Thrombin-mediated Proteolysis of Factor V Resulting in Gradual B-domain Release and Exposure of the Factor Xa-binding Site

Steen, Mårten; Dahlbäck, Björn

doi:10.1074/jbc.m204972200

Cited by 45 publications

(60 citation statements)

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“…The enzyme (E) interacts with prothrombin (P) to form either complex (E⅐P) 1 , from which meizothrombin (M) is produced, or complex (E⅐P) 2 , from which prethrombin-2 (P2) is produced. The formations of (E⅐P) 1 and (E⅐P) 2 1 , and k C2 for (E⅐P) 2 .…”

Section: Sds-page Time Course Analysis Of Prothrombinmentioning

confidence: 99%

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

Kim

Nesheim

2007

Journal of Biological Chemistry

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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.

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Section: Sds-page Time Course Analysis Of Prothrombinmentioning

confidence: 99%

“…Prothrombinase is a multicomponent enzymatic complex that catalyzes the conversion of prothrombin to the blood clotting enzyme thrombin (1)(2)(3)(4). It is composed of the serine protease factor Xa, the cofactor factor Va, a negatively charged phospholipid surface, and calcium ion.…”

mentioning

confidence: 99%

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

Kim

Nesheim

2007

Journal of Biological Chemistry

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“…This is in stark contrast to fully activated FVa that in the PTase complex binds FXa with very high (sub‐nM) affinity 8. The three cleavage sites in FV have different sensitivity to thrombin and FXa, R709 > R1018 > R1545, and intermediates are generated with partial FXa cofactor activity 7. Full activation requires the cleavage at R1545, which results in the release of the B domain and exposure of the FXa‐binding site 7, 8.…”

Section: Introductionmentioning

confidence: 96%

“…In its unactivated circulating form, FV has very low affinity for FXa and no procoagulant activity 7. This is in stark contrast to fully activated FVa that in the PTase complex binds FXa with very high (sub‐nM) affinity 8.…”

Section: Introductionmentioning

confidence: 99%

“…The three cleavage sites in FV have different sensitivity to thrombin and FXa, R709 > R1018 > R1545, and intermediates are generated with partial FXa cofactor activity 7. Full activation requires the cleavage at R1545, which results in the release of the B domain and exposure of the FXa‐binding site 7, 8. The large B domain comprises two highly charged regions that are shown to be important for keeping FV in a procofactor state 1.…”

Section: Introductionmentioning

confidence: 99%

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Factor V‐short and protein S as synergistic tissue factor pathway inhibitor (TFPIα) cofactors

Dahlbäck

Guo

Livaja-Koshiar

et al. 2018

Research and Practice in Thrombosis and Haemostasis

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Essentials FV‐Short, a normal splice isoform of Factor V, binds tissue factor pathway inhibitor (TFPIα) with high affinity.FV‐Short functions as a synergistic TFPIα cofactor with protein S in inhibition of Factor Xa.FV‐Short is much more efficient as TFPIα cofactor than full length FV.TFPIα‐cofactor activity of FV‐Short is lost upon activation of coagulation by thrombin‐mediated cleavage. Background FV‐Short is a normal splice variant of Factor V (FV) having a short B domain, which exposes a high affinity‐binding site for tissue factor pathway inhibitor α (TFPIα). FV‐Short and TFPIα circulate in complex in plasma.ObjectivesThe aim was to elucidate whether FV‐Short affects TFPIα as inhibitor of coagulation FXa and to test whether the TFPIα‐cofactor activity of protein S is influenced by FV‐Short.MethodsRecombinant FV, wild‐type FV‐Short and a FV‐Short thrombin‐cleavage resistant variant were expressed and purified. The influence of FV and FV‐Short variants and/or protein S on the FXa inhibitory activity of TFPIα was monitored both in a purified system and in a plasma‐based thrombin generation assay.Results FV‐Short had intrinsically weak TFPIα‐cofactor activity but with protein S present, FV‐Short yielded efficient inactivation of FXa. Protein S alone did not promote full TFPIα‐activity. Intact FV was inefficient at low protein S concentrations and had 10‐fold lower activity compared to FV‐Short at physiological protein S levels. Activation of FV‐Short by thrombin resulted in the loss of the TFPIα‐cofactor activity. The synergistic TFPIα‐cofactor activity of FV‐Short and protein S was also demonstrated in plasma using a thrombin generation assay.Conclusions FV‐Short and protein S are highly efficient, synergistic cofactors to TFPIα in the regulation of FXa activity, whereas full length FV has lower activity. Our results suggest the formation of an efficient FXa‐inhibitory complex between FV‐Short, TFPIα and protein S on the surface of negatively charged phospholipids.

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Structural models of the snake venom factor V activators from Daboia russelli and Daboia lebetina

Segers¹,

Rosing²,

Nicolaes³

2006

Proteins

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Blood coagulation factor V (FV) is a multifunctional protein that circulates in human plasma as a precursor molecule which can be activated by thrombin or activated factor X (FXa) in order to express its cofactor activity in prothrombin activation. FV activation is achieved by limited proteolysis after Arg709, Arg1018, and Arg1545 in the FV molecule. The venoms of Daboia russelli and Daboia lebetina contain a serine protease that specifically activates FV by a single cleavage at Arg1545. We have predicted the three-dimensional structure of these enzymes using comparative protein modeling techniques. The plasminogen activator from Agkistrodon acutus, which shows a high degree of homology with the venom FV activators and for which a high-quality crystallographic structure is available, was used as the molecular template. The RVV-V and LVV-V models provide for the first time a detailed and accurate structure of a snake venom FV activator and explain the observed sensitivity or resistance toward a number of serine protease inhibitors. Finally, electrostatic potential calculations show that two positively charged surface patches are present on opposite sides of the active site. We propose that both FV activators achieve their exquisite substrate specificity for the Arg1545 site via interactions between these exosites and FV.

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Thrombin-mediated Proteolysis of Factor V Resulting in Gradual B-domain Release and Exposure of the Factor Xa-binding Site

Cited by 45 publications

References 45 publications

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

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

Factor V‐short and protein S as synergistic tissue factor pathway inhibitor (TFPIα) cofactors

Structural models of the snake venom factor V activators from Daboia russelli and Daboia lebetina

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