Shear stress triggers von Willebrand factor (VWF) binding to platelet glycoprotein Ib␣ and subsequent integrin ␣ IIb ␤ 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 HermanskyPudlak patient lacking dense granules was restored by exogenous L-␤,␥-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-␤,␥-methylene ATP, which promoted granule centralization within platelets and stimulated P-selectin expression, which is a marker of ␣-granule release. In addition, during SIPA, platelet degranulation required both extracellular Ca 2؉ and VWF-glycoprotein Ib␣ interactions without involving ␣ IIb ␤ 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 apyrasetreated platelets coincided with intensified phosphorylation of MLC and was abrogated by ML-7. SIPAinduced 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 MLCmediated cytoskeletal rearrangements, thus contributing to SIPA and degranulation during VWF-triggered platelet activation.Blood platelets are constantly subjected to hemodynamic forces imposed by the blood flow, including fluid shear stress.High shear stress is generated at sites of arterial injury where laminar blood flow is forced through a stenosis (1, 2). Shear stress-triggered platelet activation and subsequent aggregation, termed SIPA 1 (3), may thus contribute to the pathogenesis of vascular diseases. In addition, platelets from patients with acute myocardial infarction (4) or cerebral ischemia (5) display enhanced SIPA in vitro.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. Downstream effectors that have been implicated in the VWF-dependent activation of ␣ IIb ␤ 3 include phosphatidylinositol 3-kinase (PI3K) (7), protein kinase C (PKC) (8), Syk, and Src, as well as co-associated immunoreceptor tyrosine-activated motif-containing transmembrane proteins and adaptor proteins (2). A recent study of platelet adhesion to dimeric VWF A1 domain, which recognizes only GPIb␣, showed that GPIb␣ itself can signal to activate ␣ IIb ␤ 3 through...