Concurrent signaling from Gαq- and Gαi-coupled pathways is essential for agonist-induced αvβ3 activation on human platelets

Abstract

Summary. The integrin avb3 mediates platelet adhesion to the matrix protein osteopontin and likely is the predominant integrin mediating platelet adhesion to the matrix protein vitronectin. To address the mechanism that regulates avb3 activity in platelets, we measured the effect of the P2Y 1 antagonist adenosine 3 H -phosphate-5 H -phosphate (A3P5P) and the P2Y 12 antagonist AR-C66096 on ADP-stimulated platelet adhesion to osteopontin and vitronectin. Each antagonist completely inhibited platelet adhesion, implying that concurrent stimulation of P2Y 1 and P2Y 12 was required to activate avb3. The reducing agent dithiothreitol and Mn 2 also induced platelet adhesion to osteopontin, but did so without stimulating platelet activation. Thus, these data suggest that ADP stimulation regulates avb3 activity by perturbing the conformation of its extracellular domain. The actin polymerization inhibitors cytochalasin D and latrunculin A also induced platelet adhesion to osteopontin and vitronectin. Thus, avb3 activity in resting platelets appears to be constrained by the platelet cytoskeleton. Moreover, the effect of these agents was inhibited by A3P5P and AR-C66096 at micromolar and subnanomolar concentrations, respectively, suggesting that subthreshold platelet stimulation by ADP was required. Our data suggest that signals from both Ga q -and Ga icoupled receptors converge to release cytoskeletal constraints on avb3. We propose that the release of cytoskeletal constraints and a concurrent increase in af®nity for ligands is responsible for avb3-mediated platelet adhesion.Keywords: ADP receptors, osteopontin, vitronectin receptors, vitronectin.Platelets express both known b3 integrins, aIIbb3 and avb3. Although these integrins readily interact with the macromolecular ligands ®brinogen, von Willebrand factor (VWF), ®bro-nectin, and vitronectin (VN) [1], aIIbb3 does not interact with the matrix protein osteopontin (OPN), an Arg-Gly-Asp (RGD)-containing ligand for avb3 and other av-containing integrins [2,3]. Nonetheless, despite the presence of 50-to 500-fold fewer copies of avb3 compared with aIIbb3 on platelets [4,5], avb3 mediates platelet adhesion to OPN-coated surfaces [6].The ability of aIIbb3 to interact with ligands requires platelet stimulation by agonists such as thrombin, ADP, thromboxane A 2 , serotonin, and epinephrine [1]. How these agonists regulate aIIbb3 function is uncertain. ADP binding to the Ga qcoupled purinergic receptor P2Y 1 stimulates platelet shape change, activates phospholipase Cb i and induces the release of calcium from intracellular stores [7], whereas ADP binding to Ga i -coupled purinergic receptor P2Y 12 inhibits the activity of the enzyme adenylyl cyclase [8]. Concurrent stimulation of both P2Y 12 and P2Y 1 is required for ADP-stimulated platelet aggregation [9], but how signals emanating from these receptors cooperate to induce platelet aggregation is not known. We have reported that incubating platelets previously exposed to subthreshold concentrations of ADP with cytochalasin D and la...

Quantitative Analysis of Platelet αvβ3 Binding to Osteopontin Using Laser Tweezers

“…However, whether platelet adhesion is due exclusively to the integrin-clustering that accompanies ligand binding or results from an agonist-induced increase in ␣ v ␤ 3 affinity for OPN was not clear before the results presented in this paper. Although we have found that perturbing the conformation of the extracellular portion of ␣ v ␤ 3 with either dithiothreitol or Mn 2ϩ also induces platelet adhesion to OPN (13), suggesting that a conformational change in ␣ v ␤ 3 is sufficient to support adhesion, the inability to detect binding of the ligand-mimetic ␣ v ␤ 3 antibody WOW-1 to thrombin-stimulated platelets (15) suggests that this might not be the case. Accordingly, we applied laser tweezers technology to this question, much as we had used the technology to study the interaction of fibrinogen to platelet ␣ IIb ␤ 3 (8).…”

“…are likely similar (13), sites at which OPN and fibrinogen bind to these integrins undoubtedly differ. Thus, whereas fibrinogen binds to ␣ IIb ␤ 3 via the carboxyl terminus of its ␥ chain (26), OPN binding to ␣ v ␤ 3 is mediated by its RGD motif (12), accounting, at least in part, for the measured differences in OPN and fibrinogen binding.…”

“…Second, platelet stimulation could increase the affinity of ␣ v ␤ 3 for OPN and VN by inducing a conformational change in ␣ v ␤ 3 , similar to the way in which platelet stimulation enables ␣ IIb ␤ 3 to bind soluble ligands (5). In support of the second possibility, we reported that incubating platelets with the reducing agent dithiothreitol or with the divalent cation Mn 2ϩ induces platelet adhesion to OPN, indicating that ␣ v ␤ 3 -mediated platelet adhesion can be induced by perturbing the conformation of extracellular domain of ␣ v ␤ 3 (13). On the other hand, Pampori et al (15), using a monovalent ligand mimetic antibody that selectively binds to the activated form of ␣ v ␤ 3 , were unable to detected antibody binding to resting or thrombin-stimulated platelets.…”

“…Interaction of OPN with ADP-stimulated Platelets-Platelet activation is required to induce platelet adhesion to OPNcoated surfaces (11)(12)(13) Rupture forces between OPN-coated beads and single unstimulated (Unstim.) gel-filtered platelets were collected as described under "Experimental Procedures."…”

“…Although there are 50 -500-fold fewer copies of ␣ v ␤ 3 compared with ␣ IIb ␤ 3 on platelets (9, 10), ␣ v ␤ 3 mediates the adhesion of platelets to the matrix proteins osteopontin (OPN) and vitronectin (VN), at least in vitro, and like ␣ IIb ␤ 3 does so only following platelet stimulation (11)(12)(13). There are two possible parallel explanations for the increase in ␣ v ␤ 3 -mediated platelet adhesion to OPN and VN following platelet stimulation.…”