Plasma Factor V Research Articles

Genome-Wide Search for Nonadditive Allele Effects Identifies PSKH2 as Involved in the Variability of Factor V Activity.

Factor V (FV) is a key molecular player in the coagulation cascade. FV plasma levels have been associated with several human diseases, including thrombosis, bleeding, and diabetic complications. So far, 2 genes have been robustly found through genome-wide association analyses to contribute to the inter-individual variability of plasma FV levels: structural F5 gene and PLXDC2. The authors used the underestimated Brown-Forsythe methodology implemented in the QuickTest software to search for non-additive genetic effects that could contribute to the inter-individual variability of FV plasma activity. QUICKTEST was applied to 4 independent genome-wide association studies studies (LURIC [Ludwigshafen RIsk and Cardiovascular Health Study], MARTHA [Marseille Thrombosis Association], MEGA [Multiple Environmental and Genetic Assessment], and RETROVE [Riesgo de Enfermedad Tromboembolica Venosa]) totaling 4505 participants of European ancestry with measured FV plasma levels. Results obtained in the 4 cohorts were meta-analyzed using a fixed-effect model. Additional analyses involved exploring haplotype and gene×gene interactions in downstream investigations. A genome-wide significant signal at the PSKH2 locus on chr8q21.3 with lead variant rs75463553 with no evidence for heterogeneity across cohorts was observed (P=0.518). Although rs75463553 did not show an association with mean FV levels (P=0.49), it demonstrated a robust significant (P=3.38x10-9) association with the variance of FV plasma levels. Further analyses confirmed the reported association of PSKH2 with neutrophil biology and revealed that rs75463553 likely interacts with two loci, GRIN2A and POM121L12, known for their involvement in smoking biology. This comprehensive approach identifies the role of PSKH2 as a novel molecular player in the genetic regulation of FV, shedding light on the contribution of neutrophils to FV biology.

Clinical, Laboratory, Molecular, and Reproductive Aspects of Combined Deficiency of Factors V and VIII.

Congenital combined deficiency of factor V (FV) and factor VIII (FVIII; F5F8D, OMIM 227300) is a rare hereditary coagulopathy and accounts for approximately 3% of cases of rare coagulation disorders. The prevalence of this disease in the general population is estimated to be 1:1,000,000 and is significantly higher in regions where consanguineous marriages are permitted, such as the Mideast and South Asia. The disease has an autosomal recessive mode of inheritance and therefore occurs with an equal incidence among males and females. Heterozygous mutation carriers usually do not have clinical manifestations. The molecular basis of this disease differs from that of stand-alone congenital deficiencies of FVIII and FV. F5F8D is caused by mutations in either LMAN1 or MCFD2, which encode components of a cargo receptor complex for endoplasmic reticulum to Golgi transport of FV and FVIII, leading to defects in an intracellular transport pathway shared by these two coagulation factors. Congenital combined deficiency of FV and FVIII is characterized by decreased activities of both FV and FVIII in plasma, usually to 5 to 30% of normal. Clinical manifestations in most cases are represented by mild or moderate hemorrhagic syndrome. The simultaneous decreases of two coagulation factors present complications in the diagnosis and management of the disease. In female patients, the disease requires a special approach for family planning, pregnancy management, and parturition. This review summarizes recent progress in clinical, laboratory, and molecular understanding of this disorder.

Factor V haemostatic diathesis impairing thrombin activation, membrane binding and circulating antigen level due to a novel compound heterozygous mutation, Leu1821Ser and Gly2192Cys.

Congenital factor V (FV) deficiency is a rare clotting disorder affecting ∼1 in 1,000,000, with bleeding severity that ranges broadly for poorly understood reasons. To help understand the molecular basis of the observed phenotype in FV deficient patients, the genetics and biochemistry causing a patient's FV deficiency were evaluated. A 71-year-old female, who had serious life-long bleeding upon provocation and profound menorrhagia that lead to hysterectomy, was found to have 3% of normal plasma FV antigen with normal electrophoretic mobility. Platelet FV was similarly low, although the banding pattern was less fragmented than normal. Plasma clotting activity was <1% of normal. Familial inheritance and DNA sequence analysis from peripheral blood leukocytes were consistent with novel compound heterozygosity with missense mutations in exon XVII, Leu1821 to Ser (L1821S) and exon XXV, Gly2192 to Cys (G2192C). The respective single-mutation variants were expressed and purified. Explaining why the antigen level and activity were inequivalent, thrombin activation of recombinant (r) FV/L1821S was impaired, and rFV/G2192C was unable to bind to a procoagulant phospholipid membrane. These findings are consistent with the observed phenotype, highlighting the importance of understanding FV biochemical function to rationalize clinical bleeding severity when the circulating antigen level is discordant.

Clinical Characterization and Molecular Analysis of Fourteen Chinese Patients with Factor V Deficiency.

Coagulation factor V (FV) functions as a vital cofactor that performs procoagulant roles in the coagulation system. We investigated 14 unrelated patients whose plasma FV levels were all below the reference range. FV activity (FV:C) and FV antigen were detected by one-stage clotting and ELISA, respectively. All 25 exons of the F5 gene in patients were amplified by the PCR, and they were sequenced directly. Haplotype analysis was performed with different polymorphisms on F5. Protein modeling was applied to analyze the potential molecular mechanisms. Of five patients with higher FV levels (FV:C > 10%), only one had minor bleeding symptoms. In contrast, of the remaining eight patients with lower FV levels (FV:C < 10%), six showed various bleeding manifestations. A total of 10 mutations were detected from 14 patients (6 were novel mutations). Interestingly, the homozygous p.Phe190Ser was found in five pedigrees, and haplotype analysis showed that they shared almost the same haplotype, indicating the common origin rather than a hotspot mutation. In silico analysis preliminarily investigated the potential pathogenic mechanism of the mutation. Modeling analysis showed that all six missense mutations would lead to conformational alterations in the FV protein. Among them, three (p.Gly1715Ser, p.Ser1753Arg, and p.Asp68His) would decrease hydrogen bonds. This is the largest genetic analysis of a single cohort of FV deficiency in Chinese. The study demonstrated that FV levels tended to be correlated with the probability of hemorrhage. The identification of a large number of unique FV-deficient pedigrees highlighted the screening for mutations in F5.

Analysis of 363 Genetic Variants in F5 via an Interactive Web Database Reveals New Insights into FV Deficiency and FV Leiden.

The inherited bleeding disorder Factor V (FV) deficiency and clotting risk factor FV Leiden are associated with genetic variants in the F5 gene. FV deficiency occurs with mild, moderate, severe, or asymptomatic phenotypes, and either dysfunctional or reduced amounts of plasma FV protein. Here we present an interactive web database containing 363 unique F5 variants derived from 801 patient records, with 199 FV deficiency-associated variants from 245 patient records. Their occurrence is rationalized based on the 2,224 residue sequence and new FV protein structures. The 199 FV deficiency variants correspond to 26 (13%) mild, 22 (11%) moderate, 49 (25%) severe, 35 (18%) asymptomatic, and 67 (34%) unreported phenotypes. Their variant distributions in the FV domains A1, A2, A3, B, C1 and C2 were 28 (14%), 32 (16%), 34 (17%), 42 (21%), 16 (8%), and 19 variants (10%), respectively, showing that these six regions contain similar proportions of variants. Variants associated with FV deficiency do not cluster near known protein-partner binding sites, thus the molecular mechanism leading to the phenotypes cannot be explained. However, the widespread distribution of FV variants in combination with a high proportion of buried variant residues indicated that FV is susceptible to disruption by small perturbations in its globular structure. Variants located in the disordered B domain also appear to disrupt the FV structure. We discuss how the interactive database provides an online resource that clarifies the clinical understanding of FV deficiency.

Differential Role of Plasma and Platelet-Derived Factor V In Vivo

Coagulation factor V (FV) has a profound effect on coagulation and deficiency states lead to bleeding. In blood, there are two pools of FV with 80% found in plasma and 20% contained in the α-granules of platelets. The biological basis for this separation is not clear; however, structural distinctions between the pools have been identified suggesting they may have unique effects or play different roles following injury. Broadly however, in vivo evidence for this is lacking. Transgenic mice with synthesis limited to liver or megakaryocytes have been generated to study the role of plasma and platelet FV (Sun et al, Blood 102:2856, 2003). Either pool is sufficient for basal hemostasis and tail-bleeding times were only slightly prolonged compared to wild-type (wt) mice. The impact of the two pools following different hemostatic challenges in different vessels is not known. To expand our in vivo understanding of the FV pools on clot formation, we further studied mice with only platelet FV (platelet-FV+) or plasma FV (plasma-FV+). As a control, we also generated mice with essentially little to no total FV (FV-low). To generate platelet-FV+ mice, wt mice were treated with a single dose of a liver targeted antisense oligonucleotide (ASO) to knockdown plasma FV (ASO-FV; 40 mg/kg). After 3 weeks of ASO-FV treatment, the plasma FV levels assessed with a murine FV-specific ELISA were at the lower limits of detection while platelet FV levels were normal. This confirms prior work showing platelet FV originates from megakaryocyte synthesis in mice, which is different from humans. To generate plasma-FV+ mice, we used previously established transgenic mice only expressing FV (~30% of wt) from the liver with no platelet FV. To create FV-low mice, plasma-FV+ mice were treated with ASO-FV; this resulted in mice with markedly reduced (<0.5%) plasma FV and no platelet FV. Using whole blood from these mice, ROTEM assessment showed that compared to wt mice (n = 5; 5.9 ± 0.3 min), the clot time (CT) for plasma-FV+ mice (n = 3; 5.9 ± 0.4 min) was normal while the CT was prolonged 2-fold for platelet-FV+ mice (n = 10; 13 ± 1.2 min). As expected, the almost complete lack of both pools (FV-low) substantially increased the CT (n = 3; 24 ± 2.2 min). Evaluation of these mice in several injury models probing different vessels was next pursued to examine the hemostatic potential of either pool. Using either a partial (mild) or total (severe) tail injury model, total blood loss for both wt (n = 5) and platelet-FV+ mice (n=10) were similar. In contrast, plasma-FV+ mice (n=5, p<0.001) exhibited a 2- (mild injury) or 5-fold (severe injury) increase in blood loss compared to wt mice. As expected, FV-low mice (n=5; p<0.001) had the highest levels of blood loss. Further, we used a microcirculatory model of hemostasis to image the distribution of plasma vs. platelet FV following laser injury and assess fibrin and platelet deposition. In plasma-FV+ mice, a rapid and robust fluorescence signal for murine FV/Va was detected on the vessel wall surrounding the microscopic injury and on a subset of platelets adherent at the injury site. For platelet-FV+ mice, murine FV/Va was detected only on core subset of platelets, which we speculate are fully activated. Further, analysis of the median integrated florescence as a function of time in multiple mice (n=3-5/group; 15-25 injuries) revealed that the absence of either platelet or plasma FV did not considerably alter the kinetics of platelet accumulation (~2-fold less than wt). However, in contrast to plasma-FV+ mice, platelet-FV+ mice had significantly reduced and delayed fibrin deposition following laser injury suggesting the FV released from platelets is not sufficient to produce normal amounts of fibrin in this model. Our data indicate that the overall impact and contribution of the platelet and plasma FV pools is different depending on the type of injury and/or vessel type. In the microcirculation, plasma FV appears to play an important role in binding to damaged endothelium and contributing to robust thrombin formation, while platelet FV may play a reduced role. However, in more severe injury models, platelet FV plays a prominent role. Collectively these data highlight potentially different roles for the FV pools and/or indicate that the injury models employed impact FV activation or release from platelet α-granules to different extents. DisclosuresIvanciu:Bayer: Research Funding; Novo Nordisk: Research Funding. Crosby:Ionis Pharmaceuticals: Employment, Research Funding. Revenko:Ionis Pharmaceuticals: Employment, Research Funding. MacLeod:Ionis Pharmaceuticals: Employment. Monia:Ionis Pharmaceuticals: Employment. Davidson:Spark Therapeutics: Consultancy. Camire:Bayer: Consultancy, Research Funding; Pfizer: Consultancy, Patents & Royalties, Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Research Funding.

Platelet FV Deficiency Restores Survival of TFPI Null Mice Independently of Plasma FV

Introduction: Platelets are essential for the hemostatic process, releasing “just in time” prothrombotic molecules, such as factor V (FV) and prothrombin to sites of vascular injury. Paradoxically, platelets also carry potent antithrombotic molecules, including tissue factor pathway inhibitor (TFPI), which may prevent hemostatic platelet plugs from expanding into vascular occluding thrombi. The TFPI gene undergoes alternative splicing to produce several isoforms, including TFPIα and TFPIβ. Platelets are the primary location of TFPIα in the murine circulation. TFPIα has a basic 9 amino acid sequence in its C-terminal region nearly identical to a basic sequence in the FV B domain. The TFPIα basic region tightly binds to an acidic region of the FV B domain. This binding takes place only when the FV basic region is absent, as occurs following proteolysis by factor Xa (FXa), in some forms of FV naturally present in platelet α-granules, or in genetically altered forms of FV, such as FV-short (associated with East Texas bleeding disorder). This interaction provides an essential exosite for TFPIα inhibition of the prothrombinase complex (FXa-FVa) assembled with forms of FVa retaining the B domain acidic region. Since prothrombinase assembles on the surface of activated platelets, we hypothesized that inhibition of prothrombinase via the platelet TFPIα-platelet FV interaction is a physiologically important biochemical mechanism for the inhibiting early stages of coagulation. We probed this hypothesis using TFPI K1 deleted (TFPI-/-), FV deleted (F5-/-) and FV transgenic (F5Tg) mice to uncover whether specifically reducing or removing platelet FV would rescue TFPI-/- mice from embryonic lethality.Methods/Results: We performed crossbreeding experiments using F5-/- mice carrying liver- or platelet-specific transgenes. F5LiverTg produces 50% plasma FV with no FV in platelets, whereas F5PltTg produces 3% platelet FV with no FV in plasma. F5-/- F5LiverTgand F5-/- F5PltTgmice were bred into TFPI+/- mice. Offspring from this cross were bred to determine the effects of the liver or platelet transgene on the survival of mice lacking TFPI.F5-/- is embryonic lethal in mice. F5-/- mice are rescued by transgenes expressing either liver-specific FV or platelet specific FV. When we bred F5+/- TFPI+/- x F5+/- F5LiverTg TFPI+/-, neither F5-/- TFPI-/- nor F5+/- TFPI-/- mice without the liver transgene were viable. Liver transgene expression did not rescue F5+/- TFPI-/- mice, however, it produced grossly normal pups lacking TFPI (F5-/- F5LiverTg TFPI-/-) that lived to adulthood with no overt phenotypes. We further characterized this phenotype by crossbreeding F5+/- TFPI+/- x F5-/- F5LiverTg TFPI-/-. In this cross, F5-/- F5LiverTg TFPI-/- pups survived with ~80% penetrance (45 of 234 total mice, p<5.0 x10-12). Next, we bred F5+/- TFPI+/- x F5+/- F5PltTgTFPI+/-. Platelet transgene expression also produced grossly normal pups lacking TFPI (F5-/- F5PltTg TFPI-/-) that lived to adulthood with no overt phenotypes. Further crossbreeding, as described for the liver transgene, revealed a similar ~80% survival of F5-/- F5PltTg TFPI-/- pups (56 of 291 total mice, p<9.0 x10-18). Both male and female platelet and liver specific F5-/- F5Tg TFPI-/- were fertile, and mothers supported pups until weaning.Conclusions: Neither total loss of FV nor concerted 50% reduction in plasma and platelet FV rescued TFPI-/- embryonic lethality, indicating that TFPI anticoagulant activity is not simply counterbalanced by FV procoagulant activity (or vice versa) during embryogenesis. Remarkably, either the total lack of platelet FV with 50% normal plasma FV, or the total lack of plasma FV with 3% normal platelet FV, resulted in the nearly complete rescue of F5-/- TFPI-/- mice with pups surviving to adulthood without grossly observable pathology. These observations indicate that severe reduction of platelet FV, independent of the plasma FV concentration, produces this extraordinary rescue of TFPI-/- embryonic lethality. Collectively, these findings expose the critical role for platelet TFPIα in regulating procoagulant activity of prothrombinase assembled with platelet FV. This platelet FV-platelet TFPIα interaction highlights the important biological activity of pro and anticoagulant proteins located within platelets in regulating hemostatic balance during murine development and into adulthood. DisclosuresMast:Novo Nordisk: Research Funding.

Protamine Sulfate Interacts with Partially Activated Forms of Factor V Which Contributes to Its Paradoxical Anticoagulant Effect

Unfractionated heparin (UFH) is a highly negatively charged molecule commonly used to prevent blood clotting in different clinical situations including cardiac and vascular surgeries. Its anticoagulant effect can be rapidly reversed or neutralized with protamine sulfate, a positively charged polypeptide that forms a stable interaction with UFH. Paradoxically, in vitro and in vivo studies have reported that protamine can also elicit an anticoagulant effect. Examination into this phenomenon (Ni Ainle, 2009; Bollinger, 2010) suggests that the anticoagulant activity of protamine relates to the inhibition of factor V (FV) activation and prothrombinase generation, but further mechanistic insights are lacking. FV circulates in blood as an inactive procofactor protein. Proteolytic cleavage of its central B-domain by thrombin converts it into an active cofactor (FVa) for factor Xa (FXa) in the prothrombinase complex. Our laboratory has established that the basic region (BR; residues 963-1008) and acidic region 2 (AR2; residues 1493-1537) within the B-domain are the minimal autoinhibitory sequences that keep FV inactive. Deletion of the BR yields a protein with cofactor-like properties in the absence of proteolysis, and the addition of the BR fragment in trans inhibits FV procoagulant function. Under normal physiological situations, naturally occurring ligands that mimic BR could re-engage forms of FV that harbor AR2 but lack BR (generically termed, FVAR). For example, it has been found that the C-terminus of TFPIα is homologous to FV BR and binds to physiologically relevant forms of FVAR including platelet FV as well as FV-short. FV-short represents less than 2% of plasma FV and is a splicing isoform initially found in the FV-East Texas family. It lacks most of the B-domain including BR and binds with high affinity to TFPIα. It is constitutively active, however the interaction with TFPIα converts it to a procofactor-like state, impairing its ability to bind to FXa and get proteolytically processed by thrombin. Due to its basic nature and observed anticoagulant effects, we hypothesized that protamine binds to the AR2 of FVAR species and inhibits cofactor function. We purified plasma derived FV (pd-FV), recombinant FVa, FV-short and FV-shortQQ (a thrombin resistant mutant) and employed functional assays to characterize the interaction with protamine. Using purified component assays we found that FV-short and FV-shortQQ activities are comparable to FVa when assembled in prothrombinase. However, protamine had a major inhibitory effect on FV-short cofactor function, but not FVa. Further, using fluorescence anisotropy measurements, we found that protamine could displace labeled BR bound to FV-short, indicating that the two molecules compete for similar binding sites on FV-short. In addition, western blotting analysis showed that protamine impaired proteolytic processing of FV species by thrombin or FXa. When added to pooled normal human plasma (NHP) or FV-deficient plasma (FV-DP) reconstituted with pd-FV or FV-short or the QQ variant, protamine caused a dose-dependent anticoagulant response on thrombin generation (TG). The inhibition was greater with FV-shortQQ, while protamine did not affect TG when FVa was employed. The addition of protamine (10 μg/ml) to heparinized (0.2 U/ml) plasma restored TG in NHP and FV-DP reconstituted with FV-short or FVa. Addition of higher concentrations of protamine to heparinized plasma resulted in the progressive reduction of TG for NHP and FV-short plasma, with no further effects on plasma reconstituted with FVa. We conclude that protamine, through molecular mimicry of FV BR and the C-terminal region of TFPIα, binds forms of FV that have an available AR2 such as FV-short. This interaction not only decreases procoagulant function of FVAR species and inhibits the formation of the prothrombinase complex but also delays their further processing by thrombin or FXa. These results suggest that, under physiological conditions, protamine exerts its anticoagulant effect by interacting with physiological relevant FVAR species. These include platelet-derived FV, partially cleaved FV, and FV-short. This study provides a molecular mechanism for the adverse anticoagulant effects of protamine and highlights the importance of physiologically relevant FVAR species. DisclosuresCamire:Pfizer: Consultancy, Patents & Royalties, Research Funding; Bayer: Consultancy, Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Research Funding.

Altered phenotype in LMAN1-deficient mice with low levels of residual LMAN1 expression

AbstractCombined deficiency of coagulation factors V and VIII (F5F8D) is an autosomal recessive bleeding disorder caused by loss-of-function mutations in either LMAN1 or MCFD2. The latter genes encode 2 components of a mammalian cargo receptor that facilitates secretion of coagulation factor V (FV) and factor VIII (FVIII) from the endoplasmic reticulum (ER) to the Golgi via coat protein complex II vesicles. F5F8D patients exhibit FV and FVIII levels that are ∼10% to 15% of normal. We report herein a comparative analysis for a series of murine Lman1 alleles. Consistent with previous reports, mice completely deficient in LMAN1 (Lman1−/−) exhibit ∼50% FV and FVIII levels. In contrast, mice carrying a hypomorphic Lman1 allele (Lman1cgt/cgt) that expresses ∼6% to 8% of wild-type Lman1 mRNA levels exhibit intermediate plasma FV and FVIII reductions (∼70% of wild-type levels). Lman1−/− mice exhibit ER accumulation of another LMAN1 cargo, alpha-1 antitrypsin (A1AT), with an intermediate level of A1AT ER retention observed in Lman1cgt/cgt mice. Finally, the previously reported strain-specific, partially penetrant, perinatal lethality of LMAN1-deficient mice (Lman1gt1/gt1) was confirmed in Lman1−/− mice, although it was not observed in Lman1cgt/cgt mice. Taken together, these results show a dose-dependent effect of residual LMAN1 on the secretion of its cargo proteins. The results also suggest that human subjects with hypomorphic LMAN1 mutations might present with mild bleeding phenotypes resulting from more modest reductions in FV and FVIII, which could be missed by routine clinical evaluation. Finally, these findings suggest that therapeutic targeting of LMAN1 to reduce FV and FVIII as an anticoagulant strategy may only require partial inhibition of LMAN1 function.

Platelet-Derived Factor V Is an Important Determinant of the Metastatic Potential of Circulating Tumor Cells.

Factor V (FV) is a critical component in the blood coagulation cascade. In patients, FV inhibitors have been reported to be associated with malignancy. FV is present in plasma and platelets, which exhibit physical and functional differences. However, the functions of FV in cancer progression remain poorly understood. We evaluated the impact of different levels of FV in plasma and platelets on the haematogenous mouse pulmonary metastasis model to determine whether FV determines the metastatic potential of circulating tumor cells. The role of platelet-derived FV was evaluated using a murine B16F10 pulmonary metastasis model, an assay of tumor cell adhesion to endothelial cells, and western blotting. By combining genetic models and FV inhibitory antibody, the transgenic mice with lower platelet FV expression showed significant increases in metastases compared with mice with higher platelet FV expression. In vitro, labeled B16F10 melanoma cells appeared to exhibit increased adhesion to endothelial cells that were treated with lower levels of platelet FV, but not platelet-poor plasma. Furthermore, platelets from mice with lower platelet FV levels expressed TFPIα at lower levels than with mice with higher platelet FV expression. Based on these findings, platelet-derived FV contributes to haematogenous pulmonary metastasis and is associated with the regulation of tumor cell adhesion to the vessel wall.

Low factor V level ameliorates bleeding diathesis in patients with combined deficiency of factor V and factor VIII

AbstractCombined factor V (FV) and FVIII deficiency (F5F8D) is a rare autosomal-recessive bleeding disorder caused by mutations in lectin mannose binding-1 (LMAN1) and multiple coagulation factor deficiency-2 (MCFD2). Six causative homozygous mutations (5 in LMAN1 and 1 in MCFD2) were identified in 6 patients with F5F8D. A thrombin-generation assay, triggered with tissue factor (1 pM) in F5F8D plasma, paradoxically exhibited enhanced thrombin generation compared with normal plasma. Significantly lower free tissue factor pathway inhibitor (fTFPI) was found in F5F8D patients compared with healthy controls (P < .01). Normalizing tissue factor pathway inhibitor α (TFPIα) in F5F8D plasma greatly delayed and reduced thrombin generation. Increasing FV concentrations by adding plasma FV to F5F8D plasma only caused a gradual decrease in thrombin generation, suggesting that low levels of TFPIα and FV cocontributed to the elevated thrombin generation by reducing anticoagulant effects. On the contrary, thrombin generation in F5F8D platelet-rich plasma (PRP) was significantly lower than in normal controls (P < .05); however, it was fully corrected by normalizing FVIII or after 1-deamino-8-d-arginine vasopressin (DDAVP) infusion, indicating that the hypocoagulable state of F5F8D patients is associated with low FVIII levels. In addition, plasma and platelet FV in F5F8D PRP were sufficient to support normal thrombin generation, and low TFPIα may have no effect on thrombin generation. DDAVP infusion induced a complete response in 5 F5F8D patients and a partial response in the remaining patient. Based on our findings, we suggest that DDAVP may be considered a potential substitute for FVIII concentrates, and fresh-frozen plasma (FFP) infusion may not be necessary for F5F8D patients with minor bleeding challenges.

Development of a Plasma-Based Assay to Measure the Susceptibility of Factor V to Inhibition by the C-Terminus of TFPIα.

Factor V (FV) is proteolytically activated to FVa, which assembles with FXa in the prothrombinase complex. The C-terminus of tissue factor pathway inhibitor-α (TFPIα) inhibits both the activation and the prothrombinase activity of FV(a), but the pathophysiological relevance of this anticoagulant mechanism is unknown. FV Leiden (FVL) is less susceptible to inhibition by TFPIα, while overexpression of FV splicing variants with increased affinity for TFPIα (FV-short) causes bleeding. This study aims to develop a plasma-based assay that quantifies the susceptibility of FV(a) to inhibition by the TFPIα C-terminus. FV in highly diluted plasma was preactivated with FXa in the absence or presence of the TFPIα C-terminal peptide. After adding prothrombin, thrombin formation was monitored continuously with a chromogenic substrate and prothrombinase rates were obtained from parabolic fits of the absorbance tracings. TFPI resistance was expressed as the ratio of the prothrombinase rates with and without peptide (TFPIr). The TFPIr (0.25-0.34 in 45 healthy volunteers) was independent of FV levels. The TFPIr increased from normal individuals (0.29, 95% confidence interval [CI] 0.28-0.31) to FVL heterozygotes (0.35, 95% CI 0.34-0.37) and homozygotes (0.39, 95% CI 0.37-0.40), confirming TFPI resistance of FVL. Two individuals overexpressing FV-shortAmsterdam had markedly lower TFPIr (0.16, 0.18) than a normal relative (0.29), in line with the high affinity of FV-short for TFPIα. We have developed and validated an assay that measures the susceptibility of plasma FV to the TFPIα C-terminus. Once automated, this assay may be used to test whether the TFPIr correlates with thrombosis or bleeding risk in population studies.

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.

Thrombin Generation Impairments in Platelet Function Disorders

Many platelet function disorders (PFD) present as uncharacterized disorders that impair aggregation responses to multiple agonists and/or cause non-syndromic dense granule deficiency (DGD). We postulated that some of these commonly encountered PFD might also impair the ability of platelets to support thrombin generation, given that an important subgroup are caused by mutations in transcription factors, such as RUNX1, that impair multiple aspects of platelet function. Accordingly, we initiated a study of thrombin generation (TG) in a prospective cohort of individuals presenting with an uncharacterized PFD. In addition, we reassessed how Quebec platelet disorder (QPD, previously called Factor V Quebec) affects coagulation as this disorder (which is caused by a duplication mutation of PLAU) triggers intraplatelet (but not systemic) plasmin generation and proteolysis of platelet but not plasma factor V (FV), and the normal plasma FV might potentially compensate for the loss of platelet FV in QPD.The study was conducted with written informed consent of participants and with the approval of the Hamilton Integrated Research Ethics Board and the Research Ethics Board of Centre Hospitalier Universitaire Sainte Justine. Participants included: 1) five individuals with QPD; 2) eighteen individuals presenting with an uncharacterized PFD and confirmed reduced maximal aggregation responses to ≥2 agonists and/or confirmed DGD, including five that had a pathogenic RUNX1 mutation; and 3) eighteen similarly-aged general population controls. TG was assessed by the calibrated automated thrombogram (CAT) procedure on a Fluoroskan (Thermo Fisher Scientific AG, Reinach, Switzerland) using Thrombinoscope software (Synapse BV, Maastricht, The Netherlands), manufacturer- and ISTH-recommended protocols and reagents for testing platelet poor plasma (PPP) and platelet rich plasma (PRP). Endpoints included: endogenous thrombin potential (ETP, nM·min), peak thrombin concentration (nM), time-to-peak (min) and lag time (min). Platelet lysate and plasma FV concentrations were determined by enzyme-linked immunoassay. Data were analyzed using Mann-Whitney tests with Bonferroni correction for multiple comparisons.All TGA endpoints for PPP were comparable for controls and participants with PFD, including QPD (p values ≥0.10). In TGA with PRP, most PFD participants had findings similar to controls, however, the subgroups with pathogenic RUNX1 mutations or QPD had significantly reduced ETP and peak thrombin concentration compared to controls (data as median [range]: ETP: controls: 1860 [1530-2630]; RUNX1 mutation subgroup: 1520 [805-1640], p=0.004; QPD: 1370 [981-2010], p=0.01; peak thrombin concentration: controls: 111 [65-152]; RUNX1 mutations: 66 [32-93], p=0.006; QPD: 59 [41-91], p=0.005). Plasma FV levels were similar in all subjects (µg/ml PPP, median [range]: controls: 7.7 [6.3-10.7]; QPD: 8.4 [7.0-10.2], p=0.33; other PFD: 7.5 [5.4-12], p=0.74) and showed no significant association to TG endpoints for PPP or PRP (p values ≥0.14). Only QPD subjects had platelet FV deficiency (µg FV/mg platelet protein, median [range]: controls: 0.89 [0.63-1.54]; QPD: 0.35 [0.18-0.46], p<0.001; other PFD: 0.83 [0.47-1.75], p=0.48; RUNX1 mutation subgroup: 0.73 [0.50-1.53], p=0.41). In QPD, but not other participants, platelet FV showed a significant association to ETP (R2=0.81, p=0.04) and peak TG endpoints (R2=0.88, p=0.01).Our study illustrates that platelet-dependent thrombin generation, evaluated by CAT, is abnormal in QPD but normal in many uncharacterized PFD with defective aggregation and/or DGD except for the important subgroup with pathogenic RUNX1 mutations, which impair TG but do not reduce platelet FV (unlike QPD). In QPD, the platelet-dependent TG defect shows a unique relationship to the platelet FV deficiency, which suggests that the normal levels of plasma FV are insufficient to compensate for the platelet procoagulant abnormalities in this PFD. DisclosuresNo relevant conflicts of interest to declare.

A Plasma-Based Assay to Measure the Susceptibility of Factor V(a) to Inhibition By TFPIα

Background: Coagulation factor V (FV) is the precursor of activated FV (FVa), which assembles with factor Xa (FXa) on phospholipid surfaces to form the prothrombinase complex, accelerating prothrombin activation >1000-fold. FV is activated by FXa or thrombin via limited proteolysis at Arg709, Arg1018 and Arg1545. These cleavages progressively expose the FXa-binding site, generating activation intermediates with increasing affinities for FXa. Recently, it has been shown that tissue factor pathway inhibitor α (TFPIα) inhibits both the activation of FV (by interfering with cleavage at Arg1545) and the ability of partially activated FV(a) species to enhance prothrombin activation. These effects are mediated by interactions of the C-terminus of TFPIα with an acidic region in the B-domain of FV as well as with the FV(a) heavy chain. The pathophysiological relevance of these novel anticoagulant activities of TFPIα is still unexplored, but evidence has been provided that prothrombinase complexes assembled with FV(a) Leiden are less susceptible to TFPIα inhibition. Moreover, FV splicing variants (FV-short) with increased affinity for TFPIα have recently been discovered in two unrelated families (from East Texas and Amsterdam, respectively) with bleeding tendencies.Rationale and Aim: The FV present in plasma from different individuals may differ in its sensitivity to inhibition by TFPIα, with potential implications for the risk of venous thrombosis or bleeding. Therefore, the aim of this study was to develop a plasma-based assay that measures the susceptibility of FV(a) to TFPIα inhibition.Methods: FV in 1/1000 diluted plasma was activated for 3 minutes with a suboptimal FXa concentration on 20/60/20 DOPS/DOPC/DOPE lipids in the presence or absence of a peptide mimicking the C-terminus of TFPIα (TFPIα C-term). Purified prothrombin and a chromogenic substrate for thrombin were then added, and the activity of the prothrombinase complex was monitored continuously up to 30 minutes. The parabolic absorbance curves were fitted to second-order polynomial equations and the rate of prothrombin activation was calculated from the coefficient of the x2-term. The assay outcome was expressed as residual prothrombinase ratio (RP-ratio), defined as the ratio between the rates of prothrombin activation obtained in the presence and absence of TFPIα C-term. The assay was validated using plasma from 4 FV Leiden homozygotes and 4 normal controls. In addition, we tested plasma from 3 members of the FV Amsterdam family (2 carriers of the mutation up-regulating FV-short Amsterdam and 1 non-carrier).Results: The rate of prothrombin activation in the absence of peptide was a function of plasma FV level and pre-incubation time, and was inhibited by TFPIα C-term in a dose-dependent manner. A pre-incubation time of 3 minutes and a peptide concentration of 100 nM, yielding an RP-ratio of 0.30 in normal pooled plasma, were chosen. The RP-ratio was independent of the plasma FV level in the 75-150% range. Moreover, control experiments indicated that, at this high dilution, the plasma background did not influence the assay outcome. The intra- and inter-assay coefficients of variation of the RP-ratio were 5.4% and 12%, respectively. FV Leiden homozygotes had higher RP-ratios than normal controls (0.45 ± 0.04 vs. 0.30 ± 0.03, p=0.002), indicating resistance to inhibition by TFPIα C-term. Differently, the 2 carriers of the FV Asterdam mutation, who express high levels of FV-short Amsterdam, had markedly reduced RP-ratios (0.18 and 0.16 vs. 0.29 in the non-carrier), as expected from the high affinity of FV-short Amsterdam for TFPIα.Conclusions: We have developed and validated an assay that measures the susceptibility of plasma FV(a) to inhibition by TFPIα. This assay can be used to test whether TFPIα-mediated inhibition of FV activation and prothrombinase activity differs for (genetically) different FV variants and whether it correlates with the risk of thrombosis or bleeding.Supported by grant 2014-1 from the Dutch Thrombosis Foundation. DisclosuresNo relevant conflicts of interest to declare.

Subtype‐specific clinical and prognostic relevance of tumor‐expressed F5 and regulatory F5 variants in breast cancer: the CoCaV study

Background Tumor expression of certain coagulation factors has been linked to cancer progression. Single nucleotide polymorphisms (SNPs) in F5 (encoding the FV protein) have been found to be associated with breast cancer; however, the role of coagulation factor V (FV) in cancer pathogenesis remains undiscovered. Objectives We aimed to investigate the clinical significance of FV and the regulatory role of F5 gene variants in breast cancer. Patients/Methods A Scandinavian 503-sample breast cancer cohort and three public breast cancer datasets (GOBO, TCGA and KM plotter) were used to determine associations between F5 gene expression (tumor-specific), circulating FV, F5 SNPs, clinical characteristics and breast cancer survival. Immunohistochemistry (IHC) was used to detect FV antigen in tumors. Results F5 expression was 2-fold higher in breast tumors compared with normal tissue, and the presence of FV antigen in breast tumors was confirmed by IHC staining. F5 expression was increased in patients with hormone receptor negative tumors, triple negative tumors, HER2-enriched and basal-like tumors. In patients with basal tumors, high expression of F5 was associated with improved overall survival (hazard ratio, HR = 0.52, 95% confidence interval, 0.31-0.86). SNPs in F5 were associated with tumor size and luminal A tumors. The rs6427202-rs9332542 C-G haplotype, previously associated with breast cancer, displayed a cis-regulatory effect on F5 expression in tumors and plasma FV antigen levels. In silico mining supported this regulatory function. Conclusions FV was a possible marker of aggressive breast cancer, yet also a predictor of favorable outcome. Evaluation of FV expression may be clinically useful for prognosis and treatment decisions in aggressive breast cancer.

Factor V Has Anticoagulant Activity in Plasma in the Presence of TFPIα: Difference between FV1 and FV2.

Activated factor V (FVa) is a potent procoagulant cofactor in the prothrombinase complex, whereas its precursor factor V (FV) stimulates the inhibition of factor Xa (FXa) by tissue factor pathway inhibitor-α (TFPIα), presumably by promoting TFPIα binding to phospholipids. Plasma FV comprises two glycosylation isoforms (FV1 and FV2) with low and high phospholipid-binding affinity, respectively. The FV1/FV2 ratio is increased in carriers of the FV R2 haplotype. This article demonstrates the TFPIα-cofactor function of FV in plasma and compares FV1 and FV2. Thrombin generation at low TF concentration was measured in FV-depleted plasma reconstituted with 0 to 100% FV, FV1 or FV2, and in 122 individuals genotyped for the R2 haplotype. The TFPIα-cofactor activities of FV1 and FV2 were also investigated in a model system of TFPIα-mediated FXa inhibition. In the FV titration, thrombin generation first increased (up to 5% FV) and then progressively decreased at higher FV concentrations. This anticoagulant effect of FV, which was also observed with FV2 but not with FV1, was largely abolished by anti-TFPIα antibodies, suggesting that it reflects TFPIα-cofactor activity of FV. In the model system of TFPIα-mediated FXa inhibition, FV2 was a more potent TFPIα-cofactor than FV1, in line with their respective phospholipid affinities. Accordingly, FV R2 carriers had higher thrombin generation than non-carriers, even after correction for demographics and plasma levels of coagulation factors and inhibitors. FV (and particularly its FV2 isoform) contributes to the TFPIα-dependent down-regulation of thrombin generation in plasma triggered with low TF.

Analysis of MCFD2- and LMAN1-deficient mice demonstrates distinct functions in vivo

AbstractThe LMAN1–MCFD2 complex serves as a cargo receptor for efficient transport of factor V (FV) and FVIII from the endoplasmic reticulum (ER) to the Golgi. Genetic deficiency of LMAN1 or MCFD2 in humans results in the moderate bleeding disorder combined FV and FVIII deficiency, with a similar phenotype previously observed in LMAN1-deficient mice. We now report that MCFD2-deficient mice generated by gene targeting also demonstrate reduced plasma FV and FVIII, with levels lower than those in LMAN1-deficient mice, similar to previous observations in LMAN1- and MCDF2-deficient humans. Surprisingly, FV and FVIII levels in doubly deficient mice match the higher levels observed in LMAN1-deficient mice. In contrast to the strain-specific partial lethality previously observed in LMAN1-null mice, MCFD2-null mice demonstrate normal survival in different genetic backgrounds, although doubly deficient mice exhibit partial embryonic lethality comparable to LMAN1-deficient mice. These results suggest that an alternative pathway is responsible for FV/FVIII secretion in doubly deficient mice and distinct cargo-specific functions for LMAN1 and MCFD2 within the ER-to-Golgi secretory pathway. We also observed decreased plasma levels of α1-antitrypsin (AAT) in male mice for all 3 groups of deficient mice. Comparable accumulation of AAT was observed in hepatocyte ER of singly and doubly deficient mice, demonstrating a role for LMAN1 and MCFD2 in efficient ER exit of AAT.

Factor V Inhibitors: A Diagnostic and Therapeutic Challenge.

Historically, inhibitors to coagulation factor V (FV) most often have developed in patients treated with bovine thrombin, a topical hemostatic agent used during surgical procedures. With the advent of newer hemostatic agents, and the concurrent diminished use of bovine thrombin, the incidence of FV inhibitors has fallen. Nevertheless, FV inhibitors are occasionally seen on an idiopathic basis as well as in association with medications, malignancies, autoimmune disorders, pregnancy, and infections. Factor V inhibitors may present with life-threatening bleeding or thrombosis, or they may be discovered incidentally as a coagulation screening test abnormality. Management of patients with FV inhibitors is challenging and consists of control of bleeding and eradication of the inhibitor. In this short overview we review the role of platelet and plasma FV in hemostasis and discuss the unique characteristics, clinical features, diagnosis, treatment, and prognosis associated with FV inhibitors.

Platelet-Derived Factor V Is a Critical Mediator of Arterial Thrombosis.

BackgroundCoagulation factor V (FV) plays a key role in hemostasis, is present in plasma and platelets, and has both pro‐ and anticoagulant properties; however, the contribution of platelet‐derived FV to arterial thrombosis remains undetermined.Methods and ResultsUsing transgenic mice with various levels of FV gene expression that was restricted to the plasma or platelets, the roles of platelet FV were evaluated in the regulation of arterial thrombosis and platelet activation. Mice with higher levels of platelet FV exhibited faster thrombotic occlusion of the carotid artery after injury compared with mice with lower platelet FV levels. Infusion of platelets with higher levels of FV into transgenic mice with undetectable levels of platelet FV reduced the time to carotid artery occlusion. In contrast, infusion of purified recombinant plasma FV into mice with undetectable platelet FV levels failed to reduce the carotid occlusion times following injury. Evaluation of isolated platelets revealed that platelet‐derived FV was critical for the regulation of platelet activation. These effects were associated with an increased level of expression of P‐selectin and increased cGMP in platelets.ConclusionsWe established that platelet‐derived FV is a critical mediator of arterial thrombosis that involves platelet activation.