Kinetics of the formation of thrombin-thrombospondin complexes: involvement of a 77-kDa intermediate.
Thrombin forms sodium dodecyl sulfate stable complexes of 77 and greater than 450 kDa with proteins secreted by activated platelets. The kinetics of formation of these complexes were investigated by addition of 125I-thrombin to the supernatant solution of A23187-activated platelets. Complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis either with or without reduction of disulfide bonds. When analyzed on nonreduced gels, the 77-kDa complex reached a maximum at about 3 min and then declined as the greater than 450-kDa complex increased. On reduced gels (on which there was no greater than 450-kDa complex) the 77-kDa complex approached the level of the greater than 450-kDa complex on nonreduced gels. The half-time of formation was less than 1 min for the 77-kDa complex and about 15 min for the greater than 450-kDa complex. These time courses suggested that the 77-kDa complex was incorporated into the greater than 450-kDa complex as an essential precursor. Formation of complexes was inhibited by a competitive inhibitor or a noncompetitive inhibitor of thrombin, and the pH dependence of formation of both complexes was similar to the pH dependence for catalytic activity of thrombin. Ca2+ inhibited formation of the greater than 450-kDa complex but not of the 77-kDa complex. A model is presented in which thrombin and a secreted protein form a 77-kDa complex by a process that involves the active site of thrombin. The 77-kDa complex is then incorporated into a greater than 450-kDa complex by thiol-disulfide exchange with thrombospondin, a process that is inhibited by Ca2+. Thrombin in the greater than 450-kDa complex had no catalytic activity.
Browne
P C
PC
Department of Biochemistry, State University of New York Health Science Center, Brooklyn 11203.
Miller
J J
JJ
Detwiler
T C
TC
D001667:Binding, Competitive; D001792:Blood Platelets / Q000378:metabolism*; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010972:Platelet Activating Factor / Q000032:analysis*; D011498:Protein Precursors / Q000032:analysis*; D011956:Receptors, Cell Surface / Q000032:analysis; D018179:Receptors, Thrombin; D013917:Thrombin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019699:Thrombospondins
D006023:Glycoproteins; D010972:Platelet Activating Factor; D011498:Protein Precursors; D011956:Receptors, Cell Surface; D018179:Receptors, Thrombin; D019699:Thrombospondins; D000001:Calcimycin; D013917:Thrombin; D002118:Calcium