Summary. Background: Binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates a cascade of events leading to a IIb b 3 activation and platelet aggregation. The roles of ADP and thromboxane A 2 (TXA 2 ) in agglutination-induced GPIba-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, Gaq, or a IIb b 3 , and inhibitors and chelating agents were used to investigate the roles of TXA 2 , ADP, a IIb b 3 and Ca 2+ in botrocetin/ VWF-induced signaling. Results: Our data demonstrate that botrocetin/VWF/GPIba-mediated agglutination results in calcium-independent protein kinase C (PKC) and phospholipase A2 (PLA2) activities required for GPIba-elicited TXA 2 production that in turn causes dense granule secretion. Aggregation of washed platelets requires TXA 2 -induced a IIb b 3 activation and ADP signaling. TXA 2 or ADP can activate a IIb b 3 , but both are required for a-granule secretion and aggregation. Botrocetin/VWF-induced dense granule secretion is Gaq-dependent. a-Granule secretion requires initial ADP signaling through P2Y1 and subsequent signaling through P2Y12. Signaling initiated by agglutination is propagated and amplified in an a IIb b 3 -dependent manner. Conclusions: In contrast to adhesion or shear stress-induced GPIb-elicited signaling, agglutination-elicited GPIb signaling that activates a IIb b 3 requires TXA 2 . Agglutination-elicited TXA 2 production is independent of Ca 2+ influx and mobilization of internal Ca 2+ stores. Therefore, our results demonstrate that agglutination-elicited GPIb signaling causes a IIb b 3 activation by a mechanism that is distinct from those used by adhesion, or shear stress-induced GPIb signaling.

Collagen-induced activation of platelets in suspension leads to ␣ IIb ␤ 3 -mediated outside-in signaling, granule release, thromboxane A2 (TxA2) production, and aggregation. Although much is known about collagen-induced platelet signaling, the roles of TxA2 production, adenosine diphosphate (ADP) and dense-granule secretion, and ␣ IIb ␤ 3 -mediated outside-in signaling in this process are unclear. Here, we demonstrate that TxA2 and ADP are required for collagen-induced platelet activation in response to a low, but not a high, level of collagen and that ␣ IIb ␤ 3 -mediated outside-in signaling is required, at least in part, for this TxA2 production and ADP secretion.