Thrombin binding to the A alpha-, B beta-, and gamma-chains of fibrinogen and to their remnants contained in fragment E.
In order to study thrombin interaction with fibrinogen, thrombin binding to fragments D and E (prepared by plasmin digestion of fibrinogen) and to intact S-carboxymethylated chains of fibrinogen (A alpha, B beta, and gamma) was analyzed by autoradiography, immunoblotting, and affinity chromatography. Complex formation was observed between late fragment E and thrombin but not with fragment D. The three reduced chain remnants of fragment E all formed complexes with thrombin. Also, thrombin bound to the intact, separated A alpha, B beta, and gamma chains of fibrinogen as well as to the alpha and beta chains of fibrin. In these experiments the extended substrate-binding site, but not the catalytic-binding site, was being examined because fragment E had as its amino-terminal amino acids Val20 in the alpha chain, Lys54 in the beta chain, and Tyr1 in the gamma chain. Also, thrombin inhibited in its active center by D-phenyl-alanyl-L-prolyl-L-arginine-chloromethyl ketone bound to fragment E and to the separated chains in the same manner as unmodified thrombin. A lysine residue to thrombin was essential for its binding to fibrinogen. Thrombin attached to CNBr-activated Sepharose through its amino groups did not bind to fragment E, but when thrombin was attached through its carboxyl groups, it bound fragment E.
Department of Pathology, Beth Israel Hospital, Boston, Massachusetts.
E
E
Kaczmarek
J
J
McDonagh
D000595:Amino Acid Sequence; D001345:Autoradiography; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005338:Fibrin Fibrinogen Degradation Products / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism*; D006801:Humans; D007118:Immunoassay; D008969:Molecular Sequence Data; D011485:Protein Binding; D013917:Thrombin / Q000378:metabolism*
D005338:Fibrin Fibrinogen Degradation Products; C015214:fibrinogen D fragment; C018543:fibrinogen fragment E; D005340:Fibrinogen; D013917:Thrombin