Selective inhibition of platelet macroaggregate formation by a recombinant heparin-binding domain of human thrombospondin.
Thrombospondin (TSP) is a platelet alpha-granule adhesive protein that plays a critical role in the stabilization of thrombus by promoting the formation of platelet macroaggregates. We have recently shown that a monoclonal antibody (mAb) to the NH2-terminal heparin-binding domain of TSP, MAII, inhibits platelet aggregation induced by thrombin in a dose-dependent manner. In this study, we have expressed in Escherichia coli two recombinant proteins comprising residues 1 to 174 (TSP18) and 1 to 242 (TSP28) of TSP. After purification, both proteins reacted equally well with mAb MAII, whereas the reactivity of TSP18 for heparin was lower than that of TSP28 or native TSP. At micromolar concentrations, TSP18 and TSP28 inhibited the second wave of platelet aggregation and the concomitant release of [14C]5-hydroxytryptamine induced by ADP in citrated platelet-rich plasma as well as aggregation and secretion induced by a low concentration of thrombin in washed platelet suspensions. The proteins did not inhibit surface expression of endogenous TSP on activated platelets, as measured by the binding of radiolabeled mAb 5G11, indicating that they did not interfere with the primary binding of TSP to the plasma membrane. In contrast, in a solid-phase binding assay, the proteins inhibited in a dose-dependent manner (IC50, 0.1 and 0.06 mumol/L for TSP18 and TSP28, respectively) the binding of radiolabeled TSP to surface-adsorbed fibrinogen. Furthermore, specific and saturable binding of the proteins to immobilized fibrinogen was demonstrated by enzyme-linked immunosorbent assay. The results suggest that interaction between the heparin-binding domain of TSP and membrane-bound fibrinogen may be critical in the platelet aggregation/secretion process.
Legrand
C
C
Unité 353 INSERM, Protéines Adhésives et Protéases des Cellules Vasculaires et Sanguines, Hôpital St Louis, Paris, France.
Morandi
V
V
Mendelovitz
S
S
Shaked
H
H
Hartman
J R
JR
Panet
A
A
D001792:Blood Platelets / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000494:pharmacology; D005340:Fibrinogen / Q000378:metabolism; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008562:Membrane Glycoproteins / Q000037:antagonists & inhibitors / Q000378:metabolism* / Q000494:pharmacology*; D010446:Peptide Fragments / Q000494:pharmacology*; D015539:Platelet Activation; D010974:Platelet Aggregation / Q000187:drug effects*; D010975:Platelet Aggregation Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins; D019699:Thrombospondins
D015815:Cell Adhesion Molecules; D008562:Membrane Glycoproteins; D010446:Peptide Fragments; D010975:Platelet Aggregation Inhibitors; D011994:Recombinant Proteins; D019699:Thrombospondins; D005340:Fibrinogen; D006493:Heparin