Destabilization of the Factor V B-Domain Results in Procofactor Activation.

Abstract A hallmark of hemostasis is that proteins involved in the formation of a blood clot remain in a quiescent state and are only activated following an appropriate stimulus. Blood coagulation factor V (FV), which is structurally homologous to FVIII, cannot function in the prothrombinase complex and is thus considered a procofactor. Thrombin catalyzes the conversion of FV to FVa following three cleavages (Arg709, Arg1018, and Arg1545) releasing a large heavily glycosylated central B-domain (836 amino acids). Explanations as to how bond cleavage or B-domain release facilitates the transition to the active species are incomplete. Recent studies using a partial B-domainless form of FV (FV-810 des811–1491) support a model in which removal of B-domain sequences from FV rather than specific proteolysis underlies the mechanism by which cofactor function is realized. This single-chain derivative is functionally equivalent to FVa suggesting that the deleted B-domain sequences somehow suppress cofactor activity. To investigate this further, we have expressed and purified several single-chain derivatives of FV that vary in B-domain length from 155 to 497 residues. Functional activity assays as well as direct binding fluorescent measurements revealed that elimination of most of the C-terminal half of the B-domain (residues 1034–1491; 458 out of 836 a.a. deleted) had no influence on maintaining the procofactor state. However, deletion of sequences from the N-terminal half of the B-domain resulted in derivatives with cofactor-like properties. Using progressively finer deletion variants we were able to demonstrate that either a B-domain length of at least 378 amino acids or specific sequences contained within residues 902–1033 is sufficient to suppress cofactor activity. To examine these possibilities, we constructed additional FV variants in which a B-domain length of 378 amino acids was maintained, but specific portions of 902–1033 were exchanged with FVIII B-domain. Using the FV-1033 derivative (residues 1034–1491 of B-domain deleted) as a scaffold, three constructs were prepared, s-131, s-104, and s-46, representing 131, 104, and 46 amino acids from the FV B-domain exchanged with FVIII B-domain. In activity assays and direct binding measurements, each of these variants had properties consistent with the cofactor-like form indicating that a length of ~375 residues is not sufficient to maintain the procofactor state. These findings demonstrate for the first time that there are indeed specific FV-B domain sequences between 902–1033 that directly or indirectly stabilize the procofactor state. Remarkably, simply replacing these sequences in FV-1033 resulted in activation of the proteins in the absence of proteolysis. These observations change existing ideas about FV activation and provide insight into specific regions of the B-domain that assist in preserving the procofactor state.

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Characterization of B-Domain Elements Which Maintain Factor V in a Procofactor Form.

Blood ◽

10.1182/blood.v104.11.1726.1726 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1726-1726

Author(s):

Rodney M. Camire ◽

Hua Zhu

Keyword(s):

Amino Acids ◽

Functional Properties ◽

Tandem Repeats ◽

Coagulation Factor ◽

High Yield ◽

Factor V ◽

Single Chain ◽

High Affinity ◽

Single Chain Fv ◽

Cofactor Activity

Abstract Blood coagulation factor V (FV) has little or no procoagulant activity and is activated by thrombin (IIa) to FVa following proteolytic removal of a central glycosylated B-domain (residues 710-1545; 836 a.a.). Discrete proteolysis is necessary for activation; however, recent data indicates that it is incidental to the mechanism by which cofactor function is realized. It was found that shortening the B-domain in the absence of bond cleavage to 155 amino acids also yielded an active cofactor form that assembles and functions in prothrombinase. These observations support the conclusion that macromolecular binding sites, which govern the eventual function of FVa, are concealed on FV by B-domain sequences. At present, it is unknown which elements of the B-domain contribute to maintaining the procofactor form. To begin to investigate this, we have designed eight recombinant derivatives of FV with variable amounts of the B-domain: FV-810 (des811–1491), FV-866 (des867–1491), FV-902 (des903–1491), FV-956 (des957–1491), FV-1033 (des1034–1491), FV-1053 (des1054–1491), FV-1106 (des1107–1491), and FV-1152 (des1153–1491). These constructs were stably expressed in BHK cells and purified to homogeneity in high yield (1–2 mg/L of media). The proteins migrated as single bands on SDS-PAGE, and were completely processed to FVa following incubation with IIa. Using either a purified prothrombinase assay with limiting amounts of (pro)cofactor or a one-stage PT-based clotting assay, we found that most of the derivatives were grouped into two categories: procofactor-like and cofactor-like. Factor V-810, FV-866, and FV-902 exhibited activity profiles consistent with the cofactor-like form and, as expected, bound with high affinity to FXa-membranes (Kd = 0.2–0.4 nM; n = 1). In contrast, FV-1033, FV-1053, FV-1106, and FV-1152 had very little activity and exhibited functional properties consistent with the procofactor-like form. Direct binding fluorescent measurements revealed that unlike FV, these constructs did appear to bind FXa-membranes, albeit with a much lower affinity compared to FVa. Interestingly, an outlier to either of the groups was FV-956 (301 a.a. B-domain). This derivative bound FXa-membranes with moderate affinity (Kd = 5 nM; n = 1) and it exhibited functional properties intermediate between the procofactor and cofactor forms in both the PT-based clotting and purified component assays. Control experiments indicated that all of the derivatives exhibited full cofactor activity and high affinity binding to FXa-membranes following treatment with IIa. Collectively, these data demonstrate that the single-chain cofactor-like forms can be shifted to the procofactor-like form by relatively small, systematic increases in the length of the B-domain. This transition was accompanied by a marked decrease in functional activity and FXa binding. Surprisingly, these data also show that elimination of >50% of the B-domain (1034–1491; 458 out of 836 a.a. deleted), a region that contains 31, nine amino acid tandem repeats and 16 out of 25 potential N-linked carbohydrate sites, has little, if any, influence on maintaining the procofactor form. Taken together, our data are consistent with the interpretation that a length of at least 375 amino acids or specific sequences contained within residues 902–1033 is sufficient to suppress cofactor activity of single chain FV.

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Mapping the Location of Autoinhibitory B-Domain Motifs within Factor V: New Insights Into How Factor V Activation Unfolds

Blood ◽

10.1182/blood.v120.21.495.495 ◽

2012 ◽

Vol 120 (21) ◽

pp. 495-495

Author(s):

Raffaella Toso ◽

Matthew W. Bunce ◽

Rodney M. Camire

Keyword(s):

Amino Acids ◽

Binding Site ◽

Heavy Chain ◽

3D Structure ◽

Regulatory Region ◽

Spatial Relationship ◽

Coagulation Factor ◽

Factor V ◽

High Affinity ◽

Cofactor Activity

Abstract Abstract 495 Blood coagulation factor V (FV) circulates as a procofactor with little or no procoagulant activity. Proteolytic removal of a large central B-domain converts FV (domain organization of A1-A2-B-A3-C1-C2) to an active cofactor for membrane-bound FXa to form prothrombinase. Recently we found that discrete, evolutionarily conserved sequences within the B-domain serve an autoinhibitory function and are necessary to maintain FV as an inactive procofactor. The autoinhibitory region within the B-domain (termed the procofactor regulatory region or PRR) is sequence specific and remarkably short (∼100 amino acids out of 836 from B-domain) consisting of basic (963–1008; basic region or BR) and acidic (1493–1537; acidic region or AR) sequences. Dismantling this region appears to be the driving force to unveil a high affinity binding site(s) for FXa and thus underpins the procofactor to cofactor transition. While the BR and AR seem to work together to provide “on-site” repression of cofactor activity, there is no structural information about the FV B-domain that could provide clues into how this critical region functions. Here we use B-domain fragments tethered to an artificial protease in order to map the position of the PRR relative to the rest of the FV molecule. A BR peptide with an introduced free Cys at position 990 was stoichiometrically modified with FeBABE (Fe-p-bromoacetamidobenzyl-EDTA). FeBABE is a protein cutting reagent with a sulfhydryl-reactive moiety for attachment to Cys and an EDTA-chelated iron atom which, when triggered with ascorbic acid and peroxide, generates hydroxyl radicals that cut peptide bonds in a sequence-independent fashion. When a protein labeled with FeBABE binds its target, the Fe-BABE moiety facilitates cutting if it is orientated correctly and near (<12 Å) the contact site of the target protein. In our experimental system, we employed a constitutively active B-domainless form of FV (FV-810) that harbors the AR but lacks the BR. Initial studies revealed that the BR peptide, without or tethered with FeBABE, binds to FV-810 with high affinity (nM) and inhibits cofactor activity. Proteolysis of FV-810 by BR-FeBABE, was visualized by Western blot, using monoclonal antibodies recognizing either the heavy or light chains. Using this approach, we consistently found that BR-FeBABE cut FV-810 predominantly at two sites. The first is at the C-terminal end of the B-domain. This region is enriched in acidic residues and represents the AR defined above. The second site of cleavage is within the heavy chain. While we cannot exclude the possibility that the site of contact is at the N-terminus of the A1 domain, based on the proposed 3D structure of FVa, it is more likely that the BR is binding near the C-terminal end of the A2 domain which is also enriched in acidic amino acids. Compared to a panel of FVa derivatives with variably truncated heavy chains, we suggest that BR-FeBABE cuts FV-810 near residues 675–685. Overall the data suggest that the B-domain must be folded in such a way as to place the BR in close spatial proximity to the AR at the C-terminal end of the B-domain and also to the C-terminal heavy chain region. Consistent with the idea that these interacting motifs work to keep FV as a procofactor by suppressing FXa binding, adding saturating amounts of FXa, but not the zymogen FX, prevented the BR-FeBABE induced cleavage of FV-810. Similarly, activated protein C (APC) which is thought to share a related binding site with FXa, also prevented BR-FeBABE from cutting FV-810. Addition of excess unlabeled BR peptide substantially reduced cutting of FV-810 by BR-FeBABE, indicating that proteolysis is not due to non-specific cleavage. Additionally, removing the basic charge of BR-FeBABE via acetylation eliminated binding to FV-810 and also any signs of specific cutting. Further, FV-derivatives harboring both the AR and BR in cis and hence not binding the BR peptide, were not cut by excess BR-FeBABE added in trans. In conclusion, these findings provide new insights into the spatial relationship between key regulatory B-domain motifs and the core heavy/light chain region. These interacting autoinhibitory motifs directly or indirectly obscure FXa binding and hence stabilize the inactive procofactor state of FV. These studies also document a remarkably powerful way to map the spatial relationship between regions of proteins and provide new information on coagulation factor binding sites. Disclosures: Camire: Pfizer: Patents & Royalties, Research Funding.

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Most Factor VIII B Domain Missense Mutations Are Unlikely to Be Causative Mutations for Hemophilia A: Implications for Factor VIII Genetic Analysis

Blood ◽

10.1182/blood.v112.11.513.513 ◽

2008 ◽

Vol 112 (11) ◽

pp. 513-513

Author(s):

Kyoichi Ogata ◽

Steven W. Pipe

Keyword(s):

Amino Acid ◽

Factor Viii ◽

Hemophilia A ◽

Coagulation Factor ◽

Causative Mutation ◽

Factor V ◽

Missense Mutations ◽

Severe Hemophilia ◽

Single Chain

Abstract Hemophilia A results from the quantitative or qualitative deficiency of coagulation factor VIII (FVIII). FVIII is synthesized as a single-chain polypeptide of approximately 280 kDa with the domain structure A1-A2-B-A3-C1-C2. Whereas the A and C domains exhibit ~40% amino acid identity to each other and to the A and C domains of coagulation factor V, the B domain is not homologous to any known protein and is dispensable for FVIII cofactor activity. Missense mutations in the FVIII B domain have been described in patients with variable phenotypes of hemophilia A. According to the NCBI SNPs (single nucleotide polymorphism) database, 22 SNPs are reported within FVIII, 11 of which occur within the B domain. FVIII B domain variant D1241E has been reported as a missense mutation associated with mild or severe hemophilia A, yet this mutation is also present in the NCBI SNPs database. We hypothesize that D1241E and most other reported B domain missense mutations are not the causative mutation for hemophilia A in these patients but represent SNPs or otherwise non-pathologic mutations. To investigate this, we analyzed 7 B domain missense mutations that were previously found in hemophilia A patients (T751S, V993L, H1047Y, D1241E, T1353A, P1641L and S1669L). Comparative analysis showed that the amino acids at these positions are not conserved in all species and in some cases, the amino acid substitution reported in hemophilia patients is represented in the native sequence in other species. Analysis with PolyPhen Software showed that only H1047Y mutation was considered as “possibly damaging”, while the others were considered as “benign”. To investigate this further, we constructed seven plasmid vectors containing these B domain missense mutations. The synthesis and secretion of FVIII wild-type (WT) and these seven mutants were compared after transient DNA transfection into COS-1 monkey cells in vitro. Analysis of the FVIII clotting activity and antigen levels in the conditioned medium demonstrated that all mutants had FVIII activity and antigen levels similar to FVIII WT. Further, FVIII WT, H1047Y and D1241E mutants were introduced into a FVIII exon 16 knock-out mouse model of hemophilia A by hydrodynamic tailvein injection in vivo. The mouse plasma was analyzed at 24 hrs for activity and antigen expression. Mutants H1047Y and D1241E expressed at 211 mU/mL and 224 mU/mL activity with FVIII antigen levels of 97 ng/mL and 118 ng/mL, respectively, similar to FVIII WT. These results suggested that H1047Y and D1241E mutants did not lead to impairments in secretion or functional activity. We conclude that most missense mutations within the FVIII B domain would be unlikely to lead to severe hemophilia A and that the majority of such missense mutations represent polymorphisms or non-pathologic mutations. Investigators should search for additional potentially causative mutations elsewhere within the FVIII gene when B domain missense mutations are identified.

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Conserved Structural Motifs Cooperate to Maintain Blood Coagulation Factor V in An Inactive Procofactor State.

Blood ◽

10.1182/blood.v114.22.850.850 ◽

2009 ◽

Vol 114 (22) ◽

pp. 850-850

Author(s):

Mettine H.A. Bos ◽

Rodney M. Camire

Keyword(s):

Amino Acids ◽

Blood Coagulation ◽

Coagulation Factor ◽

Factor V ◽

Vertebrate Lineage ◽

New Variant ◽

Coagulation Factor V ◽

Blood Coagulation Factor V ◽

Basic Region ◽

Constitutively Active

Abstract Abstract 850 Blood coagulation factor V (FV) is a multi-domain protein which circulates as an inactive procofactor and has high structural homology with factor VIII. To express procoagulant activity, FV must be proteolytically processed within its central B-domain (836 residues) with thrombin being considered the key physiological activator. Following liberation of the B-domain (residues 710-1545), activated FV (FVa) functions as a cofactor for factor Xa within the prothrombinase complex and dramatically enhances the rate of thrombin generation. The central role which FVa assumes in prothrombinase indicates that its activation must be a key regulatory step in hemostasis. Although the proteolytic events that lead to the activation of FV have been well studied, the molecular mechanism by which B-domain release facilitates the procofactor to cofactor transition is not well understood. Recently, we have shown that in the absence of intentional proteolysis, deletion or substitution of discrete B-domain sequences drives the expression of procoagulant function (JBC, 282, 15030-9, 2007). Conversion to the constitutively active cofactor state is related, at least in part, to a cluster of amino acids (963-1008) which is highly basic and well conserved, even though most of the B-domain has weak homology within the vertebrate lineage. In the current study, we examined if this basic B-domain region is sufficient to preserve FV as an inactive procofactor. To investigate this, the basic region (46 residues) was incorporated within the short B-domain of a previously characterized FV variant, FV-810. Factor V-810 has amino acids 811-1491 within the B-domain deleted and is a constitutively active cofactor, with functional properties equivalent to FVa. Using a PT-based clotting assay, purified prothrombinase assay, and direct fluorescent binding measurements with FXa-membranes we found that insertion of the basic region into FV-810 (inserted after residue 810) converted this cofactor-like species back to the procofactor-like state, despite >75% of the B-domain being absent. Next, using this new variant (FV+BR; B-domain of 201 residues), we assessed whether residual B-domain sequences within FV+BR contribute to maintaining FV in an inactive, procofactor state. Elimination of ∼100 residues on the N-terminal side of FV+BR was without functional consequence; that is, the procofactor state was maintained. In contrast, removal of B-domain sequences (∼50 residues, 30% of which are acidic) to the C-terminal side of the basic region shifted FV-810+BR from an inactive procofactor to an active cofactor. As expected, all purified FV derivatives exhibited full cofactor activity following treatment with thrombin. Together, these data show that B-domain sequences 963-1008 (basic region) appear to work in concert with the acidic C-terminal region of the B-domain (1492-1545) to keep FV in an inactive procofator state. These sequence elements appear to be necessary and sufficient as we were able to construct a FV variant with a B-domain length of only 103 amino acids that remarkably still had procofactor-like properties. Interestingly, these two regions of the B-domain (963-1008 and 1492-1545) are generally well conserved throughout the vertebrate lineage, while the remaining regions of the B-domain are not. We speculate that these B-domain sequences bind intramolecularly to heavy and/or light chain sequences thereby concealing critical binding sites on the FV molecule which govern the function of the active cofactor species. Disclosures: Camire: Wyeth: Patents & Royalties, Research Funding.

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The anticoagulant function of coagulation factor V

Thrombosis and Haemostasis ◽

10.1160/th11-06-0431 ◽

2012 ◽

Vol 107 (01) ◽

pp. 15-21 ◽

Cited By ~ 26

Author(s):

Thomas J. Cramer ◽

Andrew J. Gale

Keyword(s):

Protein C ◽

Activated Protein C ◽

Coagulation Factor ◽

Protein S ◽

Factor V ◽

Phospholipid Membrane ◽

Terminal Part ◽

Coagulation Factor V ◽

Cofactor Activity ◽

Anticoagulant Function

SummaryAlmost two decades ago an anticoagulant function of factor V (FV) was discovered, as an anticoagulant cofactor for activated protein C (APC). A natural mutant of FV in which the R506 inactivation site was mutated to Gln (FVLeiden) was inactivated slower by APC, but also could not function as anticoagulant cofactor for APC in the inactivation of activated factor VIII (FVIIIa). This mutation is prevalent in populations of Caucasian descent, and increases the chance of thrombotic events in carriers. Characterisation of the FV anticoagulant effect has elucidated multiple properties of the anticoagulant function of FV: 1) Cleavage of FV at position 506 by APC is required for anticoagulant function. 2) The C-terminal part of the FV B domain is required and the B domain must have an intact connection with the A3 domain of FV. 3) FV must be bound to a negatively charged phospholipid membrane. 4) Protein S also needs to be present. 5) FV acts as a cofactor for inactivation of both FVa and FVIIIa. 6) The prothrombotic function of FVLeiden is a function of both reduced APC cofactor activity and resistance of FVa to APC inactivation. However, detailed structural and mechanistic properties remain to be further explored.

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Identification of a Novel Factor V A2 Domain Tyrosine Deletion Mutation in a Patient with Factor V Deficiency and Bleeding.

Blood ◽

10.1182/blood.v104.11.4033.4033 ◽

2004 ◽

Vol 104 (11) ◽

pp. 4033-4033

Author(s):

Carol D. Jones ◽

Fernando Negro ◽

Katherine Darnell ◽

James L. Zehnder

Keyword(s):

Amino Acids ◽

Frameshift Mutation ◽

Coagulation Factor ◽

Severe Bleeding ◽

In Vitro Mutagenesis ◽

Factor V ◽

Coding Region ◽

Base Pair Deletion ◽

A2 Domain ◽

Factor Function

Abstract The gene for coagulation Factor V (FV) is located on chromosome 1q23. FV deficiency shows an autosomal recessive mode of inheritance; heterozygotes are generally not clinically affected. The homozygous clinical phenotype occurs in approximately 1 per million individuals with variable severity of bleeding. Thus, genotype-phenotype correlations are likely to shed light on functionally important residues of FV. Here we describe a case of FV deficiency with a severe bleeding phenotype. The proband is a male infant from Argentina. His parents are unrelated. He was born healthy with no bleeding from the umbilical stump or other symptoms. He presented at eight months with a CNS hemorrhage, then suffered a second massive subdural bleed at nine months of age. Both episodes required surgical drainage and treatment with fresh frozen plasma He continues to receive prophylactic FFP infusions and has some residual neurologic impairment. The proband’s FV activity ranges from 2–14%. Two siblings are unaffected. His father’s FV activity is 50% and his mother’s is 70%. We performed DNA sequencing spanning the entire coding region of the proband’s FV gene and found two heterozygous mutations: a heterozygous single base pair deletion, del 2952T in exon 13, located in the B-domain of the FV protein, causing a frameshift mutation followed by a premature termination codon 3 amino acids downstream; and a novel 3-bp deletion in exon 10. This deletion is in-frame and results in the deletion of Y478. The del 2952T frameshift mutation was present in the father, while the del Y478 mutation was present in the mother. Y478 is in the A2 domain of FV and adjacent to another tyrosine, Y477. Evidence suggests that these tyrosine residues are important for co-factor function. Tyrosine residue sulfation has been shown to be required for full activity of the homologous co-factor, FVIII, as well as for hirudin. These sulfated tyrosines and surrounding acidic amino acids have been proposed to be important in interactions with the thrombin anion binding exosite; in the case of hirudin, sulfation of a carboxy-terminal tyrosine increases the affinity for thrombin 10-fold. The homologous tyrosines, Y718 and Y719 appear to be sulfated in FVIII. FV has been shown to be sulfated, but the precise location of the FV sulfation sites has not yet been determined. One of this patient’s FV alleles is nonfunctional due to a frameshift and a premature trancation of translation. With respect to the other allele, we hypothesize that, like FVIII, one or both of FV tyrosines 477 and 478 is sulfated, and that deletion of Y478 may result in disruption of FV co-factor function. In vitro mutagenesis and expression studies to characterize the functional consequences of the del Y478 and/or del Y477 are in progress.

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Venom-Derived Factor V from the Common Brown Snake P. Textilis Is Expressed as a Constitutively Active Cofactor.

Blood ◽

10.1182/blood.v110.11.1765.1765 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1765-1765

Author(s):

Mettine H.A. Bos ◽

Michael Boltz ◽

Liam St. Pierre ◽

Martin F. Lavin ◽

Rodney M. Camire

Keyword(s):

Disulfide Bond ◽

Factor Xa ◽

Proteolytic Processing ◽

Light Chains ◽

Factor V ◽

Single Chain ◽

Processing Step ◽

Sds Page ◽

Cofactor Activity ◽

Constitutively Active

Abstract Factor V (FV) circulates as procofactor with little or no procoagulant activity and is activated upon proteolytic removal of a central B-domain. Recently, we have shown that discrete B-domain sequences stabilize the inactive procofactor state and that their deletion drives the expression of procoagulant function without the need for proteolytic processing (JBC2007;282:15033). While the B-domain length is highly conserved in most mammals (∼800 aa), recent genomic data indicates that in some vertebrates the FV B-domain is dramatically shortened. The most striking example is found in an Australian snake family (P. textilis and O. scutellatus). These snakes, which are among the most venomous in Australia, have a powerful prothrombin activating complex in their venoms consisting of FXa-like and FVa-like components. Remarkably, both plasma FV and venom FV sequences of these snakes predict a B-domain of only 45 and 46 residues, suggesting that snake FV may have lost B-domain sequences that maintain the protein as procofactor. Alternatively, snake FV could use a different strategy to preserve the procofactor state. To gain insight into this, we expressed and purified venom-derived P. textilis FV (pt-FV) in BHK cells. Pt-FV expressed very well (4mg protein/L media), and SDS-PAGE analysis revealed that it migrated as a single-chain protein with a molecular mass of ∼180 kDa. Upon incubation with thrombin (IIa), pt-FV was completely processed to pt-FVa, yielding fragments similar to the human FVa heavy and light chains. Surprisingly, pt-FVa migrated as a single band on a non-reducing gel, indicating that the heavy and light chains are held together by a disulfide bond. Sequence analysis suggests that this disulfide bond is located between the A2 and A3 domains. Functional analysis using a PT-based clotting assay with human FV-deficient plasma demonstrated that pt-FV has low activity compared to human FVa. This is consistent with previous studies which showed that venom FV is a relatively ineffective cofactor when using bovine factor Xa (FXa), suggesting that it needs snake-derived FXa to optimally function. Despite the low reactivity towards human plasma, we were able to demonstrate that processing of pt-FV to pt-FVa did not significantly increase cofactor activity (activation quotient of 2 compared to 10–15 for human FV). This suggests that unlike all nonhuman FV variants characterized so far, pt-FV is expressed as a constitutively active cofactor. Consistent with this, assessment of cofactor activity using a purified prothrombinase assay with either human FXa or transiently expressed O. scutellatus FXa revealed that the initial rates of prothrombin activation were equivalent for pt-FV or pt-FVa. To further confirm these results we expressed and purified pt-FV in which IIa cleavage sites Arg709 and Arg1545 were eliminated (pt-FV-QQ). SDS-PAGE analysis showed that pt-FV-QQ was not cleaved by IIa, yet functional measurements revealed that its activity was similar to pt-FV or pt-FVa. These data indicate that pt-FV has constitutive cofactor activity and thus bypasses the normal proteolytic processing step to function within prothrombinase. This is in agreement with our previous findings that discrete sequences within the FV B-domain are necessary to maintain FV as procofactor. Thus, venom-derived P. textilis FV represents the first example of a naturally occurring FV variant that does not require proteolytic processing to be active.

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An Assay to Quantify the Two Plasma Isoforms of Factor V

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614172 ◽

2000 ◽

Vol 84 (12) ◽

pp. 1066-1071 ◽

Cited By ~ 7

Author(s):

Lico Hoekema ◽

Guido Tans ◽

Jan Rosing

Keyword(s):

Mole Fraction ◽

Plasma Sample ◽

Coagulation Factor ◽

Model Systems ◽

Phospholipid Vesicles ◽

Factor V ◽

Increased Risk ◽

Coagulation Factor V ◽

Cofactor Activity ◽

Blood Coagulation Factor V

SummaryBlood coagulation factor V (FV) circulates in the blood in two forms, designated FV1 and FV2. In model systems containing purified proteins FV1 appears to be more thrombogenic than FV2. Recently, we reported that in plasma from carriers of the R2 haplotype, a polymorphism which encodes several amino acid changes in FV and which is associated with an increased risk of thrombosis, the FV1/FV2 ratio is shifted in favor of the more thrombogenic form FV1. Here we describe in detail the assay that enables quantification of the plasma levels of FV1 and FV2. FV present in highly diluted plasma samples was activated with thrombin and the FVa generated was subsequently quantified in two prothrombinase-based assay systems. In the first assay, which is performed at saturating amounts of FXa and phospholipid vesicles with a high mole fraction phosphatidylserine, FVa1 and FVa2 express the same cofactor activity in prothrombin activation. Hence, this assay quantifies the total FV level (FV1 + FV2) present in plasma. In the second assay, which is performed at suboptimal amounts of FXa and phospholipid vesicles with a low mole fraction phosphatidylserine, FVa2 has approximately an 8-fold higher cofactor activity than FVa1. Therefore, the response in this assay depends on the relative amounts of FV1 and FV2 in the plasma sample. Calibration curves made with samples containing known concentrations of purified FVa1 and FVa2 subsequently allowed calculation of the amounts of FV1 and FV2 present in plasma.

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Cleavage of Factor Va Heavy Chain at Arg643 by Thrombin Partially Inactivates the Cofactor.

Blood ◽

10.1182/blood.v108.11.1703.1703 ◽

2006 ◽

Vol 108 (11) ◽

pp. 1703-1703 ◽

Cited By ~ 1

Author(s):

Evrim Erdogan ◽

Michael A. Bukys ◽

Thomas Orfeo ◽

Kenneth G. Mann ◽

Michael Kalafatis

Keyword(s):

Endothelial Cells ◽

Heavy Chain ◽

Consensus Sequence ◽

Factor V ◽

Single Chain ◽

Chain Fragment ◽

Thrombin Cleavage ◽

Factor Va ◽

Plasma Factor ◽

Cofactor Activity

Abstract Prothrombinase, the enzyme complex required to activate prothrombin, is composed of the serine protease factor Xa and the cofactor factor Va, associated in 1:1 stoichiometry on a phospholipid surface in the presence of Ca2+. Incorporation of factor Va in prothrombinase is required for any meaningful rate of thrombin generation and the arrest of hemorrhage. Factor Va inactivation down-regulates thrombin production resulting in the termination of the hemostatic response. The principal enzyme involved in this down-regulation is activated protein C (APC). Factor Va is formed following enzymatic cleavage of the single chain procofactor, factor V (Mr 330,000) by thrombin. Thrombin cleaves and activates the procofactor sequentially at Arg709, Arg1018, and Arg1545. The active cofactor, factor Va, is composed of heavy (HC105, Mr 105,000) and light (Mr 74,000) chains non-covalently associated in the presence of divalent ions. Previous studies of factor Va inactivation on human umbilical vein endothelial cells (HUVEC) have shown that thrombin cleaves the heavy chain at the COOH-terminus to produce a Mr 97,000 fragment containing the NH2-terminal portion of the heavy chain and a Mr 8,000 peptide representing the COOH-terminus of the molecule which remains attached to the heavy chain by a disulfide bond. The thrombin cleavage appeared to occur between residues 586 and 654. This region contains a consensus sequence for cleavage by thrombin located between residues 640–643 (S-P-R). To evaluate the functional importance of thrombin cleavage at Arg643 for factor Va inactivation, site-directed mutagenesis was used to create recombinant factor V molecules with mutations R643→Q (factor VR643Q) and R643→A (factor VR643A). All recombinant molecules were purified to homogeneity and assayed for activity following extended activation with thrombin. Under similar experimental conditions, cleavage of HC105 and appearance of the Mr 97,000 heavy chain fragment in the wild type molecule correlated with partial loss of cofactor activity, while following incubation of factor VR643Q and factor VR643A with thrombin no cleavage of HC105 at Arg643 was observed and no presence of the Mr 97,000 heavy chain fragment was noticed. Further, no loss in cofactor activity was observed using these mutant recombinant factor Va molecules following extended incubation with thrombin. The endothelial cell surface has been presumed to be the site of PC activation and factor Va inactivation in vivo. The relative phospholipid composition of endothelial membranes has been suggested to be consistent with their ability to support factor Va inactivation in a manner analogous to the commonly used phospholipid system composed of 25% phosphatidylserine and 75% phosphatidylcholine. In the experiments conducted on the HUVEC surface incubation of 20 nM plasma factor V with 0.1 nM thrombin resulted in almost complete cleavage of HC105 over a 60 minute thrombin treatment. In the experiments presented herein much higher concentrations of thrombin were necessary to obtain a similar effect. The combined data suggest the presence of a cofactor for thrombin on the surface of endothelial cells that would facilitate cleavage of factor Va heavy chain at Arg643. Collectively, the data demonstrate that cleavage of HC105 at Arg643 by thrombin results in a partially inactive cofactor molecule and provide for an APC-independent anticoagulant effect of thrombin.

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Evolutionary Adaptation of Factor V from the Venom of the Common Brown Snake into a Potent Procoagulant

Blood ◽

10.1182/blood.v112.11.586.586 ◽

2008 ◽

Vol 112 (11) ◽

pp. 586-586

Author(s):

Mettine H.A. Bos ◽

Michael Boltz ◽

Liam St. Pierre ◽

John de Jersey ◽

Paul P Masci ◽

...

Keyword(s):

Membrane Surface ◽

Light Chains ◽

Factor V ◽

Single Chain ◽

Prothrombinase Complex ◽

High Affinity ◽

The Common ◽

Cofactor Activity ◽

Mechanistic Basis ◽

Normal Requirement

Abstract Some of the most toxic snakes in the world are those from the Australian Elapid family, including the three most venomous land snakes Inland Taipan, Coastal Taipan, and Common Brown snake. Their venom is strongly procoagulant and they are the only species known to have acquired a powerful prothrombin activator in their venom, which consists of a factor Xa (FXa)-like and factor V (FV)-like component. Venom-derived FV (pt-FV) from the Common Brown snake P. textilis shares 44% sequence homology with mammalian FV and has a similar domain organization. Remarkably, the B domain length of pt-FV is dramatically shortened compared to human FV (46 vs. 836 aa). This adaptation provides a unique opportunity to gain new insight into the function of the B domain and to examine the mechanistic basis for the strong procoagulant nature of the venom-derived prothrombinase complex. Pt-FV was expressed in BHK cells, purified, and characterized in functional assays employing FXa purified from P. textilis venom (pt-FXa). SDS-PAGE analysis revealed that pt-FV migrated as a single chain protein (~180 kDa). Thrombin completely processed pt-FV to pt-FVa, yielding the characteristic heavy and light chains. Surprisingly, pt-FVa migrated as a single band on a non-reducing gel, indicating that the heavy and light chains are connected by a unique disulfide bond. Functional analysis of prothrombin and prethrombin-1 conversion using a purified component assay in the presence of pt-FXa and negatively charged phospholipids revealed that pt-FV exhibits kinetic parameters comparable to human prothrombinase. Proteolytic processing of single chain pt-FV to the heterodimer did not significantly increase cofactor activity, indicating that pt-FV is expressed as a constitutively active cofactor that has bypassed the normal requirement for proteolytic activation. These results were confirmed using an uncleavable variant, pt-FV-QQ. We speculate that the mechanistic basis for this constitutive cofactor activity is related to the absence of a key cluster of conserved B domain residues, which we have recently shown to play an important role in maintaining FV as an inactive procofactor (JBC2007;282:15033). Additional experiments revealed that the pt-FV–pt-FXa complex does not require a membrane surface to optimally function, as the kinetics of prethrombin-1 activation were equivalent in the presence or absence of membranes. Binding measurements indicated that this was due to the high affinity interaction (Kd ~8 nM) of pt-FV with pt-FXa in solution. Interestingly, human FVa did not bind soluble pt-FXa with high affinity, suggesting that pt-FXa binding involves unique molecular features on pt-FV. Additional studies revealed that pt-FV does not lose activity following incubation with high concentrations of activated protein C (APC), even though the pt-FV heavy chain was fully proteolyzed. Collectively, our findings provide new insights into FV structure/function as well as a biochemical rationale for the powerful procoagulant nature of the prothrombinase complex from P. textilis venom. Remarkably, pt-FV has acquired at least three gain of function elements: first, it is constitutively active and as such the first example of a naturally occurring active FV variant. Second, pt-FV has a unique conformation as it bypasses the normal requirement for a membrane surface to achieve high affinity FXa binding. Finally, pt-FV is functionally resistant to APC which could be due to its unique disulfide bond. Taken together, venom-derived P. textilis FV represents an exceptional example of a protein that has adapted into a potent biological weapon for host defense and to incapacitate prey. Uncovering the mechanistic details of these gain of function elements will provide a new level of understanding of FV/FVa function.

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