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

Abstract

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.

Bruton tyrosine kinase is essential for botrocetin/VWF-induced signaling and GPIb-dependent thrombus formation in vivo

“…This aspect of GPIb signaling is important because agglutination-elicited TxA2 production is required for ␣IIb␤3 activation in this GPIb-dependent signaling system. 5,25 The results of this investigation demonstrate that Btk is essential for both bt/VWF-induced TxA2 production and stable GPIb-dependent thrombus formation in FeCl 3 -treated, mouse carotid arteries, as well as clarify, at least in part, the role of Btk in this signaling system.…”

“…Similar results are obtained using EDTA to prevent aggregation. 5,14 Agglutination-elicited signaling induces a characteristic level of TxA2 production (about 2.5 ng/mL) and ATP secretion. The level of TxA2 produced by the combination of agglutination and aggregation is about 4-fold to 5-fold greater (about 12 ng/mL) than that caused by agglutination in the absence of aggregation ( Figure 1B).…”

“…This biphasic response is composed of agglutination and agglutination-dependent aggregation. 5 The hamster anti-mouse ␣IIb␤3 monoclonal antibody (mAb) 1B5 42 allows a clear distinction between the agglutination and aggregation phases of the biphasic aggregation-like response because it prevents fibrinogen binding to ␣IIb␤3 and thereby prevents aggregation without affecting agglutination. Similar results are obtained using EDTA to prevent aggregation.…”

“…For example, ␣IIb␤3 activation resulting from botrocetin (bt)-facilitated, GPIb-VWF-mediated agglutination is (thromboxane A2) TxA2 dependent, and the agglutination-elicited TxA2 production is not dependent on Ca 2ϩ influx or mobilization of internal Ca 2ϩ stores. 5 In contrast, adhesion-independent shear stress-induced GPIb signaling (as in a cone and plate viscometer) also activates ␣IIb␤3 but requires Ca 2ϩ influx and probably mobilization of internal Ca 2ϩ stores, as well as ADP secretion, but not TxA2 production. 3,4,6,7 In a different system, adhesiondependent shear stress-induced GPIb signaling (flow) that activates ␣IIb␤3 does not require Ca 2ϩ influx (although Ca 2ϩ influx potentiates the process) 8 but does require mobilization of internal Ca 2ϩ stores 2,8 and is not dependent on either ADP secretion or TxA2 production.…”

“…[1][2][3][4][5] This behavior demonstrates that GPIb is a versatile signaling molecule. For example, ␣IIb␤3 activation resulting from botrocetin (bt)-facilitated, GPIb-VWF-mediated agglutination is (thromboxane A2) TxA2 dependent, and the agglutination-elicited TxA2 production is not dependent on Ca 2ϩ influx or mobilization of internal Ca 2ϩ stores.…”