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

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.

Download Full-text

Abstract 43: Prothrombinase Assembled with Factor V Leiden is Resistant to Inhibition by Tissue Factor Pathway Inhibitor α Lowering the Procoagulant Threshold for Initiation of Coagulation

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.36.suppl_1.43 ◽

2016 ◽

Vol 36 (suppl_1) ◽

Author(s):

Jeremy P Wood ◽

Lisa M Baumann Kreuziger ◽

Susan A Maroney ◽

Rodney M Camire ◽

Alan E Mast

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Tissue Factor Pathway Inhibitor ◽

Platelet Rich Plasma ◽

Anticoagulant Activity ◽

Factor V Leiden ◽

Factor Xa ◽

Factor V ◽

Activation Threshold ◽

Tissue Factor Pathway

Factor V (FV) assembles with factor Xa (FXa) into prothrombinase, the enzymatic complex that converts prothrombin to thrombin. Tissue factor pathway inhibitor α (TFPIα) inhibits prothrombinase by high affinity interactions with FXa-activated FV and the FXa active site, thereby blocking the initiation of coagulation. FV Leiden (FVL) is strongly linked to venous thrombosis through its resistance to degradation by activated protein C (aPC), which enhances the propagation of coagulation. FVL combined with a 50% reduction in TFPI causes severe thrombosis and perinatal lethality in mice, suggesting that FVL also promotes the initiation of coagulation. To examine this possibility, thrombin generation assays initiated with limiting FXa were performed with control or FVL plasma and platelet-rich plasma (PRP). The activation threshold for thrombin generation was 10 to 20 pM FXa in 10 control plasmas, but was 5 pM in 4 of 10 homozygous FVL plasmas. FVL PRP had a similar decrease in the activation threshold. The differences in activation threshold were totally normalized by an anti-TFPI antibody, while exogenous TFPIα and a FV-binding peptide that mimics TFPIα had reduced anticoagulant activity in FVL plasma, revealing that the procoagulant effects of FVL in these assays rely on TFPIα. Next, FVL plasmas were studied in fibrin clot formation assays, as they are sensitive to small amounts of thrombin. In reactions activated with 0.5 pM FXa, 1 of 8 control plasmas, compared to 7 of 8 homozygous FVL plasmas, clotted within 60 minutes, with differences again normalized by the anti-TFPI antibody. In prothrombinase activity assays using purified proteins, TFPIα was a 1.7-fold weaker inhibitor of prothrombinase assembled with FVL compared to FV. Thus, in addition to its aPC-mediated effect on the propagation of coagulation, FVL is resistant to TFPIα inhibition, exerting a procoagulant effect on coagulation initiation. This is evident in responses to small stimuli, where TFPIα blocks clotting in plasmas with FV but not FVL. The TFPIα-mediated modulation of the procoagulant threshold may explain the severe perinatal thrombosis in FVL mice with decreased TFPI and be clinically relevant in the clotting associated with oral contraceptives, which cause acquired TFPI deficiency.

Download Full-text

In vitro effects of human neutrophil cathepsin G on thrombin generation: Both acceleration and decreased potential

Thrombosis and Haemostasis ◽

10.1160/th09-10-0690 ◽

2010 ◽

Vol 104 (09) ◽

pp. 514-522 ◽

Cited By ~ 3

Author(s):

Thomas Lecompte ◽

Agnès Tournier ◽

Lise Morlon ◽

Monique Marchand-Arvier ◽

Claude Vigneron ◽

...

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Time Course ◽

Anticoagulant Activity ◽

Cathepsin G ◽

Coagulation Cascade ◽

Clotting Factor ◽

Factor V ◽

Clotting Factors

SummaryCathepsin G (Cath G), a serine-protease found in neutrophils, has been reported to have effects that could either facilitate or impede coagulation. Thrombin generation (CAT method) was chosen to study its overall effect on the process, at a plasma concentration (240 nM) observed after neutrophil activation. Coagulation was triggered by tissue factor in the presence of platelets or phospholipid vesicles. To help identify potential targets of Cath G, plasma depleted of clotting factors or of inhibitors was used. Cath G induced a puzzling combination of two diverging effects of varying intensities depending on the phospholipid surface provided: accelerating the process under the three conditions (shortened clotting time by up to 30%), and impeding the process during the same thrombin generation time-course since thrombin peak and ETP (total thrombin potential) were decreased, up to 45% and 12%, respectively, suggestive of deficient prothrombinase. This is consistent with Cath G working on at least two targets in the coagulation cascade. Our data indicate that coagulation acceleration can be attributed neither to platelet activation and nor to activation of a clotting factor. When TFPI (tissue factor pathway inhibitor) was absent, no effect on lag time was observed and the anticoagulant activity of TFPI was decreased in the presence of Cath G. Consistent with the literature and the hypothesis of deficient prothrombinase, experiments using Russel’s Viper Venom indicate that the anticoagulant effect can be attributed to a deleterious effect on factor V. The clinical relevance of these findings deserves to be studied.

Download Full-text

Inhibition of thrombin generation by the zymogen factor VII: implications for the treatment of hemophilia A by factor VIIa

Blood ◽

10.1182/blood.v95.4.1330.004k28_1330_1335 ◽

2000 ◽

Vol 95 (4) ◽

pp. 1330-1335 ◽

Cited By ~ 98

Author(s):

Cornelis van 't Veer ◽

Neal J. Golden ◽

Kenneth G. Mann

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Thrombin Generation ◽

Hemophilia A ◽

Factor Viia ◽

Plasma Concentrations ◽

Factor Xa ◽

Factor Vii ◽

Lag Phase ◽

Single Chain

Factor VII circulates as a single chain inactive zymogen (10 nmol/L) and a trace (∼10-100 pmol/L) circulates as the 2-chain form, factor VIIa. Factor VII and factor VIIa were studied in a coagulation model using plasma concentrations of purified coagulation factors with reactions initiated with relipidated tissue factor (TF). Factor VII (10 nmol/L) extended the lag phase of thrombin generation initiated by 100 pmol/L factor VIIa and low TF. With the coagulation inhibitors TFPI and AT-III present, factor VII both extended the lag phase of the reaction and depressed the rate of thrombin generation. The inhibition of factor Xa generation by factor VII is consistent with its competition with factor VIIa for TF. Thrombin generation with TF concentrations >100 pmol/L was not inhibited by factor VII. At low tissue factor concentrations (<25 pmol/L) thrombin generation becomes sensitive to the absence of factor VIII. In the absence of factor VIII, factor VII significantly inhibits TF-initiated thrombin generation by 100 pmol/L factor VIIa. In this hemophilia A model, approximately 2 nmol/L factor VIIa is needed to overcome the inhibition of physiologic (10 nmol/L) factor VII. At 10 nmol/L, factor VIIa provided a thrombin generation response in the hemophilia model (0% factor VIII, 10 nmol/L factor VII) equivalent to that observed with normal plasma, (100% factor VIII, 10 nmol/L factor VII, 100 pmol/L factor VIIa). These results suggest that the therapeutic efficacy of factor VIIa in the medical treatment of hemophiliacs with inhibitors is, in part, based on overcoming the factor VII inhibitory effect.

Download Full-text

Impact of V617F JAK2 Mutation on Monocyte Tissue Factor and Procoagulant Activity in Patients with Essential Thrombocythemia(ET) or Polycythemia VERA (PV)

Blood ◽

10.1182/blood.v112.11.3736.3736 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3736-3736

Author(s):

Anna Falanga ◽

Alfonso Vignoli ◽

Marina Marchetti ◽

Laura Russo ◽

Marina Panova-Noeva ◽

...

Keyword(s):

Tissue Factor ◽

Blood Coagulation ◽

Thrombin Generation ◽

Procoagulant Activity ◽

Jak2v617f Mutation ◽

Wild Type ◽

Coagulation Activation ◽

Allele Burden ◽

Generation Capacity

Abstract Clinical data suggest an increased thrombotic risk in patients with ET or PV carrying the JAK2V617F mutation. Laboratory data from our group show that ET patients carrying the JAK2V617F mutation are characterized by an enhanced platelet and neutrophil activation status (Falanga et al, Exp Hem 2007) and blood coagulation activation (Marchetti et al, Blood 2008), as compared to JAK2 wild-type ET. Since monocytes significantly contribute to blood coagulation activation as an important source of circulating tissue factor (TF), in this study we aimed to characterize the prothrombotic phenotype of monocytes from ET and PV patients and to evaluate whether and to what extent it is influenced by the JAK2V617F mutation. Twenty-four ET patients (10 JAK2 wild-type; 14 JAK2V617F carriers with 2%–35% mutant allele burden), 27 PV patients (all JAK2V617F carriers, 16 with 9%– 44% and 11 with 60%–100% allele burden, respectively), and 20 age-matched healthy subjects (controls, C) were enrolled into the study. Monocyte-associated TF antigen was measured on the cell surface by whole blood flow-cytometry, in both basal condition and after in vitro stimulation by bacterial endotoxin (lypopolysaccharide, LPS), as well as in cell lysates by ELISA. Monocyte procoagulant activity was evaluated by the Calibrated Automated Thrombogram (CAT) as the capacity of isolated monocyte lysates to induce thrombin generation in normal pool plasma. In basal conditions, significantly (p<0.05) higher surface levels of TF were measured on monocytes from ET (17.1±3.2% positive cells) and PV (24.4±3.7% positive cells) patients compared to C (8.2±1.9% positive cells). Similarly, the total TF antigen content of cell lysates was significantly increased in patients compared to C. The analysis of the data according to JAK2V617F mutational status, showed a gradient of increased TF expression starting from JAK2V617F negative patients (11.7±2.5%), versus JAK2V617F ET and PV subjects with <50% allele burden (20.3±3.6% and 23.2±2.8%, respectively), versus JAK2V617F PV patients with >50% allele burden (26.1±4.2%). The in vitro LPS stimulation significantly increased TF expression on monocytes from all study subjects and C compared to non-stimulated monocytes (p<0.05 for all groups), with a more elevated expression by monocytes from PV and ET patients compared to C. However, the relative increase in TF expression was greater in C (=3.7 fold) compared to both ET (=2.2 fold) and PV (=2 fold) patients. As observed in basal conditions, LPS-induced TF was higher in JAK2V617F positive patients as compared to negative, with the highest expression in JAK2V617F PV carriers with >50% allele load. Thrombin generation induced by monocytes from ET and PV patients was significantly increased compared to controls, as determined by significantly higher thrombin peaks (ET=145±12, PV=142±17, C=72.2±5 nM), and quantity of thrombin generated in time, i.e. the endogenous thrombin potential (ETP) (ET=1143±34, PV=1074±64, C=787±58 nM*min). The JAK2V617F PV subjects with >50% allele burden presented with the highest thrombin generation capacity (peak= 184±34 nM; ETP= 1268±32 nM). Our data indicate that the expression of the JAK2V617F mutation in ET and PV patients may confer to monocytes a different hemostatic phenotype in terms of increased expression of surface TF and thrombin generation capacity. These findings are in agreement with the previous observation of a hypercoagulable state associated with this mutation and suggest a new mechanism linking hemostatic cellular phenotypic alteration to genetic dysfunction in patients with myeloproliferative disease.

Download Full-text

The Structure and Function of Murine Factor V and Its Inactivation by Protein C

Blood ◽

10.1182/blood.v91.12.4593.412a02_4593_4599 ◽

1998 ◽

Vol 91 (12) ◽

pp. 4593-4599 ◽

Cited By ~ 1

Author(s):

Tony L. Yang ◽

Jisong Cui ◽

Alnawaz Rehumtulla ◽

Angela Yang ◽

Micheline Moussalli ◽

...

Keyword(s):

Amino Acid ◽

Protein C ◽

Mammalian Species ◽

Structure And Function ◽

Procoagulant Activity ◽

Factor V ◽

Cleavage Sites ◽

Wild Type ◽

Coding Region ◽

And Function

Factor V (FV) is a central regulator of hemostasis, serving both as a critical cofactor for the prothrombinase activity of factor Xa and the target for proteolytic inactivation by the anticoagulant, activated protein C (APC). To examine the evolutionary conservation of FV procoagulant activity and functional inactivation by APC, we cloned and sequenced the coding region of murine FV cDNA and generated recombinant wild-type and mutant murine FV proteins. The murine FV cDNA encodes a 2,183-amino acid protein. Sequence comparison shows that the A1-A3 and C1-C2 domains of FV are highly conserved, demonstrating greater than 84% sequence identity between murine and human, and 60% overall amino acid identity among human, bovine, and murine FV sequences. In contrast, only 35% identity among all three species is observed for the poorly conserved B domain. The arginines at all thrombin cleavage sites and the R305 and R504 APC cleavage sites (corresponding to amino acid residues R306 and R506 in human FV) are invariant in all three species. Point mutants were generated to substitute glutamine at R305, R504, or both (R305/R504). Wild-type and all three mutant FV recombinant proteins show equivalent FV procoagulant activity. Single mutations at R305 or R504 result in partial resistance of FV to APC inactivation, whereas recombinant murine FV carrying both mutations (R305Q/R504Q) is nearly completely APC resistant. Thus, the structure and function of FV and its interaction with APC are highly conserved across mammalian species.

Download Full-text

Tissue Factor Bearing Microparticles in the Myeloproliferative Neoplasms

Blood ◽

10.1182/blood.v118.21.5174.5174 ◽

2011 ◽

Vol 118 (21) ◽

pp. 5174-5174

Author(s):

Brady L Stein ◽

Brandon McMahon ◽

Ivy Weiss ◽

James Marvin ◽

Hau C. Kwaan

Keyword(s):

Sample Size ◽

Tissue Factor ◽

Thrombin Generation ◽

Myeloproliferative Neoplasms ◽

Flow Cytometric Analysis ◽

Jak2 V617f ◽

Lag Phase ◽

Thrombotic Risk ◽

Larger Sample

Abstract Abstract 5174 Background: Thrombosis is a well recognized complication in the myeloproliferative neoplasms (MPN), essential thrombocytosis (ET), polycythemia vera (PV), and primary myelofibrosis (PMF). The mechanism for thrombosis is not well-established, nor are there relevant biomarkers to predict risk and/or recurrence. Circulating cellular microparticles (MP) containing procoagulant tissue factor (TF) have been shown to correlate with thrombotic risk in many forms of cancer and cardiovascular diseases. To investigate the role of MP in the MPN, we studied 16 patients (ET=5; PV=6; PMF=2; post-ETMF=1, and MPN NOS=2) and compared results to 15 healthy subjects. Methods: Citrated blood samples were collected from the 16 MPN patients and 15 controls. Platelet poor plasma (PPP) was obtained by centrifuging at 1,500 G for 20 minutes. 50 μL of PPP was added to 200 μL PBS (without Mg/Ca) and centrifuged at 20,000 G for 10 minutes. The sediment containing MP was resuspended in 100μL of buffer for labeling with TF, CD41a (platelets), CD14 (monocytes), CD66b (neutrophils), and CD33 (myeloid lineage). Following incubation, PBS was added to the suspension to a volume of 1ml for flow cytometric analysis (LSR Fortessa, FlowJo software). Electronic triggering was done on side-scatter, and acquisition regions were defined based on sizing beads (0.3 to 1.0 micron) along with annexin A5 positivity. Using MP sediment, TF activity was measured using chromogenic assays (Actichrome TF ELISA, American Diagnostica,) and thrombin generation (TGT) was assayed (Technothrombin TGA, diaPharma), with results expressed as lag phase, velocity-index, peak thrombin, and area under the curve (AUC). The Wilcoxon-Rank Sum test was used to compare group differences (MPN vs. control) in median values. Results: Among the MPN patients, 7(44%) were male, and the median age was 60 years. 11 (69%) were JAK2 V617F positive, and 3 (19%) had a prior history of thrombosis (2 hepatic vein thromboses, 1 myocardial infarction). At the time of collection, 14 (93%) were on aspirin, 1 (6%) was on Coumadin, and 5 (31%) were on Hydroxyurea. The median total MP number was increased in MPN patients vs. controls (243580 vs. 83120; p=0.0057). The median percentage of TF-bearing MP's was also significantly greater in MPN patients compared to controls (35.5% vs. 12%; p=0.0003). When comparing MPN patients to controls, these TF-bearing MP were derived from CD14 (31.5% vs. 2%; p<0.001) and CD41a (24% vs. 7%; p=0.0157), respectively, reflecting monocyte and platelet origins of the MPs. The TF-bearing MPs in MPN patients (N=10) were functionally active compared to controls (N=10) (median TF activity: 1.7 pM vs. 0.03 pM; p=0.0022). Thrombin generation assays were performed in 13 MPN patients and 9 controls, and were comparable: mean lag phase (14.4 vs. 10.15 minutes; p=0.26); mean velocity index (21.13 vs. 14.78; p=0.31); mean peak thrombin generation (111.66 nM vs. 120.41 nM; p=0.75); and mean AUC (3218.77 vs. 3807.41 p=0.37). Conclusion: Compared to controls, samples of JAK2 V617F-positive and negative MPN patients revealed a higher median total number of microparticles. Further, the proportion of TF-bearing MPs was higher in MPN patients, and of monocyte and platelet origin, suggesting their possible role in thrombotic complications. Though the MPs in MPN patients appear functional, based on higher TF activity, functional assays with thrombin generation testing failed to reveal a difference between MPN patients and controls. A lack of difference in TGT may suggest the presence of one or more inhibitors present in the MPN; the nature of this inhibitor is under investigation. Future studies, with a larger sample size and prospective follow-up are indicated to determine the role of MPs in predicting incident or recurrent thrombosis in the MPN. In addition, with a larger sample size, differences by MPN disease class and JAK2 V617F status will be uncovered. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Regulation of Prothrombinase Activity by Protein S

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614633 ◽

1999 ◽

Vol 82 (07) ◽

pp. 80-87 ◽

Cited By ~ 14

Author(s):

Saulius Butenas ◽

Neal Golden ◽

Kenneth Mann ◽

Cornelis van't Veer

Keyword(s):

Tissue Factor ◽

Protein C ◽

Thrombin Generation ◽

Plasma Concentrations ◽

Factor Xa ◽

Protein S ◽

Coagulation Factors ◽

Independent Effect ◽

Factor Va ◽

Prothrombinase Activity

SummaryThe independent effect of protein S as prothrombinase inhibitor has been proposed to depend on binding to both coagulation factors Va and factor Xa or on the binding to phospholipid thereby limiting the phospholipid available for prothrombinase activity. In this study we show that plasma concentrations of protein S (300 nM) equilibrated with the prothrombinase components (factor Va, factor Xa, phospholipid) cause a profound inhibition at low phospholipid concentrations (~0.2 μM). This inhibition by protein S of prothrombinase activity is abrogated with increasing phospholipid concentrations. Modeling of the effect of protein S on prothrombinase based only on the reported affinity of protein S for phospholipids (Kd ~ 10-8 M) in an equilibrium model (Clotspeed), predicted the experimentally obtained thrombin generation rates at low phospholipid in the presence of protein S based on the diminished available phospholipid binding sites for the prothrombinase components. Consistently, initial rates of prothrombinase activity are already maximally inhibited when protein S is preincubated with the phospholipid prior to the addition of factor Xa, factor Va and pro-thrombin. The results indicate that the order of addition of prothrombinase components and the availability of phospholipid may have a profound influence on observed effects of protein S on prothrombinase activity. All prothrombinase components (factor Xa, factor Va, phospholipid) become available during the course of the physiological thrombin generation. The effect of protein S was therefore studied on tissue factor-induced, platelet-dependent thrombin generation. Protein S delayed and inhibited the rate of thrombin generation of tissue factor-induced thrombin formation when surface is provided at physiologic concentrations using isolated platelets (2 × 108/ml). In contrast, protein S hardly affected thrombin generation in this model when platelets were pre-activated with collagen. Furthermore, the observed effects of addition of protein C and thrombomodulin in the absence or presence of protein S on tissue factor-induced, platelet-dependent thrombin generation, indicate that protein S and protein C may cooperate in the regulation of prothrombinase activity through independent mechanisms.

Download Full-text

An In Vitro Analysis of the Combination of Hemophilia A and Factor VLEIDEN

Blood ◽

10.1182/blood.v90.8.3067 ◽

1997 ◽

Vol 90 (8) ◽

pp. 3067-3072 ◽

Cited By ~ 60

Author(s):

Cornelis van ‘t Veer ◽

Neal J. Golden ◽

Michael Kalafatis ◽

Paolo Simioni ◽

Rogier M. Bertina ◽

...

Keyword(s):

Factor Viii ◽

Tissue Factor ◽

Thrombin Generation ◽

Hemophilia A ◽

Factor Viia ◽

Factor V ◽

Severe Hemophilia ◽

Factor Viii Activity ◽

Thrombin Formation

Abstract The classification of factor VIII deficiency, generally used based on plasma levels of factor VIII, consists of severe (<1% normal factor VIII activity), moderate (1% to 4% factor VIII activity), or mild (5% to 25% factor VIII activity). A recent communication described four individuals bearing identical factor VIII mutations. This resulted in a severe bleeding disorder in two patients who carried a normal factor V gene, whereas the two patients who did not display severe hemophilia were heterozygous for the factor VLEIDEN mutation, which leads to the substitution of Arg506 → Gln mutation in the factor V molecule. Based on the factor VIII level measured using factor VIII–deficient plasma, these two patients were classified as mild/moderate hemophiliacs. We studied the condition of moderate to severe hemophilia A combined with the factor VLEIDEN mutation in vitro in a reconstituted model of the tissue factor pathway to thrombin. In the model, thrombin generation was initiated by relipidated tissue factor and factor VIIa in the presence of the coagulation factors X, IX, II, V, and VIII and the inhibitors tissue factor pathway inhibitor, antithrombin-III, and protein C. At 5 pmol/L initiating factor VIIa⋅tissue factor, a 10-fold higher peak level of thrombin formation (350 nmol/L), was observed in the system in the presence of plasma levels of factor VIII compared with reactions without factor VIII. Significant increase in thrombin formation was observed at factor VIII concentrations less than 42 pmol/L (∼6% of the normal factor VIII plasma concentration). In reactions without factor VIII, in which thrombin generation was downregulated by the addition of protein C and thrombomodulin, an increase of thrombin formation was observed with the factor VLEIDEN mutation. The level of increase in thrombin generation in the hemophilia A situation was found to be dependent on the factor VLEIDEN concentration. When the factor VLEIDEN concentration was varied from 50% to 150% of the normal plasma concentration, the increase in thrombin generation ranged from threefold to sevenfold. The data suggested that the analysis of the factor V genotype should be accompanied by a quantitative analysis of the plasma factor VLEIDEN level to understand the effect of factor VLEIDEN in hemophilia A patients. The presented data support the hypothesis that the factor VLEIDEN mutation can increase thrombin formation in severe hemophilia A.

Download Full-text

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 ◽

10.1182/blood.v118.21.2243.2243 ◽

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.

Download Full-text

Differential roles of factors IX and XI in murine placenta and hemostasis under conditions of low tissue factor

Blood Advances ◽

10.1182/bloodadvances.2019000921 ◽

2020 ◽

Vol 4 (1) ◽

pp. 207-216 ◽

Cited By ~ 1

Author(s):

Steven P. Grover ◽

Clare M. Schmedes ◽

Alyson C. Auriemma ◽

Emily Butler ◽

Molly L. Parrish ◽

...

Keyword(s):

Tissue Factor ◽

Thrombin Generation ◽

Critical Role ◽

Wild Type ◽

Genetic Studies ◽

Acute Hemorrhage ◽

Expected Frequency ◽

Low Levels ◽

Coagulation Pathway

Abstract The intrinsic tenase complex (FIXa-FVIIIa) of the intrinsic coagulation pathway and, to a lesser extent, thrombin-mediated activation of FXI, are necessary to amplify tissue factor (TF)-FVIIa–initiated thrombin generation. In this study, we determined the contribution of murine FIX and FXI to TF-dependent thrombin generation in vitro. We further investigated TF-dependent FIX activation in mice and the contribution of this pathway to hemostasis. Thrombin generation was decreased in FIX- but not in FXI-deficient mouse plasma. Furthermore, injection of TF increased levels of FIXa-antithrombin complexes in both wild-type and FXI−/− mice. Genetic studies were used to determine the effect of complete deficiencies of either FIX or FXI on the survival of mice expressing low levels of TF. Low-TF;FIX−/y male mice were born at the expected frequency, but none survived to wean. In contrast, low-TF;FXI−/− mice were generated at the expected frequency at wean and had a 6-month survival equivalent to that of low-TF mice. Surprisingly, a deficiency of FXI, but not FIX, exacerbated the size of blood pools in low-TF placentas and led to acute hemorrhage and death of some pregnant dams. Our data indicate that FIX, but not FXI, is essential for survival of low-TF mice after birth. This finding suggests that TF-FVIIa–mediated activation of FIX plays a critical role in murine hemostasis. In contrast, FXI deficiency, but not FIX deficiency, exacerbated blood pooling in low-TF placentas, indicating a tissue-specific requirement for FXI in the murine placenta under conditions of low TF.

Download Full-text