Exposure of Human Platelets to Plasmin Results in the Expression of Irreversibly Active Fibrinogen Receptors

Abstract

SummaryAlthough plasmin can trigger strong platelet responses such as shape change and exocytosis of internal granules, limited platelet aggregation is induced by this proteinase, owing to its capacity to rapidly proteolyse secreted adhesive proteins. In this context, we have investigated the state of activation of the fibrinogen receptor, the integrin αIIbβ3, on platelets exposed to plasmin. Following incubation with plasmin at 37 °C, washing, and resuspension, platelets exhibit a moderate, low-velocity aggregation when stirred in the presence of fibrinogen. Optimum aggregability is observed when platelets have been exposed to plasmin activity of ≈0.5 CU/ml for 20 min, and aggregation is insensitive to the presence of antagonists such as prostaglandin (PG) E1 and apyrase. Plasmin-induced platelet aggregability is associated with the expression of active fibrinogen receptors on the cell surface, which, using a l25I-fibrinogen binding assay, can be quantified to ≈2,300 molecules per platelet. Exposure of active αIIbβ3 receptors appears to depend partially, but not totally on a metabolic activation and granule exocytosis at the time of incubation with plasmin. In contrast with a-thrombin, plasmin-induced activation of αIIbβ3 is sustained and cannot be reversed by exposure of platelets to PGE1. Immunoblotting analysis of the receptor subunits shows no extensive proteolytic modification of αIIbβ3 by plasmin, and only reveals a limited proteolysis of the aminoterminal domain of the αIIb subunit. In addition to their capacity to aggregate in the presence of fibrinogen alone, plasmin-treated platelets also show a potentiated aggregability in response to low doses of ADP. Thus, plasmin has the potential to activate the platelet fibrinogen receptor in such a way that it remains irreversibly available to fibrinogen on the surface of nonaggregated cells, a feature that may participate to pathological states of in vivo platelet hyperaggregability.

Glycoprotein IIb/IIIa Receptor Antagonist (2S)-2-[(2-Naphthyl-sulfonyl)amino]-3-{[2-({4-(4-piperidinyl)-2-[2-(4-piperidinyl)ethyl] butanoyl}amino)acetyl]amino}propanoic Acid Dihydrochloride (CRL42796), in Combination with Aspirin and/or Enoxaparin, Prevents Coronary Artery Rethrombosis after Successful Thrombolytic Treatment by Recombinant Tissue Plasminogen Activator

“…The mechanism of platelet activation or inhibition by plasmin is poorly understood. Plasmin at a concentration in excess of 1 caseinolytic unit/ml triggers platelet aggregation at 37°C through direct or indirect activation of the integrin glycoprotein IIb/ IIIa (Loscalzo et al, 1995;Rabhi-Sabile and Pidard, 1995). Plasmin may promote platelet activation by cleaving the seven transmembrane thrombin receptor to expose the tethered ligand containing the SFLLRN sequence, which directly activates platelets in the same manner as thrombin (Kuliopulos et al, 1999).…”

Cardiopulmonary bypass technology transfer: musings of a cardiac surgeon

“…Other biomaterial-independent factors contributing to platelet activation (and subsequent dysfunction) after CPB include heparin at the doses used for CPB [92], activated complement [93], plasmin [94] and a variety of other soluble blood elements. Platelets adhere to binding sites located on surface-adsorbed brinogen and form aggregates [95].…”

Proteolysis of thrombospondin during cathepsin‐G‐induced platelet aggregation: functional role of the 165‐kDa carboxy‐terminal fragment

“…2. Immunoblotting sion was continuously recorded and platelet aggregation expressed as analysis of cell-associated TSP-1 performed under reducing a percentage of the maximal variation in LT [19]. At the given time conditions indicated that TSP-1 was rapidly proteolyzed durpoints, the enzymic activity of CG was blocked by the addition of 1 mmol/1 PMSF and 200 gg/ml SBTI [12], whereas cx-thrombin was ing activation ( Fig.…”

“…2 indicated that the conversion of mixed with 40 gl of a solution of 0.1 moll1 EDTA to stop exocytosis TSP-1 into TSP-1 f closely paralleled the rates of a-granule and centrifuged at 12000×g for 4 min. The supernatant, and an exocytosis and platelet aggregation (indicated at the bottom aliquot of the unsedimented platelet suspension, were kept at of the figure), with TSP-lf corresponding to 12, 40, and 81% -20°C until they were processed for the measurement of secreted I 3-thromboglobulin (13-TG) [19]. This was done using a commercial ELIof the total TSP-l-reactive material at 1, 3, and 5 min of SA (Diagnostica Stago, Asni~res, France) following the manufacactivation, respectively ( Fig.…”

“…Protein disulfide bonds were reduced ~ 1°° ~/w 40 '°°IB ~40 by heating the samples at 100°C for 5 min in the presence of 5% (v/v) ~ 2-mercaptoethanol. Separated proteins were electrotransferred onto ~ ao 80 nitrocellulose membranes and analyzed by immunoblotting as previously described [19]. Primary incubation with the monoclonal anti-0 60 60 body MAII was followed by a second incubation with a rabbit antim,, mouse IgG antibody (RAM/7S).…”