As the final enzyme in the coagulation cascade, activated fibrin stabilizing factor or factor XIII catalyzes the intermolecular cross-linking of fibrin chains. To study this enzyme in plasma, we derived a monoclonal antibody (MAb 309) against a peptide sequence (NH2-G-V-N-L-Q-E-F-C-COOH) in the thrombin activation site of factor XIII. Radioimmunoassays indicate that MAb 309 binds specifically to both platelet and plasma factor XIII. Peptide inhibition studies demonstrate that the MAb binds equally well to the factor XIII (FXIII) zymogen and the active form of FXIII (FXIIIa). In immunoblots of whole platelet lysates, MAb 309 binds only to FXIII and does not cross-react with other proteins. In saturation binding studies, the antibody shows a binding avidity of (1.75 +/- 0.35) x 10(9) M-1. MAb 309 also inhibited 99% of apparent FXIIIa activity in a standard transglutaminase assay. SDS-PAGE analysis of fibrin clots showed that MAb 309 inhibited fibrin gamma-gamma cross-linking. Moreover, MAb 309 accelerated the lysis of plasma clots, consistent with inhibition of fibrin-fibrin and fibrin-alpha 2-antiplasmin cross-linking. Immunoblotting experiments revealed that MAb 309 affected apparent FXIIIa activity by inhibiting the thrombin activation of the FXIII zymogen. In addition to its utility as a specific probe for the FXIII a-subunit, the strategy used to obtain MAb 309 may be used to generate MAbs that inhibit the activation of other coagulation factor zymogens.
SummaryAlthough known to play a critical role in thrombosis, the precise role of factor XIII in other processes like wound healing and gestation remains to be elucidated. Because a specific, potent inhibitor could help define the function of factor XIII in these processes, or determine the potential benefits of factor XIII suppression in thrombotic disease, we have derived and characterized a monoclonal antibody inhibitor of factor XIII activation. This immunoinhibitor, 9C11, reacts specifically with both plasma and platelet forms of factor XIII. When added to human plasma, either as whole immunoglobulin or as Fab fragments, 9C11 completely prevented thrombin-activated factor XIII activity with small molecular weight substrates like 14C-putrescine. In addition, when clotted with plasma, 9C11 ablated the gamma chain crosslinking of fibrin catalyzed by factor XIII and markedly accelerated the fibrinolysis of plasma clots by urokinase. Studies of the mechanism of action showed that 9C11 inhibited the cleavage and activation of factor XIII by thrombin, but did not affect the catalytic function of previously activated factor XIII. Although it inhibited thrombin activation, experiments indicated that it bound comparably to both factor XIII zymogen and the thrombin-cleaved zymogen, and did not bind to the thrombin cleavage site of the molecule. Taken together these experiments indicated that 9C11 acted as an inhibitor of the thrombin cleavage and activation of the factor XIII A-subunit. Its potency and specificity make it a useful agent for studying thrombin-activatable factor XIII function in biological systems.
SummaryTo facilitate the analysis of the catalytic subunit of factor XIII we have developed a method for the immunoaffinity purification of this protein from platelets. This method employs a monoclonal antibody that binds to the a-subunit of factor XIII. The anti-factor XIII antibody was immobilized on agarose and then incubated with platelet lysate. Subsequently factor XIII was isolated from the platelet lysate in a single step with a 41% yield as measured by enzyme assay. The purified platelet factor XIII appeared nearly homogeneous when analyzed by polyacrylamide electrophoresis and by immunoblotting with another factor XIII monoclonal antibody.
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