The roles of ADP and TXA2 in botrocetin/VWF-induced aggregation of washed platelets

Background: Binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates a cascade of events leading to alpha(11b)beta(3) activation and platelet aggregation. The roles of ADP and thromboxane A(2) (TXA(2)) in agglutination-induced GPIbalpha-mediated platelet activation have not been fully described. Methods: Botrocetin and human VWF were used to stimulate washed mouse platelets. Platelets deficient in TXA(2) receptors, or alpha(11b)beta(3), and inhibitors and chelating agents were used to investigate the roles of TXA(2), ADP, alpha(11b)beta(3) and Ca2+ in botrocetin/ VWF-induced signaling. Results: Our data demonstrate that botrocetin/VWF/GPlbalpha-mediated agglutination results in calcium-independent protein kinase C (PKC) and phospholipase A2 (PLA2) activities required for GPlbalpha-elicited TXA(2) production that in turn causes dense granule secretion. Aggregation of washed platelets requires TXA(2)-indLIced alpha(11b)beta(3) activation and ADP signaling. TXA(2) or ADP can activate alpha(11b)beta(3), but both are required for alpha-granule secretion and aggregation. Botrocefin/VWF-induced dense granule secretion is Galphaq-dependent. alpha-Granule secretion requires initial ADP signaling through P2Y1 and subsequent signaling through P2Y12. Signaling initiated by agglutination is propagated and amplified in an alpha(11b)beta(3)-dependent manner. Conclusions: In contrast to adhesion or shear stress-induced GPIb-elicited signaling, agglutination-elicited GPIb signaling that activates 04103 requires TXA(2). Agglutination-elicited TXA(2) production is independent of Ca2+ influx and mobilization of internal Ca2+ stores. Therefore, our results demonstrate that agglutination-elicited GPIb signaling causes alpha(11b)beta(3) activation by a mechanism that is distinct from those Used by adhesion, or shear stress-induced GPIb signaling.