Suboptimal Activation of Protease-activated Receptors Enhances α2β1 Integrin-mediated Platelet Adhesion to Collagen

Abstract

Thrombin and fibrillar collagen are potent activators of platelets at sites of vascular injury. Both agonists cause platelet shape change, granule secretion, and aggregation to form the primary hemostatic plug. Human platelets express two thrombin receptors, protease-activated receptors 1 and 4 (PAR1 and PAR4) and two collagen receptors, the ␣ 2 ␤ 1 integrin (␣ 2 ␤ 1 ) and the glycoprotein VI (GPVI)/FcR␥ chain complex. Although these receptors and their signaling mechanisms have been intensely studied, it is not known whether and how these receptors cooperate in the hemostatic function of platelets. This study examined cooperation between the thrombin and collagen receptors in platelet adhesion by utilizing a collagen-related peptide (␣2-CRP) containing the ␣ 2 ␤ 1 -specific binding motif, GFOGER, in conjunction with PAR-activating peptides. We demonstrate that platelet adhesion to ␣2-CRP is substantially enhanced by suboptimal PAR activation (agonist concentrations that do not stimulate platelet aggregation) using the PAR4 agonist peptide and thrombin. The enhanced adhesion induced by suboptimal PAR4 activation was ␣ 2 ␤ 1 -dependent and GPVI/FcR␥-independent as revealed in experiments with ␣ 2 ␤ 1 -or FcR␥-deficient mouse platelets. We further show that suboptimal activation of other platelet G q -linked G protein-coupled receptors (GPCRs) produces enhanced platelet adhesion to ␣2-CRP. The enhanced ␣ 2 ␤ 1 -mediated platelet adhesion is controlled by phospholipase C (PLC), but is not dependent on granule secretion, activation of ␣ IIb ␤ 3 integrin, or on phosphoinositol-3 kinase (PI3K) activity. In conclusion, we demonstrate a platelet priming mechanism initiated by suboptimal activation of PAR4 or other platelet G q -linked GPCRs through a PLC-dependent signaling cascade that promotes enhanced ␣ 2 ␤ 1 binding to collagens containing GFOGER sites.Platelets are small anuclear cellular elements that play a central role in hemostasis and contribute to vascular pathology. At sites of intravascular injury, platelets are exposed to multiple prothrombotic factors that promote thrombus formation and trigger firm adhesion of platelets to the subendothelial extracellular matrix. Thrombin and fibrillar collagen are two of the more potent stimuli (1, 2).Thrombin is an essential serine protease in the coagulation cascade that ultimately converts fibrinogen to fibrin. Thrombin also has a direct signaling effect on cells through protease-activated receptors (PARs) 2 , which are G protein-coupled receptors (GPCRs) that are activated by enzymatic cleavage of the N terminus of the receptor to produce a tethered ligand (3). Of the four known PAR isoforms, human platelets express PAR1 and PAR4. In platelets, these receptors show different signaling kinetics; PAR1 is activated at low thrombin concentrations with a quick signal shutoff, whereas, PAR4 is activated at higher thrombin concentrations with a slow signal shut off (4 -6). Thrombin-induced signaling has been shown to modulate ligand affinity of the platelet membrane ␣ IIb ...

Ibrutinib Inhibits Platelet Integrin α IIb β 3 Outside-In Signaling and Thrombus Stability But Not Adhesion to Collagen

“…The integrin α 2 β 1 mediates adhesion directly to collagen and is thought to be capable of initiating intracellular signaling, although evidence also exists that the affinity for collagen must first be enhanced via intracellular signaling mediated by another receptor. 25 The contribution of α 2 β 1 to collagenevoked platelet activation is only apparent during adhesion to immobilized collagen and not in suspension either in vitro 14 or in vivo after injection of mice with soluble collagen.26 This correlates with the observed effect of ibrutinib, which inhibits collagen-mediated platelet signaling and aggregation in suspension but only partially inhibits signaling and thrombus formation on immobilized collagen. However, further investigation is required to identify the collagen receptor capable of initiating signaling in response to collagen in the presence of ibrutinib.…”

Annexin V binding to platelets is agonist, time and temperature dependent

“…A recent study by Nagy et al showed that activation of both GPVI and PAR induced phosphorylation on PKC- [24]. Marjoram et al also recently demonstrated a PLC dependent enhanced adhesion to a collagen related peptide by PAR peptides [23].…”

“…with CRP are not clear [23]. There are significant similarities in signalling between both pathways as they activate phospholipase C (PLC), which results in the generation of inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG).…”

PI3K/Akt in platelet integrin signaling and implications in thrombosis

“…In platelets, most studies have been performed to understand the molecular mechanism behind integrin a IIb b 3 inside-out activation. Relatively limited information is available about the control of integrin a 2 b 1 Jung and Moroi, 2001;Marjoram et al, 2009;Pula and Poole, 2008;Wang et al, 2009) with conflicting observations about the possible contribution of PI3K (Jung and Moroi, 2001;Marjoram et al, 2009), and nothing is known about inside-out regulation of other integrins in platelets.…”