Plasma Factor V Research Articles

Effects of Multimerin 1 on Platelet and Plasma FV as Determined by Calibrated Automated Thrombography.

Multimerin 1 (MMRN1) is a protein that is not found in plasma and that is complexed with factor V (FV) in platelet α-granules. Following thrombin activation, platelet FV and MMRN1 are released, and FV is converted to Va, promoting its dissociation from MMRN1. Recent studies identifying functional overlap between FV/Va binding sites for phospholipids and MMRN1, and unanswered questions about the functional consequences of MMRN1 on platelet FV function, led us to compare how MMRN1 modulates platelet and plasma FV procoagulant functions using calibrated automated thrombograms (CAT). The effects of platelet and exogenous MMRN1 on FV activity were evaluated using samples of platelet poor human plasma or platelet extracts resuspended in FV deficient plasma to equivalent final FV antigen concentrations. Reactions were triggered with a commercial reagent containing saturating concentrations of tissue factor and phospholipids (60:20:20 phosphatidylcholine: phosphatidylserine: phosphatidylethanolamine). These assays were also used to compare the functional properties of FV in MMRN1-immunodepleted and sham-depleted fractions of a soluble platelet protein extract (prepared with calpain inhibitors). The effects of exogenous MMRN1 on platelet-dependent thrombin generation were also evaluated using normal platelets resuspended in FV deficient plasma. In contrast to plasma, platelet fractions (with an equivalent FV antigen content) consistently showed lower peak and total endogenous thrombin potential. Addition of 17 fold molar excess of exogenous MMRN1 (which represents the physiologic ratio of MMRN1 to FV in platelets) to plasma, or to MMRN1 depleted platelet protein extracts, induced similar reductions in thrombin generation, manifested by increases in lag times (p<0.05), and reductions in peak (p<0.05), and total thrombin generation (p<0.05). Higher concentrations of exogenous MMRN1 caused further attenuation. Analogous, dose-dependent, inhibitory effects of MMRN1 were observed on thrombin generation by normal platelets in FV deficient plasma. Further investigations of the thrombin generating potential of platelet FV were done using sham and MMRN1-depleted platelet extracts. While exogenous MMRN1 inhibited platelet FV dependent thrombin generation, sham-depleted platelet FV (containing MMRN1) consistently showed higher thrombin generation than the same quantity of FV in MMRN1-depleted platelet extracts, characterized by shorter lag times (p<0.05), higher peak thrombin generation (p<0.05), and increased endogenous thrombin potential (p<0.05). Although the sham-depleted fraction had a similar reduced mobility to the MMRN1 depleted platelet FV, it contained very large forms of platelet FV, linked to MMRN1 via the human FV B-domain cysteine. These data indicate that the pool of platelet FV associated with MMRN1 in vivo has procoagulant function and that MMRN1 has inhibitory effects on thrombin generation. The observed differences in the procoagulant properties of platelet and plasma FV in CAT assays likely result from a partial inhibition of platelet FV by MMRN1 that may be counterbalanced by partial platelet FV activation. The paradoxical effects of MMRN1 depletion on the procoagulant function of platelet FV suggests MMRN1 may have other influences that help preserve platelet FV function in vitro, and possibly also in vivo. The MMRN1 liberated by activated platelets and endothelium in vivo could have local modulating effects on thrombin generation.

Dysregulation of Alternative Splicing of Coagulation Factor V Results in Bleeding Disorder, East Texas Type.

Factor V has recently been observed to act as a synergistic anticoagulant cofactor in the activated Protein C (APC) pathway; however, the importance of factor V's anticoagulant nature in vivo is poorly understood. A novel autosomal dominant bleeding disorder, which is characterized by excessive bleeding with trauma or surgery and menorrhagia in affected women, was identified in a large family (16 affected individuals) from east Texas. Affected members had a prolongation of their PT and/or aPTT. Clinical studies indicated normal activities and levels of all coagulation factors. Linkage analysis mapped the defective gene to 1q23–24 (LODmax 7.33), which contains the gene for coagulation factor V (FV). An alteration (A2440G)in the FV gene in exon 13, which encodes the entire B-domain of the wildtype protein, segregates with disease and was not present in 62 controls. Interestingly, this alteration falls on the 5′ splice site (TA/GT) of an alternative splicing variant confirmed by RT-PCR of control liver and leukocytes. Furthermore, immunoblot analysis demonstrated that this splice variant produces a low abundance, 250kD isoform of FV in control plasma. The A2440G alteration can theoretically form a higher efficiency consensus site for splicing (TG/GT). Semi-quantitative RT-PCR confirms this improved efficiency with an increase of splicing seen in patients' RNA from leukocytes (n=3) versus controls (n=3). This translates into an approximately 20-fold increase in the 250kD isoform of FV seen in patients' plasma (n=6) versus controls (n=5), while wildtype FV levels remain equivalent. Co-precipitation with Vitamin-K dependent coagulation factors from patient plasma suggests that this isoform interacts with known FV-interacting proteins (Figure 1). A recombinant of this splicing event (FV-703) exhibited normal activation by thrombin and normal prothrombinase activity when compared to the wildtype recombinant. Thrombin-activated FV-703 also showed normal degradation by APC, while intact FV-703 displayed an increased sensitivity to APC that was more striking in the presence of PS (Figure 2). We hypothesize that this novel isoform has a stronger affinity for APC in the presence of PS than wildtype FV and works primarily as a cofactor in the APC pathway to heighten the overall anticoagulant activity in the bloodstream. These data indicate that A2440G up-regulates an alternatively spliced transcript of FV, and increases a FV isoform that hinders coagulation as opposed to promoting it like its wildtype counterpart. As verified by this unique mutation, this novel alternative splicing of coagulation FV is an important physiological event that can result in disease if its delicate balance is disrupted. [Display omitted] [Display omitted]

Severe Factor V Deficiency Associated with Chromosome 1q Deletion.

Background:Coagulation factor V (FV) is an essential cofactor of the prothrombinase complex, catalyzing conversion of prothrombin to thrombin. Severe FV deficiency is a rare coagulation defect with a prevalence in the general population of approximately 1 in 1,000,000 and a variable bleeding risk (Asselta R et al. J Thromb Haemostas 2006). Genetic mutations underlying a FV-deficient hemorrhagic diathesis have been described in a limited number of cases, display considerable allelic heterogeneity, and have been due to point mutations in the FV gene located at chromosome 1q23.Here, we report a 15 year-old girl with psychomotor delay, CNS abnormalities, and a solitary left kidney - features consistent with a congenital 1q deletion including band 1q23 - who was found to have severely reduced plasma FV activity after a routine preoperative laboratory evaluation prior to an elective spinal stabilization surgery revealed a mildly prolonged prothrombin (PT, 13.2 seconds; normal 8.4 – 12 seconds) and activated partial thromboplastin (aPTT, and 38 seconds; normal 21 – 33 seconds) time. She had undergone minor surgical procedures in the past without reports of significant bleeding, and her family members were also clinically unaffected.Methods:FV procoagulant activity level was measured in the patient with an MDA-180 automated coagulation analyser (Trinity Biotech, Ireland). The patient's peripheral blood leukocytes were separated by density centrifugation and fixed in methanol:glacial acetic acid (2:1). We then performed interphase fluorescent in-situ (FISH) analysis using probes covering the FV gene and the more telomeric HPC1 gene as a control. Genomic DNA was also isolated from the patient, and the coding region (25 exons) of the FV gene amplified by polymerase chain reaction (PCR) and sequenced.Results:FV activity assays revealed a severely reduced level of 9% (normal range: 60–130%). Factor V activity levels in the patient's family members were not assessed. Peripheral blood karyotyping revealed an interstitial deletion of 1q: 46,XX,del(1)(q24.2q25.3 or q23.3q25.1).Interphase FISH analysis was performed. Red signals corresponded to the FV probe and green signals corresponded to HPC1, which is approximately 13 MB telomeric to FV. In contrast to healthy controls, the patient had a signal pattern of only 1 green HPC1 signal and 1 red FV signal, corresponding to heterozygous loss of the FV gene and adjacent chromosomal material. Sequencing of the retained allele revealed a novel hemizygous S234W mutation (encoded by exon 6). The lost serine is part of a conserved “SGP” peptide segment predicted to be a solvent-exposed region of the heavy chain of the FV protein.Conclusion:Most mutations causing FV deficiency are autosomal recessive and introduce frame shifts resulting in the generation of a premature termination codon. Karyotypic and FISH analysis of this patient revealed a novel mechanism for FV deficiency: loss of an entire FV allele as part of a chromosomal deletion, coupled with a point mutation in the other allele. Structural modeling of the consequences of the S234W point mutation, which is predicted to alter the hydrophobicity of the protein, is ongoing. Syndromes involving an interstitial deletion of 1q have been described in association with ATIII deficiency (Pallotta, Dalpra et al. 2001), but to our knowledge, this is the first description of a 1q deletion with FV deficiency.

Von Willebrand Factor Protects Ca2+-Dependent Structure of the Factor VIII Light Chain.

Factor VIII (FVIII) cofactor function is dependent on the presence of divalent cation, since its active form is metal-linked heterotrimer as well as factor V (FV). The activity of FVIII (FVIII:C) and FV in normal plasma were completely and selectively inactivated by the treatment with divalent cation exchange resin, imidinoacetate. However, FVIII antigen (FVIII:Ag) was preserved, suggesting that deprivation of FVIII:C by the resin was not due to absorption of FVIII but due to inactivation. Both FVIII:Ag and FVIII:C of recombinant FVIII were decreased by treatment with the resin. However, FVIII:Ag but not FVIII:C, was preserved in FVIII and von Willebrand factor complex (FVIII/VWF). Sandwich ELISA using anti-A3 and anti-A2 monoclonal antibodies revealed that association with heavy and the light chains were impaired in the resin-treated FVIII. These results suggested that FVIII was inactivated by deprivation of the metal ion by the resin and that VWF protected cation-dependent FVIII structure. Therefore, we tested if the resin deprives the metal ion such as Ca2+ from FVIII or FVIII/VWF. [Ca2+] in FVIII preparation was decreased from 1.30 to 0.07 mM by the resin. Furthermore, [Ca2+] was decreased from 0.39 to 0.01 mM in FVIII/VWF preparation. [Ca2+] was recovered completely by elution with 1 N HCl from the resins used in both preparations. FVIII:C of the resin-treated FVIII/VWF was partially recovered by addition of Ca2+, whilst FVIII:C of the resin-treated FVIII was not recovered. When FVIII was treated with the resin after addition of [Ca2+], the inactivation of FVIII was dose-dependently inhibited by ~20 % (at [Ca2+]: ~75 mM). On the other hand, the inactivation of FVIII was inhibited by ~60 % (at [Ca2+]: ~25 mM) in FVIII/VWF preparation. Present results demonstrated that FVIII was selectively inactivated by the cation exchange resin due to deprivation of Ca2+. Kinetic experiments by surface plasmon resonance using BIAcore demonstrated that the resin-treated FVIII as well as treated C2 didn't interact with phospholipid and VWF. Furthermore, immunoblot analysis using the resin-treated FVIII revealed that anti-A2 monoclonal antibody reacted with the heavy chain, whilst anti-A3 and anti-C2 antibodies failed to react with the light chain. On the other hand, these antibodies reacted with the light chain in the experiment using FVIII/VWF, indicating that VWF protects antigenic conformation of the FVIII light chain. Present findings suggest another protection mechanism of VWF on FVIII through stabilization of Ca2+-dependent structure of the FVIII light chain.

Gene duplication of coagulation factor V and origin of venom prothrombin activator in Pseudonaja textilis snake

The 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 approximately 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.

Secretable Human Megakaryocyte Factor V Endocytosed from Plasma in Complex with Multimerin: The Origin of Human Megakaryocyte Factor V.

Factor V (FV) is an essential coagulation cofactor that circulates in distinct plasma and platelet pools. Unlike plasma FV, platelet FV is stored complexed to the megakaryocyte (MK) synthesized α-granule protein multimerin, which is not found in plasma. The origin of platelet FV has been controversial and recent studies have suggested human platelet factor V originates from plasma. To determine if human MK could endocytose FV and generate the FV-multimerin complexes, we investigated FV endocytosis and storage by human MK derived from cord blood CD34+ cells. Cells were cultured with or without 10% plasma in 15% BIT/ IMDM containing 5ng/ml SCF and 50ng/ml TPO. Human plasma or purified plasma FV was added on day 7 of culture. Cells were harvested to analyze α-granule proteins on day 11. Enzyme-linked immunoassays were used to evaluate MK content of FV and multimerin, and their complexes. Control α-granule proteins analyzed included albumin, IgG and fibrinogen, as they are known to originate from plasma. Multimerin was detected in all MK whereas FV, albumin, IgG, fibrinogen and FV-multimerin complexes were only detected in MK cultured with plasma. The amounts of plasma-derived proteins in MK were orders of magnitude lower than in platelets. MK cultured with purified FV had dose-dependent FV uptake. With SFLLRN stimulation, MK cultured with plasma released multimerin, FV, albumin, IgG and fibrinogen, consistent with their storage in secretion granules. Like platelet FV, MK FV showed a proteolyzed pattern. Double immunostaining showed partial colocalization of intracellular multimerin and FV in MK cultured with FV. Immunoelectron microscopy confirmed that like bone marrow MK, MK cultured with FV contained FV within demarcation membranes, small vesicles and α-granules. Analyses of FV expression by cultured MK, using semi-quantitative RT-PCR, indicated there were no significant differences in FV expression by MK cultured with or without plasma, at any of the time points evaluated. However, there was a trend to increased FV expression with MK maturation, and for MK cultured in plasma, FV expression was significantly higher at 15 compared to 7 days, raising the possibility that some FV was synthesized at late stages of megakaryopoiesis. Our study illustrates that cultured human MK can proteolyze and target endocytosed plasma FV into α-granules and that they can generate complexes of FV with multimerin.

Clinical and molecular characterization of 6 patients affected by severe deficiency of coagulation factor V: broadening of the mutational spectrum of factor V gene and in vitro analysis of the newly identified missense mutations

AbstractSevere factor V (FV) deficiency is a rare bleeding disorder, whose genetic bases have been characterized only in a limited number of cases. We investigated 6 unrelated patients with extremely reduced plasma FV levels, associated with a bleeding tendency ranging from moderately severe to severe. Clinical manifestations were substantially concordant with the previously established spectrum of hemorrhagic symptoms of the disease. Molecular analysis of FV gene identified 9 different mutations, 7 hitherto unknown, and 2 previously reported (Arg712ter and Tyr1702Cys). Four of 6 analyzed patients were compound heterozygotes, indicating the high allelic heterogeneity of this disease. Among novel mutations, 5 led to premature termination codons, because of nonsense (Arg1002ter, Arg1606ter, and Trp1854ter), or frameshift mutations (5127-5128insA and 6122-6123insAACAG). The remaining 2 were missense mutations (Cys472Gly and Val1813Met), located in FV A2 and A3 domains. Their effect on FV expression was studied by transient transfection experiments, demonstrating that the presence of each mutation impaired FV secretion. These data increase the number of severe FV deficiency–causing mutations by about 50%. The high number of “private” mutations identified in FV-deficient families indicates that full mutational screening of FV gene is still required for molecular diagnosis.

The murine platelet and plasma factor V pools are biosynthetically distinct and sufficient for minimal hemostasis

AbstractCoagulation factor V (FV) is a central regulator of the coagulation cascade. Circulating FV is found in plasma and within platelet α granules. The specific functions of these distinct FV pools are uncertain. We now report the generation of transgenic mice with FV gene expression restricted to either the liver or megakaryocyte/platelet lineage using bacterial artificial chromosome (BAC) constructs. Six of 6 independent albumin BAC transgenes rescue the neonatal lethal hemorrhage of FV deficiency. Rescued mice all exhibit liver-specific Fv expression at levels ranging from 6% to 46% of the endogenous Fv gene, with no detectable FV activity within the platelet pool. One of the 3 Pf4 BAC transgenes available for analysis also rescues the lethal FV null phenotype, with FV activity restricted to only the platelet pool (approximately 3% of the wild-type FV level). FV-null mice rescued by either the albumin or Pf4 BAC exhibit nearly normal tail bleeding times. These results demonstrate that Fv expression in either the platelet or plasma FV pool is sufficient for basal hemostasis. In addition, these findings indicate that the murine platelet and plasma FV pools are biosynthetically distinct, in contrast to a previous report demonstrating a plasma origin for platelet FV in humans.

The factor V Leiden mutation results in the in vivo loss of a critical FV anticoagulant function

OC257 The factor V Leiden mutation results in the in vivo loss of a critical FV anticoagulant function Hall 4 11:30 16th July, 2003 Session Type: Oral communications Subject area: Factor VIII, factor V Session title: Factor V and thrombotic disease Abstract: OC257 Authors: R. J. Westrick, H. Sun, D. T. Eitzman, S. L. Manning, A. Y. Yang, D. R. Siemieniak, A. Ayigari & D. Ginsburg University of Michigan, USA The Factor V Leiden (FVL) mutation is the commonest thrombotic risk factor in humans. Heterozygous carriers have an approximately 10% lifetime incidence of venous thrombosis, compared to 80% in homozygotes. FVL is resistant to degradation by Activated Protein C (APC), resulting in prolonged factor V (FV) prothrombinase cofactor activity. In vitro studies suggest that FV can serve as a cofactor for APC in the inactivation of Factor VIIIa, with FVL demonstrating a reduced cofactor activity. To address the relative in vivo contributions of these mechanisms to thrombosis associated with FVL, we examined crosses among genetically engineered mice heterozygous deficient for FV (Fv+/-) or tissue factor pathway inhibitor (TFPI, Tfpi+/-), homozygous or heterozygous for the FVL mutation (FvQ/Q or FvQ/+), or carrying either a liver specific (MalbFvTg+) or platelet specific (Pf4FvTg+) FV transgene. Analysis of offspring from an intercross between FvQ/and FvQ/Q mice demonstrated a reduction in the survival of FvQ/mice compared to FvQ/Q (49 vs. 113 out of 162 total offspring, expected ratio is 1 : 1, p < 0.05). We recently reported synthetic thrombotic lethality of the FvQ/Q and Tfpi+/genotypes. Analysis of FvQ/mice in the setting of reduced TFPI demonstrated a similar uniform lethality for the FvQ/Tfpi+/genotype, in contrast to the normal survival of FvQ/+Tfpi+/mice. Introduction of either the MalbFv or Pf4Fv transgenes into this cross resulted in partial rescue of the lethal FvQ/Tfpi+/phenotype. These data confirm the association of homozygous FVL with a more severe thrombotic phenotype than in heterozygotes. These findings identify the critical mechanism for this enhanced thrombosis as loss of a wild-type-specific activity, rather than increased APC resistance. This critical wild type FV function can be provided by either the platelet or plasma FV pool. These findings have important implications for the role of FV in the regulation of hemostatic balance. Supplement to the Journal of Thrombosis and Haemostasis July 2003 (ISSN 1740 3340) file:///E|/working/LAXMI-PRASAD/WileyML-3G/deepak/31-Jan/Wednesday/Abstract%20OC257.html [1/31/2014 3:49:44 PM]

Comparison of Activated Protein C/Protein S-Mediated Inactivation of Human Factor VIII and Factor V

The proteolytic cleavage and subsequent inactivation of recombinant human factor VIII (rhFVIII) and human factor VIIIa (rhFVIIIa) by recombinant human activated protein C (rAPC) was analyzed in the presence and absence of human protein S and human factor V (FV). Membrane-bound rhFVIIIa spontaneously looses most of its initial cofactor activity after 15 minutes of incubation at pH 7.4. The remaining activity can be eliminated after incubation with rAPC. Complete inactivation of the membrane-bound rhFVIII and rhFVIIIa by APC correlates with cleavage at Arg336. The inactivation of rhFVIII and human plasma FV by rAPC were also compared. Under similar experimental conditions, complete inactivation of membrane-bound FVIII (60 nmol/L) by rAPC (10 nmol/L) requires 4 hours of incubation, in contrast to 5 minutes for FV (60 nmol/L). The presence of protein S (100 nmol/L) enhances rhFVIII inactivation by rAPC by 6.4-fold and FVa inactivation by twofold, whereas membrane-bound FV showed no protein S dependence during inactivation. The addition of human FV to the APC/protein S inactivation mixture increases by approximately twofold the rate of inactivation of rhFVIII. The effect of FV on the rhFVIII inactivation by APC is protein S-dependent, because FV alone has no effect on the inactivation rate of rhFVIII by APC. Western blotting using a monoclonal antibody that recognizes an epitope between amino acid residues 307 and 506 of human FV showed that FV was completely cleaved by APC at the beginning of the rhFVIII inactivation process. These data suggest that FV fragments derived from the B region of the procofactor after incubation of the membrane-bound procofactor with APC, but not intact single-chain FV, stimulate APC activity in the presence of protein S. rhFVIII, FV, and rhFVIIIa were not inactivated by Glu20-->Ala-substituted rAPC (rAPCgamma20A), and membrane-bound factor Va was only partially inactivated. Our data suggest that (1) FV and FVa are the physiologically significant substrates for APC inactivation and (2) membranes-bound APC-treated FV is a cofactor for the APC inactivation of rhFVIII only in the presence of the intact form of protein S.

Characterization of Monocyte-Associated Factor V

We demonstrate that in addition to possessing binding sites for intact factor V (FV), unstimulated peripheral blood monocytes also express activated factor V (FVa) on their surfaces. FVa was identified on the monocyte surface by monoclonal antibody B38 recognizing FVa light chain and by human oligoclonal antibodies H1 (to FVa light chain) and H2 (to FVa heavy chain) using immunofluorescence microscopy and flow cytometry. On Western blots, partially cleaved FV could be identified as a 220 kDa band in lysates of monocytes. In addition to surface expression of FVa, monocytes also contain intracellular FV as detected only after permeabilization by Triton X-100 by monoclonal antibody B10 directed specifically to the Cl domain not present in FVa. We sought to determine whether the presence of FV in peripheral blood monocytes is a result of de novo synthesis. Using in situ hybridization, no FV mRNA could be detected in monocytes, while in parallel control studies, factor V mRNA was detectable in Hep G2 cells and CD18 mRNA in monocytes. In addition, using reverse transcriptase and the polymerase chain reaction, no FV mRNA was detected in mononuclear cells or in U937 cells, but mRNA for factor V was present in Hep G2 cells using the same techniques. These data suggest that FV is present in human monocytes, presumably acquired by binding of plasma FV, and that the presence of this critical coagulation factor is not due to de novo synthesis.