Further Evidence for Two Functional Forms of Prothrombinase Each Specific for Either of the Two Prothrombin Activation Cleavages

Abstract

Previous work showed that prothrombin derivatives cleavable only at Arg-320 (rMZ) or Arg-271 (rP2) are partial, rather than competitive, inhibitors of prothrombin activation by prothrombinase. A "ping-pong"-like model, which posits two equilibrating forms of prothrombinase, explained the inhibition pattern. The present studies were undertaken to further investigate this putative mechanism. Two models were developed, one allowing for one form of the enzyme and the other allowing for two forms. Both models also allowed channeling and ratcheting. The models were fit to full time courses of prothrombin, meizothrombin, prethrombin-2, and the B-chain. In the absence of ratcheting and channeling, neither model fits the data. In their presence, however, both models fit very well, and thus they could not be distinguished. Therefore, inhibition of rMZ activation by rP2 was studied. Inhibition was partial and the two-form model fit the data with randomly distributed residuals, whereas the oneform model did not. Initial rates of fluorescein-labeled prothrombin cleavage in the presence of various prothrombin derivatives reported by Brufatto and Nesheim (Brufatto, N., and Nesheim, M. E. (2003) J. Biol. Chem. 278, 6755-6764) were also analyzed using the two models. The two-form model fit the partial inhibition data well, whereas the one-form model did not. In addition, prothrombin at varying concentrations was activated, and subsequently, the initial rates were plotted with respect to the initial prothrombin concentration. When compared with the expected initial rates as determined by the simulation of the models, the two-form model fit the observed rates better than the one-form model. The results obtained here further support the existence of two functional forms of prothrombinase.

Restricted Active Site Docking by Enzyme-bound Substrate Enforces the Ordered Cleavage of Prothrombin by Prothrombinase

“…In contrast to the interpretations outlined here, it has alternately been proposed that the recognition and cleavage of the two bonds in prothrombin is accomplished by two different isoforms of prothrombinase that are differentially specific for the two sites (10,11). This alternate interpretation, which requires that the affinity of prothrombinase for the substrate is cleavage site-dependent, is inconsistent with a series of kinetic findings and with the fact that II QQ is a classical competitive inhibitor of all possible half-reactions of prothrombin activation (12).…”

“…Meizothrombin (mIIa), 3 produced as the sole detectable intermediate, accumulates transiently at concentrations in vast excess over the concentration of enzyme and cleavage of the two bonds in the opposite order is below experimental limits of detection (9,10). Although recent studies indicate that some intermediate produced via the alternate pathway can be detected (11), it is clear that both cleavage sites in prothrombin are accessible to externally added proteinase, yet prothrombinase preferentially rec-* This work was supported by National Institutes of Health Grants HL-74124 (to S. K., and R. M. C.), HL-47465 (to S. K.), HL-038779 (to P. E. B.…”

Meizothrombin Is an Unexpectedly Zymogen-like Variant of Thrombin

“…Kinetic modeling studies have led to the proposal that both ratcheting of substrate and two interconverting forms of prothrombinase are responsible for the two cleavage reactions (57). The rate constants proposed for a possible ratcheting step are Ͼ20-fold slower than those shown by the present direct measurement and represented as unidirectional (57).…”

“…Kinetic modeling studies have led to the proposal that both ratcheting of substrate and two interconverting forms of prothrombinase are responsible for the two cleavage reactions (57). The rate constants proposed for a possible ratcheting step are Ͼ20-fold slower than those shown by the present direct measurement and represented as unidirectional (57). Alternatively, others have argued that the equilibrium between E* and E detected in Na ϩ binding studies with mIIa⌬F1 need to be accounted for in explaining the pathway of prothrombin activation (51).…”

Meizothrombin Preparation and Its Role in Fibrin Formation and Platelet Aggregation

“…Active centre formed in molecule of produced MT. As was shown previously, during the first minute of clotting system activation in vitro levels of both FIIa and MT are equal and can reach 0.8 μM [2]. Moreover, MT can bind to platelet membrane [8], so local concentration of MT near platelet surface may be significantly increased during the initial activation stage.…”