title: ATP augments von Willebrand factor-dependent shear-induced platelet aggregation through Ca2+-calmodulin and myosin light chain kinase activation.
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.
authors.affiliation: Center for Molecular and Vascular Biology, University of Leuven, 3000 Leuven, Belgium.
authors.forename: Cécile
authors.initials: C
authors.lastname: Oury
authors.forename: Elsie
authors.initials: E
authors.lastname: Sticker
authors.forename: Heidi
authors.initials: H
authors.lastname: Cornelissen
authors.forename: Rita
authors.initials: R
authors.lastname: De Vos
authors.forename: Jos
authors.initials: J
authors.lastname: Vermylen
authors.forename: Marc F
authors.initials: MF
authors.lastname: Hoylaerts
mesh_terms: D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D001391:Azo Compounds / Q000737:chemistry / Q000378:metabolism*; D001792:Blood Platelets / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry*; D003094:Collagen / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D004789:Enzyme Activation; D005434:Flow Cytometry; D006801:Humans; D015151:Immunoblotting; D007477:Ions; D008854:Microscopy, Electron; D008954:Models, Biological; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D019007:P-Selectin / Q000096:biosynthesis / Q000737:chemistry; D010766:Phosphorylation; D015539:Platelet Activation; D010974:Platelet Aggregation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000737:chemistry*; D010980:Platelet Membrane Glycoproteins; D011485:Protein Binding; D011732:Pyridoxal Phosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000502:physiology*; D058469:Receptors, Purinergic P2X; D013917:Thrombin / Q000378:metabolism; D013997:Time Factors; D014841:von Willebrand Factor / Q000378:metabolism*
chemical_list: D001391:Azo Compounds; D002147:Calmodulin; D007477:Ions; C000602459:MRS2159; D019007:P-Selectin; D019038:Platelet Glycoprotein GPIb-IX Complex; D010980:Platelet Membrane Glycoproteins; D018048:Receptors, Purinergic P2; D058469:Receptors, Purinergic P2X; D014841:von Willebrand Factor; D011732:Pyridoxal Phosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D003094:Collagen; D009219:Myosin-Light-Chain Kinase; D013917:Thrombin; D002118:Calcium