Summary. Background: The stable or second wave of platelet aggregation often observed in ADP-stimulated platelet-rich plasma (PRP) with an artificially lowered extracellular calcium level has been attributed to enhanced thromboxane A 2 (TXA 2 ) generation and inhibition of ectonucleotidase activity. However, the role of thrombin in ADP-induced platelet secretion and the second wave of aggregation is unknown. Objectives and Methods: We employed aggregometry, flow cytometry, immunoblotting and ELISA to determine whether and how thrombin participates in ADP-induced platelet secretion and the second wave of aggregation. Results: ADP induces a phosphoinositide 3-kinase (PI3K) pathway-dependent thrombin generation, presumably resulting from the cleavage of a IIb b 3 -associated prothrombin. Generated thrombin subsequently activates protease-activated receptor-1 (PAR-1) and mediates dense granule secretion and the second wave of platelet aggregation in ADP-stimulated citrated PRP. Thus, ADP-induced dense granule secretion and the second wave of platelet aggregation in PRP were similarly and nonadditively blocked by thrombin inhibitor hirudin, PAR-1 antagonist SCH-79797 or PI3K inhibitor wortmannin. Moreover, ADP stimulation caused the dissociation of prothrombin from a IIb b 3 and an increased plasma thrombin level; both were prevented by wortmannin. Furthermore, the wortmannin-inhibited second wave of platelet aggregation by ADP was restored by a subaggregation concentration of PAR-1 activating peptide SFLLRN. Blocking TXA 2 production with indomethacin or restoring extracellular calcium to physiological concentration did not influence this thrombin/PAR-1 dependence. Conclusions: A PI3K-dependent thrombin generation and the resultant PAR-1 activation serve as an indispensable mechanism to relay the platelet activation process induced by ADP.

Shp2 negatively regulates thrombus stability under pathological shear rate. Shp2 suppresses TXA2 receptor‐mediated platelet dense granule secretion. Through αIIbβ3 outside‐in signaling, Shp2 targets calmodulin‐dependent activation of Akt. Shp2 may serve to prevent the formation of unwanted occlusive thrombi. Summary BackgroundPerpetuation is the final phase of thrombus formation; however, its mechanisms and regulation are poorly understood. ObjectiveTo investigate the mechanism of Shp2 in platelet function and thrombosis. Methods and resultsWe demonstrate that the platelet‐expressed Src homology region 2 domain‐containing protein tyrosine phosphatase Shp2 is a negative regulator of thrombus stability under high shear stress. In a ferric chloride‐induced mesenteric arteriole thrombosis model, megakaryocyte/platelet‐specific Shp2‐deficient mice showed less thrombi shedding than wild‐type mice, although their occlusion times were comparable. In accordance with this in vivo phenotype, a microfluidic whole‐blood perfusion assay revealed that the thrombi formed on collagen surfaces by Shp2‐deficient platelets were more stable under high shear rates than those produced by wild‐type platelets. Whereas Shp2 deficiency did not alter platelet responsiveness towards thrombin, ADP and collagen stimulation, Shp2‐deficient platelets showed increased dense granule secretion when stimulated by the thromboxane A2 analog U46619. Shp2 appears to act downstream of integrin αIIbβ3 outside‐in signaling, inhibiting the phosphorylation of Akt (Ser473 and Thr308) and dense granule secretion. Calmodulin was also shown to bind both Shp2 and Akt, linking Shp2 to Akt activation. ConclusionsPlatelet Shp2 negatively regulates thrombus perpetuation under high shear stress. This signaling pathway may constitute an important mechanism for the prevention of unwanted occlusive thrombus formation, without dramatically interfering with hemostasis.

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