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

The serine-proteinase cathepsin G (CG) is a potent agonist of platelet aggregation inducing the release and surface expression of alpha-granule adhesive proteins such as fibrinogen (Fg) and thrombospondin-1 (TSP-1). Because Fg and TSP-1 are potential substrates for the enzymatic activity of CG, we investigated the fate of these proteins during CG-induced platelet aggregation using an immunoblot technique. Only a small proportion of secreted Fg was proteolyzed by CG and platelet aggregation was efficiently inhibited by anti-fibrinogen Fab fragments. In contrast, TSP-1 was extensively proteolyzed on aggregated platelets releasing in the milieu a fragment with Mr approximately 28 000, corresponding to the amino-terminal heparin-binding domain (HBD). Several antibodies, directed against the cell-associated carboxy-terminal TSP-1f fragment (Mr approximately 165000) impaired the formation of stable macroaggregates, indicating that this fragment may contribute to platelet aggregation in the absence of the HBD.

Exposure of human platelets to plasmin results in the expression of irreversibly active fibrinogen receptors.

Although 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 alpha IIb beta 3, on platelets exposed to plasmin. Following incubation with plasmin at 37 degrees 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 approximately 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 125I-fibrinogen binding assay, can be quantified to approximately 2,300 molecules per platelet. Exposure of active alpha IIb beta 3 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 alpha-thrombin, plasmin-induced activation of alpha IIb beta 3 is sustained and cannot be reversed by exposure of platelets to PGE1. Immunoblotting analysis of the receptor subunits shows no extensive proteolytic modification of alpha IIb beta 3 by plasmin, and only reveals a limited proteolysis of the aminoterminal domain of the alpha IIb 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.(ABSTRACT TRUNCATED AT 250 WORDS)

On the mechanism of plasmin-induced aggregation of human platelets: implication of secreted von Willebrand factor.

Plasmin triggers a strong metabolic activation in human platelets, leading to shape change and granule exocytosis. However, its capacity to induce cell aggregation remains discussed and, when observed, this aggregation is preceded by a remarkable lag phase. We have thus investigated the effect of plasmin on the adhesive proteins which can be secreted by isolated platelets and mediate cell-to-cell interactions, but are also substrates for the enzyme. Immunoblot analysis of fibrinogen (Fg), thrombospondin-1 (TSP-1), fibronectin (Fn) and von Willebrand factor (vWf) was performed on extracts of platelets exposed under stirring to increasing concentrations of plasmin for up to 10 min at 37 degrees C. Under conditions leading to formation of large aggregates, Fg, Fn and TSP-1 are extensively degraded concomitantly with their secretion, and readily lost from the surface of aggregated cells. Part of the monomers in the platelet vWf are cleaved during secretion into two main fragments with Mr approximately 180,000 and approximately 145,000. However, multimer distribution analysis shows only a slight decrease in the very high molecular weight multimers, and most of the fragmented as well as intact vWf remains associated with the platelet surface when aggregation is maximal. That indeed vWf largely supports plasmin-induced aggregation is suggested by the observation that platelets from a patient with type 3 von Willebrand's disease, who totally lacks vWf, show little aggregation in response to the enzyme. Finally, plasmin-induced aggregation can be totally inhibited by antagonists of the alpha(IIb)beta3 integrin. The present study thus indicates a major role for secreted vWf in platelet aggregation induced by plasmin, through its likely interaction with the multifunctional receptor alpha(IIb)beta3.

Plasmodium falciparum-infected erythrocytes: a mutational analysis of cytoadherence via murine thrombomodulin.

The pathophysiologic events leading to organ damage in Plasmodium falciparum malaria infections involve adhesion and sequestration of parasite-infected erythrocytes (PRBC) to the vascular endothelium and syncytiotrophoblast. Several potential receptors to which the PRBCs may bind have recently been identified, one of which is thrombomodulin (TM). TM has been implicated particularly in mediating sequestration of P. falciparum-infected erythrocytes in the placenta and brain, two sites of disease associated with high morbidity. In order to establish that binding of parasite-infected red blood cells to TM is dependent on its containing chondroitin-4-sulfate (CSA), we have mutated the CSA-attachment site of murine TM, and expressed this mutant form (TMsergly) in COS-7 cells. In cytoadhesion assays, we demonstrate that, in contrast to wild-type TM which contains CSA and supports the adhesion of 1466 PRBCs/mm2, TMser-gly does not contain CSA and adhesion of PRBCs to those cells expressing TMser-gly is entirely abrogated (200 PRBCs/mm2). These studies further confirm that the CSA of TM may play a role in the pathophysiology of malaria by providing a binding site for PRBCs.

Thrombospondin peptides inhibit the secretion-dependent phase of platelet aggregation.

Thrombospondin (TSP) is a platelet alpha-granule adhesive glycoprotein (M(r) = 450,000) that is released in large amounts from activated platelets and participates in thrombus formation. The aim of the present study was to assess the effect of peptides corresponding to sequences within the NH2-terminal region and type 1 repeats of TSP on platelet aggregation induced by thrombin in washed platelet suspensions. We found that TSP18 (amino acids 1-174), used at micromolar concentrations, inhibited platelet aggregation by 30-50%, reducing the size of the aggregates formed. Similar results were obtained with the hexapeptide Cys-Ser-Val-Thr-Cys-Gly (amino acids 429-434 and 486-491) used at 1.2 mM. The shorter peptide Val-Thr-Cys-Gly was even more inhibitory whereas the peptide Val-Thr-Lys-Gly, which lacks a cysteine, had no effect. Interestingly, we have constantly found that inhibition of platelet aggregation by these peptides was accompanied by an inhibition of alpha and dense granule secretion, suggesting that the binding of secreted TSP to the plasma membrane may participate in the platelet signaling process. We conclude that peptides of TSP may prove useful in the treatment of thrombosis by impairing both the release of proaggregating substances and platelet macroaggregate formation.