Factor V Leiden paradox in a middle-aged Swedish population: A prospective study.

Few prospective studies have examined the factor V paradox: factor V Leiden (FVL) is a stronger risk factor for deep venous thrombosis (DVT) than for pulmonary embolism (PE). The present study, to the best of our knowledge, is the first population-based study aimed to examine the relationship between FVL and incidence of venous thromboembolism (VTE), DVT and PE in a prospective cohort study of middle-aged Swedish individuals. FVL was determined in 4890 subjects (aged 46-68 years, 57% women) from the general population without previous VTE or cancer, who participated in the Malmö Diet and Cancer study between 1991 and 1994. Incident cases of VTE were identified from the Swedish patient register during a mean follow-up of 15.6 years. Of 4890 subjects with determination of FVL (10.2% carriers), 220 had VTE during follow-up (113 DVT, 78 PE, 29 both). Incidence of VTE was significantly higher in subjects with heterozygous and homozygous FVL: adjusted hazard ratios (HR) were 1.8 (95% CI 1.3-2.6, p=0.001) and 6.5 (2.1-21, p=0.001), respectively. The population attributable fraction was 8.7% for FVL. Adjusted HRs for DVT were 2.2 (1.4-3.3, p<0.001) for heterozygotes and 3.3 (0.5-24, p=0.233) for homozygotes. Adjusted HRs for PE were 1.2 (0.65-2.2, p=0.582) for heterozygotes and 8.7 (2.1-36, p=0.003) for homozygotes. The FVL paradox was confirmed for heterozygotes for FVL. However, homozygotes for FVL had a high risk for PE, suggesting that the FVL paradox is related to the carriership of one wild type and one mutated factor V allele.

Protease-activated receptor (PAR) 1 and PAR4 differentially regulate factor V expression from human platelets.

With the recent interest of protease-activated receptors (PAR) 1 and PAR4 as possible targets for the treatment of thrombotic disorders, we compared the efficacy of protease-activated receptor (PAR)1 and PAR4 in the generation of procoagulant phenotypes on platelet membranes. PAR4-activating peptide (AP)-stimulated platelets promoted thrombin generation in plasma up to 5 minutes earlier than PAR1-AP-stimulated platelets. PAR4-AP-mediated factor V (FV) association with the platelet surface was 1.6-fold greater than for PAR1-AP. Moreover, PAR4 stimulation resulted in a 3-fold greater release of microparticles, compared with PAR1 stimulation. More robust FV secretion and microparticle generation with PAR4-AP was attributable to stronger and more sustained phosphorylation of myosin light chain at serine 19 and threonine 18. Inhibition of Rho-kinase reduced PAR4-AP-mediated FV secretion and microparticle generation to PAR1-AP-mediated levels. Thrombin generation assays measuring prothrombinase complex activity demonstrated 1.5-fold higher peak thrombin levels on PAR4-AP-stimulated platelets, compared with PAR1-AP-stimulated platelets. Rho-kinase inhibition reduced PAR4-AP-mediated peak thrombin generation by 25% but had no significant effect on PAR1-AP-mediated thrombin generation. In conclusion, stimulation of PAR4 on platelets leads to faster and more robust thrombin generation, compared with PAR1 stimulation. The greater procoagulant potential is related to more efficient FV release from intracellular stores and microparticle production driven by stronger and more sustained myosin light chain phosphorylation. These data have implications about the role of PAR4 during hemostasis and are clinically relevant in light of recent efforts to develop PAR antagonists to treat thrombotic disorders.

Interpretation of correlations between coagulation factors FV, FVIII and vWF in normal and type 2 diabetes mellitus patients.

Increased risk of vascular diseases in hypercoagulable states is assumed to be the major cause for prevalence of vascular problems in type 2 diabetes mellitus patient. The present study was undertaken to explore the basis underlying the correlation between the aforesaid three coagulation factors with reference to the relevant mechanistic findings using statistical calculation. Our findings showed that the correlation between each pairs of coagulation factors, FV, FVIII and vWF could be easily interpreted using previously characterized interactions between these factors. Also, given the sequence similarities between FV and FVIII as well as the results of this study, it was indicated, for the first time, that FV may bind to vWF in the same way as FVIII binds. This finding seems to be of great importance to be evaluated through experimental techniques.

Evaluation of factor V mRNA to define the residual factor V expression levels in severe factor V deficiency.

We evaluated FV mRNA in severe factor V deficiency caused by the -12T/A IVS18 mutation, activating a cryptic splice site and leading to premature translation termination. Quantitative evaluation of factor V cDNA from homozygous and heterozygous subjects, and correction for nonsense mediated decay, suggested the presence of 0.1% of normal factor V mRNA.

Blood coagulation at the site of microvascular injury in healthy and coumadin-treated subjects heterogenous for factor V Leiden mutation.

There is little knowledge regarding the in-vivo course of coagulation reactions in subjects with factor (F)V Leiden (G1691A, R506Q). The aim of the current study was to evaluate the effect of FV Leiden on coagulant reactions triggered by vascular injury. At the site of microvascular injury, prothrombin activation and FVa formation and inactivation have been evaluated in 16 apparently healthy subjects and 16 patients on long-term anticoagulation with acenocoumarol, with eight heterogenous carriers of FV Leiden in the healthy and in the anticoagulated group. Thrombin formation, measured by levels of thrombin-antithrombin complexes (TAT) and thrombin B-chain, proceeded at similar rates in the bleeding-time blood in carriers and non-carriers of FV Leiden. The onset and rate of FV activation did not differ significantly among the four groups. When healthy carriers of FV Leiden were compared to healthy non-carriers, the onset of FVa inactivation by activated protein C (APC) was delayed by 60 to 90 seconds (p=0.01). Anticoagulated individuals also showed the same pattern of FV Leiden associated differences in the time of occurrence of the 30 kD FVa heavy-chain fragment (p=0.021). Our results indicate that when blood clotting is triggered by microvascular injury, the heterozygous expression of FV Leiden has no effect on thrombin generation in healthy or coumadin-treated subjects. Inactivation of FVa by the APC mechanism is attenuated significantly in carriers of FV Leiden but the magnitude of this effect is smaller than that observed in most purified systems.

[Impact of inherited thrombophilia on the development of some pregnancy complications].

AIM: To assess the clinical significance of inherited thrombophilia [IT] for the development of some pregnancy complications. MATERIAL AND METHODS: The incidence of the following factors was studied in 97 pregnant women with pregnancy complications and in 103 controls: R506Q mutation encoding Factor V Leiden [FVL] synthesis, Prothrombin G20210A mutation, T677 methylenetetrahydropholate reductase mutation [MTHFR], 4G/4G polymorphism of the plasminogen activator inhibitor [PAI 4G/4G]. Among 97 patients in the group studied 39 had early onset severe preeclampsia [PE], 14--placental abruption [AP] without PE, 18--intrauterine growth restriction [IUGR] without PE, 12--stillbirth [SB] without PE, 14--habitual spontaneous abortions [HSA]. The control group included 103 clinically healthy pregnant women with at least one previous uneventful pregnancy, without history of thromboembolic disorders. In addition, patients with severe PE with and without IT were compared regarding g. a. and birthweight at delivery and intrauterine fetal loss rate. DNA analysis was performed according to internationally accepted standards. Pregnancy outcomes were ascertained from hospital records. Statistical significance (p < 0.05) was assessed by means of Student's t-test. RESULTS: FVL mutation was found in 23.7% (23/97) of the patients from the studied group and in 5.8% (6/103) of the controls. Prothrombin G20210A carriers were 11% (11/97) of the studied and 3.8% (4/103) of the controls, while with PAI 4G/4G polymorphism they were 30.9% (30/97) and 14.5% (15/103) respectively. MTHFR T677 was not more frequent in the studied group (8.2%) compared to the control one (29%). Eight of the patients (9.6%) were carriers of more than one mutation. In 22 cases with early onset severe PE and IT gestational age and birthweight at delivery were lower than in the cases with severe PE without IT while intrauterine fetal loss rate did not differ significantly between the two groups. CONCLUSIONS: Inherited thrombophilia is found more frequently in women with pregnancy complications like PE, IUGR, AP, SB, HSA. The incidence of homozygous MTHFR T677 is not higher in these cases. IT worsens the prognosis of severe PE. The diagnosis of IT is important since anithrombotic therapy has to be considered to protect the mother and the fetus.

Increased prevalence of factor V Leiden in patients with retinal vein occlusion and under 60 years of age.

Retinal vein occlusion (RVO) is a multifactorial disease involving vessel damage, stasis, viscosity and thrombosis. Conflicting findings on hereditary thrombophilic risk factors have been reported and their impact on RVO features remains to be defined. The aim of the present study was to evaluate the prevalence of hereditary thrombophilic risk factors (HTRF) and characteristics of RVO in patients with or without HTRF. The design of the study was a prospective, observational case series. Two hundred and thirty-four patients with RVO were included consecutively. A French healthy population of the same region was studied as control group. The HTRF studied were protein C (PC), protein S (PS) and antithrombin (AT) deficiencies, factor V Leiden (FVL) and factor II 20210A polymorphisms. Chi-Square was used for comparison with the healthy subjects and between RVO patient with and without HTRF according to localisation (branch vs. central), type of RVO (ischemic or non-ischemic), recurrence, age at first event and classical vascular risk factors. Twenty-two patients had HTRF (12 FV Leiden heterozygotes, 9 FII 20210A heterozygotes and 1 PS deficiency). No AT or PC deficiency was detected. Frequencies of PS deficiency, FVL and FII 20210A allele were similar to the reference population as well as to published data in the general caucasian population. Eighty-six patients experienced their first episode before the age of 60 years. Systemic hypertension, glaucoma and angina were significantly less frequent in patients with RVO before 60 years. Fourteen of the 22 patients with one HTRF (64%) experienced their first episode of RVO before the age of 60 years compared to 72 of 212 without HTRF (34%) (p = 0.006). Heterozygote status for FV Leiden was significantly more frequent in patients who had experienced their first episode of RVO before 60 years (p = 0.027). In conclusion, this study suggests a role of FV Leiden in the occurrence of RVO in patients younger than 60 years who exhibit fewer acquired vascular risk factors than in older patients.

Lipoprotein (a) and other prothrombotic risk factors in Caucasian women with unexplained recurrent miscarriage. Results of a multicentre case-control study.

From 1998 to 2003, 133 Caucasian women aged 17-40 years (median 29 years) suffering from unexplained recurrent miscarriage (uRM) were consecutively enrolled. In patients and 133 age-matched healthy controls prothrombotic risk factors (factor V (FV) G1691A, factor II (FII) G20210A, MTHFR T677T, 4G/5G plasminogen activator inhibitor (PAI)-1, lipoprotein (Lp) (a), protein C (PC), protein S (PS), antithrombin (AT), antiphospholipid/anticardiolipin (APA/ACA) antibodies) as well as associated environmental conditions (smoking and obesity) were investigated. 70 (52.6%) of the patients had at least one prothrombotic risk factor compared with 26 control women (19.5%; p<0.0001). Body mass index (BMI; p=0.78) and smoking habits (p=0.44) did not differ significantly between the groups investigated. Upon univariate analysis the heterozygous FV mutation, Lp(a) > 30 mg/dL, increased APA/ACA and BMI > 25 kg/m(2) in combination with a prothrombotic risk factor were found to be significantly associated with uRM. In multivariate analysis, increased Lp(a) (odds ratio (OR): 4.7/95% confidence interval (CI): 2.0-10.7), the FV mutation (OR:3.8/CI:1.4-10.7), and increased APA/ACA (OR: 4.5/CI: 1.1-17.7) had independent associations with uRM.

Assembly and regulation of prothrombinase complex on B16F10 melanoma cells.

A number of studies indicate that coagulation proteases play significant roles in cancer biology. Melanoma is a highly metastatic cancer, and there is evidence that thrombin contributes to this aggressive pattern. However, few studies correlate this type of cancer with formation of the prothrombinase complex, which is responsible for conversion of prothrombin into thrombin in the coagulation system. The aim of this study was to investigate the assembly and regulation of prothrombinase complex on the murine melanoma cell line, B16F10. B16F10 cells were unable to activate prothrombin except when previously incubated with factor Xa. This effect was dependent on factor Xa binding to cell membranes, since no activation was detected with Gla-domainless factor Xa. The thrombin formation by B16F10-bound factor Xa was enhanced approximately 10 fold in the presence of factor Va, indicating the assembly of prothrombinase complex. Differently from platelets, B16F10-assembled prothrombinase complex was inhibited by prothrombin fragment 1 but not by fragment 2. In addition, bothrojaracin, a specific ligand of proexosite I on prothrombin, caused a significant decrease in the zymogen activation. Our data demonstrate that B16F10 melanoma cells generate thrombin by promoting assembly of the prothrombinase complex. This ability might be correlated with the increased metastatic potential of this cell line. Moreover, B16F10-assembled prothrombinase complex seems to be modulated in a different way from that found for the physiological complex assembled on platelets.

Factor-V expression in platelets from human megakaryocytic culture.

The origin of platelet-factor-V has long been discussed. To elucidate whether and when human platelet-factor-V is synthesized by megakaryocytes, we utilized in vitro-generated megakaryocytes capable of producing platelets. Factor-V gene was silent in purified progenitors and megakaryocytic precursors but was expressed in late culture phase and maintained also in platelets. Similarly, factor-V protein was expressed in mature proplatelet-bearing megakaryocytes (immunofluorescence analysis); it was also detectable in cultured megakaryocytes and platelets (Western blotting) and within permeabilized cultured platelets (flow cytometry). The absence of other cells in our culture system indicates conclusively that human megakaryocytes synthesize factor-V.

Human platelets contain forms of factor V in disulfide-linkage with multimerin.

Factor V is an essential cofactor for blood coagulation that circulates in platelets and plasma. Unlike plasma factor V, platelet factor V is stored complexed with the polymeric alpha-granule protein multimerin. In analyses of human platelet factor V on nonreduced denaturing multimer gels, we identified that approximately 25% was variable in size and migrated larger than single chain factor V, the largest form in plasma. Upon reduction, the unusually large, variably-sized forms of platelet factor V liberated components that comigrated with other forms of platelet factor V, indicating that they contained factor V in interchain disulfide-linkages. With thrombin cleavage, factor Va heavy and light chain domains, but not B-domains,were liberated from the components linked by interchain disulfide bonds, indicating that the single cysteine in the B-domain at position 1085 was the site of disulfide linkage. Since unusually large factor V had a variable size and included forms larger than factor V dimers, the data suggested disulfide-linkage with another platelet protein, possibly multimerin. Immunoprecipitation experiments confirmed that unusually large factor V was associated with multimerin and it remained associated in 0.5 M salt. Moreover, platelets contained a subpopulation of multimerin polymers that resisted dissociation from factor V by denaturing detergent and comigrated with unusually large platelet factor V, before and after thrombin cleavage. The disulfide-linked complexes of multimerin and factor V in platelets, which are cleaved by thrombin to liberate factor Va, could be important for modulating the function of platelet factor V and its delivery onto activated platelets. Factor Va generation and function from unusually large platelet factor V is only speculative at this time.

Endogenous factor V synthesis in megakaryocytes contributes negligibly to the platelet factor V pool.

BACKGROUND AND OBJECTIVES: Coagulation factor V (FV) is distributed between two pools: 80% circulates in plasma and 20% is stored in platelets. The aim of the study was to determine the origin of platelet FV. DESIGN AND METHODS: We investigated a FV Leiden heterozygous patient who had received an allogeneic bone marrow transplant from a normal donor. The patient had been referred to our laboratory for his marked activated protein C (APC) resistance in the apparent absence of FV Leiden. Analysis of the DNA from a buccal swab showed that the patient was indeed a heterozygous carrier of FV Leiden. The difference in FV genotype between the hepatocytes (heterozygous FV Leiden) and the blood cells (homozygous normal) of the patient provided a good model to investigate the origin of platelet FV. Platelets were isolated from the patient and the bone marrow donor and activated with thrombin and ionomycin to release and activate FV. APC was then added and the inactivation of platelet FVa was followed over time with a highly sensitive prothrombinase-based assay. RESULTS: While the donor's platelet FVa showed a normal inactivation time course, the patient's platelet FVa was considerably resistant to APC. The kinetic pattern of APC-catalyzed inactivation of the patient's platelet FVa was indistinguishable from that of plasma FVa from a FV Leiden heterozygote. INTERPRETATION AND CONCLUSIONS: These data indicate that platelet FV is derived from plasma and that endogenous FV synthesis by megakaryocytes contributes negligibly to the platelet FV pool.

Co-segregation of thrombophilic disorders in factor V Leiden carriers; the contributions of factor VIII, factor XI, thrombin activatable fibrinolysis inhibitor and lipoprotein(a) to the absolute risk of venous thromboembolism.

BACKGROUND AND OBJECTIVES: The clinical expression of factor V Leiden varies widely within and between families and only a minority of carriers will ever develop venous thromboembolism. Co-segregation of thrombophilic disorders is a possible explanation. Our aim was to assess the contributions of high levels of factor VIII:C, factor XI:C, thrombin activatable fibrinolysis inhibitor (TAFI) and lipoprotein (a) (Lp(a)) to the risk of venous thromboembolism in factor V Leiden carriers. DESIGN AND METHODS: Levels of the four proteins were measured, in addition to tests of deficiencies for antithrombin, protein C and protein S, and the prothrombin G20210A mutation, in 153 factor V Leiden carriers, derived from a family cohort study. The (adjusted) relative risk and absolute risk of venous thromboembolism for high levels of each protein were calculated. RESULTS: Of carriers, 60% had one or more concomitant thrombophilic disorders. Crude odds ratios (95% CI) of venous thromboembolism for high protein levels were: 3.2 (1.1-9.3) (factor VIII:C); 1.7 (0.6-4.9) (factor XI:C); 3.0 (1.1-8.2) (TAFI); and 1.9 (0.7-5.7) (Lp(a)). Adjusted for age, sex, other concomitant thrombophilic disorders and exogenous risk factors, the odds ratio for venous thromboembolism were 2.7 (0.8-8.7) for high factor VIII:C levels and 1.8 (0.6-5.3) for high TAFI levels. Annual incidences in subgroups of carriers were 0.35% (0.09-0.89), 0.44% (0.05-1.57) and 0.94% (0.35-2.05) for concomitance of high levels of factor VIII:C, TAFI and both, respectively, as compared to 0.09% (0.00-0.48) in single factor V Leiden carriers and 1.11% (0.30-2.82) for other concomitant disorders. INTERPRETATION AND CONCLUSIONS: High levels of factor VIII:C and TAFI, in contrast with factor XI:C and Lp(a), are mild risk factors for venous thromboembolism, and substantially contribute to the risk of venous thromboembolism in factor V Leiden carriers. Our data support the hypothesis that the clinical expression of factor V Leiden depends on co-segregation of thrombophilic disorders.

Successful extracorporeal porcine liver perfusion for 72 hr.

BACKGROUND: Improvements in extracorporeal perfusion technology and the production of transgenic pigs resistant to hyperacute rejection have stimulated several groups to re-explore the possibility of supporting patients in hepatic failure with extracorporeal porcine livers. The success of organ transplantation has also stimulated interest in using extracorporeal perfusion as a means of organ preservation and resuscitation of organs from marginal donors. The present study describes a method by which livers can be maintained in a viable condition for a minimum of 72 hr of normothermic, extracorporeal perfusion. METHODS: Five extracorporeal porcine liver perfusions were performed, each with a duration of 72 hr. Hepatectomy was performed, followed by cold preservation, cannulation of vessels, and initiation of perfusion with normothermic, oxygenated porcine blood. Organ viability was assessed by metabolic, synthetic, hemodynamic, and histologic parameters. RESULTS: After 72 hr of normothermic, extracorporeal perfusion, the isolated livers demonstrated maintenance of normal physiological levels of pH and electrolytes. Continued hepatic protein synthesis (complement and factor V) was maintained throughout the perfusion. Hemodynamic parameters remained within normal physiological range. Histology demonstrated good preservation of the liver with no overall architectural change. CONCLUSION: It is possible to maintain a liver in a viable condition for a minimum of 72 hr of extracorporeal perfusion. This technique has been developed primarily as a preclinical model of extracorporeal liver support with the intention of proceeding to a clinical trial in patients with fulminant liver failure. However, it also has potential applications in organ preservation or resuscitation before transplantation and in the experimental study of isolated liver physiology.

Factor V and thrombotic disease: description of a janus-faced protein.

The generation of thrombin by the prothrombinase complex constitutes an essential step in hemostasis, with thrombin being crucial for the amplification of blood coagulation, fibrin formation, and platelet activation. In the prothrombinase complex, the activated form of coagulation factor V (FVa) is an essential cofactor to the enzyme-activated factor X (FXa), FXa being virtually ineffective in the absence of its cofactor. Besides its procoagulant potential, intact factor V (FV) has an anticoagulant cofactor capacity functioning in synergy with protein S and activated protein C (APC) in APC-catalyzed inactivation of the activated form of factor VIII. The expression of anticoagulant cofactor function of FV is dependent on APC-mediated proteolysis of intact FV. Thus, FV has the potential to function in procoagulant and anticoagulant pathways, with its functional properties being modulated by proteolysis exerted by procoagulant and anticoagulant enzymes. The procoagulant enzymes factor Xa and thrombin are both able to activate circulating FV to FVa. The activity of FVa is, in turn, regulated by APC together with its cofactor protein S. In fact, the regulation of thrombin formation proceeds primarily through the upregulation and downregulation of FVa cofactor activity, and failure to control FVa activity may result in either bleeding or thrombotic complications. A prime example is APC resistance, which is the most common genetic risk factor for thrombosis. It is caused by a single point mutation in the FV gene (factor V(Leiden)) that not only renders FVa less susceptible to the proteolytic inactivation by APC but also impairs the anticoagulant properties of FV. This review gives a description of the dualistic character of FV and describes the gene-gene and gene-environment interactions that are important for the involvement of FV in the etiology of venous thromboembolism.

Single vs. dual vessel porcine extracorporeal liver perfusion.

BACKGROUND: The use of porcine extracorporeal liver perfusion (PECLP) to provide temporary hepatic support for patients in fulminant hepatic failure has been limited by the fact that individual perfusions can be sustained for only a few hours. Inadequate liver function and/or hemodynamic instability are the major contributing factors for early interruption of PECLP. Recent reports suggest that the choice of single (portal vein only) vs dual (portal vein and hepatic artery) vessel perfusion may influence the duration of perfusion. We hypothesize that PECLP with single vessel perfusion (SVP) is associated with worse liver function and greater hemodynamic instability than PECLP with dual vessel perfusion (DVP). MATERIALS AND METHODS: To eliminate the potentially confounding influences of liver failure and xenograft rejection, liver isografts procured from White-Landrace pig donors were perfused by either SVP or DVP via an extracorporeal circuit established with normal White-Landrace pig recipients. The function of perfused livers was evaluated by measuring production of bile and Factors V and VIII, clearance of ammonia and lactate, and extraction of O(2) at baseline and at 0, 1, 3, 6, 12, and 24 h after initiation of PECLP. The impact of PECLP on recipient hemodynamic status was assessed by monitoring BP, heart rate, urine output, O(2) saturation, etc. Among other parameters evaluated were serum albumin and total protein and hepatic release of IL-1beta and nitric oxide to assess their possible contributions to hemodynamic instability. RESULTS: DVP and SVP livers cleared ammonia and lactate similarly. Both approaches were associated with progressive hypoalbuminemia and hypoproteinemia. DVP livers produced more bile and Factor V and were associated with less recipient hypotension and IL-1beta and NO release than SVP livers. CONCLUSIONS: Livers with DVP function better than livers with SVP. The duration of PECLP can be limited by recipient hypotension, although this complication is less severe with DVP than with SVP.