Lactadherin's Multistate Binding Predicts Stable Membrane-Bound Conformations of Factors V and VIII's C Domains.

Protein binding to negatively charged lipids is essential for maintaining numerous vital cellular processes where its dysfunction can lead to various diseases. One such protein that plays a crucial role in this process is lactadherin, which competes with coagulation factors for membrane binding sites to regulate blood clotting. Despite identifying key binding regions of these proteins through structural and biochemical studies, models incorporating membrane dynamics are still lacking. In this study, we report on the multimodal binding of lactadherin and use it to gain insight into the binding mechanisms of its C domain homologs, factor V and factor VIII. Molecular dynamics simulations enhanced with the highly mobile mimetic model enabled the determination of lactadherin's multimodal binding on membranes that revealed critical interacting residues consistent with prior NMR and mutagenesis data. The binding occurred primarily via two dynamic structural ensembles: an inserted state and an unreported, highly conserved side-lying state driven by a cationic patch. We utilized these findings to analyze the membrane binding domains of coagulation factors V and VIII and identified their preferred membrane-bound conformations. Specifically, factor V's C domains maintained an inserted state, while factor VIII preferred a tilted, side-lying state that permitted antibody binding. Insight into lactadherin's atomistically resolved membrane interactions from a multistate perspective can guide new therapeutic opportunities in treating diseases related to blood coagulation.

Regulation of factor V and factor V-short by TFPIα: Relationship between B-domain proteolysis and binding.

Coagulation factor V (FV) plays an anticoagulant role but serves as a procoagulant cofactor in the prothrombinase complex once activated to FVa. At the heart of these opposing effects is the proteolytic removal of its central B-domain, including conserved functional landmarks (basic region, BR; 963-1008 and acidic region 2, AR2; 1493-1537) that enforce the inactive FV procofactor state. Tissue factor pathway inhibitor α (TFPIα) has been associated with FV as well as FV-short, a physiologically relevant isoform with a shortened B-domain missing the BR. However, it is unclear which forms of FV are physiologic ligands for TFPIα. Here, we characterize the binding and regulation of FV and FV-short by TFPIα via its positively charged C-terminus (TFPIα-BR) and examine how bond cleavage in the B-domain influences these interactions. We show that FV-short is constitutively active and functions in prothrombinase like FVa. Unlike FVa, FV-short binds with high affinity (Kd ∼1 nM) to TFPIα-BR, which blocks procoagulant function unless FV-short is cleaved at Arg1545, removing AR2. Importantly, we do not observe FV binding (µM detection limit) to TFPIα. However, cleavage at Arg709 and Arg1018 displaces the FV BR, exposing AR2 and allowing TFPIα to bind via its BR. We conclude that for full-length FV, the detachment of FV BR from AR2 is necessary and sufficient for TFPIα binding and regulation. Our findings pinpoint key forms of FV, including FV-short, that act as physiologic ligands for TFPIα and establish a mechanistic framework for assessing the functional connection between these proteins.

Identification of a potent regulatory T cell epitope in factor V that modulates CD4+ and CD8+ memory T cell responses.

Identification of T cell epitopes that are recognized by Tregs may elucidate the relative contributions of thymic Tregs and induced Tregs to control of autoimmune diseases and allergy. One such T regulatory cell epitope or 'Tregitope', derived from blood Factor V, is described here. Tregs responding to Tregitope FV621 are potent suppressors of CD4+ T effector responses to Tetanus Toxoid in an in vitro bystander suppression assay, strongly inhibit proliferation of effector CD8+ T cells, down-modulate CD86 and HLA DR on antigen-presenting cells, and enhance expression of granzyme B in Tregs. Tregitope FV621 also suppresses anti-OVA immune responses in vivo. The immunomodulatory effect of Tregitope FV621 is enhanced when conjugated to albumin, suggesting that the short half-life of Tregitope peptides can be prolonged. The in silico tools used to prospectively identify the FV Tregitope described here, when combined with in vitro /in vivo validating assays, may facilitate future Tregitope discoveries.

Protein Z-dependent protease inhibitor (ZPI) is a physiologically significant inhibitor of prothrombinase function.

Current thought holds that factor Xa (FXa) bound in the prothrombinase complex is resistant to regulation by protein protease inhibitors during prothrombin activation. Here we provide evidence that, contrary to this view, the FXa-specific serpin inhibitor, protein Z-dependent protease inhibitor (ZPI), complexed with its cofactor, protein Z (PZ), functions as a physiologically significant inhibitor of prothrombinase-bound FXa during prothrombin activation. Kinetics studies showed that the rapid rate of inhibition of FXa by the ZPI-PZ complex on procoagulant membrane vesicles (ka(app) ∼107 m-1 s-1) was decreased ∼10-fold when FXa was bound to FVa in prothrombinase and a further ∼3-4-fold when plasma levels of S195A prothrombin were present (ka(app) 2 × 105 m-1 s-1). Nevertheless, the ZPI-PZ complex produced a major inhibition of thrombin generation during prothrombinase-catalyzed activation of prothrombin under physiologically relevant conditions. The importance of ZPI-PZ complex anticoagulant regulation of FXa both before and after incorporation into prothrombinase was supported by thrombin generation assays in plasma. These showed enhanced thrombin generation when the inhibitor was neutralized with a PZ-specific antibody and decreased thrombin generation when exogenous ZPI-PZ complex was added whether prothrombin was activated directly by FXa or through extrinsic or intrinsic pathway activators. Moreover, the PZ antibody enhanced thrombin generation both in the absence and presence of activated protein C (APC) anticoagulant activity. Taken together, these results suggest an important anticoagulant role for the ZPI-PZ complex in regulating both free FXa generated in the initiation phase of coagulation as well as prothrombinase-bound FXa in the propagation phase that complement prothrombinase regulation by APC.

Danger of false negative (exclusion) or false positive (diagnosis) for 'congenital thrombophilia' in the age of anticoagulants.

Background Most guidelines and experts recommend against performance of thrombophilia testing in general, and specifically against testing patients on pharmacological anticoagulants, due to substantially increased risk of false positive identification. For example, vitamin K antagonist (VKA) therapy affects protein C (PC) and protein S (PS), as well as some clotting assays (e.g. as used to investigate activated PC resistance [APCR]). Although heparin may also affect clotting assays, most commercial methods contain neutralisers to make them 'insensitive' to therapeutic levels. Direct oral anticoagulants (DOACs) also affect a wide variety of thrombophilia assays, although most reported data has employed artificial in vitro spiked samples. Methods In the current report, data from our facility for the past 2.5 years has been assessed for all 'congenital thrombophilia' related tests, as evaluated against patient anticoagulant status. We processed 10,571 'thrombophilia' related test requests, including antithrombin (AT; n=3470), PC (n=3569), PS (n=3585), APCR (n=2359), factor V Leiden (FVL; n=2659), and prothrombin gene mutation (PGM; n=2103). Results As expected, VKA therapy affected PC and PS, and despite manufacturer claims, also APCR. Most assays, as suggested by manufacturers, were largely resistant to heparin therapy. DOACs' use was associated with falsely low APCR ratios (i.e. FVL-like effect) and somewhat unexpectedly, anti-Xa agents apixaban and rivaroxaban were also associated with lower AT and higher PS values. Conclusions It is concluded that ex-vivo data appears to confirm the potential for both false positive and false negative 'thrombophilia' events in patients on anticoagulant (including DOAC) treatment.

Understanding the Impact of Aberrant Splicing in Coagulation Factor V Deficiency.

Rare inherited coagulation disorders (RICDs) are congenital deficiencies of the plasma proteins that are involved in blood coagulation, which generally lead to lifelong bleeding manifestations. These diseases are generally qualitative and/or quantitative defects that are associated with monoallelic or biallelic mutations in the relevant gene. Among RICDs, factor V (FV) deficiency is one of the least characterized at the molecular level. Here, we investigated four unrelated patients with reduced plasma FV levels (three severe, one mild), which were associated with a moderately severe bleeding tendency. Sequence analysis of the FV gene identified seven different variants, five hitherto unknown (p.D1669G, c.5789-11C>A, c.5789-12C>A, c.5789-5T>G, and c.6528G>C), and two previously reported (c.158+1G>A and c.5789G>A). The possible pathogenic role of the newly identified missense variant was studied by in silico approaches. The remaining six genetic defects (all putative splicing mutations) were investigated for their possible effects on pre-mRNA splicing by transient transfection experiments in HeLa cells with plasmids expressing appropriate hybrid minigenes. The preparation of minigene constructs was instrumental to demonstrate that the two adjacent variants c.5789-11C>A and c.5789-12C>A are indeed present in cis in the analyzed FV-deficient patient (thus leading to the c.5789-11_12CC>AA mutation). Ex vivo experiments demonstrated that each variant causes either a skipping of the relevant exon or the activation of cryptic splice sites (exonic or intronic), eventually leading to the introduction of a premature termination codon.

[Phenotypic and mutational analysis of a pedigree affected with hereditary coagulation factor â ¤ deficiency].

OBJECTIVE: To explore the molecular pathogenesis for a pedigree affected with coagulation factor Ⅴ (FⅤ) deficiency. METHODS: Prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen (FIB), coagulation factor Ⅱ activity (FⅡ: C), FⅤ activity (FⅤ: C), coagulation factor Ⅶ activity (FⅦ: C), and coagulation factor Ⅹ activity (FⅩ: C) were determined with a STAGO automatic coagulometer. FⅤ antigen (FⅤ: Ag) was detected with enzyme linked immunosorbent assay (ELISA). All exons and their flanking regions, and 5' and 3' untranslated regions of the F5 gene were analyzed by direct sequencing. Suspected mutation was verified by reverse sequencing as well as testing of family members. ClustalX software was used to analyze the conservative property of the mutation sites. PROVEAN and MutationTaster online software was used to predict the effect of the mutation on the protein function. Swiss-pdbViewer was used to analyze the protein model and interaction of amino acids. RESULTS: The PT and APTT of the proband were slightly prolonged to 15.2 s and 41.8 s, respectively. And the FⅤ: C and FⅤ: Ag measured 55% and 62%, respectively. The FⅤ: C and FⅤ: Ag of his father and son were decreased to various extent (60%, 65% and 31%, 40%, respectively). A c.911G>A heterozygous mutation (Gly276Glu) was detected in exon 6 of the proband, for which her father and son were heterozygotes. The same mutation was not found in her mother, brother and husband. Conservation analysis showed that the Gly276 is highly conserved across various species. By bioinformatic analysis, the PROVEAN (scored -6.214) indicated Gly276Glu was harmful, and MutationTaster (scored 0.976) suggested that it is pathogenic. Model analysis suggested there are two hydrogen bonds between Gly276 and Ile298 in the wild type protein. When Gly276 was replaced by Glu276, the original hydrogen bond did not change, but the side chain of Glu was extended, which added steric hindrance with the surrounding amino acids, which resulted in decreased protein stability. CONCLUSION: The heterozygous c.911G>A (Gly276Glu) mutation of the F5 gene probably underlies the decreased level of FⅤin the proband.

A novel mutation in the F5 gene (factor V Amsterdam) associated with bleeding independent of factor V procoagulant function.

We investigated a small Dutch family with a bleeding diathesis, prolonged prothrombin, and activated partial thromboplastin times, in whom no classifying diagnosis was made. The 2 affected relatives had severely decreased in vitro thrombin generation, and levels of tissue factor pathway inhibitor (TFPI) were strongly increased. To identify the genetic cause of the bleeding diathesis, we performed whole exome sequencing analysis of all living relatives. We found a novel gain-of-function mutation in the F5 gene (c.C2588G), which leads to an aberrant splicing of F5 and ultimately to a short factor V protein (missing 623 amino acids from the B domain), which we called factor V Amsterdam. Factor V Amsterdam binds to TFPI, prolonging its half-life and concentration. This is the second report of an association between a shorter form of factor V and increased TFPI levels, resulting in severely reduced thrombin generation and a bleeding tendency.

Factor VIII/V C-domain swaps reveal discrete C-domain roles in factor VIII function and intracellular trafficking.

Factor VIII C-domains are believed to have specific functions in cofactor activity and in interactions with von Willebrand factor. We have previously shown that factor VIII is co-targeted with von Willebrand factor to the Weibel-Palade bodies in blood outgrowth endothelial cells, even when factor VIII carries mutations in the light chain that are associated with defective von Willebrand factor binding. In this study, we addressed the contribution of individual factor VIII C-domains in intracellular targeting, von Willebrand factor binding and cofactor activity by factor VIII/V C-domain swapping. Blood outgrowth endothelial cells were transduced with lentivirus encoding factor V, factor VIII or YFP-tagged C-domain chimeras, and examined by confocal microscopy. The same chimeras were produced in HEK293-cells for in vitro characterization and chemical foot-printing by mass spectrometry. In contrast to factor VIII, factor V did not target to Weibel-Palade bodies. The chimeras showed reduced Weibel-Palade body targeting, suggesting that this requires the factor VIII C1-C2 region. The factor VIII/V-C1 chimera did not bind von Willebrand factor and had reduced affinity for activated factor IX, whereas the factor VIII/V-C2 chimera showed a minor reduction in von Willebrand factor binding and normal interaction with activated factor IX. This suggests that mainly the C1-domain carries factor VIII-specific features in assembly with von Willebrand factor and activated factor IX. Foot-printing analysis of the chimeras revealed increased exposure of lysine residues in the A1/C2- and C1/C2-domain interface, suggesting increased C2-domain mobility and disruption of the natural C-domain tandem pair orientation. Apparently, this affects intracellular trafficking, but not extracellular function.

Rethinking events in the haemostatic process: role of factor V and TFPI.

Regulatory mechanisms responsible for limiting blood clot formation are critical for maintaining normal haemostasis. Dysregulation can lead to bleeding (e.g. haemophilia) or thrombosis. New findings showing that tissue factor pathway inhibitor-alpha (TFPIα) binds coagulation factor V(a) and inhibits prothrombinase assembly highlights that our understanding of the initiation of coagulation is evolving. Work over the past decade on the biochemistry of FV activation has laid the groundwork for deciphering the mechanistic bases that may underpin how TFPIα mediates these anticoagulant effects. Collectively, these new findings are re-shaping our thinking about how coagulation is initiated at the site of injury. These ideas could have important clinical implications and help identify new ways to bias the coagulation response for the treatment of haemophilia and other disorders of the haemostatic process.

Efficacy of solvent/detergent plasma after storage at 2-8 °C for 5 days in comparison to other plasma products to improve factor V levels in factor V deficient plasma.

OBJECTIVES: Factor V (FV) plays an important role in coagulation. As no purified concentrate is available to restore critical FV levels, the main blood product used to replace FV is plasma. The aim of the present in vitro study was to compare the efficacy of the different available plasma products on the reversal of moderate and severe FV deficiency as assessed by ROTEM® and FV levels. METHODS: Five different plasma products (6 batches of each) were compared to determine their effectiveness in replacing FV in plasma moderately or severely deficient in FV. Effectiveness was measured using the ROTEM® EXTEM clotting time (CT) and a factor V assay. RESULTS: FFP, plasma frozen within 24 hours (FP24), Octaplas (solvent/detergent treated pooled plasma), as well as Octaplas and FP24 thawed and stored for 5 days (Octaplas TP and TP), were all used for in vitro replacement of FV. TP was significantly less effective at reversing a prolonged EXTEM CT and FV levels in FV deficient plasma than other tested products. There were no significant differences in EXTEM CT between Octaplas and Octaplas TP, while factor V activity was significantly lower in the Octaplas TP. There was no significant difference between Octaplas and FFP for EXTEM CT or FV activity. CONCLUSIONS: Octaplas and Octaplas TP appear to have an equivalent ability to improve the EXTEM CT and could be considered as a treatment alternative to FFP in patients with FV deficiency.

Factor Xa dimerization competes with prothrombinase complex formation on platelet-like membrane surfaces.

Exposure of phosphatidylserine (PS) molecules on activated platelet membrane surface is a crucial event in blood coagulation. Binding of PS to specific sites on factor Xa (fXa) and factor Va (fVa) promotes their assembly into a complex that enhances proteolysis of prothrombin by approximately 105. Recent studies demonstrate that both soluble PS and PS-containing model membranes promote formation of inactive fXa dimers at 5 mM Ca²âº. In the present study, we show how competition between fXa dimerization and prothrombinase formation depends on Ca²âº and lipid membrane concentrations. We used homo-FRET measurements between fluorescein-E-G-R-chloromethylketone (CK)-Xa [fXa irreversibly inactivated by alkylation of the active site histidine residue with FEGR (FEGR-fXa)] and prothrombinase activity measurements to reveal the balance between fXa dimer formation and fXa-fVa complex formation. Changes in FEGR-fXa dimer homo-FRET with addition of fVa to model-membrane-bound FEGR-fXa unambiguously demonstrated that formation of the FEGR-fXa-fVa complex dissociated the dimer. Quantitative global analysis according to a model for protein interaction equilibria on a surface provided an estimate of a surface constant for fXa dimer dissociation (K(fXa×fXa)(d, σ)) approximately 10-fold lower than K(fXa×fVa)(d,σ) for fXa-fVa complex. Experiments performed using activated platelet-derived microparticles (MPs) showed that competition between fXa dimerization and fXa-fVa complex formation was even more prominent on MPs. In summary, at Ca²âº concentrations found in the maturing platelet plug (2-5 mM), fVa can compete fXa off of inactive fXa dimers to significantly amplify thrombin production, both because it releases dimer inhibition and because of its well-known cofactor activity. This suggests a hitherto unanticipated mechanism by which PS-exposing platelet membranes can regulate amplification and propagation of blood coagulation.

Crystal structure of the prothrombinase complex from the venom of Pseudonaja textilis.

The prothrombinase complex, composed of the protease factor (f)Xa and cofactor fVa, efficiently converts prothrombin to thrombin by specific sequential cleavage at 2 sites. How the complex assembles and its mechanism of prothrombin processing are of central importance to human health and disease, because insufficient thrombin generation is the root cause of hemophilia, and excessive thrombin production results in thrombosis. Efforts to determine the crystal structure of the prothrombinase complex have been thwarted by the dependence of complex formation on phospholipid membrane association. Pseutarin C is an intrinsically stable prothrombinase complex preassembled in the venom gland of the Australian Eastern Brown Snake (Pseudonaja textilis). Here we report the crystal structures of the fX-fV complex and of activated fXa from P textilis venom and the derived model of active pseutarin C. Structural analysis supports a single substrate binding channel on fVa, to which prothrombin and the intermediate meizothrombin bind in 2 different orientations, providing insight into the architecture and mechanism of the prothrombinase complex-the molecular engine of blood coagulation.

Membrane binding by prothrombin mediates its constrained presentation to prothrombinase for cleavage.

Long-standing dogma proposes a profound contribution of membrane binding by prothrombin in determining the rate at which it is converted to thrombin by prothrombinase. We have examined the action of prothrombinase on full-length prothrombin variants lacking γ-carboxyglutamate modifications (desGla) with impaired membrane binding. We show an unexpectedly modest decrease in the rate of thrombin formation for desGla prothrombin but with a major effect on the pathway for substrate cleavage. Using desGla prothrombin variants in which the individual cleavage sites have been singly rendered uncleavable, we find that loss of membrane binding and other Gla-dependent functions in the substrate leads to a decrease in the rate of cleavage at Arg(320) and a surprising increase in the rate of cleavage at Arg(271). These compensating effects arise from a loss in the membrane component of exosite-dependent tethering of substrate to prothrombinase and a relaxation in the constrained presentation of the individual cleavage sites for active site docking and catalysis. Loss of constraint is evident as a switch in the pathway for prothrombin cleavage and the intermediate produced but without the expected profound decrease in rate. Extension of these findings to the action of prothrombinase assembled on platelets and endothelial cells on fully carboxylated prothrombin reveals new mechanistic insights into function on physiological membranes. Cell-dependent enzyme function is probably governed by a differential ability to support prothrombin binding and the variable accumulation of intermediates from the two possible pathways of prothrombin activation.

Amino acid region 1000-1008 of factor V is a dynamic regulator for the emergence of procoagulant activity.

Single chain factor V (fV) circulates as an Mr 330,000 quiescent pro-cofactor. Removal of the B domain and generation of factor Va (fVa) are vital for procoagulant activity. We investigated the role of the basic amino acid region 1000-1008 within the B domain of fV by constructing a recombinant mutant fV molecule with all activation cleavage sites (Arg(709)/Arg(1018)/Arg(1545)) mutated to glutamine (fV(Q3)), a mutant fV molecule with region 1000-1008 deleted (fV(ΔB9)), and a mutant fV molecule containing the same deletion with activation cleavage sites changed to glutamine (fV(ΔB9/Q3)). The recombinant molecules along with wild type fV (fV(WT)) were transiently expressed in COS-7L cells, purified, and assessed for their ability to bind factor Xa (fXa) prior to and following incubation with thrombin. The data showed that fV(Q3) was severely impaired in its interaction with fXa before and after incubation with thrombin. In contrast, KD(app) values for fV(ΔB9) (0.9 nM), fVa(ΔB9) (0.4 nM), and fV(ΔB9/Q3) (0.7 nM) were similar to the affinity of fVa(WT) for fXa (0.3 nM). Two-stage clotting assays revealed that although fV(Q3) was deficient in its clotting activity, fV(ΔB9/Q3) had clotting activity comparable with fVa(WT). The kcat value of prothrombinase assembled with fV(ΔB9/Q3) was minimally affected, whereas the Km value of the reaction was increased 57-fold compared with the Km value obtained with prothrombinase assembled with fVa(WT). These findings strongly suggest that amino acid region 1000-1008 of fV is a regulatory sequence protecting the organisms from spontaneous binding to fXa and unnecessary prothrombinase complex formation, which in turn results in catastrophic physiological consequences.

Homology model of human prothrombinase based on the crystal structure of Pseutarin C.

Thrombin is generated from prothrombin through cleavage at two sites by the prothrombinase complex. Prothrombinase is composed of a protease, factor (f) Xa, and a cofactor, fVa, which interact on negatively charged phospholipid surfaces and cleave prothrombin into thrombin 300 000 times faster than fXa alone. The balance between bleeding and thrombosis depends on the amount of thrombin produced, and this in turn depends on the function of the prothrombinase complex. How fXa and fVa interact and how improved prothrombin processing is conferred are of critical importance for understanding healthy and pathological blood clotting. Until recently, little structural information was available, and molecular models were built on partial structures with assembly guided by biochemical data. Last year our group published a crystal structure of a prothrombinase complex from the venom of the Australian Eastern Brown snake (known as Pseutarin C). Here we use the crystal structure of Pseutarin C as a starting point for homology modelling and assembly of the full human prothrombinase complex. The interface is complementary in shape and charge, and is consistent with much of the published biochemical data. The model of human prothrombinase presented here provides a powerful resource for contextualizing previous data and for designing future experiments.

DockAFM: benchmarking protein structures by docking under AFM topographs.

UNLABELLED: Proteins can adopt a variety of conformations. We present a simple server for scoring the agreement between 3D atomic structures and experimental envelopes obtained by atomic force microscopy. Three different structures of immunoglobulins (IgG) or blood coagulation factor V activated were tested and their agreement with several topographical surfaces was computed. This approach can be used to test structural variability within a family of proteins. AVAILABILITY AND IMPLEMENTATION: DockAFM is available at http://biodev.cea.fr/dockafm.

Conservative mutations in the C2 domains of factor VIII and factor V alter phospholipid binding and cofactor activity.

Factor VIII and factor V share structural homology and bind to phospholipid membranes via tandem, lectin-like C domains. Their respective C2 domains bind via 2 pairs of hydrophobic amino acids and an amphipathic cluster. In contrast, the factor V-like, homologous subunit (Pt-FV) of a prothrombin activator from Pseudonaja textilis venom is reported to function without membrane binding. We hypothesized that the distinct membrane-interactive amino acids of these proteins contribute to the differing membrane-dependent properties. We prepared mutants in which the C2 domain hydrophobic amino acid pairs were changed to the homologous residues of the other protein and a factor V mutant with 5 amino acids changed to those from Pt-FV (FV(MTTS/Y)). Factor VIII mutants were active on additional membrane sites and had altered apparent affinities for factor X. Some factor V mutants, including FV(MTTS/Y), had increased membrane interaction and apparent membrane-independent activity that was the result of phospholipid retained during purification. Phospholipid-free FV(MTTS/Y) showed increased activity, particularly a 10-fold increase in activity on membranes lacking phosphatidylserine. The reduced phosphatidylserine requirement correlated to increased activity on resting and stimulated platelets. We hypothesize that altered membrane binding contributes to toxicity of Pt-FV.

Asp68His mutation in the A1 domain of human factor V causes impaired secretion and ineffective translocation.

Congenital factor V (FV) deficiency is a rare inherited disorder. We determined the mechanism of a missense mutation, Asp68His, in the A1 domain of the FV protein, is associated with severe FV deficiency. We characterized the mutant FV-Asp68His protein using in vitro expression studies by using specific secretion and degradation pathway inhibitors and analysed the intracellular translocation of the mutant protein by immunofluorescence staining. The Asp68His mutation caused very low levels of FV protein in the conditioned media, with normal specific FV activity. Similar mRNA degradation rates between FV-wild-type (wt) and FV-Asp68His mRNA showed that the Asp68His mutation does not affect FV expression at the transcriptional level. A specific secretion pathway inhibitor, brefeldin A, was used to demonstrate that the lower efficiency of transport to the outside of the cell for FV-Asp68His mutant protein compared with that of the FV-wt protein. Furthermore, we showed that the Asp68His mutation resulted in increased intracellular degradation through a MG132-mediated proteasomal degradation pathway. In the transfected cell lysates, FV-wt protein had multiple posttranslational modified forms, but the FV-Asp68His protein was not completely glycosylated. We further observed that the FV-Asp68His protein was retrieved in the endoplasmic reticulum only and did not undergo transport to the Golgi apparatus, leading to impaired secretion. These results strongly suggest that the Asp68His mutation may result in intracellular defective trafficking and enhanced degradation, and impaired secretion of FV protein.

A bipartite autoinhibitory region within the B-domain suppresses function in factor V.

Activation of blood coagulation factor V (FV) is a key reaction of hemostasis. FV circulates in plasma as an inactive procofactor, and proteolytic removal of a large central B-domain converts it to an active cofactor (FVa) for factor Xa (FXa). Here we show that two short evolutionary conserved segments of the B-domain, together termed the procofactor regulatory region, serve an essential autoinhibitory function. This newly identified motif consists of a basic (963-1008) and an acidic (1493-1537) region and defines the minimal sequence requirements to maintain FV as a procofactor. Our data suggest that dismantling this autoinhibitory region via deletion or proteolysis is the driving force to unveil a high affinity binding site(s) for FXa. These findings document an unexpected sequence-specific role for the B-domain by negatively regulating FV function and preventing activity of the procofactor. These new mechanistic insights point to new ways in which the FV procofactor to cofactor transition could be modulated to alter hemostasis.