Redundancy and interaction of thrombin- and collagen-mediated platelet activation in tail bleeding and carotid thrombosis in mice.

OBJECTIVE: Current antiplatelet strategies to prevent myocardial infarction and stroke are limited by bleeding risk. A better understanding of the roles of distinct platelet-activating pathways is needed. We determined whether platelet activation by 2 key primary activators, thrombin and collagen, plays distinct, redundant, or interacting roles in tail bleeding and carotid thrombosis in mice. APPROACH AND RESULTS: Platelets from mice deficient for the thrombin receptor protease-activated receptor-4 (Par4) and the collagen receptor glycoprotein VI protein (GPVI) lack responses to thrombin and collagen, respectively. We examined tail bleeding and FeCl3-induced carotid artery occlusion in mice lacking Par4, GPVI, or both. We also examined a series of Par mutants with increasing impairment of thrombin signaling in platelets. Ablation of thrombin signaling alone by Par4 deficiency increased blood loss in the tail bleeding assay and impaired occlusive thrombus formation in the carotid occlusion assay. GPVI deficiency alone had no effect. Superimposing GPVI deficiency on Par4 deficiency markedly increased effect size in both assays. In contrast to complete ablation of thrombin signaling, 9- and 19-fold increases in EC50 for thrombin-induced platelet activation had only modest effects. CONCLUSIONS: The observation that loss of Par4 uncovered large effects of GPVI deficiency implies that Par4 and GPVI made independent, partially redundant contributions to occlusive thrombus formation in the carotid and to hemostatic clot formation in the tail under the experimental conditions examined. At face value, these results suggest that thrombin- and collagen-induced platelet activation can play partially redundant roles, despite important differences in how these agonists are made available to platelets.

Protein kinase C isoform ε negatively regulates ADP-induced calcium mobilization and thromboxane generation in platelets.

OBJECTIVE: Members of the protein kinase C (PKC) family are shown to positively and negatively regulate platelet activation. Although positive regulatory roles are extensively studied, negative regulatory roles of PKCs are poorly understood. We investigated the mechanism and specific isoforms involved in PKC-mediated negative regulation of ADP-induced functional responses. METHODS AND RESULTS: A pan-PKC inhibitor, GF109203X, potentiated ADP-induced cPLA(2) phosphorylation and thromboxane generation as well as ERK activation and intracellular calcium (Ca(2+)(i)) mobilization, 2 signaling molecules, upstream of cPLA(2) activation. Thus, PKCs inhibit cPLA(2) activation by inhibiting ERK and Ca(2+)(i) mobilization. Because the inhibitor of classic PKC isoforms, GO-6976, did not affect ADP-mediated thromboxane generation, we investigated the role of novel class of PKC isoforms. ADP-induced thromboxane generation, calcium mobilization, and ERK phosphorylation were potentiated in PKCε null murine platelets compared with platelets from wild-type littermates. Interestingly, when thromboxane release is blocked, ADP-induced aggregation in PKCε knockout and wild-type was similar, suggesting that PKCε does not affect ADP-induced aggregation directly. PKCε knockout mice exhibited shorter times to occlusion in an FeCl(3)-induced arterial injury model and shorter bleeding times in tail-bleeding experiments. CONCLUSIONS: We conclude that PKCε negatively regulates ADP-induced thromboxane generation in platelets and offers protection against thrombosis.

Protein kinase C - possible therapeutic target to treat cardiovascular diseases.

Cardiovascular diseases (CVDs) such as atherosclerosis, hypertension and diabetes, are major global health problems and one of the leading causes of death. Thrombosis associated with multiple CVDs such as atherosclerosis and diabetes further increase morbidity by causing myocardial infarction or stroke. The members of Protein Kinase C (PKC) family are serine threonine kinases, abundantly expressed in cells that maintain cardiovascular health. Studies done using pharmacological tools that block wide range of PKCs or specific PKC isoforms and PKC gene knockout animals revealed that these enzymes regulate critical functional responses in cardiovascular cells. Interestingly, PKC isotype activity is context specific and PKC isotypes may have opposing functional roles depending on cell type and cellular environment (eg., cardiomyocytes, platelets). Furthermore, precise structural differences that occur amongst these isoforms have lead to development of compounds that inhibit or activate specific PKC isoforms. Thus, it is feasible to enhance the protective effects of a PKC isoform, while minimizing the damage caused by other members of PKC family. In this review, we summarize the role of each of these PKC isoforms in various cardiovascular diseases. In addition, we detail the specific PKC isoform modulators, their mechanism of action and ability to treat cardiovascular diseases, as evaluated in animal models or human subjects.