Expression and characterization of recombinant human factor V and a mutant lacking a major portion of the connecting region

Kane, William H.; Devore-Carter, Denise; Ortel, Thomas L.

doi:10.1021/bi00481a003

Cited by 52 publications

(68 citation statements)

References 47 publications

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“…Because the B-domain of FV has no crucial role in the APC-catalyzed inactivation of FVa, we used mutants lacking a major part of the B-domain in order to increase the yield of rFV mutants. The expression of these B-domainless mutants was about 10 times higher than that of the full-length constructs, which is in line with previous studies (35,40). Initial inactivation experiments with the purified re- FIG.…”

Section: Discussionsupporting

confidence: 90%

Factor Va Is Inactivated by Activated Protein C in the Absence of Cleavage Sites at Arg-306, Arg-506, and Arg-679

Kolfschoten

Dirven

Vos

et al. 2004

Journal of Biological Chemistry

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Section: Discussionsupporting

confidence: 90%

Factor Va Is Inactivated by Activated Protein C in the Absence of Cleavage Sites at Arg-306, Arg-506, and Arg-679

Kolfschoten

Dirven

Vos

et al. 2004

Journal of Biological Chemistry

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“…15,29 Studies using an FV derivative with a shortened B-domain (FVdes 811-1491 ; FV-810) support a model in which this region serves an inhibitory function by rendering binding sites on the heavy and/or light chain inaccessible to FXa and/or prothrombin. 16,30,31 Using a panel of progressively finer FV B-domain truncated variants, we also found that a key cluster of B-domain residues plays an important role in maintaining the inactive procofactor state. 17 Strikingly, part of this sequence cluster (963-1008) is unusually basic (18 of 46 residues are Arg or Lys) and remarkably well conserved among mammals and even lower vertebrates, pointing to its functional importance.…”

Section: Discussionmentioning

confidence: 78%

Venom factor V from the common brown snake escapes hemostatic regulation through procoagulant adaptations

Bos

Boltz

Pierre

et al. 2009

Blood

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Venomous snakes produce an array of toxic compounds, including procoagulants to defend themselves and incapacitate prey. The Australian snake Pseudonaja textilis has a venom-derived prothrombin activator homologous to coagulation factors V (FV) and Xa (FXa). Here we show that the FV component (pt-FV) has unique biologic properties that subvert the normal regulatory restraints intended to restrict an unregulated procoagulant response. Unlike human FV, recombinant pt-FV is constitutively active and does not require proteolytic processing to function. Sequence comparisons show that it has shed a large portion of the central B-domain, including residues that stabilize the inactive procofactor state. Remarkably, pt-FV functions in the absence of anionic membranes as it binds snake-FXa with high affinity in solution. Furthermore, despite cleavage in the heavy chain, pt-FV is functionally resistant to activated protein C, an anticoagulant. We speculate this stability is the result of noncovalent interactions and/or a unique disulfide bond in pt-FV linking the heavy and light chains. Taken together, these findings provide a biochemical rationale for the strong procoagulant nature of venom prothrombinase. Furthermore, they illustrate how regulatory mechanisms designed to limit the hemostatic response can be uncoupled to provide a sustained, disseminated procoagulant stimulus for use as a biologic toxin. IntroductionThe circulation of blood coagulation factors as precursors and their localization to the site of vascular injury after activation are important factors that maintain normal hemostasis and restrict indiscriminate clotting. 1 Bypassing these regulatory paradigms can be life-threatening, yet organisms have evolved strategies that exploit these systems for a selective advantage. For example, the venom of numerous snake species comprises a diverse array of proteases that activate and overwhelm the host hemostatic system in an uncontrolled fashion. 2 Some of the most potent venoms come from the Australian elapid family, including Pseudonaja textilis, Oxyuranus microlepidotus, and Oxyuranus scutellatus. 3 Their venom is considered the most toxic in the world and is unusual as it contains 2 coagulation proteins: factor V (FV) and a factor Xa (FXa)-like enzyme. [4][5][6][7][8][9][10] These proteins make up approximately 20% to 40% of the total venom and form a powerful procoagulant complex. 5,7 This complex converts prothrombin to thrombin, and its activity is enhanced, to various degrees, by calcium and phospholipids, but not by the cofactor FVa. [4][5][6][7]11 Venom-derived FV from these snakes share approximately 44% homology with mammalian FV and have a similar domain structure (A1-A2-B-A3-C1-C2). 8,10,12 However, we were surprised to learn that their B-domains are remarkably short, approximately 46 versus approximately 800 residues in mammals. (Amino acid numbering for pt-FV is as follows: the mature sequence starts at ϩ1 and the remaining sequence is numbered consecutively to 1430. The pt-FV B-domain is defined ...

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“…1). We The recombinant Factor V deletion mutants used in this study were prepared from the Factor V cDNA (16) (Fig. 1).…”

Section: Introductionmentioning

confidence: 99%

Characterization of an acquired inhibitor to coagulation factor V. Antibody binding to the second C-type domain of factor V inhibits the binding of factor V to phosphatidylserine and neutralizes procoagulant activity.

Ortel

Quinn-Allen²,

Charles³

et al. 1992

J. Clin. Invest.

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Coagulation Factor V is an essential component of the prothrombinase complex, which activates the zymogen prothrombin to thrombin. A patient was described who developed a Factor V inhibitor that neutralized the procoagulant activity of Factor V and resulted in a fatal hemorrhagic diathesis (Coots, M. C., A. F. Muhleman, and H. I. Glueck. 1978. Am. J. Hematol. 4:193-206). This inhibitor was shown to be an IgG antibody that bound to the light chain of Factor V. Using a series of light chain deletion mutants, we have found that this antibody binds to the second C-type domain of the light chain. Both inhibitor IgG and Fab fragments rapidly neutralized the procoagulant activity ofFactor Va, implying that the neutralization resulted from specific binding to the C2 domain. We have previously demonstrated that deletion of the C2 domain results in loss of procoagulant activity, as well as loss of phosphatidylserine-specific binding. Confirming these results, both inhibitor IgG and Fab fragments interfered with phosphatidylserinespecific binding of Factor V. Conversely, preincubation of Factor Va with procoagulant phospholipids protected the cofactor from inactivation by the inhibitor. Our results suggest that this inhibitor neutralizes the procoagulant activity of Factor Va by interfering with the C2-mediated interaction with phospholipid surfaces, thereby disrupting formation of the prothrombinase complex. (J. Clin. Invest. 1992. 90:2340-2347

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Expression and characterization of recombinant human factor V and a mutant lacking a major portion of the connecting region

Cited by 52 publications

References 47 publications

Factor Va Is Inactivated by Activated Protein C in the Absence of Cleavage Sites at Arg-306, Arg-506, and Arg-679

Factor Va Is Inactivated by Activated Protein C in the Absence of Cleavage Sites at Arg-306, Arg-506, and Arg-679

Venom factor V from the common brown snake escapes hemostatic regulation through procoagulant adaptations

Characterization of an acquired inhibitor to coagulation factor V. Antibody binding to the second C-type domain of factor V inhibits the binding of factor V to phosphatidylserine and neutralizes procoagulant activity.

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