COMPLEX FORMATION,BETWEEN THROMBIN AND FIBRINOGEN OR FIBRINOGEN DEGRADATION PRODUCTS (FDP)

Abstract

To identify the part of the fibrinogen molecule which interacts with thrombin binding of human thrombin to plasmic FDP was analyzed.125I-thrombin was incubated with FDP, purified fibrinogen fragment D or fragment E in the presence of 0.2% glutaraldehyde. Incubation mixtures were analyzed by SDS-PAGE and autoradiography. Under non-reducing conditions, the autoradiogram from the thrombin and fibrinogen fragment D incubation showed only one dark band, the molecular weight (Mr) of which was identical to that of thrombin, indicating no complex formation between thrombin and fragment D. With thrombin and fibrinogen fragment E, two dark bands were observed: the electrophoretic mobility of the first was the same as that of thrombin and the Mr of the second was equal to the sum of the Mr of thrombin and fragment E. This shows that human thrombin Forms a complex with fibrinogen fragment E. Hence, we can conclude that only the N-terminal part of the fibrinogen molecule is necessary for interaction with thrombin. Under reducing conditions, the complex of thrombin with fragment E produced four bands on gel electrophoresis. One was thrombin; the remaining three were complexes of thrombin with fragment E chain remnants. To investigate this further, carboxymethylated human fibrinogen chains Aα, Bβ and γ were purified and coupled to Sepharose 4B. 125I-thrombin was applied on the three columns. Nearly all radioactivity was bound to the three affinity columns and was eluted with higher NaCl concentration. We can infer that complex formation between thrombin and fibrinogen requires interaction between thrombin and all three fibrinogen chains. To find which thrombin amino acid residues are responsible for interaction with fibrinogen, human thrombin was coupled to Affi-Gel 102 and Affi-Gel 202 through thrombin's carboxyl and amino groups, respectively. We observed binding of fibrinogen and fibrinogen fragment E only to Affi-Gel 102 column, indicating that lysine residues and perhaps the N-terminal of the thrombin molecule interact with fibrinogen. When thrombin was bound to the gel through its amino groups, there was no interaction between thrombin and fibrinogen or fragment E.