Platelets play a central role in mediating atherothrombosis and are therefore the target of numerous therapies aimed at reducing their activity, particularly in the prevention of coronary artery thrombosis in heart attacks (45
). PKC is established, largely by pharmacological studies, as a major regulator of multiple platelet activities (2
), and it is increasingly clear that the different isozymes of PKC expressed in platelets perform distinct functions. There is a difficulty of interpretation of data from some pharmacological studies, however, because of the lack of selectivity of the reagents available to target specific PKC isozymes. Here, we used a genetic approach to demonstrate definitively, for what we believe is the first time, the role played by PKCα in regulating platelet function and thrombus formation. Importantly, the study revealed a key role for PKCα in regulating granule biogenesis and exocytosis, which was essential for thrombus formation, since ablation of thrombus formation in Prkca–/–
platelets could be rescued by addition of exogenous ADP. The findings reveal PKCα to be a potential drug target for antithrombotic therapy, since selective inhibitors would exert a major effect upon thrombus formation while sparing primary platelet adhesive functions.
It has recently been shown pharmacologically that PKC isoforms exert dual control of thrombus formation by mediating secretion and integrin activation under flow while suppressing phosphatidylserine exposure and subsequent thrombin generation and coagulation (7
). There is incomplete and largely indirect evidence that, of the various isoforms of PKC expressed in platelets, PKCα plays a role in regulation of integrin αIIb
). The study by Han et al. (33
) elegantly showed that PKCα was required for integrin αIIb
activation in a reconstituted cell system rather than in the platelet. The study by Tabuchi et al. (18
) used an in vitro permeabilized platelet system to investigate receptor-independent calcium-mediated activation of platelet aggregation. Although this latter study demonstrated that added purified PKCα was able to support a calcium-induced platelet aggregation response, the approach has limitations in that receptor-mediated activation is lost. Nonetheless, these studies provided indications that PKCα may regulate platelet integrin activation and aggregation, and it was therefore essential to perform a genetic-based definitive study of the role played by PKCα in mediating these events.
The marked knockdown in activation of integrin αIIb
in response either to thrombin or CRP in platelets lacking PKCα paralleled a deficit in the ability of these platelets to undergo aggregation at submaximal concentrations of agonist. This was consistent with the impaired ability of Prkca–/–
platelets to form a thrombus in blood flowing over a collagen-coated surface. The effect on aggregation could, however, be overcome by increasing the concentration of agonist, and the reasons for this may derive from several factors. First, it is known that integrin αIIb
activation in response to strong agonists is in part regulated independently of PKC (3
). Further, studies with integrin blockers have shown that not all αIIb
receptors are required to be activated (e.g., by the PKC-independent pathway) for a full platelet aggregation response in the platelet aggregometer (47
). Thus, it may be possible to achieve a maximal aggregation response with a markedly reduced expression of activated integrin. In contrast, in thrombus formation in flowing whole blood, stable platelet aggregation critically depends on limited levels of autocrine-produced ADP and hence limited and reversible integrin activation (48
). Under these more physiological conditions, the number of activated integrin receptors may be low, and therefore the marked reduction in integrin activation seen in Prkca–/–
platelets may cause a significant reduction in the ability of platelets to form thrombi. Additionally, we provide evidence for redundancy between PKCα and PKCβ in Figure , which shows that selective inhibition of PKCβ only significantly attenuated aggregation responses in Prkca–/–
platelets, not WT platelets, indicating redundant functions of these 2 isoforms. It is also significant to note that in assays for occult blood in feces, no gastrointestinal bleeding was detected in either WT (n
= 5) or Prkca–/–
= 6) mice. Together with the data shown in Figure , where no difference in tail bleeding time is seen in Prkca–/–
compared with WT controls, this also implies redundancy of signaling molecules for regulation of hemostasis in vivo.
There is significant contribution to the effects of PKCα ablation on aggregation and thrombus formation by regulation of granule secretion, since these processes greatly rely upon autocrine ADP release (49
). Clearly, both dense- and α-granule secretion are markedly disrupted in platelets lacking PKCα, and evidence that this is functionally critical is provided by the rescue experiments depicted in Figure for aggregation and Figure for in vitro thrombus formation, where addition of exogenous ADP recovers the deficits in these responses seen in Prkca–/–
The role of unspecified PKC isoforms as a family in the regulation of secretion has been shown pharmacologically by several groups (3
). The role of the PKCα isoform in agonist-independent secretion has been demonstrated previously in an artificial permeabilized platelet system (17
). Although valuable to indicate the role of PKCα, it was important to address the issue by a definitive genetic approach. Indeed, the only study to date to address specific PKC isoform function in secretion in platelets in a combined genetic and pharmacological approach had shown a major discrepancy in results obtained by the 2 approaches for PKCδ. Pula et al. (15
) showed that genetic ablation of PKCδ had no significant effect upon dense-granule secretion, whereas in that study and previous studies (9
), the PKCδ-selective inhibitor rottlerin had been shown to enhance dense-granule secretion in response to collagen and alboaggregin, operating through the GPVI receptor. The present results provide direct evidence that PKCα is a major regulator of secretion of α-granules, since P selectin expression is reduced almost to basal levels in Prkca–/–
platelets in response either to CRP or thrombin. Importantly, we show that α-granule numbers are equivalent between Prkca–/–
and WT platelets and therefore the secretion defect for this granule type reflects a genuine deficiency in the secretory pathway. For dense granules, this study shows an additional knockdown in numbers of granules in Prkca–/–
platelets. This suggests a role for PKCα in biogenesis of dense granules and is therefore an area that requires further analysis. A number of different elements of the secretory machinery, including SNAP23, SNAP25, Munc18a and Munc18b, syntaxin 2, and Rab6, have been reported to be PKC substrates (40
), and therefore their phosphorylation may influence secretion. In this study then, phosphorylation of SNAP23 serves as a potential marker for these events and may contribute functionally to the process of secretion. However, although SNAP23 phosphorylation on Ser95 is markedly reduced in Prkca–/–
platelets and this may play a role in regulating α-granule secretion, its role in dense-granule secretion cannot be clarified from this study.
In summary, we have shown PKCα to play major roles in regulating platelet secretion of α-granules, biogenesis and secretion of dense granules, regulation of integrin αIIb
, and thrombus formation in vitro and in vivo. PKCα also regulates platelet aggregation, although the defect in Prkca–/–
platelets is relative, since high concentrations of agonists overcome the deficit and there is evident redundancy of the action of PKCα with the other expressed classical PKC isoform in platelets, PKCβ. PKCα plays no significant role in regulating adhesion of platelets to collagen-coated surfaces under static or flow conditions and does not regulate platelet spreading response or outside-in signaling through integrin αIIb
. It could be argued that, because PKCα is the most highly expressed of the PKC isoforms in platelets, its role in platelets may be wide ranging and effectively mask that of other PKC isoforms that are expressed at lower levels. The evidence presented here, however, suggests highly specific roles for PKCα, since, for instance, its absence has no significant effect upon platelet adhesion and spreading on collagen but markedly suppresses thrombosis. Additionally, if PKCα were so predominant functionally in platelets, knockout of the other PKC isoforms would be predicted not to have functional effects. This is clearly not the case, since we have shown absence of PKCδ to enhance platelet responses to collagen through enhanced filopodia formation (15
) and we have more recently shown absence of PKCθ to enhance α-granule secretion and integrin αIIb
activation in response to GPVI agonists (53
). Shattil’s group has also shown absence of PKCs β and θ to ablate outside-in signaling through integrin αIIb
). These data therefore demonstrate specific functional roles for the different PKC isoforms, including for PKCα in regulating secretion in particular, such that redundancy of activity is not apparent for specific functions.
Genome-wide association analyses of major human diseases have been conducted recently, based upon technical advances in high throughput microarray analyses of SNPs. These studies are introducing major new leads in genes that may be related to disease, and although PKCα is not in the top ranking of genes associated with coronary artery disease, a cluster of SNPs in this gene with a maximal P
value of just over 10–3
(SNP rs12600582, intronic, minor allele frequency of 0.233 in Europeans) (34
) may indicate some significance in this disease of polygenic cause. For these reasons, PKCα may represent a drug target for antithrombotic therapy, with inhibitors exerting an effect upon thrombus formation but sparing primary platelet adhesive functions. PKCα is already a target for the drug aprinocarsen, an antisense oligonucleotide therapy used in the treatment of specific neoplastic conditions (54
), and it will now be important to assess whether this or other small molecule–based approaches may represent opportunities in the development of platelet-based antithrombotic drugs in the management of coronary artery disease and other arterial thrombotic diseases.