Exposure of fibrinogen receptors in human platelets by surface proteolysis with elastase.

Abstract

Human platelets that were preincubated with porcine elastase aggregated spontaneously upon the addition of fibrinogen. Maximal aggregation to fibrinogen was observed with platelets pretreated with an elastase concentration of 111 tg/ml, and halfmaximal aggregation occurred after treatment with 11 ,ug/ml elastase. Binding of radiolabeled fibrinogen to elastase-treated platelets was specific, saturable, and showed a single class of 48,400±9,697 fibrinogen-binding sites per platelet with a dissociation constant of 6.30±1.48 X i0-M. ATP, apyrase, and the stimulators of platelet adenylate cyclase forskolin, prostaglandin El, prostacyclin, and N6, 2'-O-dibutyryl cyclic AMP did not inhibit the fibrinogen-induced aggregation of elastase-treated platelets. EDTA completely blocked the initiation of aggregation and reversed the fibrinogen-induced aggregation of elastasetreated platelets. Monoclonal and polyclonal antibodies directed against glycoproteins (GP) Ilb and II1a completely blocked the fibrinogen-induced aggregation of elastase-treated platelets. Immunoprecipitates with these antibodies obtained from detergent extracts of surface-radiolabeled, intact, and elastase-treated platelets contained the glycoproteins IUb and IIIa. We conclude that surface proteolysis by low concentrations of elastase can expose fibrinogen-binding sites associated with GPIIb and GPIIIa on the platelet surface, resulting in spontaneous aggregation upon the addition of fibrinogen. These findings may be relevant to hemostatic changes observed in patients with increased levels of circulating elastase.

Association of Plasma Levels of F11 Receptor/Junctional Adhesion Molecule-A (F11R/JAM-A) With Human Atherosclerosis

“…One potential mechanism is that F11R is shed from endothelial cells and/or platelets through a pathway involving enzymatic cleavage at a site in close proximity to the membrane, resulting in the release of the extracellular domain of F11R into the circulation. Such cleavage mechanisms have been reported for L-selectin (19,20) and for the beta-integrin glycoprotein IIIa (21), resulting in the shedding of fragments of these molecules. Whether the platelets or the endothelium are the source of the circulating F11R is not known.…”

Human Neutrophil Elastase Proteolytically Activates the Platelet Integrin αIIbβ3 through Cleavage of the Carboxyl Terminus of the αIIb Subunit Heavy Chain

“…Thus, treatment of platelets with chymotrypsin (28 -30) or elastases (26,27) results in a proteolytic-dependent exposure of fibrinogen-binding sites at the platelet surface. In accordance with these studies, measurements of the binding of PAC-1 and AP-5 antibodies allowed us to demonstrate that NE rapidly induces the high affinity and ligand-occupied conformers of native ␣ IIb ␤ 3 .…”

“…Activation of the Platelet Fibrinogen Receptor by NE and Cathepsin G-Previous reports have shown that platelet exposure to various serine proteinases, including elastases, induces expression of fibrinogen binding sites (26,27). This, together with the requirement for exogenous fibrinogen for the potentiation of platelet aggregation (the present work) led us to consider that the synergism resulting from the combination of NE and threshold of cathepsin G could be exerted at the level of the ␣ IIb ␤ 3 integrin, the platelet fibrinogen receptor.…”

“…Finally, an alternative pathway for activation of ␣ IIb ␤ 3 at the surface of platelets could be a proteolytic modification of the extracellular regions of this receptor. Thus, exposure of platelets to pancreatic or leukocyte elastases (26,27) or to ␣-chymotrypsin (28 -30) has been reported to induce the irreversible expression of fibrinogen-binding sites in the absence of intracellular activation.…”

The role of neutrophils in vascular injury: a summary of signal transduction mechanisms in cell/cell interactions

“…The requirement for activation is eliminated when platelets are treated with proteases [101], which suggests that proteolytic cleavage of gpIIb/IIIa mimics the necessary physiological change. Recent studies have attempted to identify the RGD binding region within the gpIIb/IIIa molecule by chemically cross-liking synthetic radiolabeled RGD peptides to the receptor.…”