Molecular requirements for the interaction of thrombospondin with thrombin-activated human platelets: modulation of platelet aggregation.
We have investigated the molecular requirements for thrombospondin (TSP) to bind to the platelet surface and to support the subsequent secretion-dependent platelet aggregation. For this, we used two distinct murine monoclonal antibodies (MoAbs), designated MAI and MAII, raised against human platelet TSP, and three polyclonal antibodies, designated R3, R6, and R5, directed against fusion proteins containing the type 1 (Gly 385-Ile 522), type 2 (Pro 559-Ile 669), and type 3 (Asp 784-Val 932) repeating sequences, respectively. Among them, R5 and R6, but not R3, inhibited thrombin-induced aggregation of washed platelets and the concomitant secretion of serotonin. These antibodies, however, did not inhibit the expression of TSP on thrombin-activated platelets, as measured by the binding of a radiolabeled MoAb to TSP, suggesting that they may inhibit platelet aggregation by interfering with a physiologic event subsequent to TSP binding. In contrast, MoAb MAII, which reacts with an epitope located within the heparin-binding domain of TSP, inhibited both TSP surface expression and platelet aggregation/secretion induced by thrombin. In addition, this MoAb inhibited in a dose-dependent manner (IC50 approximately 0.5 mumol/L) the interaction of 125I-TSP with immobilized fibrinogen and platelet glycoprotein IV, both potential physiologic receptors for TSP on thrombin-activated platelets. These results indicate that the interaction of TSP with the surface of activated platelets can be modulated at the level of a specific epitope located within the amino terminal heparin-binding domain of the molecule. Thus, selective inhibition of the platelet/TSP interaction may represent an alternative approach to the inhibition of platelet aggregation.
Legrand
C
C
Unité 150 INSERM, Hôpital Lariboisière/Hôpital St Louis, Paris, France.
Thibert
V
V
Dubernard
V
V
Bégault
B
B
Lawler
J
J
D000906:Antibodies; D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D015153:Blotting, Western; D006801:Humans; D007700:Kinetics; D046911:Macromolecular Substances; D015539:Platelet Activation; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis / Q000276:immunology / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000276:immunology; D013917:Thrombin / Q000494:pharmacology*; D019699:Thrombospondins
D000906:Antibodies; D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D010980:Platelet Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D019699:Thrombospondins; D013917:Thrombin