PECAM-1 Inhibits Signaling Pathways That Amplify Platelet Aggregation.

Abstract

Though protein-tyrosine kinases get most of the attention, there is growing evidence that serine/threonine kinases (S/TKs) also play an important role in amplifying platelet activation responses following exposure to low-dose platelet agonists. For example, granule secretion is markedly impaired in platelets from mice deficient in either isoform of the STKs Akt1 (Blood104:1703, 2004) or Akt2 (J Clin Invest113:441, 2004), and pharmacological inhibition of the mitogen-activated protein kinase, p38, results in greatly reduced platelet aggregation and secretion (JBC271:6586, 1996; Blood107:965, 2006). Similarly, the integrin-linked kinase, ILK, is an S/TK that has recently been implicated in regulating both integrin activation (Thromb Haemost88:115, 2002; J Thromb Haemost2:1443, 2004) and cytoskeletal reorganization (Biochem J365:79, 2002; BBRC297:1324, 2002). We and others have previously shown that PECAM-1, a 130 kDa member of the immunoglobulin (Ig)-superfamily expressed on the surface of circulating platelets, leukocytes, and at the intercellular junctions of all continuous endothelium, functions to suppress platelet activation, though the mechanism by which this occurs has not been defined. To determine whether PECAM-1 might suppress S/TK-mediated platelet activation pathways, wild-type and PECAM-1 deficient murine platelets were stimulated with the GPVI-specific agonist, collagen-related peptide (CRP), and the activation state of Akt, p38, and ILK assessed biochemically. As previously reported, platelets from PECAM-1-deficient mice aggregated to a significantly greater extent in response to low-dose CRP than did their wild-type, PECAM-1-positive counterparts. Immunoblot analysis using phospho-specific antibodies revealed that both Akt and p38 existed in a hyper-activated state in CRP-stimulated, PECAM-1-deficient platelets - becoming phosphorylated both earlier and more intensively during platelet aggregation. ILK was similarly hyperactive, as assessed by determining the phosphorylation state of its substrate, glycogen synthase kinase 3β (GSK3β). To determine whether the inhibitory effects of PECAM-1 on S/TK-mediated platelet activation were dependant on prior fibrinogen binding to the major platelet integrin, GPIIb-IIIa, platelets were activated with low-dose CRP in the presence or absence of 2 mM RGDW. Pretreatment of platelets with RGDW had no effect on the ability of PECAM-1 to suppress activation of Akt,. p38, or ILK. Taken together, these data suggest that PECAM-1 functions as an inhibitory receptor in platelets, at least in part, by suppressing the activity of at least three different S/TKs, and thereby their ability to amplify early platelet activation responses.