Integrin αIIbβ3-dependent calcium signals regulate platelet-fibrinogen interactions under flow: Involvement of phospholipase Cγ2

Australian Centre for Blood Diseases, Department of Medicine, Monash University, Box Hill Hospital, Victoria 3128, Australia.
Journal of Biological Chemistry (Impact Factor: 4.57). 10/2003; 278(37):34812-22. DOI: 10.1074/jbc.M306504200
Source: PubMed


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 alpha IIb beta 3 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 (IP3) 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 PLC gamma 2-/- 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 PLC gamma 2-/- mice formed less stable adhesive interactions with fibrinogen, particularly in the presence of ADP antagonists. Our studies define an important role for PLC gamma 2 in integrin alpha IIb beta 3-dependent calcium flux, necessary for stable platelet adhesion and spreading on fibrinogen. Furthermore, they establish an important cooperative signaling role for PLC gamma 2 and ADP in regulating platelet adhesion efficiency on fibrinogen.

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    • "This contrasts with the present findings where appropriate concentrations of different integrin blockers all had similar effects. Another published finding that αIIbβ3 blockage reduces shear-dependent Ca2+ responses and microparticle release [19, 44] can be explained by increased fibrinogen secretion of platelets subjected to a high shear rate. "
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    ABSTRACT: The fibrin(ogen) receptor, integrin αIIbβ3, has a well-established role in platelet spreading, aggregation and clot retraction. How αIIbβ3 contributes to platelet-dependent coagulation is less well resolved. Here, we demonstrate that the potent suppressing effect of clinically used αIIbβ3 blockers on tissue factor-induced thrombin generation is linked to diminished platelet Ca2+ responses and phosphatidylserine (PS) exposure. The same blockers suppress these responses in platelets stimulated with collagen and thrombin receptor agonists, whereas added fibrinogen potentiates these responses. In platelets spreading on fibrinogen, outside-in αIIbβ3 signaling similarly enhances thrombin-induced Ca2+ rises and PS exposure. These responses are reduced in αIIbβ3-deficient platelets from patients with Glanzmann’s thrombasthenia. Furthermore, the contribution of αIIbβ3 to tissue factor-induced platelet Ca2+ rises, PS exposure and thrombin generation in plasma are fully dependent on Syk kinase activity. Tyrosine phosphorylation analysis confirms a key role of Syk activation, which is largely but not exclusively dependent on αIIbβ3 activation. It is concluded that the majority of tissue factor-induced procoagulant activity of platelets relies on Syk activation and ensuing Ca2+ signal generation, and furthermore that a considerable part of Syk activation relies on αIIbβ3 signaling. These results hence point to a novel role of Syk in integrin-dependent thrombin generation. Electronic supplementary material The online version of this article (doi:10.1007/s00018-012-1033-2) contains supplementary material, which is available to authorized users.
    Full-text · Article · Jun 2012 · Cellular and Molecular Life Sciences CMLS
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    • "Activation of integrin αIIbβ3 by ‘inside-out’ signals leads to binding of fibrinogen and platelet aggregation. In turn, clustering of αIIbβ3 as a consequence of engagement of αIIbβ3 by fibrinogen mediates ‘outside-in’ signals that stimulate tyrosine phosphorylation of β3-integrin tail [1] and activation of a Src-based signalling cascade [2] that involves Syk [3,4], SLP-76 [5] and PLCγ2 [6,7]. Binding of Syk to the αIIbβ3-tail is inhibited by tyrosine phosphorylation of the β3-integrin tail [8], suggesting that these two integrin-dependent signalling cascades are distinct. "
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    ABSTRACT: The integrin alpha(IIb)beta(3) plays a critical role in mediating clot retraction by platelets which is important in vivo in consolidating thrombus formation. Actin-myosin interaction is essential for clot retraction. In the present study, we demonstrate that the structurally distinct Src kinase inhibitors, PP2 and PD173952, significantly reduced the rate of clot retraction, but did not prevent it reaching completion. This effect was accompanied by abolition of alpha(IIb)beta(3)-dependent protein tyrosine phosphorylation, including PLCgamma2. A role for PLCgamma2 in mediating clot retraction was demonstrated using PLCgamma2-deficient murine platelets. Furthermore, platelet adhesion to fibrinogen leads to MLC phosphorylation through a pathway that is inhibited by PP2 and by the PLC inhibitor, U73122. These results demonstrate a partial role for Src kinase-dependent activation of PLCgamma2 and MLC phosphorylation in mediating clot retraction downstream of integrin alpha(IIb)beta(3).
    Full-text · Article · Feb 2007 · Thrombosis Research
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