Abstract
While calcium signaling is known to play vital roles in platelet function, the mechanisms underlying its receptor-operated calcium entry component (ROCE) remain poorly understood. It has been proposed, but never proven in platelets, that the canonical transient receptor potential channel-6 (TRPC6) mediates ROCE. Nonetheless, we have previously shown that the mouse TRPC6 regulates hemostasis, thrombogenesis by regulating platelet aggregation. In the present studies, we used a pharmacological approach to characterize the role of TRPC6 in human platelet biology. Thus, interestingly, we observed that a TRPC6 inhibitor exerted significant inhibitory effects on human platelet aggregation in a thromboxane receptor (TPR)-selective manner; no additional inhibition was observed in the presence of the calcium chelator BAPTA. This inhibitor also significantly inhibited human platelet secretion (dense and alpha granules), integrin IIb-IIIa, Akt and ERK phosphorylation, again, in a TPR-selective manner; no effects were observed in response to ADP receptor stimulation. Furthermore, there was a causal relationship between these inhibitory effects, and the capacity of the TRPC6 inhibitor to abrogate elevation in intracellular calcium, that was again found to be TPR-specific. This effect was not found to be due to antagonism of TPR, as the TRPC6 inhibitor did not displace the radiolabeled antagonist [3H]SQ29,548 from its binding sites. Finally, our studies also revealed that TRPC6 regulates human clot retraction, as well as physiological hemostasis and thrombus formation, in mice. Taken together, our findings demonstrate, for the first time, that TRPC6 directly regulates TPR-dependent ROCE and platelet function. Moreover, these data highlight TRPC6 as a novel promising therapeutic strategy for managing thrombotic disorders.
Highlights
Platelets are anucleate cells that play an important role in hemostasis and thrombosis [1]
Our initial experiments revealed that the 5 μM of the transient receptor potential channel-6 (TRPC6) inhibitor exerted significant inhibitory effect on platelet aggregation induced by 0.5 μM of the thromboxane receptor (TPR) agonist U46619, when compared to vehicle control (Fig 1)
These results indicate that the inhibition of aggregation in the presence of the TRPC6 inhibitor is agonist/receptor (i.e., TPR)-specific
Summary
Platelets are anucleate cells that play an important role in hemostasis and thrombosis [1]. Functional and physical coupling of platelet TPRs has only been documented with two GPs, i.e., Gq [8, 16] and G13 [11, 16], with the GqPLCβ-inositol triphosphate (IP3)-Ca2+-signaling cascade being the most characterized of the two In this regard, experiments by Offermanns group have provided evidence that platelet shape change can be stimulated through G12/13 pathway [18]. While research efforts have attempted to define the channels involved in the Gqdependent, receptor-operated calcium entry (ROCE) and store-operated calcium entry (SOCE) [27], the underlying mechanism at the molecular level, especially that for ROCE, is still poorly understood In this regard, the transient receptor potential channel (TRPC) proteins, were suggested to be mostly receptor-activated, and an ideal candidate for ROCE [28]
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