Src family kinase-mediated and Erk-mediated thromboxane A2 generation are essential for VWF/GPIb-induced fibrinogen receptor activation in human platelets

Abstract

The binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein Ib-IX (GPIb-IX) results in platelet activation. In this study, we sought to clarify previous conflicting reports and to elucidate the mechanism of activation and the precise role of extracellular signal-regulated kinase (Erk) in VWF-induced platelet activation. Erk2 is activated in platelets on stimulation with VWF/ristocetin in a time-dependent manner. VWF-induced Erk2 phosphorylation and thromboxane A2 (TXA2) release were completely blocked by PP2, an Src family kinase inhibitor, suggesting that Erk is downstream of Src family kinases. U73122, a phospholipase C inhibitor, also abolished TXA2 generation and Erk phosphorylation. Although VWF fostered the agglutination of platelets regardless of any additional treatment, the inhibition of mitogen-activated protein kinase kinase (MEK) with U0126 abolished VWF-induced platelet aggregation and thromboxane production in non-aspirin-treated washed platelets. However, in platelets treated with aspirin, VWF failed to cause any aggregation. Thus, we conclude that VWF stimulation of platelets results in phospholipase A2 activation through Erk stimulation and that Src family kinases and phospholipase C play essential roles in this event. We further conclude that VWF-induced platelet aggregation does not directly depend on Erk activation but has an absolute requirement for Src/Erk-mediated TXA2 generation.

Figure 1.

VWF-mediated Erk phosphorylation in platelets. Platelets were stimulated with VWF/ristocetin (8 and 300 μg/mL, respectively) for various amounts of time (A) or for 1 minute in the absence (vehicle) or presence of the Src kinase inhibitor PP2 or its inactive analog, PP3 (B). Both figures were analyzed to detect either phospho-Erk (top panels) or total Erk (bottom panels), as described in “Materials and methods.” Arrows denote the 2 molecular forms of Erk in platelets.

Figure 2.

Inhibition of PLC decreases VWF-mediated Erk phosphorylation. Platelets were stimulated with VWF/ristocetin (8 and 300 μg/mL, respectively) in the absence (vehicle) or presence of the PLC inhibitor U73122. Erk activation was assayed as described in “Materials and methods.”

Figure 3.

Inhibition of Src kinases and PLC decreases VWF-mediated thromboxane production. Platelets were unstimulated (control) or stimulated with VWF/ristocetin (8 and 300 μg/mL, respectively) in the absence or presence of either (A) the Src kinase inhibitor PP2 (10 μM) or (B) the PLC inhibitor U73122 (4 μM). Platelets were then processed for measurements of thromboxane production, as described in “Materials and methods.”

Figure 4.

Inhibition of MEK decreases Erk phosphorylation and VWF-mediated thromboxane production. Platelets were treated with the MEK inhibitor U0126 (10 μM) or its vehicle (control) before stimulation with VWF/ristocetin (8 and 300 μg/mL, respectively). Platelets were then processed for measurement of thromboxane production (A) or detection of phospho-Erk (B), as described in “Materials and methods.”

Figure 5.

Inhibition of MEK abolishes VWF-mediated platelet aggregation. Platelets were treated with the MEK inhibitor U0126 (10 μM [+] or its vehicle [–] before stimulation with ristocetin (300 μg/mL) and VWF (8 μg/mL) in the absence (–) or presence (+) of previous treatment with 1 mM aspirin. Platelet aggregation was measured as described in “Materials and methods.”

Figure 6.

Inhibition of Src kinases or phospholipase C blocks VWF-mediated platelet aggregation. Platelets were treated with the Src kinase inhibitor PP2 (10 μM), its inactive analog PP3 (10 μM), or the PLC inhibitor U73122 (10 μM) before stimulation with ristocetin (300 μg/mL) and VWF (8 μg/mL). Platelet aggregation was measured as described in “Materials and methods.”

Figure 7.

Mechanism of thromboxane production and integrin αIIbβ3 activation after GPIb stimulation in human platelets. Stimulation of GPIb with VWF activates Src, which in turn causes successive activation of phospholipase Cγ2 (PLC2), MEK, and Erk. Erk activation by MEK-mediated phosphorylation activates phospholipase A2 (PLA2), leading to the liberation of arachidonic acid (AA) and its conversion to thromboxane A2 (TXA2) by cyclooxygenase (which is inhibited by aspirin). The released TXA2 then interacts with the G-protein–coupled TP receptor, whose stimulation ultimately contributes to GPIIb–IIIa activation and platelet aggregation. Dotted lines indicate the inhibitory action of various compounds against their respective targets. Note that Src-dependent PLCγ2 activation in response to VWF stimulation in platelets has been previously reported.,