Platelet-agglutinating protein p37 from a thrombotic thrombocytopenic purpura plasma forms complexes with platelet membrane glycoprotein IV (CD36).
We have previously reported the purification of a 37 kDa platelet agglutinating protein (PAPp37) from the plasma of a patient with Thrombotic Thrombocytopenic purpura (TTP), and have shown recently that p37 causes platelet agglutination through its binding to membrane glycoprotein IV (GPIV). To gain further insight into the mechanism of p37 binding to GPIV, we have studied the interaction between p37 and GPIV. We now demonstrate specific complex formation of p37 with GPIV. In Western immunoblotting p37 binds to purified GPIV and the complex formed between the two proteins was detected by polyclonal antibody to p37 and peroxidase conjugated second antibody. No binding of p37 was noticed with the purified GPIIIa. A solid phase binding assay was developed to study the complex formation. Microtiter wells were coated with GPIV and the control proteins; 125I-p37 was added, allowed to bind and bound radioactivity was measured. Several lines of evidence indicate that the binding of p37 to GPIV was specific. a) GPIV immobilized on Immulon-2 wells bound 10-30 fold more p37 than immobilized fibrinogen, GPIIIa, and BSA. b) Polyclonal antibodies against p37 and GPIV inhibited the binding by 39-68% as compared with control IgG. c) GPIIIa antibody did not inhibit the binding. Molecular sieve chromatography of a mixture of 125I-p37 and GPIV also revealed the fluid phase complex formation ranging in molecular weight from 132,000 to over 350,000 daltons. These results show the specific interaction between p37 and GPIV and suggest that GPIV functions as a p37 receptor during platelet agglutination.
Siddiqui
F A
FA
Hemophilia and Thrombosis center, W.J. Harrington Center for Blood Diseases, University of Miami School of Medicine, Florida.
Lian
E C
EC
D015703:Antigens, CD / Q000378:metabolism*; D001798:Blood Proteins / Q000378:metabolism*; D018955:CD36 Antigens; D006801:Humans; D010974:Platelet Aggregation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D011485:Protein Binding; D011697:Purpura, Thrombotic Thrombocytopenic / Q000378:metabolism*
D015703:Antigens, CD; D001798:Blood Proteins; D018955:CD36 Antigens; D010980:Platelet Membrane Glycoproteins; C046935:platelet agglutinating protein, thrombotic thrombocytopenic purpura