Functional Divergence of Platelet Protein Kinase C (PKC) Isoforms in Thrombus Formation on Collagen

Abstract

Arterial thrombosis, a major cause of myocardial infarction and stroke, is initiated by activation of blood platelets by subendothelial collagen. The protein kinase C (PKC) family centrally regulates platelet activation, and it is becoming clear that the individual PKC isoforms play distinct roles, some of which oppose each other. Here, for the first time, we address all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet adhesion to collagen and their subsequent activation under physiological flow conditions. Using mouse gene knock-out and pharmacological approaches in human platelets, we show that collagen-dependent α-granule secretion and thrombus formation are mediated by the conventional PKC isoforms, PKCα and PKCβ, whereas the novel isoform, PKCθ, negatively regulates these events. PKCδ also negatively regulates thrombus formation but not α-granule secretion. In addition, we demonstrate for the first time that individual PKC isoforms differentially regulate platelet calcium signaling and exposure of phosphatidylserine under flow. Although platelet deficient in PKCα or PKCβ showed reduced calcium signaling and phosphatidylserine exposure, these responses were enhanced in the absence of PKCθ. In summary therefore, this direct comparison between individual subtypes of PKC, by standardized methodology under flow conditions, reveals that the four major PKCs expressed in platelets play distinct non-redundant roles, where conventional PKCs promote and novel PKCs inhibit thrombus formation on collagen.

Glucose and collagen regulate human platelet activity through aldose reductase induction of thromboxane

“…Looking further downstream (Figure 13), in HG-and collagen-stimulated platelets, PKCα, PKCβII, and PKCδ were translocated from cytosol to membrane, with concomitant increase in their phosphorylated forms, thus supporting a role of PKCα, PKCβII, and PKCδ in this process. These findings are also supported by previous studies that demonstrate PKCα, PKCβII, and PKCδ as playing a crucial role in collagen-induced platelet aggregation (21,22). Thus, a complex web of signaling components is involved in platelet activation upon exposure to collagen and hyperglycemia ( Figure 13).…”

“…Since the subcellular translocation of PKCα, PKCβII, and PKCδ was prevented by AR inhibitor, we next determined whether inhibition of AR prevents the phosphorylation of PKCα, PKC βII, and PKCδ by using phosphospecific antibodies that correlate with kinase activity in total extracts. Consistent with the previous genetic knockout and inhibitor studies on the role of PKC isoforms in platelet aggregation (21,22), the phosphorylation of different PKC isoforms was in line with the findings in their translocation (Supplemental Figure 4), indicating that PKCα, PKCβII, and PKCδ translocation from cytoplasm to membrane and phosphorylation are required for collagen-induced aggregation. In addition, these results suggest that HG potentiates the activation of PKCβII and PKCδ similarly to the effect of collagen in an AR-dependent manner.…”

Conventional protein kinase C isoforms differentially regulate ADP- and thrombin-evoked Ca2+ signalling in human platelets

“…In contrast, treating platelets with an inhibitor of novel PKC isoforms appears to suppress the Ca 2+ signals elicited by ADP and thrombin. Interestingly, previous work has demonstrated that collagen-evoked Ca 2+ signalling in human platelets is negatively regulated by novel PKC isoforms and positively regulated by conventional PKC isoforms [44]. An agonist-specific effect of isoform-selective PKC inhibition has also been reported for platelet dense granule secretion [45].…”

Dual Mechanism of Integrin αIIbβ3 Closure in Procoagulant Platelets

“…Thrombus Formation on Collagen under Flow-Collagen-induced thrombus formation was assayed as described before (20,31). In brief, PPACK/fragmin-anticoagulated mouse blood was flowed over a coverslip coated with collagen type I in a transparent parallel-plate perfusion chamber, at shear rate of 1000 s Ϫ1 for 4 min.…”