On the location in the thrombin B chain of substrate recognition sites for fibrinopeptide release and factor XIII activation.
Thrombin, a serine proteinase comprised of two disulfide-linked subunits (A chain and B chain), induces clotting by releasing fibrinopeptide A from fibrinogen and then influences the character of the resulting fibrin by releasing fibrinopeptide B and by activating factor XIII. While the active center of thrombin is known to reside in its B chain, the subunit location of the structural determinants that govern the specific release of fibrinopeptides A and B and the activation of factor XIII have not been established. We have investigated the subunit location within the thrombin molecule of the determinants of substrate specificity for these actions using an isolated, immobilized B-chain preparation. Isolated B chain was prepared by covalently linking the intact thrombin molecule to Sepharose beads via the carbohydrate chain attached to asparagine 53 of its B chain, then reducing the single interchain disulfide bond to release the A chain, and finally reoxidizing the intrachain disulfide bonds of the immobilized B chain, allowing it to refold. The isolated, immobilized B chain of thrombin induced clotting of purified fibrinogen, releasing both fibrinopeptides A and B as demonstrated by HPLC and by electrophoresis of reduced fibrin chains. In addition, the B-chain preparation activated added factor XIII, yielding electrophoretically characteristic cross-linked fibrin chains.
Pirkle
H
H
Department of Pathology, University of California, Irvine 92717.
Theodor
I
I
Christofferson
M
M
Vukasin
P
P
Miyada
D
D
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D004800:Enzymes, Immobilized; D005176:Factor XIII / Q000378:metabolism*; D005340:Fibrinogen / Q000378:metabolism*; D005344:Fibrinopeptide A / Q000378:metabolism*; D005345:Fibrinopeptide B / Q000378:metabolism*; D066298:In Vitro Techniques; D013379:Substrate Specificity; D013917:Thrombin / Q000502:physiology*
D004800:Enzymes, Immobilized; D005344:Fibrinopeptide A; D005345:Fibrinopeptide B; D005340:Fibrinogen; D005176:Factor XIII; D013917:Thrombin