Abstract
Excessive accumulation of platelets at sites of athero-sclerotic plaque rupture leads to the development of arterial thrombi, precipitating clinical events such as the acute coronary syndromes and ischemic stroke. The major platelet adhesion receptor glycoprotein (GP) IIb-IIIa (integrin alpha(IIb)beta3) plays a central role in this process by promoting platelet aggregation and thrombus formation. We demonstrate here a novel mechanism down-regulating integrin alpha(IIb)beta3 adhesive function, involving platelet factor XIII (FXIII) and calpain, which serves to limit platelet aggregate formation and thrombus growth. This mechanism principally occurs in collagen-adherent platelets and is induced by prolonged elevations in cytosolic calcium, leading to dramatic changes in platelet morphology (membrane contraction, fragmentation, and microvesiculation) and a specific reduction in integrin alpha(IIb)beta3 adhesive function. Adhesion receptor signal transduction plays a major role in the process by sustaining cytosolic calcium flux necessary for calpain and FXIII activation. Analysis of thrombus formation on a type I fibrillar collagen substrate revealed an important role for FXIII and calpain in limiting platelet recruitment into developing aggregates, thereby leading to reduced thrombus formation. These studies define a previously unidentified role for platelet FXIII and calpain in regulating integrin alpha(IIb)beta3 adhesive function. Moreover, they demonstrate the existence of an autoregulatory feedback mechanism that serves to limit excessive platelet accumulation on highly reactive thrombogenic surfaces.
Highlights
The ability of platelets to adhere to the injured vessel wall and recruit other platelets is critical for hemostatic plug formation and vascular repair
We demonstrate that platelets undergoing a high level of prolonged cytosolic calcium flux exhibit factor XIII (FXIII) and calpain-dependent morphological changes that are associated with a selective down-regulation in the adhesive function of integrin ␣IIb3
Calcium-induced Decrease in Platelet Surface Reactivity—We have previously established in vitro flow-based assays that enable real time analysis of cytosolic calcium flux during the development of platelet-platelet adhesion contacts [9, 25]. Using these experimental systems we have established a critical role for intercellular calcium communication in promoting platelet aggregate formation and thrombus growth [11]
Summary
General Reagents and Antibodies—Fura Red-AM was from Molecular Probes Inc. Thapsigargin, ionophore A23187, thimerosal, ADP, monodansylcadaverine (MDC), and p190 –230 were from Sigma. In experiments examining the effect of calpain or FXIII inhibition on the rate of SCIP (sustained calcium-induced platelet morphology, see “Results”) formation, platelets (1 ϫ 107/ml) in Tyrode’s buffer were pretreated with vehicle (Me2SO), 50 g/ml calpeptin, 50 M ALLN, 50 M E64-d, 25 M p190 –230, or the indicated concentrations of MDC for 30 min, prior to being applied to glass or collagen. Formation of SCIP Monolayers—Several independent approaches were investigated to achieve efficient (Ͼ95%) conversion of spread platelets to SCIPs in a preformed platelet monolayer These include: 1) allowing platelets to form monolayers in the presence of extracellular calcium for prolonged periods of time (up to 120 min); 2) initially depleting platelet cytosolic calcium in a preformed monolayer with thapsigargin/EGTA for 15 min followed by exposure to thapsigargin/ CaCl2 for a further 30 min; and 3) exposing preformed monolayers to 1 M calcium ionophore A23187 in the presence of 1 mM CaCl2 for 5–10 min. Percentage of SCIP monolayers were performed using ionophore A23187 as an inducer of SCIP formation
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