Identification of platelet membrane thrombospondin binding molecules using an anti-thrombospondin antibody.
A rat monoclonal IgG2a antibody, 5G11, was raised against native human platelet thrombospondin (TSP). Western blot analysis revealed that 5G11 bound (i) to TSP before and after disulfide reduction, and (ii) to a 15-kDa fragment released after prolonged trypsin digestion. Crossed immunoelectrophoresis confirmed that the binding epitope was expressed in the presence of Ca2+ and after treatment of TSP with EDTA. Since 5G11 had no effect on platelet aggregation, the antibody was used to immunoprecipitate Ca2+-dependent and Ca2+-independent TSP-binding molecules on the surface of thrombin-activated surface-labeled 125I-platelets. The experimental basis was that ligand-receptor interactions are of high affinity and that anti-ligand antibodies should precipitate the ligand-receptor complex. With platelets activated in the presence of EDTA, 5G11 predominantly precipitated a 125I-labeled band of Mr 88,000, identified as glycoprotein (GP) IV. In contrast, in the presence of 2 mM Ca2+ and 1 mM Mg2+, 5G11 precipitated a complex of five radiolabeled proteins, among which GPIIb, GPIIIa and GPIV were the most prominent.
Kieffer
N
N
INSERM U91/CNRS UA607, Hôpital Henri Mondor, Créteil, France.
Nurden
A T
AT
Hasitz
M
M
Titeux
M
M
Breton-Gorius
J
J
D000911:Antibodies, Monoclonal; D001792:Blood Platelets / Q000378:metabolism*; D018955:CD36 Antigens; D002462:Cell Membrane / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007123:Immunoelectrophoresis, Two-Dimensional; D011975:Receptors, Mitogen / Q000032:analysis*; D019699:Thrombospondins
D000911:Antibodies, Monoclonal; D018955:CD36 Antigens; D006023:Glycoproteins; D011975:Receptors, Mitogen; D019699:Thrombospondins