Integrin αIIbβ3-dependent Calcium Signals Regulate Platelet-Fibrinogen Interactions under Flow

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 ␣ IIb ␤ 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 (IP 3 ) 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␥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␥2 ؊/؊ mice formed less stable adhesive interactions with fibrinogen, particularly in the presence of ADP antagonists. Our studies define an important role for PLC␥2 in integrin ␣ IIb ␤ 3 -dependent calcium flux, necessary for stable platelet adhesion and spreading on fibrinogen. Furthermore, they establish an important cooperative signaling role for PLC␥2 and ADP in regulating platelet adhesion efficiency on fibrinogen.Platelet adhesion and aggregation at sites of vascular injury are critical for the arrest of bleeding in traumatized vessels and also for the development of arterial thrombi, precipitating diseases such as acute myocardial infarction and stroke. The two principle adhesive ligands promoting platelet aggregation are von Willebrand factor (vWf) 1 and fibrinogen, with each having distinct, complementary roles in this process (1-5). Fibrinogen acts as a bridging molecule between adjacent activated platelets and is the principle adhesive ligand promoting platelet aggregation under low and intermediate shear flow conditions (1, 2). In addition, it also contributes to stable aggregate formation under high shear (1,5,6). It is well established that the binding of fluid-phase fibrinogen to the platelet surface is dependent on the activation of integrin ␣ IIb ␤ 3 (GPIIb-IIIa) through intracellular signaling processes linked to various G protein-coupled and tyrosine kinase-linked receptors (7).Platelets can also adhere to immobilized fibrinogen, a process that is important for primary platelet adhesion onto artificial surfaces, including vascular prostheses (8, 9), and for normal thrombus development (5, 6). In the case of thrombus formation, active integrin ␣ IIb ␤ 3 on the surface of firmly adherent platelets adsorbs soluble fibrinogen to the thrombus surface, thereby providing a reactive substrate for the recruitment of additional platelets. In contrast to soluble fibrinogen, the adhesion of platelets onto...

Membrane Targeting and Coupling of NHE1-IntegrinαIIbβ3-NCX1 by Lipid Rafts following Integrin-Ligand Interactions Trigger Ca2+ Oscillations

“…1b, light blue bars). Also, Goncalves et al (20) had previously reported that platelets isolated from PLC knock-out mice still exhibited calcium oscillation when triggered by Fg, despite having a delayed onset. Taken together, these findings suggested the presence of additional pathway(s), other than PLC, that might be involved in triggering the calcium oscillations downstream of integrin ␣ IIb ␤ 3 .…”

Phospholipase Cγ2 contributes to stable thrombus formation on VWF

“…Though PLCc2 is known to play an important role in platelet activation by collagen [5,12,13] and fibrinogen [14,15] (via GPVI and the GPIIb-IIIa complex, respectively), the importance of PLC-dependent phosphoinositide turnover in GPIb-mediated platelet activation is just now beginning to be unraveled. Mangin et al [16] recently reported that the small amount of IP 3 that is generated downstream of GPIb-mediated platelet adhesion to immobilized VWF under static conditions, as well as the low-level calcium transients and cytoskeletal changes that follow, can be completely inhibited by the pan-PLC pharmacological antagonist, U73122.…”

Dok-2 Adaptor Protein Regulates the Shear-dependent Adhesive Function of Platelet Integrin αIIbβ3 in Mice

“…Analysis of Calcium Flux-Calcium flux in isolated platelets under static and shear conditions was quantified as described previously (31). In some experiments, platelets were stimulated with thrombin (0.1-1.0 units/ml), collagen-related peptide (1-10 g/ml), or ADP (2-25 M) in the presence or absence of EDTA (1 mM), EGTA (1 mM), or 2-APB (10 M), either alone or in combination, prior to measurement of calcium concentrations.…”

“…Changes in phosphatidylinositol 3-kinase lipid products lead to dysregulated integrin ␣ IIb ␤ 3 adhesive function and calcium signaling under flow (31). To examine potential alterations in cytosolic calcium levels in Dok-2 Ϫ/Ϫ platelets, washed platelets were loaded with the calcium indicator dyes Oregon green 1,2-bis(o-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid and FuraRed, and fluorescence changes were monitored by confocal microscopy (31,40).…”

“…To examine potential alterations in cytosolic calcium levels in Dok-2 Ϫ/Ϫ platelets, washed platelets were loaded with the calcium indicator dyes Oregon green 1,2-bis(o-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid and FuraRed, and fluorescence changes were monitored by confocal microscopy (31,40). As demonstrated in Fig.…”