Conservative Mutations in the Membrane-Binding Motif of Factor V C2 Domain to Residues of Pseudonaja Textilis Venom Factor V Confer Phosphatidylserine-Independent Activity

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2243-2243
Author(s):  
Valerie A Novakovic ◽  
Hongzhi Miao ◽  
Steven Pipe ◽  
Gary E. Gilbert
Keyword(s):  
Amino Acids ◽  
Human Factor ◽  
Membrane Binding ◽  
Factor V ◽  
C2 Domain ◽  
Wild Type ◽  
Hydrophobic Membrane ◽  
Prothrombinase Complex ◽  
Membrane Affinity ◽  
Prothrombinase Activity

Abstract Abstract 2243 Toxicity of venom from the eastern brown snake (Pseudonaja textilis) is related to a prothrombin activator protein complex (pseutarin C) that is homologous to the factor Va/factor Xa complex. A previous study has found that the factor V-homologous subunit of this protein (pt-fV) is constitutively active and does not require anionic membranes to function (Bos et al. 2010, Blood). We have previously found that conservative mutation of the amino acids on the hydrophobic membrane binding regions (called spikes) of factor V (W2063M/W2064F/S2117L) can produce increased prothrombinase activity, increased membrane binding affinity and apparent phospholipid-independent prothrombinase activity. However, the membrane-independent activity is caused by retention of phospholipid by factor V through the purification process. We hypothesized that the P.textilis venom-derived factor V has an increase in lipid affinity due to differences in the membrane-interactive spikes. Sequence alignment of the P.textilis venom-derived factor V with bovine and human factor V revealed 5 amino acids located in the putative membrane-binding region (four on spike 3 and one in a region targeted by a small-molecule inhibitor of membrane binding for both factor VIII and factor V) that differed in the venom-derived factor V versus the consensus sequence of mammalian factor V. A mutant factor V that incorporated these five mutations (L2116M, S2117T, S2118T, E2119S, and S2183Y) (factor VMTTS/Y) was expressed in COS cells. After purification utilizing ion exchange chromatography, factor VMTTS/Y showed phospholipid-independent activity that could be inhibited with phospholipase A2. Subsequently, factor VMTTS/Y was washed extensively with CHAPS during purification to prevent phospholipid from co-purifying. Activity was measured with a prothrombin time assay with plasma lacking factor V. Specific activity was 1183 units/mg vs. 676 units/mg for wild type human factor V. Steady state kinetics of the prothrombinase complex with factor VMTTS/Y were assessed with varying concentrations of phospholipid vesicles. In the presence of membranes containing excess phosphatidylserine (15:20:65 PS:PE:PC), factor VMTTS/Y (5 pM) showed 39% greater Vmax than wild type human factor V and 3-fold higher apparent membrane affinity. With limiting phosphatidylserine (2:20:78 PS:PE:PC), factor VMTTS/Y (10 pM) showed 64% greater Vmax and 2-fold higher apparent membrane affinity. Factor VMTTS/Y, purified with a CHAPS wash, did not show lipid-independent activity but did support prothrombinase activity on membranes lacking PS or other negatively charged lipid (20:80 PE:PC). On these vesicles factor VMTTS/Y (50 pM) had a Vmax that was 8-fold higher than wild type factor V (see figure). These data indicate that the apparent phospholipid-independent activity results from higher membrane affinity or from greater activity on minimal phospholipid retained by factor V during purification. They imply that toxicity of pseutarin C may result, in part, from procoagulant activity on cell membranes that do not support the mammalian prothrombinase complex. Furthermore, they indicate that the precise manner in which the C2 domain of factor V binds to a phospholipid membrane influences the Vmax of the prothrombinase complex. Disclosures: No relevant conflicts of interest to declare.

The Mutations SER2117→LEU, and TRP/TRP2063/64→MET/PHE in the Hydrophobic Spikes of the Factor V C2 Domain Lead to Tenacious Phospholipid Binding and Suggest a Mechanism through Which Phosphatidylserine Activates the Prothrombinase Complex.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2703-2703
Author(s):  
Gary E. Gilbert ◽  
Valerie A. Novakovic ◽  
Randal J. Kaufman ◽  
Hongzhi Miao ◽  
Steven W. Pipe
Keyword(s):  
Specific Activity ◽  
Phospholipid Membranes ◽  
Unexpected Result ◽  
Factor V ◽  
C2 Domain ◽  
Wild Type ◽  
Hydrophobic Residues ◽  
Phospholipid Binding ◽  
Prothrombinase Complex ◽  
Type Factor

Abstract Factor V (fV) binds to phospholipid (PL) membranes via a motif localized to the C2 domain. We and others have shown that PL binding is mediated by two pairs of hydrophobic residues, each displayed at the tips of β-hairpin turns. The homologous hydrophobic residues in the C2 domain of factor VIII also contribute to PL binding. We hypothesized that the solvent-exposed hydrophobic residues of the fV C2 domain make specific contacts that influence membrane affinity and activity of fV. To test this hypothesis we have prepared fVIII/fV hybrid mutants in which amino acid(s) of the fV C2 domain were changed to the homologous residues of fVIII (Mutants #1 W/W 2063/2064 M/F, #2 L/S 2116/2117 L/L, and #3 W/W/S 2063/2064/2117 M/F/L). Mutants were expressed in COS-1 cells and purified by FPLC. The specific activity of the fV/FVIII hybrids #2 and #3 exceeded those of wild type factor V by approx. 10-fold and approx. 4-fold, respectively, in a prothrombin time assay with factor V deficient plasma. Apparent PL affinites were evaluated in a prothrombinase complex assay with limiting phospholipid. The apparent affinities are 9.8 and 21-fold higher than wild type factor V for mutants #2 and #3 which contain the S→L change in the second hydrophobic spike. An unexpected result was that mutants 1–3 supported prothrombinase activity in the absence of added phospholipid, in contrast to wild type fV. We hypothesized that this activity resulted from phospholipid that was not dissociated from fV during purification. This hypothesis was supported because activity was eliminated by incubation of the factor V mutants with phospholipase A2 or by incubation with lactadherin. The tenacity of the phospholipid binding was further investigated by washing immobilized wild type fV and fV mutants with CHAPS prior to elution from an FPLC column. fV mutants #2 and #3 retained activity after the CHAPS wash, free of added PL and activity remained inhibitable by lactadherin, further illustrating the tenacious phospholipid affinity. We utilized Mutants #1 & 2 in the absence of added PL to evaluate the mechanism through which soluble phosphatidylserine with 6-carbon acyl chains (C6PS) enhances activity of the prothrombinase complex. In the absence of phospholipid vesicles C6PS enhanced activity of Mutant 1 equivalent to wild type fV (> 20 fold). However, enhancement of mutant 2 activity, which presumably retained phospholipid via residues 2116/2117, was < 3-fold. This suggests that C6PS functions to activate wild type fV, in part, by engaging the free LS 2116/2117 hydrophobic spike. Together, these data indicate that the hydrophobic spikes of factor V influence the specific activity of factor V, that the high affinity reversible binding of fV to phospholipid membranes is readily perturbed by mutations, and that activation of the prothrombinase complex by C6PS and phospholipid membranes likely involves engagement of amino acids 2116/2117.

Lactadherin C2 Domain Exhibits Ptd-L-Ser Specificity and Anticoagulant Properties Distinct From Homologous Factor VIII C2 Domain and Full-Length Lactadherin

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1105-1105
Author(s):  
Valerie A Novakovic ◽  
Eugene R Gilbert ◽  
Jialan Shi ◽  
Gary E. Gilbert
Keyword(s):  
Factor Viii ◽  
Hela Cells ◽  
Binding Sites ◽  
Metal Ion ◽  
Membrane Binding ◽  
Full Length ◽  
Crystallographic Structure ◽  
Factor V ◽  
C2 Domain ◽  
Prothrombinase Activity

Abstract Abstract 1105 Background: Lactadherin (aka mfg-e8) is a milk-fat globule membrane protein with a domain structure of EGF1-EGF2-C1-C2, where the lectin-like C1 and C2 domains are homologous to the membrane binding domains of factor VIII and factor V. Like factor VIII and factor V, lactadherin exhibits calcium-independent membrane binding that is selective for phosphatidyl-L-serine (Ptd-L-Ser). Lactadherin also binds preferentially to convex membranes, competes efficiently for binding sites of factor VIII and factor V, and can function as an anticoagulant via competition for these binding sites. On stressed endothelial cells lactadherin binds to filopodia and the cell margins, identifying sites that have exposed Ptd-L-Ser and support assembly of the prothrombinase complex. The crystallographic structure of the lactadherin C2 domain (Lact-C2) and structure-function studies have shown that membrane binding is mediated by a longer β-hairpin turn, with different residues than fVIII-C2 and fV-C2. Further, we have shown that Lact-C2 maintains specificity for phosphatidylserine, in contrast to fVIII-C2, and that fluorescent fusion proteins containing Lact-C2 can be used as intracellular phosphatidylserine probes (Yeung et al. Science 2008;319:210). However, the extent to which Lact-C2 retains the membrane binding and anticoagulant properties of full-length lactadherin, has not been studied. Methods: Lact-C2 was produced in E. coliand purified by metal ion chromatography followed by gel filtration. Competition experiments were performed by flow cytometry using phospholipid bilayers supported by glass microspheres to determine Lact-C2's ability to block binding sites of FITC-labeled lactadherin, or fluorescein-labeled factor VIII and factor V. Lact-C2 was also labeled with FITC to measure its binding to sonicated or 100 nm diameter, extruded vesicles with varying PS content in order to assess Ptd-L-Ser selectivity and membrane curvature sensitivity. Two-step amidolytic factor Xase and prothrombinase assays were used to assess the ability of Lact-C2 to block activity. Fluorescence microscopy experiments were used to compare the binding of Alexa 647-labeled Lact-C2 vs. FITC-labeled lactadherin on staurosphorine-treated HeLa cells. Results: Lact-C2 showed stereospecific binding to Ptd-L-Ser vs. Ptd-D-Ser in vesicles of 4% and 10% PS. Lact-C2 was sensitive to vesicle curvature, detecting as little as 1% Ptd-L-Ser on sonicated vesicles but requiring 4% Ptd-L-Ser on extruded vesicles. Lact-C2 competed for 89% of lactadherin binding sites and 84% of factor VIII binding sites. Inhibition of factor Xase activity plateaued at 89% reduction vs. >99% reduction for lactadherin. Lact-C2 also competed for 61% of factor V binding sites corresponding to an 82% reduction in prothrombinase activity. We are currently comparing the distribution of binding sites for Lact-C2 vs. lactadherin on stressed HeLa cells, with preliminary data showing distinct, but overlapping, binding site distribution. Discussion: Lact-C2 exhibits stereospecific Ptd-L-Ser binding and convex curvature preference similar to full-length lactadherin. Lact-C2 contrasts with fVIII-C2 in Ptd-L-Ser specificity and capacity to compete with factor VIII and inhibit factor Xase activity and prothrombinase activity. These results provide a framework for interpreting experiments in which Lact-C2 is used as an anticoagulant or as a calcium-independent probe for exposed membrane Ptd-L-Ser. Lact-C2 is able to bind to only a subset of lactadherin binding sites, highlighting the importance of the lactadherin C1 domain for high affinity binding and underscoring the largely unappreciated complexity of phospholipid membrane binding sites. Disclosures: Shi: Brigham and Women's Hospital: Use of Lactadherin to detect phosphataidylserine, Use of Lactadherin to detect phosphataidylserine Patents & Royalties.

High Resolution X-Ray Structure of Snake Venom Factor V: Evolution of a Hemostatic Cofactor to a Toxin Poised to Inflict Maximal Damage to Mammalian Blood Coagulation

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 375-375
Author(s):  
Shekhar Kumar ◽  
Steven Stayrook ◽  
James A. Huntington ◽  
Rodney M. Camire ◽  
Sriram Krishnaswamy
Keyword(s):  
High Resolution ◽  
Blood Coagulation ◽  
Human Factor ◽  
Membrane Binding ◽  
Factor V ◽  
C2 Domain ◽  
X Ray ◽  
High Affinity ◽  
Mammalian Blood ◽  
A2 Domain

Abstract Abstract 375 Poisonous snakes frequently harbor activators of mammalian coagulation as part of the toxin repertoire in their venom. The venom of Pseudonaja textilis (Ptex, common brown snake) contains an efficient activator of human prothrombin comprised of a Xa-like protein tightly bound to a Va-like protein. The constituents of this complex exhibit high sequence homology to the corresponding activated coagulation factors in mammalian blood. Factors Xa and Va are produced in blood upon proteolytic activation of their precursors, complex with each other in membrane-dependent reactions to form prothrombinase and catalyze thrombin formation at the site of vascular damage. In contrast, the venom proteins are constitutively active, form a complex in solution and can efficiently catalyze prothrombin activation in the absence of membranes. These properties likely drive the disseminated and consumptive coagulopathy associated with evenomation by P. textilis. The Va-like component (Ptex-Va) of P. textilis venom is a single chain glycoprotein of 1430 residues with 53% identity to human factor V (hV) and a common A1-A2-B-A3-C1-C2 domain organization. The B-domain of Ptex-Va is significantly shorter than its counterpart in hV (46 vs. 836 residues). We now report a high resolution x-ray structure of recombinant Ptex-Va collected at 1.9 Å resolution and solved by molecular replacement. The resulting structure closely mimics those seen at lower resolution for inactivated bovine factor Va lacking the A2 domain and full length B-domainless human factor VIII (hVIII). Each A domain is formed by two cupredoxin-like β barrels with the A domains arranged in a pseudo-three-fold axis of symmetry. The two C domains are roughly cylindrical and oriented side-by-side to form a pedestal for the A1-A2-A3 rosette. The A3 domain makes extensive contacts with the C1 and A2 domains. The structure also reveals a disulfide bond unique to Ptex-Va, linking Cys642 in the A2 domain with Cys1002 in the A3. These features likely account for the high stability of the molecule even after proteolytic processing of the B domain and/or cleavage between the A1 and A2 domains. Although the C2 domain is significantly more disordered than the other domains, both C1 and C2 each contain protruding loops at their base with hydrophobic residues pointing outward. These structural features replicate those found in inactivated bovine Va and hVIII considered critical for membrane binding by the hemostatic cofactors. Surprisingly, despite the presence of these structures, light scattering measurements revealed negligible binding of Ptex-Va to synthetic membranes composed of phosphatidylcholine and phosphatidylserine with an estimated 103-fold weaker affinity than that of hV. We reasoned that this unexpected property of the venom protein, not conducive to regulated coagulation, was unlikely to be replicated in factor V from the plasma of the snake. Sequence alignment of Ptex-Va with factor V from snake plasma revealed 11 differences in the 328 residues of the C1 and C2 domains. Of these, 9 were located on the distal region of these domains, occupying a band approximately 7 Å thick across the molecule. Mutagenesis of Ptex-Va to introduce these 9 substitutions followed by its expression and purification yielded a derivative that bound to membranes with high affinity in a manner equivalent to hV. This striking gain in function sheds new and unexpected light on the structural determinants of high affinity membrane binding in Ptex-Va and by extension, its homologues hV and hVIII. Our high resolution structure of this hV-like species with a series of unusual properties provides a unique platform to address major but unresolved questions related to the structural correlates of hV function. It also reveals the basis for molecular mimicry whereby a cofactor essential for regulated blood coagulation has served as a scaffold for the evolution of a potent toxin by simultaneous loss in the ability to bind membranes and a gain in the ability to bind its proteinase with high affinity in a membrane-independent fashion. Disclosures: No relevant conflicts of interest to declare.

The R2-haplotype associated Asp2194Gly mutation in the light chain of human factor V results in lower expression levels of FV, but has no influence on the glycosylation of Asn2181

2003 ◽  
Vol 89 (03) ◽  
pp. 429-437 ◽  
Author(s):  
Richard Dirven ◽  
Hans Vos ◽  
Rogier Bertina ◽  
Marijn Kolfschoten
Keyword(s):  
Human Factor ◽  
Light Chains ◽  
Factor V ◽  
Missense Mutations ◽  
Wild Type ◽  
Molecular Weights ◽  
Apc Resistance ◽  
Increased Risk ◽  
The Individual ◽  
Lower Expression

SummaryThe R2 haplotype of the FV gene spans from exon 8 through 25 and comprises several strongly linked polymorphisms in the FV gene, including some missense mutations. Carriership of the R2-FV allele has been associated with reduced plasma FV levels, increased FV1/FV2 ratios and mild APC resistance. Some studies have reported that carriership of the R2-FV allele is associated with an increased risk of venous thombosis. At this moment, the individual contribution to the R2-associated phenotypes of the different mutations linked to the R2 haplotype of FV is unclear. The main objective of our study was to obtain insight in the influence of the R2-related Asp2194Gly mutation on FV expression, FV structure and FV function using B-domainless rFV mutants. Replacing Asp at position 2194 by Gly resulted in a more than threefold reduction of rFV expression compared to rFV wild-type. Therefore, we propose that the R2-linked Asp2194Gly mutation is an important determinant of the association of the R2-FV allele with lower FV levels. Furthermore, the light chains from Asp2194Gly containing rFV mutants showed similar molecular weights as the light chains of the non-glycosylated rFVwt or the plasma FV2 isoform, indicating that glycosylation at Asn2181 is not stimulated by the presence of a glycine in position 2194. Finally, the apparent K d for dissociation of the FXaVa complex (K 1/2Xa) was not higher in rFV mutants with the Asp2194Gly mutation than for rFVwt, suggesting that also the affinity for negatively charged phospho-lipids is not affected by substitution of Asp into Gly at position at 2194.

Partial Glycosylation at Asparagine-2181 of the Second C-Type Domain of Human Factor V Modulates Assembly of the Prothrombinase Complex†

Biochemistry ◽  
1999 ◽  
Vol 38 (35) ◽  
pp. 11448-11454 ◽  
Author(s):  
Suhng Wook Kim ◽  
Thomas L. Ortel ◽  
Mary Ann Quinn-Allen ◽  
Lina Yoo ◽  
Laura Worfolk ◽  
...  
Keyword(s):  
Human Factor ◽  
Factor V ◽  
Prothrombinase Complex

Membrane-Interactive Amino Acids in the Factor VIII C1 Domain Are Cooperative with C2 Domain Epitopes for Membrane Binding and Cofactor Function.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 848-848
Author(s):  
Junhong Lu ◽  
Steven W. Pipe ◽  
Hongzhi Miao ◽  
Marc Jacquemin ◽  
Gary E. Gilbert
Keyword(s):  
Amino Acids ◽  
Factor Viii ◽  
Membrane Binding ◽  
Binding Motif ◽  
C2 Domain ◽  
Factor X ◽  
Apparent Affinity ◽  
Factor Ixa ◽  
C1 Domain ◽  
Membrane Interactive

Abstract Abstract 848 Background: Factor VIII functions as a cofactor in blood coagulation. When released from a non-covalent complex with von Willebrand factor (vWf), activated factor VIII assembles with factor IXa on phosphatidylserine (PS)-containing membranes to form the factor Xase complex. Binding to PS-containing membranes amplifies the activation of factor X by several orders of magnitude. Factor VIII is composed of three A domains, one B domain and two C domains (C1 and C2). The role of C2 domain, including the orientation with respect to membrane surface, vWf-binding motif, and protein-protein contact sites among Xase complex, are relatively well-documented. Recently, the position of the C domains in the factor VIII crystal structure suggested a possible role for the C1 domain in membrane binding. We recently confirmed the participation of K2092 and F2093 of the factor VIII C1 domain in membrane binding (Meems et al. Blood 2009 First edition Aug 18). This work explores the participation of additional C1 domain amino acids and the way the corresponding motif(s) cooperate with motifs of the C2 domain for membrane binding. Methods: Four factor VIII C1 domain mutants encompassing the lower surface of the C1 domain (Arg2090/GLy2091, Lys 2092/Phe2093, Gln2042/Tyr2043, and Arg2159) had individual or paired amino acids mutated to alanine. Mutants were produced in COS-1 cells and purified by immunoaffinity chromatography. The specific activities of these mutants were assessed in a commercial PTT assay as well as phospholipid-limiting and phospholipid-saturating factor Xase assay. Their affinities to factor IXa and factor X were measured by titration experiments using different concentrations of factor IXa and factor X, respectively. Binding to plasma vWf was evaluated in a competition, solution phase enzyme-linked immunosorbent assay (ELISA). The cooperative role of C1 and C2 domains in membrane-binding for cofactor activity was carried out using C1 mutants and antibodies against established membrane-interactive C2 domain motifs, ESH4 and BO2C11. Results: In a competition ELISA for vWf, the affinity of Arg2159 was reduced more than 50-fold, while the other mutants were normal. All mutants had reduced specific activity (range 24-61% of wild type) in a commercial PTT assay containing excess phospholipid. All mutants had decreased apparent affinity for vesicles with limiting (4%) PS by 33, 5, 20, and 18-fold for Arg2090/GLy2091, Gln2042/Tyr2043, Arg2159, and Lys 2092/Phe20933, respectively. However, addition of excess vesicles led to near normal activity for Arg2159. Mutants Arg2090/GLy2091 and Gln2042/Tyr2043 both had 4-fold decreased apparent affinity for factor X and 77% and 84% reduction in Vmax even when phospholipid and factor X were in excess. Mutant Lys 2092/Phe2093 had normal apparent affinity for factor IXa and factor X but > 91% reduction in Vmax. These results indicate that the C1 domain affects interaction with factor X and the Vmax of the factor Xase complex aside from the effect on membrane affinity. To further explore the role of membrane-binding motif in the Xase complex, the activities of mutants were tested with the C2 domain membrane-interactive epitopes blocked by mAb's BO2C11 or ESH4. For WT factor VIII, ESH4 and B02C11 decreased apparent affinity for vesicles of 15% PS by 6-fold and 5-fold, and decreased the Vmax by 0 and 89%, respectively. BO2C11 completely inhibited the activity of Arg2090/GLy2091, Lys 2092/Phe2093, and Arg2159 while ESH4 decreased apparent affinity 2-7-fold for the three mutants. ESH4 decreased the Vmax by 2-5-fold for the mutants. Thus, the intact membrane-binding motif in C1 can independently support Xase activity although the C1 motifs and both C2 membrane-interactive epitopes are required for full activity. Conclusion: Amino acids Arg2090/GLy2091, Lys2092/Phe2093 , Gln2042/Tyr2043, and Arg2159 of the factor VIII C1 domain participate in membrane binding. Our data suggest that engagement of the C1 domain through these residues, together with the ESH4 and the BO2C11 epitopes of the C2 domain, cooperatively influence alignment or an allosteric effect that alters activity for the assembled factor Xase complex. Disclosures: Pipe: Baxter: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Wyeth: Speakers Bureau; Inspiration Biopharmaceuticals: Research Funding; CSL Behring: Honoraria.

Secretable Human Platelet-Derived Factor V Originates From the Plasma Pool

Blood ◽  
1998 ◽  
Vol 92 (9) ◽  
pp. 3035-3041 ◽  
Author(s):  
Rodney M. Camire ◽  
Eleanor S. Pollak ◽  
Kenneth Kaushansky ◽  
Paula B. Tracy
Keyword(s):  
Western Blotting ◽  
Platelet Rich Plasma ◽  
Activated Protein C ◽  
Allogeneic Transplantation ◽  
Factor V ◽  
Wild Type ◽  
Prothrombinase Complex ◽  
Specific Association ◽  
American Society ◽  
Genetic Pools

Factor Va (FVa), derived from plasma or released from stimulated platelets, is the essential protein cofactor of the prothrombinase complex. Plasma-derived factor V (FV) is synthesized by the liver, whereas the source of the platelet-derived cofactor has not been unambiguously identified. Megakaryocytes, platelet precursors, are known to synthesize platelet proteins and to endocytose proteins from plasma (ie, fibrinogen) and then package these proteins into -granules. To determine which mechanism accounts for FV presence in platelets, two patients heterozygous for FVLeiden who underwent allogeneic transplantation from homozygous FV wild-type donors (bone marrow [BM] or liver) were studied. Patient JMW, whose skin biopsy specimen showed heterozygous FVLeiden, received a BM transplant from a wild-type homozygous FV donor as analyzed from posttransplant peripheral blood cells. Patient FW, whose native liver is heterozygous for FVLeiden, received a homozygous wild-type FV liver. Because each individual has two distinct genetic pools of factor V in liver and megakaryocytes, it was possible to determine whether secretable platelet-derived FV was normal or contained the FVLeiden mutation. Platelet-derived FVa released from thrombin-activated platelets from a normal individual, an individual heterozygous for the FVLeiden mutation, and the two patients was incubated with phospholipid vesicles and activated protein C (APC). Western blotting analyses using a monoclonal antibody that allows distinction between platelet-derived FVa and FVaLeiden subsequent to APC-catalyzed cleavage were then performed. Based on the accumulation of proteolytic fragments derived from APC-induced cleavage, analyses of platelet-derived FVa from JMW demonstrated both normal FVa and FVaLeiden consistent with a plasma-derived origin of the secretable platelet-derived FVa. Western blotting analyses of the APC-cleaved platelet-derived FVa from FW showed a wild-type phenotype, despite the presence of a FVLeiden allele in her megakaryocyte genome, also consistent with a plasma origin of her secretable platelet-derived FVa. Platelets do not appear to endocytose the plasma cofactor, because a 35-hour incubation of platelet-rich plasma with 125I-factor V showed no specific association/uptake of the radiolabeled ligand with the platelet pellet. Collectively, these results show for the first time that the majority of secretable platelet-derived factor V is endocytosed by megakaryocytes from plasma and is not exclusively synthesized by these cells, as previously believed. © 1998 by The American Society of Hematology.

Cleavage Requirements of Factor V in Tissue-factor Induced Thrombin Generation

1998 ◽  
Vol 80 (07) ◽  
pp. 92-98 ◽  
Author(s):  
Elisabeth Thorelli ◽  
Randal Kaufman ◽  
Björn Dahlbäck
Keyword(s):  
Tissue Factor ◽  
Thrombin Generation ◽  
Lag Phase ◽  
Factor V ◽  
Relative Importance ◽  
Cleavage Sites ◽  
Wild Type ◽  
Generation System ◽  
Subsequent Phase ◽  
Prothrombinase Activity

SummaryFactor V (FV) activation is the result of cleavages at Arg709, Arg1018 and Arg1545 by thrombin or FXa. The relative importance of these cleavages in tissue factor (TF) induced thrombin generation in plasma and in a purified system was elucidated with recombinant FV in which the three sites had been eliminated one by one or in combinations. The mutants were analyzed with a clotting assay using FV-deficient plasma and in a TF induced thrombin generation system using plasma or purified components. Surprisingly, in the standard FV clotting assay, all mutants gave similar clotting activities and the thrombin generation curves obtained with wild-type and thrombin-resistant FV were similar. Differences in clotting activities and thrombin generation patterns between wild-type and thrombin-resistant FV were only observed when lower TF concentrations were used. The thrombin generation curve obtained in plasma containing wt FV was characterized by a short lag phase and a subsequent phase of rapid thrombin generation (propagation phase). The Arg709 to Gln mutation yielded a slightly prolonged lag phase and the rate of thrombin generation during the propagation phase was approximately 5-fold lower than that observed with wt FV. The Arg1018 to Ile mutation only slightly affected the thrombin generation curve, whereas the Arg1545 to Gln mutation yielded a prolonged lag phase and decreased maximum thrombin activity. Thrombin-resistant FV (mutated at all three sites) yielded a prolonged lag phase and poor thrombin generation during the propagation phase. The purified system further demonstrated the importance of the three cleavage sites for rapid and sustained thrombin generation. The results demonstrate that cleavages at positions 709, 1018 and 1545 are not required for assembly of a FXa-FV complex expressing low but significant prothrombinase activity but that all three sites in different ways are important for the creation of a FVa which maximally supports the FXa-mediated activation of prothrombin.

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

2019 ◽  
Vol 120 (01) ◽  
pp. 055-064
Author(s):  
Peter van Doorn ◽  
Jan Rosing ◽  
Elena Campello ◽  
Saskia Middeldorp ◽  
Paolo Simioni ◽  
...  
Keyword(s):  
Tissue Factor Pathway Inhibitor ◽  
Bleeding Risk ◽  
Factor V ◽  
Prothrombinase Complex ◽  
C Terminus ◽  
Normal Individuals ◽  
Splicing Variants ◽  
Tissue Factor Pathway ◽  
Fv Leiden ◽  
Prothrombinase Activity

Abstract Background 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. Objective This study aims to develop a plasma-based assay that quantifies the susceptibility of FV(a) to inhibition by the TFPIα C-terminus. Materials and Methods 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). Results 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. Conclusion 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.

THE COMPLETE AMINO ACID SEQUENCE OF HUMAN FACTOR V

1987 ◽  
Author(s):  
Richard J Jenny ◽  
Debra D Pittman ◽  
John J Toole ◽  
Ronald W Kriz ◽  
Randal J Kaufman ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Factor Viii ◽  
Untranslated Region ◽  
Human Factor ◽  
Leader Peptide ◽  
Cdna Clones ◽  
Factor V ◽  
Human Factor Viii

cDNA clones encoding human factor V have been isolated and sequenced. The cDNA sequence of factor V obtained from overlapping clones includes a 6672 bp coding region, a 90 bp 5'-untranslated region and a 163 bp 3’-untranslated region including a poly-A tail. The deduced amino acid sequence consists of 2224 amino acids including a 28 amino acid leader peptide. A direct comparison to human factor VIII reveals considerable homology between both proteins with respect to amino acid sequence and domain structure. A triplicated "A" domain and duplicated "C" domain show an approximate 40% identity to the corresponding domains in factor VIII. Factor V and Factor VIII both possess a heavily glycosylated B domain that separates the heavy and light chains of the activated cofactors, although no significant homology is observed in this region. The B domain of factor V contains 35 tandem and approximately 9 additional semi - conserved repeats of nine amino acids of the form (D-L-S-Q-T-T-L-S-P) and 2 additional semi-conserved repeats of 17 amino acids. Factor V contains 37 potential N-linked glycosylation sites, 25 of which are in the B domain, and a total of 19 cysteine residues. By direct comparison to amino acid sequence obtained from both human and bovine factor V, the thrombin (IIa) cleavage sites have been assigned as Arg-709/Ser-710, Arg-1018/Thr-1019, and Are-1545/Ser-1546.(Supported by NIH Grant HL-34575)

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