Importance of individual activated protein C cleavage site regions in coagulation Factor V for Factor Va inactivation and for Factor Xa activation

SummaryThe origin and evolution of venom toxins is a mystery that has evoked much interest. We have recently shown that pseutarin C, a prothrombin activator from Pseudonaja textilis venom, is structurally and functionally similar to mammalian coagulation factor Xa – factor Va complex. Its catalytic subunit is homologous to factor Xa while the nonenzymatic subunit is homologous to factor Va. P.textilis therefore has two parallel prothrombin activator systems: one expressed in its venom gland as a toxin and the other expressed in its liver and released into its plasma as a haemostatic factor. Here we report the complete amino acid sequence of factor V (FV) from its liver determined by cDNA cloning and sequencing. The liver FV shows 96% identity to pseutarin C nonenzymatic subunit. Most of the functional sites involved in its interaction with factor Xa and prothrombin are conserved. However, many potential sites of post-translational modifications and one critical cleavage site for activated protein C are different. The absence of the latter cleavage site makes pseutarin C nonenzymatic subunit resistant to inactivation and enhances its potential as an excellent toxin. By PCR and real-time quantitative analysis, we show that pseutarin C nonenzymatic subunit gene is expressed specifically in the venom gland at ~280 fold higher than that of FV gene in liver. These two are thus encoded by two separate genes that express in a highly tissue-specific manner. Our results imply that the gene encoding pseutarin C nonenzymatic subunit was derived by the duplication of plasma FV gene and they have evolved to perform distinct functions.

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Coagulation Factor V Leiden Mutation Was Detected in the Patients with Activated Protein C Resistance in Thailand

Thrombosis and Haemostasis ◽

10.1055/s-0037-1615201 ◽

1998 ◽

Vol 80 (08) ◽

pp. 344-345 ◽

Cited By ~ 2

Author(s):

Pasra Arnutti ◽

Motofumi Hiyoshi ◽

Wichai Prayoonwiwat ◽

Oytip Nathalang ◽

Chamaiporn Suwanasophon ◽

...

Keyword(s):

Protein C ◽

Activated Protein C ◽

Factor V Leiden ◽

Coagulation Factor ◽

Factor V ◽

Activated Protein C Resistance ◽

Factor V Leiden Mutation ◽

Coagulation Factor V

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Cryo-EM structures of human coagulation factors V and Va

Blood ◽

10.1182/blood.2021010684 ◽

2021 ◽

Author(s):

Eliza A Ruben ◽

Michael J Rau ◽

James Fitzpatrick ◽

Enrico Di Cera

Keyword(s):

Protein C ◽

Activated Protein C ◽

Factor Xa ◽

Coagulation Factor ◽

Coagulation Factors ◽

Proteolytic Processing ◽

Coagulation Cascade ◽

Factor V ◽

Prothrombinase Complex ◽

A2 Domain

Coagulation factor V is the precursor of factor Va that, together with factor Xa, Ca2+ and phospholipids, defines the prothrombinase complex and activates prothrombin in the penultimate step of the coagulation cascade. Here we present cryo-EM structures of human factors V and Va at atomic (3.3 Å) and near-atomic (4.4 Å) resolution, respectively. The structure of fV reveals the entire A1-A2-B-A3-C1-C2 assembly but with a surprisingly disordered B domain. The C1 and C2 domains provide a platform for interaction with phospholipid membranes and support the A1 and A3 domains, with the A2 domain sitting on top of them. The B domain is highly dynamic and visible only for short segments connecting to the A2 and A3 domains. The A2 domain reveals all sites of proteolytic processing by thrombin and activated protein C, a partially buried epitope for binding factor Xa and fully exposed epitopes for binding activated protein C and prothrombin. Removal of the B domain and activation to fVa exposes the sites of cleavage by activated protein C at R306 and R506 and produces increased disorder in the A1-A2-A3-C1-C2 assembly, especially in the C-terminal acidic portion of the A2 domain responsible for prothrombin binding. Ordering of this region and full exposure of the factor Xa epitope emerge as a necessary step for the assembly of the prothrombin-prothrombinase complex. These structures offer molecular context for the function of factors V and Va and pioneer the analysis of coagulation factors by cryo-EM.

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The R306G and R506Q mutations in coagulation Factor V reveals additional cleavage sites for Activated Protein C in the R313-R321 region and at R505

Thrombosis Research ◽

10.1016/j.thromres.2009.12.005 ◽

2010 ◽

Vol 125 (5) ◽

pp. 444-450 ◽

Cited By ~ 3

Author(s):

Richard J. Dirven ◽

Hans L. Vos ◽

Rogier M. Bertina

Keyword(s):

Protein C ◽

Activated Protein C ◽

Coagulation Factor ◽

Factor V ◽

Cleavage Sites ◽

Coagulation Factor V

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Molecular Detection of a Common Mutation in Coagulation Factor V Causing Thrombosis via Hereditary Resistance to Activated Protein C

Diagnostic Molecular Pathology ◽

10.1097/00019606-199509000-00006 ◽

1995 ◽

Vol 4 (3) ◽

pp. 191-197 ◽

Cited By ~ 34

Author(s):

Xiao-Yuan Liu ◽

Diana Nelson ◽

Chris Grant ◽

Virginia Morthland ◽

Scott H. Goodnight ◽

...

Keyword(s):

Protein C ◽

Molecular Detection ◽

Activated Protein C ◽

Coagulation Factor ◽

Common Mutation ◽

Factor V ◽

Coagulation Factor V

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Activation of Bovine Blood Coagulation Factor V, A Prerequisite for it of Bind Both Prothrombin and Factor Xa.

10.1055/s-0039-1682675 ◽

1977 ◽

Author(s):

M.C. Guillin ◽

A. Bezeaud ◽

J.P. Freeman ◽

C.M. Jackson

Keyword(s):

Factor Xa ◽

Coagulation Factor ◽

Factor V ◽

Factor Va ◽

Bovine Plasma ◽

Coagulation Factor V ◽

Russell’S Viper ◽

Blood Coagulation Factor V ◽

Bovine Factor ◽

Plasma Heparin

It is known that prior to bind bovine prothrombin and to become fully functional, bovine Factor V must itself be “activated” by either thrombin or an enzyme isolable from Russell’s viper venom. The purpose of this work was to determine if Factor V activation is also required in order for it to bind bovine Factor Xa.This has been investigated by measuring the binding of both “native” (unactivated) Factor V and Factor V activated by the Russell’s viper venom activating enzyme, to a column of agarose-bound Factor Xa. The experiments were also performed using diisopropylfluorophosphate (DFP) inhibited Factor Xa covalently bound to agarose. Both purified bovine Factor V (Va) and bovine plasma were used and gave the same results. In order to prevent initiation of clotting in bovine plasma, heparin wad added to the plasma to promote inactivation of Factor Xa by antithrombin III.The results indicate that Factor V activation is a prerequisite for it to bind Factor Xa ; Factor Va binds both Factor Xa and DFP inhibited Factor Xa, unmodified Factor V does not.These experiments suggest that Factor V may not participate in prothrombin activation at all, until after some thrombin has been formed. If this is so, an alternate pathway by which the first thrombin is generated must be considered and may be proposed to be simply that involving Factor Xa, phospholipid and Ca2+ alone.

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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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