Effect of Stimulation on the Stabilization of Platelet-Fibrinogen Interactions

Abstract

SummaryThe interaction between fibrinogen and stimulated platelets is a multiphasic process that culminates in the stabilization of ligand binding and reduced accessibility of bound fibrinogen to exogenous antibody. The present study was designed to further explore platelet-fibrinogen interactions by examining the effect of agonist on bound fibrinogen expression and interaction with stimulated platelets as a function of time after ligand binding. Two agents were identified, Zn2+ and phorbol myristate acetate (PMA), which support progressive decreases in bound fibrinogen expression on platelets, but fail to support the stabilization of fibrinogen binding. Sixty min after binding to platelets, approximately 80% of bound fibrinogen remained reversibly associated with Zn2+- or PMA-treated platelets and failed to associate with the Triton X-100 insoluble cytoskeleton. In contrast, polyclonal anti-fibrinogen antibody binding decreased by more than 66%. Over the same time course, fibrinogen binding to control platelets, stimulated with thrombin or ADP, was not only accompanied by a 70% decrease in antifibrinogen antibody binding, but also an inability of EDTA or excess exogenous fibrinogen to dissociate more than half of platelet-associated fibrinogen, as well as the progressive association of bound fibrinogen with the platelet cytoskeleton. Costimulation of platelets with ZnCl2 and thrombin or ZnCl2 and ADP enhanced overall fibrinogen binding but not the EDTA-resistant component, and prevented the recovery of irreversibly bound fibrinogen with the Triton X-100 insoluble cytoskeleton. Costimulation of PMA- or Zn2+-treated platelets with low doses of A23187, however, restored the stabilization of platelet-fibrinogen interactions. These data suggest that modulation of bound fibrinogen expression on stimulated platelets can occur independently of the development of EDTA-resistant fibrinogen binding, and that stabilization of platelet-fibrinogen interactions is sensitive to stimulation-specific intracellular signal transduction.

Exposure of Platelet Fibrinogen Receptors by Zinc Ions: Role of Protein Kinase C

“…This region is essential for phorbol ester and for diacylglycerol binding to protein kinase C. The tandem repeats of a cysteine-rich motif resemble the "Zn2+ finger domain" that is found in some DNA-binding proteins. Most recently, Peerschke (38) demonstrated that Zn2+ (concentration 300 p M ) or PMA (500 nM) prevent formation of irreversible bonds between activated platelets and fibrinogen and inhibit incorporation of fibrin into cytoskeleton. Ono et al (9) studied various mutants of recombinant protein kinase C and demonstrated that phorbol ester binding to protein lunase C requires a cysteine-rich Zn2+ fingerlike sequence.…”

Vascular homeostasis, adhesion molecules, and macrovascular disease in non-insulin-dependent diabetes mellitus

“…Evidence indicates that these peptides are produced by a variety of cell lines and have been platelets and is cleaved by thrombin to produce soluble and, ultimately, cross-linked fibrin. 30 Fibrinogen and implicated in a variety of disorders as diverse as autoimmune insulin-dependent (Type 1) diabetes and fibrin(ogen) degradation products have both been identified as components of atherosclerotic plaques. 31 Fibrino-atherothrombotic vascular disease.…”

Regulation of Platelet Aggregation by Post-fibrinogen Binding Events

“…The platelet membrane GPIIb-IIIa complex can be activated by a va riety of stimuli, and experimental evidence suggests the development of antigenically (1,2) and functionally (3,4) distinct GPIIb-IIIa activation states. In addition to physiologic compounds such as ADP, epinephrine and thrombin, GPIIb-IIIa can be enzymatically activated (chymotryp sin, pronase, elastase) to bind fibrinogen (1,5,6).…”

“…Regulation of both GPIIb-IIIa activation (1,5), and fibrinogen conformation (16) are essential for modulating ligand-receptor interac tions. Post-fibrinogen binding events such as the stabilization of fi brinogen binding (3,17) and bound fibrinogen processing and/or re distribution by stimulated platelets (18), however, may also play a role in regulating ligand binding and subsequent platelet cohesion. A variety of evidence suggests that fibrinogen binding to GPIIb-IIIa while neces sary is not sufficient for platelet aggregation (19)(20)(21).…”

“…Fibrinogen binding to GPIIb-IIIa on activated platelets is a multi phasic process (22). Bound fibrinogen becomes progressively less reversible (3,23) and loses its accessibility to antibodies (17). More recently, fibrinogen redistribution both on the platelet surface and se questration into inaccessible internal compartments has been suggested (24).…”