Purinergic P2Y 12 Receptor Blockade Inhibits Shear-Induced Platelet Phosphatidylinositol 3-Kinase Activation JULIO C. RES ´ ENDIZ, SHUJU FENG, GUILAN JI, KETIA A. FRANCIS, MICHAEL C. BERNDT, and MICHAEL H. KROLL VA Medical Center, Baylor College of Medicine and Rice University, Houston, Texas (J.C.R., S.F., G.J., K.A.F., M.C.B., M.H.K.); Wihuri Research Institute, Helsinki, Finland (J.C.R.); and Monash University, Clayton, Victoria, Australia (M.C.B.) Received June 6, 2002; accepted November 21, 2002 This article is available online at http://molpharm.aspetjournals.org ABSTRACT Pathologically elevated shear stress triggers aspirin-insensitive platelet thrombosis. Signaling mechanisms involved in shear-in- duced platelet thrombosis are not well understood. To investigate these, we examined the hypothesis that functionally important platelet phosphatidylinositol 3-kinase (PI3-K) activity is stimulated by an in vitro shear stress of 120 dynes/cm 2 (shear rate of 6000 sec -1 ). Phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) production was examined in washed human platelets subjected to patholog- ical shear stress in a cone-plate viscometer. PIP 3 production peaks 30 s after shear begins and is initiated by von Willebrand factor (VWF) binding to the glycoprotein (Gp) Ib-IX-V complex. Inhibiting PI3-K with wortmannin or 2-(4-morpholinyl)-8-phenyl- 4H-1-benzopyran-4-one (LY294002) results in the inhibition of shear-induced platelet aggregation. In resting platelets, class IA PI3-K associates with the tyrosine kinase Syk. Within 30 s of beginning shear, PI3-K-associated Syk becomes tyrosine phos- phorylated. Inhibiting Syk activation with piceatannol results in the inhibition of PIP 3 production and aggregation. Selective blockade of the P2Y 12 receptor results in the inhibition of Syk phosphory- lation, PIP 3 production, and aggregation. These results indicate that shear-induced VWF binding to platelet GpIb-IX-V stimulates functionally important PI3-K activity. PI3-K activation is signaled by rapid feedback amplification that involves P2Y 12 receptor- mediated activation of Syk. High levels of wall shear stress are generated at sites of arterial injury, such as a ruptured atherosclerotic plaque, where laminar blood flow is forced through a narrowed lumi- nal diameter (Berndt et al., 2001). Such pathological shear stress causes platelet-dependent thrombosis (Kroll et al., 1996). The trigger for shear-dependent platelet adhesion is von Willebrand factor (VWF) in the plasma and vessel wall binding to platelet glycoprotein (Gp) Ib-IX-V, which causes a transient tethering of platelets to the damaged vessel wall and signals the activation of IIb3. Further adhesion and IIb3 activation develop via platelet GpVI and 21 bind- ing exposed collagen fibrils (Moroi et al., 1997; Savage et al., 1998). Shear-dependent platelet thrombus formation is ADP- dependent (Turner et al., 2001), but it is not affected by inhibiting platelet cyclooxygenase with aspirin or other agents (Maalej and Folts, 1996). Mechanisms by which shear-induced VWF binding to GpIb-IX-V activates IIb3 have not been established. There is direct evidence that pathological shear causes VWF-depen- dent platelet calcium, protein kinase C, and tyrosine kinase signaling responses, and each of these signals may contribute to IIb3 activation (Kroll et al., 1993; Razdan et al., 1994; Shattil et al., 1998; Kuwahara et al., 1999). There is also pharmacological evidence that shear-induced VWF binding to platelet GpIb-IX-V causes phosphatidylinositol 3-kinase (PI3-K)-dependent IIb3 activation (Yap et al., 2002), al- though shear-induced D3-phosphorylation of platelet polyphosphoinositides has not been reported. Such shear- induced phosphorylations are likely to occur, however, be- cause PI3-K-mediated synthesis of phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) from membrane phosphatidylino- sitol 4,5-bisphosphate (PIP 2 ) is known to be an important signal for the up-regulation of IIb3 function under low shear conditions (Kovacsovics et al., 1995). There are several types (or classes) of platelet PI3-K (Rit- tenhouse, 1996). The type IA PI3-Ks encompass several iso- forms varying in their 110-kDa catalytic (designated p110  or ) and 85-kDa regulatory subunits (designated p85  or ) (Fruman et al., 1998; Wymann and Pirola, 1998). Minimal requirements for the activation of type IA PI 3-kinases are: 1) This work was supported through grants by the Research Service of the Department of Veterans’ Affairs, the National Institutes of Health (HL18454 and HL65967), and The National Heart Foundation of Australia. This work was presented in part at the XVIIIth Congress of the Interna- tional Society on Thrombosis and Hemostasis, 2001 July; Paris, France; and was published in abstract form (Thrombosis Hemostasis 86:1860A, 2001). ABBREVIATIONS: VWF, von Willebrand factor; PI3-K, phosphatidylinositol 3-kinase; PIP 3 , phosphatidylinositol 3,4,5-trisphosphate; PIP 2 , phosphatidylinositol 4,5-bisphosphate; PI, phosphatidylinositol; AR-C69931MX, N 6 -(2-methylthioethyl)-2-(3,3,3-trifluoropropylthio)-,-dichlo- romethylene ATP; A3P5P, adenosine 3',5' diphosphate; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; PRP, platelet-rich plas- ma; Gp, glycoprotein. 0026-895X/03/6303-639 –645 MOLECULAR PHARMACOLOGY Vol. 63, No. 3 U.S. Government work not protected by U.S. copyright 1904/1043798 Mol Pharmacol 63:639–645, 2003 Printed in U.S.A. 639 at ASPET Journals on September 17, 2016 molpharm.aspetjournals.org Downloaded from