Here, for the first time, we have addressed all four of the major platelet-expressed PKC isoforms, determining their comparative roles in regulating platelet activation by collagen under physiological flow conditions. The study is also the first to determine the role of individual PKC isoforms in regulating calcium responses, at the single cell level, in growing thrombi visualized in real time. Importantly, the study reveals major positive roles for the conventional isoforms, PKCα and PKCβ, in mediating thrombus formation. The absence of either isoform leads to marked suppression of secretion of α-granules, aggregate formation, calcium signaling, and PS exposure under flow. The data therefore suggest essential but non-redundant roles for these kinases in regulating these events. In contrast, the absence of the novel isoforms PKCθ and PKCδ, however, leads to enhanced thrombus formation on collagen. The mechanisms for these isoforms are also distinct and non-redundant because although the absence of PKCθ also leads to enhanced secretion, calcium signaling, and phosphatidylserine exposure, the absence of PKCδ does not potentiate any of these functions. We conclude that all four major expressed PKC isoforms play distinct non-redundant roles, where the conventional PKCs promote and the novel PKCs inhibit thrombus formation on collagen, by a variety of mechanisms (summarized in C).
The studies here with mice lacking PKCα or PKCβ demonstrated that platelet thrombi formed on collagen were significantly smaller in comparison with wild-type thrombi. This reduced thrombus formation was associated with impaired GPVI-induced α-granule secretion. A key role for murine PKCα in exocytosis of platelet α-granules and dense granules has recently been demonstrated (18
). Importantly, these effects were not caused by diminished primary adhesion to collagen because time lapse studies with Fluo-4-loaded platelets showed normal adhesion under shear in case of PKCα or PKCβ deficiency. Instead, our data suggest that the defect is in the ability of platelets to form aggregates on platelets that have already adhered.
In addition, in both PKCα−/−
mice, the procoagulant activity of collagen-adhered platelets was diminished, consistent with the reduced Ca2+
signal. Earlier, it had been shown that the platelet procoagulant response is a direct consequence of GPVI-induced activation of collagen-adhered platelets (42
). How PKCα and PKCβ regulate Ca2+
signaling is not yet understood, although a role for conventional PKCs in store-operated calcium entry has previously been proposed on the basis of pharmacological studies in human platelets (44
). Together, these data support the concept that both conventional PKC isoforms positively regulate thrombus formation by enhancing GPVI-induced platelet activation, leading to secretion and procoagulant activity. Interestingly, there is a high degree of non-redundancy in this process, indicating that PKCα and PKCβ may each play essential, but distinct, roles.
It was important to extend the studies in mouse platelets by studies with human platelets, using the compounds Gö6976 and PKCβ inhibitor. Dose-response curves showed an inhibitory effect of both compounds on collagen-induced platelet aggregation as well as on GPVI-induced integrin activation and P-selectin expression. In addition, these compounds diminished GPVI-induced Ca2+ signal generation. The reduction in aggregation is likely to be mainly caused by loss of granule secretion as full aggregation could be restored by co-stimulation with ADP. This is consistent with our previous report that co-infusion with ADP could restore thrombus formation on collagen in PKCα−/− deficient platelets. By contrast, enhancing cytosolic Ca2+ by co-stimulation with ionomycin did not rescue collagen-inducing aggregation.
For mouse platelets, the present data indicate that both isoforms are required for full GPVI-dependent activation, suggesting a non-redundancy in function, particularly for regulation of P-selectin expression and annexin V binding, which are both markedly suppressed in the absence of either PKCα or PKCβ. For human platelets, the studies with Gö6976 and PKCβ inhibitor point to a substantial reduction in platelet responses. The PKCβ inhibitor is, however, much less effective than Gö6976 on convulxin-induced responses (, A–C
). This may suggest that in human platelets, there is more redundancy of function between PKCα and PKCβ than in mouse platelets. It may, however, be a reflection of the selectivity profile of these inhibitors and may also reflect differences between the effects of gene deletion versus
pharmacological inhibition of a kinase. In support of the concept that both PKCα and PKCβ positively contribute to human platelet activation are the findings in the literature that purified PKCα mediates granule secretion (19
), that PKCα is phosphorylated upon GPVI stimulation (45
), and that PKC in general and PKCβ in particular are implicated in αIIb
In contrast, the current studies with mice deficient in PKCθ point to a negative role in collagen-dependent granule secretion and thrombus formation. In addition, increased GPVI-induced activation of PKCθ−/−
platelets was apparent from the higher Ca2+
responses of adhered single platelets and the increased numbers of procoagulant, PS-exposing cells. Consistent with these data, the PKCθ inhibitor increased GPVI-induced human platelet aggregation as well as αIIb
mobilization, and α-granule secretion. There are discrepancies in the literature about negative or positive roles for PKCθ in regulating platelet function (24
), which may be a result of differences in platelet preparation conditions. We have, however, previously shown platelet responses to GPVI activation to be enhanced in the absence of PKCθ (24
), and taken together, these data would support the concept that PKCθ may down-regulate rather than up-regulate GPVI-mediated granule secretion and aggregate formation under physiological flow conditions. Mouse platelets express relatively high amounts of PKCθ when compared with the other novel PKC isoforms, PKCδ and PKCϵ (7
). Furthermore, all three isoforms are phosphorylated on tyrosine following GPVI stimulation (7
In addition, the present data suggest that PKCθ is the principal isoform mediating the earlier recognized effect of PKC in down-regulating GPVI-induced platelet Ca2+
signaling and procoagulant activity (17
). The mechanism is currently unknown, although PKC may reduce phospholipase C activation (51
) or increase Ca2+
extrusion via the plasma membrane Ca2+
The enhanced thrombus formation that we report in PKCθ−/−
blood is in apparent contrast to a previous report (25
), in which PKCθ−/−
mice showed reduced thrombus formation in vivo
in a FeCl3
-induced carotid injury model. This is likely to reflect the additional contribution of thrombin generation in vivo
. Importantly, although PKCθ negatively regulates collagen-dependent platelet activation, it appears to have a positive role in thrombin-induced signaling (25
). This agonist-dependent difference is similar to that proposed by Kunapuli and colleagues (23
) for PKCδ. The relative importance of collagen and thrombin in vivo
appears to depend on the injury model used and the extent of injury (53
). We therefore suggest that the effect seen in vivo
is a combination of enhanced collagen-dependent platelet activation and reduced thrombin-dependent signaling.
A tendency to increased thrombus formation was also seen with PKCδ−/−
mice in the present study. Although some reports suggest that GPVI-induced granule secretion is increased in PKCδ−/−
), we have not been able to show this (present study and Ref. 8
) but rather demonstrate a potentiated aggregation response to collagen through enhanced filopodia formation in the absence of PKCδ. Consistent with this, we found with human platelets that rottlerin had little effect on collagen-induced integrin αIIb
activation and α-granule secretion but enhanced platelet aggregation. Therefore, although PKCθ and PKCδ negatively regulate collagen-dependent thrombus formation, they act through distinct mechanisms.
Treatment of human platelets with broad spectrum PKC inhibitors blocks granule secretion and formation of large aggregates but also enhances platelet Ca2+
signaling and PS exposure (17
). This suggests that the major regulators of granule secretion and thrombus formation are PKCα and PKCβ because inhibition of all PKC isoforms replicates the phenotype of PKCα−/−
platelets. In contrast, the major regulator of Ca2+
signaling and PS exposure appears to be PKCθ because the absence or pharmacological inhibition of PKCθ has a similar effect on Ca2+
signaling and PS exposure to broad spectrum PKC inhibition.
We tested this directly by examining the effect of PKCθ inhibitor on PKCα−/− platelets. Importantly, combined loss of PKCα and PKCθ signaling produced a phenotype that resembled PKCα−/− platelets for secretion and PKCθ−/− platelets for Ca2+ signaling. Moreover, the phenotype resembled that of platelets treated with a broad spectrum PKC inhibitor. We propose that the conventional PKC isoforms are essential for collagen-induced granule secretion, whereas PKCθ negatively regulates the extent of this secretion. This means that in the absence of conventional PKC signaling, there is no granule secretion for PKCθ to regulate (C). In contrast, we suggest that collagen-induced sustained Ca2+ signals and subsequent PS exposure are positively regulated by PKCα and PKCβ and negatively regulated by PKCθ. Unlike secretion, however, the conventional isoforms are not essential for generation of the Ca2+ signal.
The data presented here are the first comparative analysis of platelet PKCs in thrombus formation on collagen and the first to determine calcium responses, secretion, and procoagulant activity at the single cell level in the growing thrombus in platelets lacking specific PKC isoforms. Together, the data form a comprehensive analysis of the roles played by PKCα, PKCβ, PKCδ, and PKCθ and reveal important distinctions in mechanism and function of these kinases. The data will provide a platform for future exploitation of these different family members in modulation of platelet function and thrombus formation.