Abstract
Shear stress triggers von Willebrand factor (VWF) binding to platelet glycoprotein Ibalpha and subsequent integrin alpha(IIb)beta(3)-dependent platelet aggregation. Concomitantly, nucleotides are released from plateletdense granules, and ADP is known to contribute to shear-induced platelet aggregation (SIPA). We found that the impaired SIPA of platelets from a Hermansky-Pudlak patient lacking dense granules was restored by exogenous l-beta,gamma-methylene ATP, a stable P2X(1) agonist, as well as by ADP, confirming that in addition to ADP (via P2Y(1) and P2Y(12)), ATP (via P2X(1)) also contributes to SIPA. Likewise, SIPA of apyrase-treated platelets was restored upon P2X(1) activation with l-beta,gamma-methylene ATP, which promoted granule centralization within platelets and stimulated P-selectin expression, which is a marker of alpha-granule release. In addition, during SIPA, platelet degranulation required both extracellular Ca(2+) and VWF-glycoprotein Ibalpha interactions without involving alpha(IIb)beta(3). Neither platelet release nor SIPA was affected by protein kinase C inactivation, even though protein kinase C blockade inhibits platelet responses to collagen and thrombin in stirring conditions. In contrast, inhibiting myosin light chain (MLC) kinase with ML-7 reduced platelet release and SIPA by 30%. Accordingly, the potentiating effect of P2X(1) stimulation on the aggregation of apyrase-treated platelets coincided with intensified phosphorylation of MLC and was abrogated by ML-7. SIPA-induced MLC phosphorylation occurred exclusively through released nucleotides and selective antagonism of P2X(1) with MRS2159-reduced SIPA, ATP release, and potently inhibited MLC phosphorylation. We conclude that the P2X(1) ion channel induces MLC-mediated cytoskeletal rearrangements, thus contributing to SIPA and degranulation during VWF-triggered platelet activation.
Highlights
High shear stress is generated at sites of arterial injury where laminar blood flow is forced through a stenosis [1, 2]
These studies have shown that platelet activation initiated by von Willebrand factor (VWF)-GPIb␣ interaction requires a transmembrane Ca2ϩ influx independent of released ADP and VWF binding to ␣IIb3 [14, 15], which promotes dense granule secretion of ADP and activates integrin ␣IIb3 through engagement of the P2 receptors for ADP
Both VWF-GPIb␣ signaling and Ca2ϩ contribute to platelet release under shear stress, a process that is independent of ␣IIb3 outside-in signals
Summary
Shear stress triggers von Willebrand factor (VWF) binding to platelet glycoprotein Ib␣ and subsequent integrin ␣IIb3-dependent platelet aggregation. High shear stress is required for the interaction between von Willebrand factor (VWF) and platelet glycoprotein Ib␣ (GPIb␣) (for review, see Ref. 6), but the effects of shear forces on GPIb␣ signaling are only just beginning to be defined. Shear stress causes the release of high levels of nucleotides, including ATP and ADP, into the extracellular environment; these nucleotides mainly originate from endothelial cells [20] and platelet-dense granules [21]. We have investigated the role of the P2X1-mediated Ca2ϩ influx and the associated downstream signaling pathways in VWF-dependent shear-induced granule release and aggregation of washed human platelets
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