Cardiovascular diseases are a major cause of mortality and morbidity in the developed countries. Anti-platelet therapy is a cornerstone treatment for patients with cardiovascular diseases. Patients are routinely managed with a combination therapy consisting of aspirin and clopidogrel. Aspirin inhibits cyclooxygenase 1 (COX 1) a crucial intermediate enzyme involved in thromboxane biosynthesis. Clopidogrel on the other hand antagonizes ADP receptor P2Y12. ADP is a weak platelet agonist stored in platelet dense granules and is released upon platelet activation. ADP activates platelets through two purinergic receptors namely P2Y1 and P2Y12 these receptors couple to Gq and Gi class of G-proteins, respectively. P2Y1 causes calcium mobilization through activation of PLC-β. P2Y12 inhibits adenylyl cyclase, causes activation of Rap1B and Akt. Signaling from both the receptors is required for complete integrin activation, thromboxane generation and Erk activation. Previous studies have shown that P2Y12 potentiates fibrinogen receptor activation, secretion, thrombi stabilization, thrombin generation, platelet leukocyte aggregation formation. ThromboxaneA2 (TXA2) is a potent platelet agonist generated through arachidonic acid metabolism in platelets. TXA2 thus, generated after platelet activation acts as a positive feedback mediator along with ADP. Under physiological conditions, platelet activation leads to thrombin generation through coagulation cascades. Generated thrombin activates PAR receptors and ADP is released from dense granules, which further potentiates thromboxane generation downstream of PARs. Current anti-platelet therapy regimens often include P2Y12 antagonists and aspirin in management of patients with acute coronary syndrome (ACS) and in those undergoing percutaneous coronary intervention (PCI) with stent implantation. However, there still exists a need for improved treatment strategies as not all patients benefit from this dual combination therapy. Reasons include, poor responders either to P2Y12 antagonists or to aspirin, or if aspirin is contraindicated in these patient populations. In the current study we evaluated the role of P2Y12 in thromboxane generation under physiological conditions. We studied serum thromboxane generation in a model system wherein P2Y12 was antagonized or deficient. Using pharmacological approaches we show that dosing mice with 30mg/Kg/body weight clopidogrel or 3mg/Kg/body weight prasugrel decreased serum thromboxane levels when compared to the control mice. Pre-treatment of human blood ex vivo with active metabolites of clopidogrel (R361015) or prasugrel (R138727) also led to reduction in thromboxane levels. We also evaluated serum thromboxane levels in P2Y receptor null mice, serum thromboxane levels in P2Y1 null mice were similar to those in wild type littermates, and were inhibited in P2Y12 null mice. Furthermore, serum thromboxane levels in P2Y12 deficient patients, previously described in France and Japan, were also evaluated and these patients had lower serum thromboxane levels compared to normal controls. In a pilot study, serum thromboxane levels were radically reduced in healthy human volunteers upon dosing with clopidogrel, compared to the levels before dosing. In conclusion, P2Y12 antagonism alone can decrease physiological thromboxane levels. Thus P2Y12 regulates physiological thromboxane levels. Further it is known that ADP-induced thromboxane generation is integrin-dependent. However it is not clear if other potent platelet agonists like thrombin require outside-in signaling for thromboxane generation. Our results show that thrombin-induced thromboxane generation was independent of integrins i.e. when platelets were stimulated with PAR agonists in presence of fibrinogen receptor antagonist thromboxane generation was not affected. Since PAR agonists, unlike ADP, activate G12/13 signaling pathways. Hence, we hypothesized that these pathways might play a role in TXA2 generation. Our results show, that inhibition of ADP-induced thromboxane generation by fibrinogen receptor antagonist SC57101 was rescued by costimulation of G12/13 pathways with YFLLRNP. This observation suggested an existence of a common signaling effector downstream of integrins and G12/13 pathways. Next, we evaluated role of three potential tyrosine kinases; c-Src, Syk and FAK (Focal Adhesion Kinase) that are known to be activated by integrins. Our results showed that c-Src and Syk kinase did not play a role in ADP-induced functional responses in platelets. We observed differential activation of FAK downstream of integrins and G12/13 pathways. ADP-induced activation of FAK was integrindependent and SFK-independent. On the other hand selective activation of G12/13 pathway lead to FAK activation, in SFK and Rho dependent manner. We also evaluated specificity of new FAK inhibitor TAE-226 to understand the role of FAK in TXA2 generation. Our results showed that TAE-226 exhibited non-specific effects at higher concentrations. Furthermore, in comparison to WT mice, FAK null mice did not show any difference in TXA2 generation. Therefore, we concluded that differential activation of FAK occurs downstream of Integrins and G12/13 pathways. However, the common effector molecule downstream of integrins and G12/ 13 pathways contributing to TXA2 generation in platelets remains elusive. / Molecular and Cellular Physiology

Platelets are primary components of hemostasis. However, incongruous activation of platelets lead to thrombosis, which result in multiple cardio-vascular and cerebrovascular complications. Thus, platelet activation is tightly regulated. Molecular components that aid in activation of platelets have been extensively studied. However, molecular pathways that negatively regulate platelet activation and prevent accidental activation of platelets are poorly understood. In this study we investigated the molecular mechanisms that negatively regulate platelet activation. Protein Kinase C isforms (PKCs) are serine threonine kinases that regulate various platelet functional responses leading to hemostasis. Positive regulatory role of PKCs towards platelet aggregation and secretion has been extensively studied. However, we have recently demonstrated that PKCs negatively regulate ADP- induced thromboxane generation. The PKC isoforms and mechanism involved in this process have not been known. Thus, in this study we investigated the mechanism by which PKCs negatively regulate ADP-induced thromboxane generation and identified PKC isoforms that regulate thromboxane generation. Thromboxane generation in platelets is a multi-step process beginning with cPLA2 activation. cPLA2 activation is the rate limiting step in the process of thromboxane generation. Furthermore, cPLA2 activation is regulated by ERK and calcium in various cell systems including platelets. PKC inhibition potentiated both cPLA2 and ERK activation, suggesting that PKCs negatively regulate thromboxane generation by regulating ERK activation, which in turn regulates cPLA2 activation. Furthermore, we have also shown that PKCs negatively regulate ADP-induced calcium mobilization. ADP activates platelets via P2Y1 and P2Y12 receptors. P2Y12 receptor-mediated signaling is shown to positively regulate P2Y1-mediated calcium mobilization in platelets. Furthermore, PKCs are shown to negatively regulate P2Y12 receptor desensitization in platelets. Thus, we investigated if PKCs regulate calcium mobilization indirectly by regulating P2Y12 receptor function. However, PKCs regulate calcium mobilization independent of P2Y12 receptor signaling. In summary we have shown that PKC isoforms negatively regulate ADP-induced thromboxane generation by regulating calcium mobilization and ERK activation that in turn regulates cPLA2 activity. We further investigated the PKC isoforms involved in this process. Based on our results with Go-6976, a classical PKC inhibitor and GF109203X, a pan PKC inhibitor, we identified that that novel or atypical PKC isoforms are involved in negative regulation of ADP-induced thromboxane generation. Thus, we investigated the role of various novel class of PKC isoforms (nPKCs) in platelets. We first investigated the nPKCs activated by ADP. In aspirin-treated platelets, ADP failed to activate nPKC θ and δ non-stirring conditions. Thus, we conclude that these isoforms are not involved in negative regulation of thromboxane generation. We further investigated if other non-classical PKC isoforms i. e nPKC η and ε or atypical PKC isoforms could be involved in this process. We began our investigation with the mechanism of activation and functional role of nPKC η in platelets. The mechanism of activation of PKCs has been extensively studied in various cell systems including platelets. However, the mechanism by which they are inactivated is not completely understood. In this study, we demonstrate a novel mechanism of inactivation of nPKC η isoform by integrin associated serine/threonine phosphatase. we demonstrated that ADP activates nPKC η via P2Y1 receptor coupled to Gq. As expected, Gi pathway, which does not generate DAG or mobilize calcium, has no role in regulation of nPKC η. Interestingly, we show that upon activation of platelets, αIIbβ3 mediated outside-in signaling dephosphorylates nPKCη through PP1γ phosphatase. We have also evaluated the role of nPKC η using η-RACK antagonistic peptides that interfere with enzyme-substrate interaction. Similar antagonistic peptides have been successfully used in various cell systems such as cardiomyocytes and neuronal cell. Using η-RACK antagonists we have demonstrated that nPKC η positively regulates agonist- induced thromboxane generation with no effect on agonist- induced platelet aggregation. The peptides were targeted in to the cell using TAT carrier protein, which is also used as a negative control for these experiments. The specificity of η-RACK antagonistic peptides is further elucidated by the fact that they do not affect the platelet aggregation. In summary, nPKC η is activated by ADP via P2Y1 receptor and is dephosphorylated by integrin αIIbβ3 via PP1γ phosphatase. Furthermore, activated nPKC η positively regulates ADP- induced thromboxane generation with no effect on aggregation. Since, our aim was to investigate the nPKC isoforms that negatively regulate ADPinduced thromboxane generation we investigated if nPKC ε is involved in this process. We made use of PKC ε knockout mice (PKC ε KO) for this process. We observed potentiated thromboxane generation in ADP-induced PKC ε murine platelets compared to witd type murine platelets. Thus, PKC ε might be one of the PKC isoforms involved in negative regulation of ADP-induced thromboxane generation. However, we failed to detect PKC ε in human platelets using western blot analysis. Since, PKC ε has been reported to be a part of platelet kinase repertoire, it could be limitation of our technique that we failed to detect it in western blot analysis. Since, PKCs negatively regulate ADP-induced thromboxane generation, we also investigated if PKCs also regulate PAR-mediated thromboxane generation. Similar to ADP, PAR-mediated thromboxane generation is not affected by Classical PKC isoforms. Furthermore, although non-classical PKC isoforms negatively regulate thromboxane generation, the extent of negative regulation is smaller and non-significant compared to ADP. Thus, we investigated if activation of nPKC isoforms were different between ADP and AYPGKF (PAR4 agonist). While, ADP fails to activate nPKC δ and θ, PARs activate Them. Furthermore, we have recently demonstrated that nPKC δ and θ are positive regulators of PAR-mediated platelet functional responses. Therefore, PKCinduced potentiation of thromboxane generation by ADP and PAR agonist are different due to differential activation of PKCs. This data lead to our final project, where we investigated the reason for differential activation of nPKC isoforms by various platelet agonists. Using strong and weak platelet agonists and DAG analogue, DiC8, we demonstrated that different platelet agonists differentially regulate nPKC activation due to variable amounts of DAG generated by them. Furthermore, we also have demonstrated that nPKC η and ε have higher affinities to DAG compared to nPKC δ and θ. / Molecular and Cellular Physiology