Purinergic P2Y12 receptor blockade inhibits shear-induced platelet phosphatidylinositol 3-kinase activation.
Pathologically elevated shear stress triggers aspirin-insensitive platelet thrombosis. Signaling mechanisms involved in shear-induced 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 6,000 sec(-1)). Phosphatidylinositol 3,4,5-trisphosphate (PIP(3)) production was examined in washed human platelets subjected to pathological 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 phosphorylated. 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 phosphorylation, 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.
Reséndiz
Julio C
JC
VA Medical Center, Baylor College of Medicine, Texas, USA.
Feng
Shuju
S
Ji
Guilan
G
Francis
Ketia A
KA
Berndt
Michael C
MC
Kroll
Michael H
MH
D001792:Blood Platelets / Q000187:drug effects* / Q000201:enzymology; D002867:Chromones / Q000494:pharmacology; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009025:Morpholines / Q000494:pharmacology; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D010766:Phosphorylation; D019038:Platelet Glycoprotein GPIb-IX Complex / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D058919:Purinergic P2 Receptor Antagonists; D058925:Receptors, Purinergic P2Y12; D015398:Signal Transduction; D013314:Stress, Mechanical; D000072377:Syk Kinase; D014443:Tyrosine / Q000378:metabolism; D014841:von Willebrand Factor / Q000378:metabolism
D002867:Chromones; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D009025:Morpholines; C548576:P2RY12 protein, human; D018129:Phosphatidylinositol Phosphates; D019038:Platelet Glycoprotein GPIb-IX Complex; D058919:Purinergic P2 Receptor Antagonists; D058925:Receptors, Purinergic P2Y12; C060974:phosphatidylinositol 3,4,5-triphosphate; D014841:von Willebrand Factor; C085911:2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; D014443:Tyrosine; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase