The Contribution of Amino Acid Region Asp695-Tyr698 of Factor V to Procofactor Activation and Factor Va Function

Beck, Daniel O.; Bukys, Michael A.; Singh, Lisam Shanjukumar; Szabo, Katalin A.; Kalafatis, Michael

doi:10.1074/jbc.m306850200

Cited by 43 publications

(81 citation statements)

References 82 publications

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“…The C-terminal region of the A2 domain of factor V also contains two acidic sequences within residues 659 -698 (60). A recent study demonstrated that a pentapeptide to an acidic segment comprising factor V residues 695-699 blocked its activation by thrombin (61). That study also demonstrated that mutations within this sequence yielded a factor V molecule that was partially resistant to thrombin-but not factor Xa-catalyzed activation.…”

Section: Discussionmentioning

confidence: 75%

Exosite-interactive Regions in the A1 and A2 Domains of Factor VIII Facilitate Thrombin-catalyzed Cleavage of Heavy Chain

Nogami

Zhou

Myles

et al. 2005

Journal of Biological Chemistry

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Thrombin catalyzes the proteolytic activation of factor VIII, cleaving two sites in the heavy chain and one site in the light chain of the procofactor. Evaluation of thrombin binding the reaction products from heavy chain cleavage by steady state fluorescence energy transfer using a fluorophore-labeled, active site-modified thrombin as well as by solid phase binding assays using a thrombin Ser 205 3 Ala mutant indicated a high affinity site in the A1 subunit (K d ϳ5 nM) that was dependent upon the Na ؉ -bound form of thrombin, whereas a moderate affinity site in the A2 subunit (K d ϳ100 nM) was observed for both Na ؉ -bound and -free forms. The solid phase assay also indicated that hirudin blocked thrombin interaction with the A1 subunit and had little, if any, effect on its interaction with the A2 subunit. Conversely, heparin blocked thrombin interaction with the A2 subunit and showed a marginal effect on A1 binding. Evaluation of the A2 sequence revealed two regions rich in acidic residues that are localized close to the N and C termini of this domain. Peptides encompassing these clustered acidic regions, residues 373-395 and 719 -740, blocked thrombin cleavage of the isolated heavy chain at Arg 372 and Arg 740 and inhibited A2 binding to thrombin Ser 205 3 Ala, suggesting that both A2 domain regions potentially support interaction with thrombin. A B-domainless, factor VIII double mutant Asp 392 3 Ala/Asp 394 3 Ala was constructed, expressed, and purified and possessed specific activity equivalent to a severe hemophilia phenotype. This mutant was resistant to cleavage at Arg 740 , whereas cleavage at Arg 372 was not affected. These data suggest the acidic region comprising residues 389 -394 in factor VIII A2 domain interacts with thrombin via its heparin-binding exosite and facilitates cleavage at Arg 740 during procofactor activation.

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Section: Discussionmentioning

confidence: 75%

Exosite-interactive Regions in the A1 and A2 Domains of Factor VIII Facilitate Thrombin-catalyzed Cleavage of Heavy Chain

Nogami

Zhou

Myles

et al. 2005

Journal of Biological Chemistry

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“…Previous biochemical and mutagenesis data have indicated that the complementary substrate interactive site on factor Va may reside in a hirudin-like sequence within the C-terminal heavy chain of the cofactor involving residues 659 -698 (22)(23)(24). In support of this hypothesis, the recent mutagenesis of the 695 DYDY 698 motif of the factor Va heavy chain dramatically impaired the cofactor function of factor Va in the prothrombinase complex (14). Neither the sequence of the activator nor the cofactor of taipan venom has been determined to understand whether the same substrate interactive site is also conserved in the venom cofactor.…”

Section: Discussionmentioning

confidence: 90%

“…Thus, the charge reversal mutants of proexosite-1 were activated normally by FXa alone, but their activation by the protease in the presence of factor Va was dramatically impaired (13). Further support for a direct interaction between basic residues of proexosite-1 with a complementary site of factor Va was provided by the observation that the mutagenesis of a hirudin-like sequence on the C-terminal heavy chain of factor Va dramatically compromised the cofactor function of factor Va in accelerating the FXa activation of prothrombin by the prothrombinase complex (14).…”

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confidence: 80%

Proexosite-1-dependent Recognition and Activation of Prothrombin by Taipan Venom

Chen

Rezaie

2004

Journal of Biological Chemistry

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An activator complex from the venom of Oxyuranus scutellatus scutellatus (taipan venom) is known to rapidly activate prothrombin to thrombin. To determine whether, similar to prothrombinase, taipan venom utilizes proexosite-1 on prothrombin for a productive complex assembly, the activation of proexosite-1 mutants of prethrombin-1 by the partially purified venom was studied. It was discovered that basic residues of this site (Arg 35 Interestingly, an ϳ50-kDa activator, isolated from the taipan venom complex, catalyzed the activation of prothrombin in a factor Va-dependent manner and exhibited identical activation kinetics toward the substrate in the presence of the hirudin peptide. These results suggest that, similar to prothrombinase, proexosite-1 is a cofactor-dependent recognition site for taipan venom.Prothrombin is a vitamin K-dependent serine protease zymogen that can be proteolytically converted to thrombin by factor Xa (FXa) 1 that has been assembled into the prothrombinase complex (cofactor Va, negatively charged phospholipid vesicles and Ca 2ϩ ) (1-5). Factor Xa must cleave two peptide bonds at the P1 Arg 273 and P1 Arg 322 sites to convert prothrombin to thrombin (1). Although FXa can by itself catalyze the cleavage of both peptide bonds on the substrate, its catalytic efficiency is improved by greater than 5 orders of magnitude when it is assembled into the prothrombinase complex (1-5). Previous kinetic data have indicated that such a dramatic improvement in the rate of prothrombin activation by the prothrombinase complex is derived from an ϳ100-fold decrease in the apparent K m and a greater than 1000-fold enhancement in the k cat of the activation reaction (2, 6). In addition to FXa, several activators from different snake venoms have been isolated that can also cleave the two peptide bonds on prothrombin to generate thrombin (7-11). One such an activator, which also exhibits FXa-like properties, has been isolated from the venom of Oxyuranus scutellatus scutellatus (taipan venom) (7,8,10). Similar to FXa, it is previously shown that the N terminus of the taipan venom activator contains several ␥-carboxyglutamic acid residues that can interact with negatively charged membrane surfaces in the presence of Ca 2ϩ (10). Moreover, similar to FXa, the venom activator is by itself a very poor activator of prothrombin unless it is in complex with a venomic protein cofactor containing an apparent molecular mass of ϳ220 kDa (10). Similar to factor Va, the snake venom cofactor dramatically accelerates the activation of prothrombin by the taipan venom activator on negatively charged membrane surfaces (10).The mechanism by which factor Va improves the catalytic efficiency of FXa in the prothrombinase complex is not very well understood. However, it was recently demonstrated that the interaction of proexosite-1 of prothrombin with a recognition site on the prothrombinase complex is required for the efficient activation of the substrate (12). Thus, a proexosite-1-specific peptide ligand derived from the C-terminal...

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“…On the basis of extensive investigations, the prothrombin complex is known to consist of phospholipid surface, prothrombin, Ca 2+ and factor Va (FVa). (8)(9)(10)(11)(12) This construct facilitates the activation of prothrombin by ∼3 × 10 5 -fold over catalysis by FXa alone. (13;14) The activation of human prothrombin involves the hydrolysis of two peptide bonds, Arg 322 -Ile 323 followed by Arg 273 -Thr 274 .…”

Section: Introductionmentioning

confidence: 99%

“…(10) Both chains are required for the interaction with FXa, but only the heavy chain binds prothrombin without Ca 2+ . (11) The binding sites for FXa and prothrombin have been located in the middle section (11) and the COOH terminus (12) of the heavy chain of FVa, respectively. (15) In fact, an exosite of FXa, which is exposed upon interaction with FVa for prothrombin tethering, seems to contain amino acids near the active site of the enzyme.…”

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confidence: 99%

Deuterium Solvent Isotope Effect and Proton-Inventory Studies of Factor Xa-Catalyzed Reactions

Zhang

Kovach

2006

Biochemistry

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Kinetic solvent isotope effects (KSIE) for the Factor Xa (FXa)-catalyzed activation of prothrombin in the presence and absence of Factor Va (FVa) and 5.0 ×10 −5 M phospholipid vesicles, are slightly inverse, 0.82−0.93, when substrate concentrations are at 0.2 K m . This is consistent with ratedetermining association of the enzyme-prothrombin assembly, rather than rate-limiting chemical transformation. FVa is known to effect a major conformational change to expose the first scissile bond in prothrombin, which is the likely event triggering a major solvent rearrangement. At prothrombin concentrations >5 K m the KSIE is 1.6 ± 0.3, when FXa is in 1 : 1 ratio with FVa, but becomes increasingly inverse, 0.30 ± 0.05 and 0.19 ± 0.04, when FXa : FVa 1:4, with increasing FXa concentration and substrate concentration. The rate-determining step changes with the conditions, but the chemical step is not limiting under any circumstance. This corroborates the proposed predominance of the meizothrombin pathway when FXa is well saturated with the prothrombin complex. In contrast, the FXa-catalyzed hydrolysis of N-α-Z-D-Arg-Gly-ArgpNA·2HCl (S-2765) and H-D-Ile-L-Pro-L-Arg-pNA.HCl (S-2288) is most consistent with twoproton bridges forming at the transition state between Ser 195 OγH and His 57 Nε2 and His 57 Nδ1 and Asp 102 COOβ-at the active site, with transition state fractionation factors of ϕ 1 = ϕ 2 = 0.57 ± 0.07 and ϕ S = 0.78 ± 0.16 for solvent rearrangement for S-2765, and ϕ 1 = ϕ 2 = 0.674 ± 0.001 for S-2288 under enzyme saturation with substrate at pH 8.40 and 25.0 ± 0.1 °C. The rate-determining step(s) in these reactions is most likely the cleavage of the C-N bond and departure of the leaving group.Factor Xa (FXa) is one of the best characterized of the serine proteases in the cardiovascular system.(1-6) It catalyzes the efficient activation of a large precursor protein, prothrombin, to form α-thrombin.(1;7;8) Although several proteolytic enzymes can bring about this reaction, it is FXa that performs this key function under physiological conditions. On the basis of extensive investigations, the prothrombin complex is known to consist of phospholipid surface, prothrombin, Ca 2+ and factor Va (FVa). (8)(9)(10)(11)(12) This construct facilitates the activation of prothrombin by ∼3 × 10 5 -fold over catalysis by FXa alone.(13;14) The activation of human prothrombin involves the hydrolysis of two peptide bonds, Arg 322 -Ile 323 followed by Arg 273 -Thr 274 . In contrast, when FXa acts alone, bond cleavage occurs in the opposite order. Different intermediates are formed in the two pathways that have also been studied independently.(9; 14) More in-depth kinetic studies lately revealed that an appreciable fraction of prothrombin is channeled directly to thrombin product without releasing intermediate products. (14)

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The Contribution of Amino Acid Region Asp695-Tyr698 of Factor V to Procofactor Activation and Factor Va Function

Cited by 43 publications

References 82 publications

Exosite-interactive Regions in the A1 and A2 Domains of Factor VIII Facilitate Thrombin-catalyzed Cleavage of Heavy Chain

Exosite-interactive Regions in the A1 and A2 Domains of Factor VIII Facilitate Thrombin-catalyzed Cleavage of Heavy Chain

Proexosite-1-dependent Recognition and Activation of Prothrombin by Taipan Venom

Deuterium Solvent Isotope Effect and Proton-Inventory Studies of Factor Xa-Catalyzed Reactions

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