Generation and Mechanism of Action of a Potent Inhibitor of Factor XIII Function

Abstract

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.

The Contribution of Activated Factor XIII to Fibrinolytic Resistance in Experimental Pulmonary Embolism

“…We used a monoclonal antibody (9C11), which is capable of quenching all factor XIIIa-mediated cross-linking, 28 and verified that during clotting it completely inhibited both fibrin ␥-chain cross-linking and ␣2-antiplasmin-fibrin crosslinking. To selectively inhibit only factor XIIIa-mediated ␣2-antiplasmin-fibrin cross-linking, we used a peptide corresponding to the amino terminus of human ␣2-antiplasmin, which contains the cross-linking site.…”

“…28 The hybridoma producing 9C11 was cloned by limiting dilution 28 and expanded into ascites in pristane-primed BALB/c mice. Antibody was purified from filtered ascites by precipitation with 40% ammonium sulfate.…”

“…36 In previous studies, we derived a monoclonal antibody (9C11) that fully inhibits all factor XIIIa-mediated cross-linking in primate plasmas. 28 To determine the amount of 9C11 required for inhibiting factor XIII activity in the present studies, we examined its dose-related effects on fibrin-fibrin ␥ chain and ␣2-antiplasmin-fibrin cross-linking during clotting. In comparison with clots formed in the absence of inhibitor or in the presence of the nonspecific alkylating agent iodoacetamide, clots formed with 9C11 at doses of 5 and 10 g/clot showed no significant fibrin-fibrin ␥ chain (Figure 1, top panel) or ␣2-antiplasminfibrin cross-linking (Figure 1, bottom panel).…”

Transglutaminases as Targets for Pharmacological Inhibition

“…Pursuing this approach, Reed and coworkers [208] developed Mab 9C11, a murine monoclonal antibody against human platelet FXIII. The antibody reacted specifically with both plasma and platelet FXIII but it did not bind well to SDS-denatured FXIII indicating that it recognized a conformationally determined epitope.…”

Sol Sherry Lecture in Thrombosis

“…[81][82][83] Positive results were obtained with a monoclonal antibody directed against the thrombin cleavage site of factor XIII, which blocked the activation of the zymogen. 84 Another interesting approach utilizes a monoclonal antibody against ␣ 2 PI. 85 As mentioned before, the factor XIIIa-catalyzed covalent attachment of ␣ 2 PI to fibrin significantly contributes to lytic resistance.…”