a dynamic model was developed which allowed the assessment of platelet adhesion by measurement of "Cr-labelled platelets and platelet a-granule and lysosomal secretion by flow cytometry, after labelling with anti- GMPl40 and anti-GPS3 antibodies, in whole blood after the perfusion of the blood along the tubing at physiologically relevant shear rates (up to 1000 s") at 37OC. In addition, contact phase activation was assessed by measuring the time taken for an aliquot of plateletfree plasma to clot after contact with catheter material (partial thromboplastin time or P'IT) and the ability of the materials to cause factor XII activation by measuring the quantity of FXIIa-Cl-Inh complexes formed by enzyme-linked immunosorbent assay after the contact of platelet-free plasma with catheter tubing. An attempt was made at finding the identity of the proteins adsorbed onto silicone using a number of electrophoretic techniques. The ability of the materials to cause haemolysis and the cytotoxicity of an extract derived from the materials after SO days incubation in PBS including the identification of these potential leachables by supercritical fluid extraction was also investigated. In addition, these data were discussed in relation to parameters of surface roughness, as viewed by SEM and the ratio of hard and soft segments appearing at the material surface by XPS. It was found that significant differences could be detected in (i) platelet adhesion, where Pellethane was shown to have poor performance; (ii) a-granule release, where all the polyurethanes displayed better performance than any of the other materials and (iii) lysosomal granule release where most materials fared similarly, except for glass which was much worse. Silicone was shown to be best in the PIT assay, Pellethane worst. Surprisingly, no correlation was found with these results and those from FXIIa assay, where Desmopan and Davathane were highly active. New and important data on the initial activation kinetics and the ability of materials to activate factor XII are shown. Silicone produced the greatest haemolysis, PVC the greatest extract toxicity. No correlation was found between the physico-cbemical data and any of the biocompatibility data.

The Protein kinase C (PKC) superfamily is a key regulator in platelet activation with individual isoforms playing distinct roles. This thesis focuses on the role of the novel PKC isoforms downstream of several agonists using both pharmacological and genetic approaches and human and mouse platelets. Quantification of the protein levels of PKC isoforms identified different levels of the five major PKC isoforms expressed in human platelets and also differences between levels of the same isoform in human and mouse platelets. Use of a selection of broad spectrum and isoform-specific inhibitors, identified both positive and negative novel roles for PKC in the regulation of human and mouse platelets. A net positive role for PKC was found in GPVI, Clec-2, and PAR receptor signalling, with classical isoforms of PKC playing a major role in aggregation and dense granule secretion. A novel negative regulatory role was also identified in the regulation of ADP-induced platelet activation for PKC~, and both PKCE and PKC~ in human and mouse platelets respectively. Gene knock-out mouse models confirmed a positive regulatory role for PKCe in allb~3 outside-in signalling but identified no other regulatory role for PKCe in agonist induced platelet activation. Despite this relatively minor role, functional redundancy was identified between PKCe and PKCE isoforms in haemostasis, as tail bleeding was significantly increased in mice deficient in both novel isoforms. The work presented here identifies key roles for the PKC superfamily in the complex regulation of platelet activation, with different isoforms supporting and limiting the process of thrombus formation and haemostasis.

Pharmacology
Ph.D.
Activation of platelets is essential for hemostasis. Following damage to the vascular endothelium collagen is exposed, to which platelets stably adhere. After adhesion on collagen, a signaling cascade is initiated, mediated by Glycoprotein VI (GPVI), which results in platelet activation. A major signaling protein in GPVI signaling is Spleen tyrosine kinase (Syk). It undergoes phosphorylation and activation following GPVI stimulation. Syk's central role in this physiological process suggests regulation of its activity is required to maintain the platelets response to collagen within physiological limits. The regulation of Syk activation is the focus of this work. Previously published reports implicate the phosphatases SHP-1, SHP-2 and TULA-2 in the negative regulation of Syk. Therefore, we tested these phosphatases possible role in platelets. We show that SHP-1 can dephosphorylate Syk in vitro, but is unable to bind Syk. Also, Syk is hypophosphorylated in GPVI-stimulated SHP-1 deficient platelets and platelet functional responses are minimally affected compared to wild-type platelets. SHP-2 is unable to bind Syk and Syk is not a good substrate for SHP-2 in vitro. TULA-2 dephosphorylated Syk in vitro and associated with Syk in platelets. In TULA-2 deficient platelets, Syk and PLCγ2 were hyperphosphorylated compared to wild-type platelets. Deletion of TULA-2 resulted in enhanced GPVI-dependent platelet functional responses and a prothrombotic phenotype. c-Cbl has been shown to be a negative regulator of GPVI signaling, possibly by regulating Syk phosphorylation. Thus, SHP-1, SHP-2 and TULA-2’s role in c-Cbl regulation of GPVI was also investigated. We show that TULA-2 is able to bind c-Cbl in platelets. SHP-1 and SHP-2 do not. Furthermore, we show a striking similarity between the phenotype of TULA-2 and c-Cbl deficient platelets. However, in vitro binding studies show TULA-2 is able to bind Syk independently of c-Cbl. Thus, the exact role of c-Cbl in regulating Syk dephosphorylation is unclear. In conclusion, we show SHP-1 and SHP-2 are probably not involved in the negative regulation of Syk. However, TULA-2 is the major phosphatase responsible for the negative regulation of Syk in GPVI signaling. This serves to negatively regulate GPVI-mediated platelet function and prevent uncontrolled platelet activation that could lead to thrombosis.
Temple University--Theses

The oxidation of low density lipoproteins (LDL) may contribute to platelet hyperactivity and thrombotic events in cardiovascular diseases. Although these pathological particles are known to stimulate platelet activation, the exact signalling mechanisms involved are poorly defined. The aim of this study was to explore the mechanisms underpinning platelet activation by oxidised LDL (oxLDL). OxLDL but not native LDL (nLDL) induced a modest degree of aggregation, which was ADP dependent. However, oxLDL was able to induce shape change, one of the earliest stages of platelet activation, independently of secondary agonists. Platelet shape change requires the protein myosin, the activity of which is controlled by the phosphorylation of its regulatory light chains (MLC) at serine¹⁹. Physiological platelet agonists activating platelets through G protein coupled receptors stimulate MLCSer¹⁹ phosphorylation by the activation of dual pathways that lead to a Ca²⁺ dependent activation of MLC kinase (MLCK) and the Rho kinase dependent inhibition of MLC phosphatase (MLCP). OxLDL induced platelet shape change correlated with MLCSer¹⁹ phosphorylation. OxLDL also stimulated a tyrosine kinase pathway involving the proteins Syk and PLCγ2, which were confirmed to be involved in MLCSer¹⁹ phosphorylation. Furthermore, oxLDL triggered a second pathway involving the activation of RhoA leading to the activation of Rho kinase and subsequent inhibition of MLCP. A receptor that has been continuously linked to oxLDL induced platelet activation is the scavenger receptor CD36. The expression of this scavenger receptor was found to increase upon stimulation with a range of platelet agonists. Furthermore, platelet shape change was discovered to be downstream of CD36. The findings of this report therefore elucidate two novel signalling pathways in response to CD36 ligation with oxLDL. Enhancements in understanding how this pathological ligand can contribute to platelet activation may lead to improvements in the treatments available for patients who suffer with hyperlipidaemia and cardiovascular diseases.

There has been renewed scientific interest in the role of oral bacteria in systemic disease in recent years. The incidence of infective endocarditis (lE) in the UK has been rising lately. Dental plaque bacteria of the genus Streptococcus are recognised causative agents of lE. Oral Streptococcus gordonii produces several factors that are thought to be involved in promoting lE. One of these, designated surface platelet adherence protein A (PadA), has been only recently identified. This study aimed to determine mechanisms by which PadA functional domains interacted with platelets, and platelet integrin GPIIbIIIa, under static or shear conditions. The role of PadA in platelet interactions in relation to a well-characterised platelet adhesin (Hsa) was clarified. Furthermore, the interactions of PadA protein with extracellular matrix (ECM) components were investigated. This work identified PadA as a protein that binds activated platelets and confers shear resistance. A two-step S. gordonii-platelet adherence model is proposed, whereby Hsamediated platelet activation then enables PadA platelet adhesion and activation. This is the result of, at least in part, the presence of an integrin-like motif NGR within PadA binding to platelets under static conditions. PadA also binds components of the ECM including fibronectin, vitronectin and von Willebrand factor. However, PadA-mediated bacterial cell adherence to vitronectin and activated platelets occurs only in the presence of Hsa. PadA and Hsa together modulate biofilm formation on salivary pellicle, but in this case Hsa only functions in the presence of PadA. Thus it is proposed that PadA and Hsa may form a macromolecular complex on the surface of S. gordonii. PadA-like family proteins appear to be structurally conserved and are found in a range of lE pathogens. Increased knowledge of the molecular interactions occurring between streptococci and platelets will enable translation into new strategies for the prevention or treatment of infection-associated cardiovascular diseases .

According to the World Health Organization (WHO), Cardiovascular Disease (CVD) is the leading cause of death in the world. Biomechanics and fluid dynamics of blood flow play an important role in CVD mediation. Shear stress plays a major role in platelet-substrate interactions and thrombus formation and growth in blood flow, where under both pathological and physiological conditions platelet adhesion and accumulation occur. In this study, a three-dimensional dynamic model of platelet-rich thrombus growth in stenosed vessels using computational fluid dynamics (CFD) methods is introduced. Platelet adhesion, aggregation and activation kinetics are modeled by solving mass transport equations for blood components involved in thrombosis. The model was first verified under three different shear conditions and at two heparin levels. Three-dimensional simulations were then carried out to evaluate the performance of the model for severely damaged (stripped) aortas with mild and severe stenosis degrees. For these cases, linear shear-dependent functions were developed for platelet-surface and platelet-platelet adhesion rates. It was confirmed that the platelet adhesion rate is not only a function of Reynolds number (or wall shear rate) but also the stenosis severity of the vessel. General correlations for adhesion rates of platelets as functions of stenosis and Reynolds number were obtained based on these cases. The model was applied to different experimental systems and shown to agree well with measured platelet deposition. Then, the Arbitrary Lagrangian Eulerian (ALE) formulation was used to model dynamic growth by including geometry change in the simulation procedure. The wall boundaries were discretely moved based on the amount of platelet deposition that occurs on the vessel wall. To emulate the dynamic behavior of platelet adhesion kinetics during thrombus growth, the validated model for platelet adhesion, which calculates platelet-surface adhesion rates as a function of stenosis severity and Reynolds number, was applied to the model. The model successfully predicts the nonlinear growth of thrombi in the stenosed area. These simulations provide a useful guide to understand the effect of growing thrombus on platelet deposition rate, platelet activation kinetics and occurrence of thromboembolism (TE) in highly stenosed arteries.
Ph. D.

Platelet activation and subsequent thrombus formation are important for preventing excessive blood loss at sites of vascular injury, a process termed haemostasis. However, excessive platelet activation at sites of atherosclerotic plaque rupture can lead to thrombus formation, which may occlude the vessel and cause heart attack or stroke. The platelet collagen receptor GPVI is essential for thrombus formation, but is largely dispensable for haemostasis. Tetraspanins are transmembrane proteins which compartmentalise the membrane through formation of dynamic tetraspanin-enriched microdomains. Due to regulation of a wide range of partner proteins, tetraspanins have been implicated in many cellular processes, including platelet activation, though most platelet tetraspanins have not been characterised. The aim of this thesis was to investigate the novel platelet tetraspanin Tspan18, using the Tspan18 knockout mouse. Tspan18 was shown to have a role in platelet activation and platelet Ca\(^2\)\(^+\) signalling specifically downstream of GPVI. Tspan18 also appeared to have a role in haemostasis, as Tspan18 deficient mice displayed a severe bleeding phenotype. The bleeding was shown to be driven by non-haematopoietic cells and is therefore unlikely to be platelet-driven. Additionally, Tspan18-induced Ca\(^2\)\(^+\) mobilisation was shown to be dependant on functioning Orai1 Ca\(^2\)\(^+\) channels and a novel interaction between Tspan18 and the Orai family was identified. Together, these findings suggest a role for Tspan18 in platelet activation and regulation of Ca\(^2\)\(^+\) mobilisation, potentially via interaction with Orai proteins.

Arterial thrombus forms from the capture and accumulation of circulating platelets on a stenosis. As the thrombus grows, the lumen becomes further stenotic producing very high shear rates as the blood velocities increase through the narrowed cross-section. This study explores the molecular binding conditions that may occur under these pathologic shear conditions where circulating platelets must adhere quickly and with strong bonds. Platelets binding in an arterial stenosis of >75% are subject to drag forces exceeding 10,000 pN. This force can be balanced by 100 simultaneous GPIb-vWFA1 bonds of 100 pN each. The number and density of GPIb on platelets is sufficiently high; however, platelet capture under high shear would require the density of A1 receptors to be increased to over 416 per square micron. A computational model is used to determine platelet capture as a function of shear rate, surface receptor density, surface contact and kinetic binding rate. A1 density could be increased by a combination of vWF events of: i) plasma vWF attach to the thrombus surface and elongate under shear; ii) the elongated vWF strands create a net with 3-D pockets; and iii) additional vWF is released from mural platelets by activation under shear. With all three events, A1 density matches the existing high GPIbα densities to provide sufficient multivalency for capture at 100,000 s-1 with greater than 170 bonds per platelet. If the on-rate is greater than 108 M-1s-1, then a platelet could be captured within 15 microseconds, the amount of time available to form bonds before the platelet is swept away. This mechanism of platelet capture allows for the rapid platelet accumulation in atherothombosis seen clinically and in high shear experiments.

Le PAF (facteur d'activation des plaquettes) est un médiateur lipidique de l'inflammation très puissant impliqué dans plusieurs conditions pathophysiologiques.Le PAF agit principalement via un seul récepteur, le PAFR qui appartient à la famille des récepteurs couplés aux protéines G, les GPCRs. Le"two state model" assume que les GPCRs existent dans un état d'équilibre entre un état inactif (R) et un état actif (R*). L'isomérisation de R vers R* peut arriver de façon spontanée, c'est à dire indépendamment de la liaison d'un agoniste. Dans ces travaux de recherche, nous avons tenté de déterminer la propriété antagoniste et agoniste inverse des molécules orthostériques (WEB2086, PCA4248, FR49175, bromure d'octylonium, CV3988 et le Trans BTP dioxolane) à activer la voie des MAPK ainsi que le cycle biochimique des inositols phosphates dans la lignée cellulaire HEK 293 transfectée de façon stable avec le récepteur du PAF. De plus, l'activité potentiellement allostérique sur le PAFR de modulateurs synthétiques tels le THG-315, le THG-316 et MAREK a également été investiguée dans la même lignée cellulaire. Finalement, des surnageants d'hybridome 9H1/1C1, 9F5/1H4, 9F5/1H4, 9F5/1F8, 9F5/2B3 et 9F5/2E4 contenant des anticorps monoclonaux, dirigés tous contre un peptide qui équivaut à la région C-terminale de la troisième boucle extracellulaire du PAFR: GFQDSKfHQA ont également été utilisés, afin : (1) de déterminer le meilleur clone en terme d'affinité et de spécificité et (2) effectuer des tests pour savoir s'ils possèdent des propriétés agonistes ou antagonistes sur le PAFR. En conclusion, les résultats obtenus nous indiquent que : (1) l'efficacité des molécules orthostériques à antagoniser les réponses induites par le PAF dépend de leur nature et de leur concentration, (2) les modulateurs potentiellement allostériques utilisés ne modulent aucune des voies majoritairement connues pour être activées par le PAFR, et (3) qu'il n'y a aucun marquage spécifique du PAFR avec les surnageants d'hybridomes utilisés.

Introduction: Inflammation and platelet activation play a pivotal role in the pathogenesis of acute coronary syndrome (ACS). Mounting evidence indicates that microparticles (MP) are potent pro-coagulant molecules modulating inflammatory processes. Aims: The aim of my study was to investigate the intracoronary and systemic microparticle expression in patients with symptomatic coronary heart disease (CHD) and their relationship with markers of inflammation, platelet activation and microvascular dysfunction (MvD). Methods: Forty eight patients with CHD (37 with ACS [23 patients with ST segment myocardial infarction (STEMI) and 14 with non-ST segment elevation myocardial infarction (NSTEMI)] and 11 with stable angina (SA) treated with percutaneous coronary intervention (PCI) were recruited. Blood samples were aspirated sequentially from the right atrium (RA) and the coronary (CO) artery (distal to the culprit lesion). AnnexinV+ MP (AnV+MP) from platelet poor plasma were measured using fluorescent monoclonal antibodies and flow cytometry. Markers of inflammation (hs-CRP, IL-6, TNF-α, serum amyloid antigen (SAA) and platelet activation (soluble p-selectin and platelet monocyte aggregates (PMA))) were measured using ELISA. MvD was assessed by measuring the index of microvascular resistance (IMR) and coronary wedge pressure (Pw). Results: The main novel findings of this study are the demonstration of: 1) differences between MP levels in patients with varying severity of CHD i.e. MP levels were overall higher in those with ACS (more specifically the highest MP levels were observed in the STEMI patients) compared with SA; 2. differential local and systemic AnV+ MP expression in human coronary artery disease; 3. differential local and systemic expression of inflammatory markers in ACS patients; 4. differential local and systemic expression of markers of platelet activation in ACS patients; 5. positive correlation between AnV+MP and markers of inflammation, platelet activation and myocardial necrosis in ACS patients both in the CO and RA; and 6. positive correlation between invasive markers of microvascular dysfunction and PMA in human coronary artery disease. Conclusions: High levels of AnV+MP occur in the coronary artery of patients with ACS. Levels of AnV+MP correlate with severity of the ischaemic lesion since higher MPs were detected in the STEMI versus the NSTEMI and SA groups. Markers of platelet activation and inflammation in ACS patients, both at the site of the culprit lesion and in the systemic circulation, strongly correlated with total and cell specific AnV+MP. The interaction between activated platelets and monocytes with endothelial cells and the subsequent formation of AnV+MP and PMA during ACS would be compatible with a direct pathogenic link between inflammatory and prothrombotic pathways in the pathogenesis of ACS and myocardial necrosis. This may suggest a novel role for MPs as effectors of the inflammatory response and cellular injury in ACS. Our observations also support the hypothesis that PMA formation may be important determinants of platelet activation, inflammation and microvascular dysfunction in coronary artery disease. Whether these MP merely reflect the severity of the ischaemic lesion, and/or are active participants in the pathogenesis of ACS now warrants further study, since these could be important, novel therapeutic targets.

Les plaquettes sanguines sont des cellules qui ont un rôle majeur au cours des processus de l’hémostase primaire et jouent un rôle primordial dans l’immunité innée mais aussi adaptative. Ces cellules anucléées ont une capacité sécrétoire très importante de facteurs solubles notamment de cytokines, de chimiokines (CK/CH) et de facteurs immunomodulateurs. L’émergence du rôle inflammatoire des plaquettes sanguines dans la communauté scientifique a soulevé de nombreuses questions auxquelles nous essayons de répondre dans ce manuscrit. La majorité de ces questions repose sur la capacité de ces cellules anucléées à répondre de manière régulée à des stimuli complexes. Nos investigations pour répondre à ces questions ont été réalisées dans un contexte transfusion sanguine. Au cours de nos travaux, nous avons mis en évidence la corrélation des profils de sécrétion plaquettaire avec les récepteurs membranaires et les voies de signalisations intraplaquettaires engagées. Les plaquettes expriment plusieurs récepteurs immunitaires sur leur surface notamment les « Pattern recognition receptors » (PRR) et des récepteurs aux CK/CH. Nous avons démontré et caractérisé la fonction d’un nouveau récepteur plaquettaire, le Siglec-7. Ce récepteur est localisé dans les granules a ; son expression sur la membrane est corrélée avec l’état d’activation plaquettaire. Le Siglec-7 a une avidité élevée avec les molécules composées d’α2,8-disialyl (NeuAcα2,8NeuAcα2,3Gal) et de α2,6-sialyl (Gal-b1,3[NeuAcα2,6]HexNAc) (comme les gangliosides GD2, GD3 et GT1b). L’engagement de ce récepteur peut induire l’apoptose plaquettaire par la voie intrinsèque et extramitochondriale. Ce processus nécessite l’engagement du récepteur GPIIbIIIa et P2Y1 et la signalisation de la voie de PI3k. Nous avons également étudié et mis en évidence une composante inflammatoire multifactorielle dans les effets indésirables des receveurs (EIR) et trouvé dans les concentrés plaquettaires (CP), plusieurs facteurs solubles ayant une valeur prédictive élevée pour la survenue des EIR, notamment le sCD40L et l’IL-13. Nous avons confirmé que la concentration de ces facteurs augmente au cours de temps de stockage des CP, étant, en partie, responsable du taux élevé de l’EIR des CP âgés. Enfin, en plus de la conservation, les processus de préparation des CP peuvent aussi avoir des impacts sur les propriétés inflammatoires des plaquettes. Ces travaux montrent que la réponse inflammatoire plaquettaire est régulée en fonction du stimulus, permettant d’argumenter sur le rôle présumé de sentinelle des plaquettes sanguines humaines. Ainsi, mes travaux s’inscrivent dans la ré-exploration de la fonction inflammatoire des plaquettes sanguines et l’étude du rôle des plaquettes comme cellules de l’immunité à composante inflammatoire
Blood platelets are non-nucleated cells and play a major role in primary hemostasis and a key role in inflammation, innate and adaptive immunity. They secrete a large variety of soluble factors including cytokines/chemokines (CK/CH) and immunomodulator factors. The emergence of their inflammatory role has raised numerous questions based on the ability of platelets to respond to complex stimuli. Our investigations to answer these questions were realized in the context of platelet component transfusion. In our study, we demonstrated the correlation between the platelet secretion of soluble factors with their membrane receptors and the signaling pathways involved. Platelets express many immune receptors on their surface, including "Pattern recognition receptors" (PRRs) and receptor for CK/CH. We discovered and characterized the function of a new platelet receptor, the Siglec-7. This receptor is located in the granules a and its expression is correlated to the platelet activation level. The Siglec -7 has a high avidity with the molecules composed of α2,8-disialyl (NeuAcα2,8NeuAcα2,3Gal) and of α2,6-sialyl (Gal-b1,3[NeuAcα2,6]HexNAc) (ganglioside GD2 , GD3 and GT1b). Stimulation of this platelet receptor may induce platelet apoptosis by the intrinsic and extramitochondrial pathway. This process requires the engagement of GPIIbIIIa and P2Y1 receptor and the PI3K pathway. We also demonstrated a multifactorial inflammatory component in adverse effects issuing from platelets transfusion, and identified many soluble factors which have a high predictive value of Acute Transfusion Reactions (ATR) occurrence, such as sCD40L and IL- 13. We confirmed that the concentration of these factors increases during storage time of platelet component (PC), being partly responsible for the high rate of ATR by old PC. Finally, in addition to the PC conservation, the process of PC preparation may also have impacts on the inflammatory properties of platelets. These studies showed that the platelet inflammatory response is regulated by the stimulus, explaining the sentinel role of human blood platelets. Therefore, my work contributes to the re-exploration of inflammatory function of these cells and studies their role as an immune cell with an inflammatory component