Activation of GPVI by collagen is regulated by alpha2beta1 and secondary mediators.

We have compared the roles of adenosine diphosphate (ADP), thromboxanes and the integrin alpha(2)beta(1) in the activation of washed platelets by collagen in the presence of the alpha(IIb)beta(3) antagonist lotrafiban. The stimulation of protein tyrosine phosphorylation by a collagen suspension is markedly delayed in the presence of the above inhibitors but shows substantial recovery with time. In comparison, activation of phospholipase C (PLC), Ca(2+) elevation and dense granule secretion are more severely suppressed by the above inhibitors. alpha(2)beta(1) blockade has a slightly greater inhibitory effect on all of the above responses than a combination of ADP receptor antagonists and cyclooxygenase inhibitor. Platelets exposed to a collagen monolayer show robust elevation of Ca(2+) that is delayed in the presence of the above inhibitors and which is accompanied by alpha-granule secretion. These results demonstrate that secondary mediators and alpha(2)beta(1) modulate collagen-induced intracellular signaling but have negligible effect on GPVI signaling induced by the specific agonist convulxin. This work supports the postulate that the major role of alpha(2)beta(1) is to increase the avidity of collagen for the platelet surface and by doing so enhance activation of GPVI. Therefore we propose an important role of secondary mediators in collagen-induced signaling is the indirect regulation of GPVI signaling via activation of alpha(2)beta(1).

Signalling events underlying platelet aggregation induced by the glycoprotein VI agonist convulxin.

We have investigated the role of secretion and intracellular signalling events in aggregation induced by the glycoprotein (GP)VI-selective snake venom toxin convulxin and by collagen. We demonstrate that aggregation induced by threshold concentrations of convulxin undergoes synergy with ADP acting via the P2Y12 receptor whereas there is no synergy via the P2Y1 receptor or with thromboxanes. On the other hand, apyrase, the P2Y12 receptor antagonist, AR-C67085, and indomethacin only marginally inhibit aggregation induced by convulxin. In comparison, these inhibitors severely attenuate the response to collagen. In order to investigate whether the weak inhibitory action against convulxin is due to release of agonists other than ADP from dense granules, experiments were performed on murine platelets deficient in this organelle (pearl mice platelets). A slightly greater reduction in aggregation induced by convulxin was observed in pearl platelets than in the presence of inhibitors of ADP, but a maximal response was still attained. Importantly, inhibition of protein kinase C further reduced the response to convulxin in pearl platelets demonstrating a direct role for the kinase in aggregation. Chelation of intracellular Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N,N',N'-tetraacetic acid (acetoxymethyl)ester (BAPTA-AM) abolished aggregation induced by convulxin under all conditions. Activation of phospholipase C by convulxin was potentiated by ADP acting through the P2Y12 receptor. In conclusion, we show that Ca2+ and protein kinase C, but not release of the secondary agonists ADP and thromboxane A2, are required for full aggregation induced by convulxin, whereas the response induced by collagen shows a much greater dependence on secretion of secondary agonists.

Signalling components underlying platelet aggregation to a Ca2+ ionophore and a phorbol ester.

Although it is well established that G(q)- and G(i)-coupled receptors can combine to mediate platelet aggregation, the signalling events underlying the synergy are not fully characterised. This study has used the calcium ionophore, A23187, and phorbol ester, PMA, to investigate this question. We show that aggregation to submaximal but not maximal concentrations of ionophore is partially inhibited by antagonism of the P2Y(12) ADP receptor or PKC blockade. However, a full aggregation response can be restored under these conditions by addition of PMA or ADP. Studies using PI 3-kinase inhibitors demonstrate that this is the second messenger pathway that restores aggregation by the G(i)-coupled receptor in the presence of PKC blockade. However, under normal circumstances, PI 3-kinase activity is not a major requirement for aggregation to the ionophore. PMA stimulates a weak aggregation which takes several minutes to reach a maximum. Threshold concentrations of PMA and a G(i)-coupled receptor agonist when added alone show no effect on aggregation, but when combined induce aggregation responses. This study demonstrates that calcium and PKC interact synergistically with a G(i)-coupled receptor agonist to mediate aggregation, and also with each other. Activation of G(i) supports aggregation in part through the PI 3-kinase pathway. High concentration of ionophore on their own can induce aggregation independent of PKC and activation of G(i). Multiple signalling pathways mediate platelet aggregation and their relative importance depends on experimental conditions.