The interaction of human platelet thrombospondin with fibrinogen. Thrombospondin purification and specificity of interaction.
Human platelet thrombospondin (TSP) was purified to homogeneity by chromatography on fibrinogen coupled to cyanogen bromide-activated Sepharose. The yield of TSP was 1.3 mg or approximately 22% of that present in platelet-rich plasma as determined by radioimmunoassay. It analyzed on discontinuous sodium dodecyl sulfate gels as a single band having apparent molecular weights of 180,000 and greater than 400,000 under reducing and nonreducing conditions, respectively. Amino acid analysis gave results similar to previously published values. Antibodies raised in rabbits were monospecific as evaluated by radioimmunoassay. In double immunodiffusion tests, these antibodies gave one line of identity against TSP purified by this procedure and TSP purified by published procedures, confirming the identity of the material isolated. The protein possesses no lectin-like activity. The specificity of the TSP-fibrinogen interaction was investigated. TSP binding to fibrinogen-Sepharose occurred in the presence of EDTA, indicating that calcium and magnesium ions are not required for interaction of TSP with fibrinogen. The binding of TSP to fibrinogen-Sepharose was quantitatively blocked by pretreatment with an antibody to the cyanogen bromide cleavage fragment composed of residues 241-476 of the carboxyl-terminal end of the alpha chain of fibrinogen. Antibodies against the D and E domains of fibrinogen had no effect on the binding. Excess fibrinogen (30 mg/ml) added to platelet extract quantitatively inhibited binding of TSP to fibrinogen-Sepharose. TSP preferentially bound to uncross-linked fibrin, suggesting that the TSP-fibrinogen binding site is unavailable in cross-linked fibrin. These results indicate that TSP binds specifically to immobilized fibrinogen or uncross-linked fibrin through determinants present in the carboxyl-terminal portion of the alpha chain and that these interactions do not require calcium or magnesium ions.
D000596:Amino Acids / Q000032:analysis; D001792:Blood Platelets / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005340:Fibrinogen / Q000378:metabolism*; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D005779:Immunodiffusion; D007700:Kinetics; D037102:Lectins; D008970:Molecular Weight; D019699:Thrombospondins
D000596:Amino Acids; D006023:Glycoproteins; D037102:Lectins; D019699:Thrombospondins; D000001:Calcimycin; D005340:Fibrinogen