Abstract
Platelet adhesion to fibrinogen is important for platelet aggregation and thrombus growth. In this study we have examined the mechanisms regulating platelet adhesion on immobilized fibrinogen under static and shear conditions. We demonstrate that integrin α<sub>IIb</sub>β<sub>3</sub> engagement of immobilized fibrinogen is sufficient to induce an oscillatory calcium response, necessary for lamellipodial formation and platelet spreading. Released ADP increases the proportion of platelets exhibiting a cytosolic calcium response but is not essential for calcium signaling or lamellipodial extension. Pretreating platelets with the Src kinase inhibitor PP2, the inositol 1,4,5-trisphosphate (IP<sub>3</sub>) receptor antagonist 2-aminoethoxydiphenyl borate (APB-2), or the phospholipase C (PLC) inhibitor U73122 abolished calcium signaling and platelet spreading, suggesting a major role for Src kinase-regulated PLC isoforms in these processes. Analysis of PLCγ2<sup>–</sup><sup>/</sup><sup>–</sup> mouse platelets revealed a major role for this isoform in regulating cytosolic calcium flux and platelet spreading on fibrinogen. Under flow conditions, platelets derived from PLCγ2<sup>–</sup><sup>/</sup><sup>–</sup> mice formed less stable adhesive interactions with fibrinogen, particularly in the presence of ADP antagonists. Our studies define an important role for PLCγ2 in integrin α<sub>IIb</sub>β<sub>3</sub>-dependent calcium flux, necessary for stable platelet adhesion and spreading on fibrinogen. Furthermore, they establish an important cooperative signaling role for PLCγ2 and ADP in regulating platelet adhesion efficiency on fibrinogen.
Highlights
Platelet adhesion to fibrinogen is important for plate- distinct, complementary roles in this process [1,2,3,4,5]
We examined the effect of apyrase on integrin ␣IIb3 activation during platelet adhesion to fibrinogen by performing indirect immunofluorescence studies using the activation-specific ␣-integrin ␣IIb3 antibody, PAC-1
The inability of apyrase to inhibit PAC-1 binding and spreading was unlikely to be the result of incomplete inhibition of ADP, as blocking the two major ADP purinergic receptors, P2Y1 and P2Y12, with AR-C69931MX and A3P5PS, respectively, did not inhibit these platelet responses. These findings suggest that integrin ␣IIb3 engagement of fibrinogen can modulate the affinity status of integrin ␣IIb3 and induce lamellipodial extensions independent of ADP
Summary
Platelet adhesion to fibrinogen is important for plate- distinct, complementary roles in this process [1,2,3,4,5]. Src Kinases are Essential for Integrin ␣IIb3 Activation and Calcium Mobilization following Platelet Adhesion to Fibrinogen—The demonstration that ADP antagonists did not eliminate platelet activation induced by immobilized fibrinogen raised the possibility that fibrinogen engagement of integrin ␣IIb3 was sufficient to induce cytosolic calcium flux through outside-in signaling processes.
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