The Pro33 isoform of integrin β3 enhances outside-in signaling in human platelets by regulating the activation of serine/threonine phosphatases

Abstract

Integrin β3 is polymorphic at residue 33 (Leu33 or Pro33), and the Pro33-positive platelets display enhanced aggregation, P-selectin secretion, and shorter bleeding times. Because outside-in signaling is critical for platelet function, we hypothesized that the Pro33 variant provides a more efficient signaling than the Leu 33 isoform. When compared with Pro33-negative platelets, Pro33-positive platelets demonstrated significantly greater serine/threonine phosphorylation of extracellular signal-regulated kinase (ERK2) and myosin light chain (MLC) but not cytoplasmic phospholipase A2 upon thrombin-induced aggregation. Tyrosine phosphorylation of integrin β3 and the adaptor protein Shc was no different in the fibrinogen-engaged platelets from both genotypes. The addition of Integrilin (αIIbβ3-fibrinogen blocker) or okadaic acid (serine/threonine phosphatase inhibitor) dramatically enhanced ERK2 and MLC phosphorylation in the Pro33-negative platelets when compared with Pro33-positive platelets, suggesting that integrin engagement during platelet aggregation activates serine/threonine phosphatases. The phosphatase activity of myosin phosphatase (MP) that dephosphorylates MLC is inactivated by phosphorylation of the myosin binding subunit of MP at Thr696, and aggregating Pro33-positive platelets exhibited an increased Thr 696 phosphorylation of MP. These studies highlight a role for the dephosphorylation events via the serine/threonine phosphatases during the integrin outside-in signaling mechanism, and the Leu33 → Pro polymorphism regulates this process. Furthermore, these findings support a mechanism whereby the reported enhanced α granule secretion in the Pro33-positive platelets could be mediated by an increased phosphorylation of MLC, which in turn is caused by an increased phosphorylation and subsequent inactivation of myosin phosphatase. © 2005 by The American Society for Biochemistry and Molecular Biology, Inc.