Pseudohomozygosity for activated protein C resistance is a risk factor for venous thrombosis

Pseudohomozygosity for activated protein C resistance (APC-r) is a rare condition due to the association of heterozygous FV Leiden mutation and partial type I FV deficiency. To assess the risk of venous thromboembolism in these subjects, seven families including 11 pseudohomozygotes and 45 relatives were examined. Among the relatives, 16 were heterozygous FV Leiden carriers, nine showed partial FV deficiency and 20 no abnormalities. Deep vein thrombosis occurred in 4/11 (36.3%) pseudohomozygous patients versus 6/16 (37. 4%) FV Leiden carriers and 1/20 (5%) normal relatives. Pseudohomozygotes and FV Leiden carriers had a significantly increased risk of venous thrombosis in comparison to normal relatives (RR 8.8 and 5.7, respectively). There was no difference between the thrombotic risk of pseudohomozygous subjects and of FV Leiden carriers (RR 1.6, 95% CI 0.43-5.7). Furthermore, there was no difference in thrombosis-free survival between pseudohomozygotes and 45 consecutive FV Leiden heterozygous outpatients, suggesting that a referral bias may explain the apparent younger age of thrombosis in the pseudohomozygotes in comparison to relatives with FV Leiden heterozygosity (27 years v 54 years; P = 0.01). Pseudohomozygosity for APC resistance carries a significantly higher risk for venous thromboembolism in comparison to normal subjects, but probably not in comparison to heterozygous FV Leiden carriers.     

Activated protein C resistance: a comparison between two clotting assays and their relationship to the presence of the factor V Leiden mutation

Resistance to the anticoagulant effect of activated protein C (APC resistance), a frequent abnormality in patients with a history of venous thrombosis, is known to be due, in the large majority of cases, to the presence of an abnormal factor V: the factor V Leiden. It is reasonable to surmise that screening for this abnormality should be performed with a clotting method for APC resistance, before submitting the patients with abnormal results to DNA analysis. The present study was performed on 216 individuals enrolled at the Bologna centre, of which 189 were unrelated patients with a history of juvenile venous thromboembolism and 27 were relatives with or without thrombosis. APC resistance was first measured in Bologna by a standard commercial method and then, in Leiden, by an in-house method; DNA analysis was performed in those cases in which at least one of the clotting methods was abnormal. The data obtained confirm the good performance and the optimal positive predictive value for the Leiden mutation (100%) of the Leiden in-house clotting method. Performance of the commercial method was less satisfactory but markedly improved by expressing the data in relation to the values simultaneously obtained with a normal plasma pool. Even with optimal data expression, however, the positive predictive value of the commercial method, versus DNA analysis, did not exceed 88%.
It is concluded that further standardization of the commercial method here evaluated is necessary before it can be widely adopted for the screening of APC resistance and prediction of the presence of factor V Leiden.     

Low risk of venous thrombosis in two families with combined type I plasminogen deficiency and factor V R506Q mutation

Two families with type I plasminogen deficiency and APC resistance are reported. The proposita of family A suffered from ischemic stroke when taking estrogen-progesterone-containing oral contraceptive. Several hemostatic challenges in the past (ovariectomy, appendectomy, and two pregnancies) were without thrombosis. Plasminogen activity and antigen (60 and 58%, normal range 72–136 and 69–135%, respectively) were reduced, and an increased APC resistance (APC-SR = 1.55; normal range 1.8–3.00) associated with G → A change at 1,691 nucleotide position in exon 10 of FV gene (FV Leiden) was observed. The asymptomatic son had isolated plasminogen deficiency (activity 57% and antigen 60%) whereas the asymptomatic daughter had isolated APC resistance (APC-SR = 1.61) and FV Leiden mutation. The proposita of family B, referred for superficial thrombophlebitis, had low plasminogen levels (activity 55% and antigen 53%) and APC resistance (APC-SR = 1.5) whereas the asymptomatic mother and the brother had isolated APC resistance (APC-SR = 1.62 and 1.8, respectively) and the asymptomatic father isolated plasminogen deficiency (activity 61% and antigen 62%). These data suggest that the combination of plasminogen deficiency and APC resistance probably does not significantly increase the risk of venous thrombosis. However, larger experience with additional cases is needed to definitely assess the magnitude of thrombotic risk in these families. Am. J. Hematol. 57:344–347, 1998. © 1998 Wiley-Liss, Inc.     

Evaluation of a tissue factor dependent factor V assay to detect factor V Leiden: demonstration of high sensitivity and specificity for a generally applicable assay for activated protein C resistance

Resistance to the anticoagulant effects of activated protein C (APC) is now considered the most prevalent cause of inherited thrombophilia. The great majority of patients with activated protein C resistance (APCR) have a missense mutation in the factor V molecule (factor V Leiden, FVR506Q) resulting in defective inactivation of factor Va due to a loss of an APC cleavage site. The diagnosis of APCR has been based upon the inability of APC to prolong the activated partial thromboplastin (aPTT) clotting time in subjects with APCR. However, this assay has a number of deficiencies which limit its general use. We have evaluated a newly described one-stage tissue factor dependent factor V coagulation assay for APCR in 117 patients and controls and compared the results of this assay in a blinded manner to a polymerase chain reaction (PCR) based assay for the molecular defect of factor V Leiden. 43% (50 /117) of the patients studied were receiving coumadin or heparin, or had a lupus anticoagulant. The tissue factor dependent factor V assay had 100% specificity and sensitivity for factor V Leiden and successfully predicted a homozygous state in the three documented homozygotes. The PCR-based assay for factor V Leiden resulted in a single false positive assay due to a silent A to C transition at nucleotide 1692 resulting in the loss of the Mnl restriction endonuclease cleavage site. The single-stage tissue factor dependent factor V assay is a highly sensitive and generally applicable assay for APCR.     

Two novel factor V null mutations associated with activated protein C resistance phenotype/genotype discrepancy

Activated protein C (APC) resistance phenotype/genotype discrepancy is a very rare event. The objective of this study was to characterize the molecular mechanisms in two cases of APC phenotype/genotype discrepancy. An approach using direct sequencing of each exon and splicing junctions of the factor V gene showed that two novel factor V null mutations combined with heterozygous factor V Leiden mutation were responsible for this discrepancy. Our results suggest the necessity to use both phenotypic and genotypic analyses in some cases to determine an accurate diagnosis.     

Prothrombin fragment 1+2 and thrombin–antithrombin complex levels in patients with inherited APC resistance due to factor V Leiden mutation

Inherited activated protein C (APC) resistance is a newly described pathological condition associated with familial thrombophilia. A recent report on a family with APC resistance showed increased levels of prothrombin fragment 1+2 (F1+2) in the affected individuals. No data concerning thrombin–antithrombin complex (TAT) levels in patients with inherited APC resistance are presently available. The aim of this study was to assess the plasma levels of F1+2 and TAT in patients with inherited APC resistance due to factor V (F.V) Leiden mutation and to evaluate F1+2 and TAT levels in symptomatic and asymptomatic patients with the defect (‘carriers’) as compared to their family members having no evidence of F.V Leiden mutation (‘non-carriers’). One hundred and twenty-nine individuals belonging to 30 families with inherited APC resistance due to F.V Leiden mutation were studied. F1+2 and TAT levels were determined using two commercially available ELISA kits and cut-off values were defined as the higher limits of normal ranges obtained in healthy volunteers. Out of the 129 family members investigated, 36 were non-carriers, 85 were heterozygous and eight homozygous for F.V Leiden mutation. Thrombosis had occurred in 2/36 (6%) non-carriers, in 36/85 (42.3%) heterozygous and in 5/8 (63%) homozygous. Median levels of F1+2 and TAT were above cut-off values in carriers, whereas they were below in non-carriers. An overall percentage of 68.8% of carriers exhibited F1+2 levels above the cut-off value as compared to 38.9% of non-carriers. For TAT, an overall percentage of 63.4% of carriers presented with levels above the cut-off compared with 28% of non-carriers. In conclusion, patients with inherited F.V Leiden mutation may exhibit increased levels of F1+2 and TAT. There are no differences in F1+2 and TAT median levels among symptomatic and asymptomatic carriers. The percentage of carriers of F.V Leiden with levels of F1+2 and TAT above cut-off appears to be higher than that found in other clotting inhibitors defects and in this respect the defect might be considered different. However, these findings and the presence of a high percentage of non-carriers presenting with increased F1+2 and TAT levels may suggest the possible coexistence in these families of other unknown defects predisposing to thrombosis.     

Development of resistance to activated protein C during pregnancy

Summary. We measured activated protein C (APC) anticoagulant activity in 20 healthy women at 14-20, 28 and 36 weeks gestation, and at 1 d post-partum. Significant reductions in the mean APC sensitivity ratio (APC-SR) were observed at all stages of pregnancy studied compared with the mean APC-SR obtained for baseline measurements carried out at > 8 weeks post-partum. APC resistance was seen in 8/19 (42%) and in 11/20 (55%) women at 14-20 and 28 weeks gestation respectively. The development of resistance to APC may contribute to the increased risk of thrombosis during pregnancy.     

A comparison between two activated protein C resistance methods as routine diagnostic tests for factor V Leiden mutation

The most common commercially available test measuring activated protein C (APC) resistance relies on the the anticoagulant response to added APC in an activated partial thromboplastin time (APTT) based method. Another method is a Russell Viper venom time (RVVT) based system. To improve the specificity for factor V Leiden of the APTT based method, pre-dilution of test plasma in FV-deficient plasma has recently been recommended. In this study we tested the relative suitabilities of the APTT-based system, the RVVT-based system and their corresponding assays modified by pre-dilution in FV-deficient plasma, for screening asymptomatic subjects, a group of thrombophilic patients (in particular those with low APC ratios), patients on oral anticoagulants, and patients with lupus anticoagulant (LAC). We found the RVVT-based assay to be superior to the APTT-based method in the separation of normals from those with FV Leiden mutation both in asymptomatic subjects and in the thrombophilic patient group. Both modified assays demonstrated a sensitivity and specificity of 100% for FV Leiden, as verified by genotyping in asymptomatic subjects, thrombophilic patients and patients on oral anticoagulants, with the modified RVVT-based assay giving better separation between normals and FV Leiden. Inhibition of phospholipid-dependent coagulation by LAC antibodies rendered the APTT-based system less suitable than the phospholipid-rich RVVT-based one, and as nine of the 20 LAC-positive patients were on warfarin, we showed only the modified RVVT assay to be a reliable predictor of factor V Leiden in this patient group.     

Venous thromboembolism after hip fracture surgery in a patient with haemophilia B and factor V Arg506Gln (factor V Leiden).

We describe a patient with mild haemophilia B who developed symptomatic venous thromboembolism after hip arthroplasty for a traumatic fracture. A deep vein thrombosis developed in the operated leg while he was receiving a high-purity factor IX concentrate. Subsequently, he was determined to be a heterozygous carrier for the factor V Arg506Gln (Leiden) mutation. This case illustrates the importance of providing thromboprophylaxis for all patients with haemophilia receiving coagulation factor replacement and who undergo surgical procedures known to be associated with a high risk of venous thromboembolism. In patients with haemophilia and a family history of venous thromboembolism, preoperative screening for the presence of the factor V Arg506Gln mutation and other thrombophilias may be useful.     

The risk of pregnancy-related venous thromboembolism in women who are homozygous for factor V Leiden

The risk of venous thromboembolism (VTE) is increased in pregnancy and during the post-partum period. The absolute risk for pregnancy-related VTE in heterozygous women with the factor V Leiden mutation is approximately 2%, but studies on this risk for homozygous women show conflicting results. In a retrospective family study, we found that the risk of pregnancy-related VTE in women with a symptomatic first-degree relative was 17% per pregnancy (95%CI 4.7-37.4). Anticoagulant prophylaxis during the post-partum period appears to be indicated in asymptomatic homozygous women from symptomatic kindred, whereas this could be decided on an individual basis during pregnancy.     

Activated protein C resistance and the factor V Leiden mutation in children with thrombosis

To determine the prevalence of activated protein C resistance and the factor V Leiden mutation (position 1691, arginine 506 to glutamine substitution) in children with thrombosis, plasma samples from children with thrombosis were tested for activated protein C resistance. DNA was analyzed for the factor V Leiden mutation. Five of 34 children (15%) had activated protein C resistance; each was heterozygous for the factor V Leiden mutation. All 5 children heterozygous for the factor V Leiden mutation suffered non-CNS venous thromboses comprising 21% of the group of children (5/24) with non-CNS venous thrombotic events. Each of these 5 children had a family history of thrombosis. In conclusion, children with non-CNS venous thrombosis should be evaluated for the factor V Leiden mutation. Children most likely affected are those with a family history of thrombosis. Am. J. Hematol. 57:29–32, 1998. © 1998 Wiley-Liss, Inc.     

Arterial and venous thrombosis in two Italian families with the factor V Arg506→ Gln mutation

Abstract: APC resistance, due to a point mutation in factor V at amino acid position Arg⁵⁰⁶, has been identified as a major cause of inherited thrombophilia. Here we report the presence of the factor V Arg⁵⁰⁶→Gln mutation in 2 Italian families. In 1 family 3 subjects heterozygous and 2 subjects homozygous for the factor V Arg⁵⁰⁶→ Gln mutation were identified. The only subject who developed a thrombotic event was a 20-yr-old girl who was found to be homozygous for the factor V Arg⁵⁰⁶→Gln mutation. In the second family 10 subjects were identified to be heterozygous for the factor V Arg⁵⁰⁶ →Gln mutation; among them 2 developed a thrombotic event. In the same family 2 individuals were found to be homozygous for the mutation: the first had a myocardial infarction at age 25 yr and the second suffered from multiple episodes of deep venous thrombosis and had a stroke at age 24 yr. These data show that the risk of developing deep venous thrombosis for the carriers of the factor V Arg⁵⁰⁶→Gln mutation is high in the families investigated. Furthermore our data imply that the factor V Arg⁵⁰⁶ →Gln mutation in its homozygous form may relate to myocardial infarction and stroke.     

Heritability of elevated factor VIII antigen levels in factor V Leiden families with thrombophilia

Factor VIII activity (factor VIII:C) and factor VIII antigen (factor VIII:Ag) levels above 150 IU/dl are associated with a five- to sixfold increased risk of venous thrombosis compared with levels < 100 IU/dl. These high levels are present in 25% of patients with a first episode of deep-vein thrombosis and in 11% of healthy controls. von Willebrand factor (VWF) and blood group are important determinants of the factor VIII level in plasma and therefore contribute to thrombotic risk, while factor VIII appears to be the final effector. Previously, we found familial clustering of factor VIII:C levels in women, which remained after adjustment for VWF and blood group. In the present study, we analysed the familial influence on factor VIII:Ag levels exceeding 150 IU/dl in 12 large families with thrombophilia in which high factor VIII:Ag levels contribute to thrombotic risk. As expected, blood group was a main determinant of the plasma factor VIII level: 58 relatives (32%) had factor VIII levels above 150 IU/dl and 50 (86%) of these had blood group non-O. After adjustment for blood group and age, we found an association between factor VIII:Ag levels in sister pairs (0.35, P = 0.003), brother pairs (0.35, P = 0.003), brother-sister pairs (0.35, P < 0.001) and in mother-son pairs (0.45, P = 0.02), but not in father-daughter or father-son pairs. The familial aggregation test was strongly positive for factor VIII:Ag levels (P < 0.001) and remained so after adjustment for the influence of blood group. We conclude that high factor VIII:Ag levels are a highly prevalent risk factor for venous thrombosis and contribute to risk in families with thrombophilia, and that these high levels are likely to be genetically determined by factors other than just blood group.     

Intraindividual consistency of the activated protein C resistance phenotype

Resistance to activated protein C (APC) has been demonstrated to be a risk factor for venous thromboembolism, but it is not known whether this phenotype is consistent over time. We reinvestigated 2580 subjects from the Vicenza Thrombophilia and Atherosclerosis (VITA) Project to evaluate the prevalence of a consistent APC resistance phenotype in the population. Among the 433 subjects with an APC resistance at first visit, the phenotype was confirmed in all the 74 factor V (FV) Leiden carriers and in 124 of 359 FV Leiden negative subjects (34%). The prevalence of a confirmed phenotype, not associated with FV Leiden, was 4.8% in our population. In a subgroup of subjects previously investigated for heritability of the APC resistance, we confirmed the APC resistance phenotype in seven of 39 (17.9%) subjects with an APC resistant sibling but only in 20 of 408 (4.9%) subjects without a sibling with the same phenotype (P = 0.005). Among the 124 FV Leiden negative subjects with a persistent APC resistance phenotype, 40 (32%) had a plasma factor VIII coagulant activity level above 150 IU/dl and eight (6.4%) were carriers of the G20210A prothrombin allele. APC resistance not due to FV Leiden is a frequent and consistent phenotype in the general population, with a possibly strong genetic influence.     

Budd-Chiari syndrome, portal vein and mesenteric vein thrombosis in a patient homozygous for factor V Leiden mutation treated by TIPS and thrombolysis

We present the first case of Budd-Chiari syndrome in association with portal and mesenteric vein thrombosis in a patient homozygous for the factor V Leiden mutation. She was treated by transjugular intrahepatic portosystemic stent (TIPS) placement followed by local thrombolytic therapy. Venous outflow from the liver was established and the thrombi in the portal and mesenteric veins were lysed completely. This therapeutic approach may be used for such patients with this severe thrombotic event, who generally have a poor prognosis.     

Factor V Leiden (FVQ 506) is common in a Brazilian population

Resistance to activated protein C (APC) is the most common risk factor in venous thrombosis. A missense mutation in the factor V gene (factor V Leiden) is the molecular basis for this phenotype. Factor V Leiden was previously found in about 20% of unselected patients with thrombosis but also occurred in 3-5% of healthy Dutch controls. In the present study of Brazilian patients with venous thrombosis we also observed a frequency of 20% for this factor. In addition, in healthy Brazilian controls the frequency of the mutation was 2%. These data suggest that factor V Leiden has a similar distribution worldwide irrespective of the ethnic origin of the population. © 1995 Wiley-Liss, Inc.     

Influence of three potential genetic risk factors for thrombosis in 43 families carrying the factor V Arg 506 to Gln mutation

The factor V (FV) Arg 506 to Gln mutation is the most common abnormality observed in familial thrombophilia. Many studies have shown that its clinical expression differs among families and among carriers. Some thrombotic patients carry an additional genetic risk factor such as protein C, protein S or antithrombin deficiency. We sought to identify other genetic risk factors potentially favouring expression of the thrombotic phenotype in 370 members of 43 families with the FV Arg 506 to Gln mutation. We analysed three candidate polymorphisms in genes involved in the PC anticoagulant pathway, consisting of two polymorphic sites in the 5' non-transcribed region of the PC gene, -1654 C/T and -1641 A/G, with three known combinations (TA, CA and CG) that influence the protein C plasma level; one polymorphic site (4070 A/G) in exon 13 of the FV gene, which influences the plasma factor V concentration, and one polymorphic site (677 C/T) in the methylenetetrahydrofolate reductase gene, which is often associated with moderate hyperhomocysteinaemia. The distribution of these different polymorphisms was similar in patients with a history of thrombosis and those who remained asymptomatic, ruling out the possibility that each of these polymorphisms alone can play a role in the onset of thrombosis in carriers of the FV Arg 506 to Gln mutation.     

Thrombophilic risk of individuals with rare compound factor V Leiden and prothrombin G20210A polymorphisms: an international case series of 100 individuals

The risk of thrombosis in individuals with rare compound thrombophilias, homozygous factor V Leiden (FVL) plus heterozygous prothrombin G20210A (PTM), homozygous PTM plus heterozygous FVL, and homozygous FVL plus homozygous PTM, is unknown. We identified, worldwide, individuals with these compound thrombophilias, predominantly through mailing members of the International Society on Thrombosis and Haemostasis. Physicians were sent a clinical questionnaire. Confirmatory copies of the genetic results were obtained. One hundred individuals were enrolled; 58% were female. Seventy‐one individuals had a venous thrombosis (includes superficial and deep vein thrombosis, and pulmonary embolism), 4 had an arterial thrombosis and 6 had both. Nineteen individuals had never had a thrombotic event. Thrombosis‐free survival curves demonstrated that 50% of individuals had experienced a thrombotic event by 35 yrs of age, while 50% had a first venous thromboembolic event (VTE; includes all venous thrombosis except superficial thrombosis) by 41 yrs of age; 38.2% of first VTEs were unprovoked. 37% of patients had at least one VTE recurrence. Seventy percent of first pregnancies carried to term and not treated with anticoagulation were thrombosis‐free. In conclusion, patients with these rare compound thrombophilias are not exceedingly thrombogenic, even though they have a substantial risk for VTE.