FV Leiden Mutation Research Articles

The Frequency of Factor V Leiden and Concomitance of Factor V Leiden With Prothrombin G20210A Mutation and Methylene Tetrahydrofolate Reductase C677T Gene Mutation in Healthy Population of Denizli, Aegean Region of Turkey

Factor V Leiden causing activated protein C resistance is the most common inherited form of thrombophilia leading to thrombosis. Its frequency shows great ethnic and geographic variations. The aim of this study was to determine the frequency of FV Leiden and coinheritance of FV Leiden with two other frequent hereditary thrombophilia causes, namely, prothrombin G20210A and methylene-tetrahydrofolate reductase (MTHFR) C677T mutation in the Aegean region of Turkey. The study population consisted of 1030 (500 men and 530 women) apparently healthy subjects. Functional resistance to activated protein C (APC) was measured by using the test kit STA staclot APC-R ((Diagnostica Stago, Asnieres, France, Cat. No. 00721). In subjects with APC resistance, molecular analyses of FV Leiden and of prothrombin G20210A and MTHFR C677T mutation were performed by using FV-PTH-MTHFR StripA (Vienna Lab, Labordiagnostika GmbH, Austria) kit, which was based on hybridization of polymerase chain reaction (PCR) amplified DNA products with mutation-specific oligonucleotide probes. Functional APC resistance was present in 93 subjects (9%). FV Leiden mutation was found in 87 of 93 subjects with APC resistance by PCR method. The FV Leiden carrier frequency was found to be 8.4% (87/1030). Seventy-six individuals were heterozygous (7.3%), and 11 were homozygous (1.06%). Among the 87 subjects with FV Leiden mutation, 45 subjects had MTHFR C677T gene mutation (7 homozygous, 38 heterozygous) and 4 subjects had heterozygote prothrombin G20210A gene mutation. A combination of FV Leiden and prothrombin G20210A and MTHFR C677T gene mutation was detected in 3 subjects. The results indicate that FV Leiden prevalence is quite high and coexistence of FV Leiden with other hereditary causes of thrombosis such as prothrombin G20210A mutation and MTHFR enzyme defect is not rare in healthy population of Aegean region of Turkey.

Venous thromboembolism in carriers of the Factor V Leiden mutation and in patients without known thrombophilic risk factor; prediction of recurrence and APC–PCI complex concentration and/or soluble thrombomodulin antigen and activity

Background The complex between activated protein C (APC) and the protein C inhibitor (PCI) is a sensitive indicator of the degree of activation of blood coagulation and higher concentrations have been measured in carriers of the FV Leiden mutation who were in the recovery phase after treatment for venous thromboembolism (VTE). Objectives The main purpose of this study was to correlate the APC–PCI complex concentration to thrombomodulin activity and antigen concentration in the same group of patients. We also add a prospective clinical follow-up of the VTE recurrence after 1 year to investigate if the markers can predict the risk for a new VTE. Patients/Methods Blood samples were collected from 50 patients with the FV Leiden mutation and 132 without any known risk factor for thrombophilia after finished treatment. Results The APC–PCI complex, s-TM activity and the quotient (s-TM activity) / (s-TM antigen) were higher in VTE patients with FV Leiden. In total, there were 19 VTE recurrences (10%) after 1 year. The OR for recurrence was 1.9 (95% CI 0.68–5.0) in all VTE patients with elevated APC–PCI complex (above 75th percentile) and 3.6 (95% CI 1.1–12) in VTE patients without any known risk factor for thrombophilia and with elevated s-TM activity. Conclusion The APC–PCI complex concentration, s-TM activity and the quotient (s-TM activity) / (s-TM antigen) were higher in VTE patients with FV Leiden. The s-TM activity showed higher OR for recurrence of VTE in patients without known thrombophilic risk factor. Both methods could be sensitive markers of increased risk for venous thrombosis.

JAK2 V617F in 3 Patients with Venous Thromboembolism (VTE) Followed during a Long Period of Time.

Hereditary thrombophilia and myeloproliferative disorders (MPD) are risk factors of VTE. The JAK2 V617F mutation may be found in polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis. We report three women referred to our out-patient clinic for a thrombophilia screening. JAK2 mutation was studied using wild type FRET probes and subsequent analysis of the melting curve (James et al, Leukemia 2006; 20: 350–353). Patient 1, 75 year-old (yo), had pulmonary embolism (PE) and proximal deep vein thrombosis (DVT) at 68. We detected heterozygous FV Leiden (FVL) and FII 20210A mutations. In 2005, she had internal jugular vein thrombosis after surgery (platelet count > 600 G/L). ET was suspected in 2006. Patient 2, 44 yo, on oral contraception, had PE and proximal DVT at 33 after sclerotherapy, recurrent massive PE requiring emergency embolectomy and proximal DVT during pregnancy. Heterozygous FVL was detected in 1995 (platelet count was 432 G/L) and ET in 2000. Patient 3, 65 yo, with PV detected in 1995, had a proximal DVT one year later. Heterozygous FVL was detected. She is the only patient treated with hydroxyurea. Results of blood cell counts and JAK2 V617F/JAK2 WT ratios are shown in the table. Conclusion 1. JAK2 mutation was found in 3 patients with VTE, platelet count >400 G/L and hereditary thrombophilia; 2. the risk of VTE related to JAK2 needs further investigation; 3. JAK2 V617F increased over time in 2 of 3 cases; 4. its early detection might improve the diagnosis and treatment of MPD and VTE.PatientYearPlatelet (G/L)Hemoglobin (g/dl)Hematocrit (%)Leukocytes (G/L)JAK2 ratio1200043115.345.98.610%200644813.943.310.450%2199754114.142.66.125%200579614.042.87.415%3199750114.142.64.615%200360913.642.15.820%200648512.837.94.850%

Microparticles Are a Major Determinant of Thrombin Generation Measured by Technothrombin®TGA.

We have recently shown that a thrombin generation assay using low concentrations of phospholipids (3,2 mM; 20% PS) can discriminate patients with an increased risk for recurrent venous thromboembolism after cessation of anticoagulant therapy from those with a lower risk (Hron et al JAMA 2006). Patients with peak thrombin of ≥400nM had a ~3-fold higher relative risk of recurrence than those with peak thrombin <400nM. This difference was not explained by a larger proportion of patients with clotting factor abnormalities among those with high peak thrombin. We therefore investigated which other factors might influence peak thrombin generation. For this purpose 35 healthy male plasma donors were analyzed. Thrombin generation was measured in standard platelet poor plasma and in plasma made microparticle (MP) free by centrifugation for 120 minutes at 35,250 x g using low (3,2μM) and high (32μM ) phospholipids concentrations at the same tissue factor concentration (71,6 pM). For control purposes, peak thrombin was measured under similar conditions in 12 individuals each having either heterozygous FVLeiden mutation, being on vitamin K-antagonist therapy (VKA; mean INR=3.2) or having a lupus anticoagulant. In healthy plasma donors peak thrombin as generated by tissue factor and low phospholipids (RCL) in platelet poor plasma was 110±40 nM and increased to 306±99 nM in the presence of high phospholipids (RCH). When MP free plasma was used, however, peak thrombin dropped to 23±13 nM (RCL) and 109±43 nM thrombin (RCH), respectively. When MPs were re-added to MP-free plasma samples, a dose dependent increase in peak thrombin was found both for RCL and RCH reagents. At a MP concentration of 25,000 added per 1 ml MP free plasma, peak thrombin was 381±24 nM for RCL and 399±63 nM for RCH. The effects of MPs on peak thrombin using RCL were much more pronounced than the differences in peak thrombin seen between plasmas from individuals with FVLeiden, during anticoagulant therapy or with lupus anticoagulant as compared to controls (FVLeiden : 161±50 nM; VKA: 50±12 nM; lupus: 37±23 nM; controls: 110±40 nM). These results indicate that MPs contained in platelet poor plasma are a major determinant of peak thrombin in thrombin generation assays when low phospholipids are used and might cause increased peak thrombin levels found in patients with recurrent venous thromboembolism.

Peptidomimetic inhibitors for activated protein C: implications for hemophilia management.

Several clinical studies and experiments with transgenic mice have suggested that the severity of the bleeding phenotype in hemophilic patients is substantially reduced in association with impaired inactivation of factor (F) Va by activated protein C (APC) in the presence of the FV Leiden mutation. Experiments using a synthetic coagulation proteome model showed that the presence of FV Leiden significantly increased thrombin generation in the absence of FVIII or FIX. To test the effect of APC inhibition on thrombin generation in hemophilia. Prothrombinase and a synthetic coagulation proteome model of tissue factor-triggered thrombin generation were used. Peptide-based APC inhibitors, which mimic the P4-P4' residues surrounding the APC cleavage site at Arg306 of FVa, were synthesized. These compounds are specific and reversible inhibitors of APC, with Ki values as low as 1-2 microM; most have insignificant affinity for FXa or thrombin. The affinity for APC is dependent upon the location and character of the protecting groups. Representatives of this group of compounds inhibit FVa inactivation by APC and prolong FVa functional activity in the prothrombinase complex. When evaluated in a synthetic coagulation proteome model, one inhibitor partially compensated for the absence of FVIII. Synthetic APC inhibitors may be useful as adjuvants for hemophilia treatment.

Effects of Factor Xa and Protein S on the Individual Activated Protein C-mediated Cleavages of Coagulation Factor Va

Effects of Factor Xa and Protein S on the Individual Activated Protein C-mediated Cleavages of Coagulation Factor Va

Arterial disease and venous thrombosis: are they related, and if so, what should we do about it?

Arterial disease and venous thrombosis: are they related, and if so, what should we do about it?

Is antithrombotic prophylaxis required in cancer patients with central venous catheters? Yes for special patient groups.

Is antithrombotic prophylaxis required in cancer patients with central venous catheters? Yes for special patient groups.

Factor V Level Affects the Host Susceptibility to Group A Streptococcal Infection.

Group A streptococci (GAS), a common human pathogen, secrete streptokinase (SK), which activates the host's plasminogen (PLG). SK is highly specific for human PLG, exhibiting little or no activity against other mammalian species. We demonstrated the major role of the PLG/SK interaction in GAS pathogenicity using a transgenic murine model expressing human plasminogen with increased susceptibility to human pathogenic streptococci. We hypothesize that GAS hijack the host fibrinolytic system in order to circumvent local thrombosis for systemic spread. Markedly increased mortality was also observed following GAS injection in C57BL/6J mice treated with the snake venom Ancrod, which proteolytically degrades plasma fibrinogen, supporting the critical roles of coagulation in host/pathogen interaction.However, fibrinogen also plays important roles in inflammation and immune response, it is necessary to use independent genetic models to further test the impact of coagulation in host defense against bacterial infection. The effect of variations in FV on mouse susceptibility to streptococcal infection was tested. We established a mouse model with low plasma FV level. These mice have a slightly increased bleeding time, though otherwise phenotypically normal. Subjected to streptococcal infection, these mice exhibited significantly increased mortality than wildtype controls, suggesting that the decreased thrombotic tendency in the low FV mice increases host susceptibility to infection. FV Leiden is a common prothrombotic mutation among Caucasian population with an incidence between 4% and 6%. Previous studies from Kerlin et al demonstrated the FV Leiden conferred survival advantage in patients with severe sepsis and in mice challenged with endotoxin. This may be an example of balanced gene polymorphism that maintains the FV Leiden mutant in the general gene pool by selection of bacterial infections. In order to identify the selective agents responsible for the prevalence of FV Leiden mutation, we took advantage of our plasminogen transgenic murine model for streptococcal infection to test whether streptococci is one of the selective agents. Human plasminogen transgene was introduced into FV Leiden background and the susceptibility to streptococcal infection was measured. No significant improvement of survival was observed in the FV Leiden mouse comparing with the wildtype control. Thus, streptococcal infection is not the selective agent for the prevalence of FV Leiden mutation.These observations highlight the potential role of variations in blood coagulation factors in host susceptibility to bacterial infection.

The factor V Glu1608Lys mutation is recurrent in familial thrombophilia

Co-inheritance of heterozygous factor V deficiency with FV Leiden enhances the activated protein C resistance (APCR) associated with this mutation, resulting in pseudo-homozygous APCR. The role of FV deficiency in modulating thrombotic risk in this rare condition is poorly understood. We have identified in thrombophilic patients with FV deficiency a novel FV gene mutation (c. 4996G>A), predicting the Glu1608Lys substitution in the A3 domain. The heterozygous mutation was detected in three unrelated patients, two carriers of the FV Leiden mutation, and one of the FVHR2 haplotype. The Glu1608Lys change was also present in two subjects with mild FV deficiency, and absent in 200 controls. The FV1608Lys carriers showed reduced mean FV activity (42% +/- 12%) and antigen (53% +/- 18%) levels and, in Western blot analysis, reduced amounts of intact platelet FV. The restriction fragment length polymorphism (RFLP) study identified two haplotypes underlying the mutation, which suggests that it is recurrent. In heterozygous subjects the amount of FV1608Lys mRNA in white blood cells was similar to that produced by the counterpart alleles (FVWt or FVHR2). Recombinant FV1608Lys (rFV1608Lys), detected by Western blot in the conditioned medium, was indistinguishable from rFVWt and FV antigen and activity were found to be respectively 44% +/- 20% and 13% +/- 4% of rFVWt. Our data indicate that FVGlu1608Lys predicts a CRM (plasma)/CRMred (cell culture) FV deficiency, and may contribute to thrombophilia in carriers of FV Leiden and FVHR2 haplotype via a pseudo-homozygosity mechanism. Our findings help to define the molecular bases of FV deficiency and thrombophilia.

Predictors of the post‐thrombotic syndrome during long‐term treatment of proximal deep vein thrombosis

The post-thrombotic syndrome is a chronic, poorly understood complication of deep venous thrombosis (DVT). To evaluate predictors of the post-thrombotic syndrome, including intensity of long-term anticoagulation, and to assess the impact of the post-thrombotic syndrome on quality of life. The setting was 13 Canadian hospitals and one US hospital. One hundred and forty-five patients with an unprovoked episode of proximal DVT who were initially treated with 3 months of conventional-intensity warfarin [target International Normalized Ratio (INR) of 2.5] then participated in a trial comparing two intensities of long-term warfarin therapy (target INR 2.5 vs. INR 1.7). Post-thrombotic syndrome was assessed at the end of the trial using a validated clinical scale. Generic and venous disease-specific quality of life was compared in patients with and without the post-thrombotic syndrome. Multivariable regression analyses were performed to identify predictors of the post-thrombotic syndrome and of its severity. After an average follow-up of 2.2 years, the prevalence of post-thrombotic syndrome was 37% and of severe post-thrombotic syndrome was 4%. Quality of life was worse in patients with the post-thrombotic syndrome compared with patients who did not have it. The presence of factor (F)V Leiden or the prothrombin gene mutation was an independent predictor of both a lower risk (P = 0.006) and reduced severity (P = 0.045) of the post-thrombotic syndrome. Intensity of anticoagulation did not influence the risk of developing the post-thrombotic syndrome. The post-thrombotic syndrome is a frequent and burdensome complication of proximal DVT, even among patients maintained on long-term oral anticoagulation. While the presence of FV Leiden or prothrombin gene mutation appears to be associated with a reduced risk of post-thrombotic syndrome, this finding requires further evaluation in prospective studies.

Pathogenic antibodies to coagulation factors. Part II. Fibrinogen, prothrombin, thrombin, factor V, factor XI, factor XII, factor XIII, the protein C system and von Willebrand factor.

Pathogenic antibodies to coagulation factors. Part II. Fibrinogen, prothrombin, thrombin, factor V, factor XI, factor XII, factor XIII, the protein C system and von Willebrand factor.

Factor V Leiden, FII G20210A, MTHFR C677T mutations as risk factors for venous thrombosis during pregnancy and puerperium

Venous thrombosis is the most common cause of obstetric morbidity and mortality during pregnancy and puerperium. The incidence of pregnancy-associated venous thrombosis varies from 1 in 1000 to 1 in 2000 deliveries. Factor V G1691A (FV Leiden), FII G20210A and MTHFR C677T mutations are the most common genetic risk factors for thromboembolism. The aim of this study was to establish the presence of these risk factors in a group of women with an episode of deep venous thrombosis during pregnancy or puerperium. The study was carried in a group of 45 women with the first episode of deep venous thrombosis during pregnancy or puerperium. The patients with antiphospholipid antibodies, antithrombin III, protein C or protein S deficiency, and autoimmune and malignant diseases were excluded from the study. FV Leiden, FII G20210A, and MTHFR C677T mutations were detected by polymerase chain reaction, followed by digestion with specific restriction enzymes. Twenty heterozygous carriers of the FV Leiden mutation and one homozygous carrier were detected, which represents the frequencies of 44.4% and 2.2%, respectively. For the FII G20210A mutation, six heterozygous carriers were identified, giving the frequency of 13.3%. The MTHFR C677T mutation was observed in 31 patients (22 heterozygous and 9 homozygous carriers) which represents the frequencies of 48.9% and 20%, respectively. Our study suggested that the obligatory testing for FV Leiden and FII G20210A mutations was strongly recommended in women with history of venous thrombosis during pregnancy and puerperium. We found a slight effect of MTHFR 677T allele, but it should be considered in association with other risk factors.

Impact of acquired and genetic factors on thrombophilic phenotype in FV Leiden mutation carriers

FV Leiden mutation is an important genetic risk factor for venous thromboembolsm (VTE). In this study we have analyzed clinical manifestation and the impact of other genetic and acquired risk factors in 100 patients (95 heterozygous and 5 homozygous) carriers of FV Leiden mutation. Among these patients, 91 experienced VTE, with down limb deep vein thrombosis as the most frequent manifestation. An acquired risk factor was present in 68.6% of women, whereas this was the case in 28.6% of men. FIIG20210A was present in 9.5%, MTHFR 677TT in 8.4% and both mutations in 2.1% of the heterozygous FV Leiden carriers. Our results suggest that knowledge of coexisting factors predisposing to VTE is very important for FV Leiden mutation carriers and may contribute to the prevention of VTE episodes.

The multiple faces of the partial thromboplastin time APTT

In the historical sketch by G. C. White [1], the importance of the activated partial thromboplastin time (APTT) for research on coagulation and hemophilia is emphasized. I wish to add that the APTT method has also had a major impact on research on the anticoagulant pathways and on thrombophilia. In this brief comment, I reflect upon the role played by the APTT in the discovery of activated protein C (APC) resistance and on the importance of the method for the elucidation of the underlying molecular mechanisms of this condition. The observation initiating the work on APC resistance was made while performing a functional protein C assay on plasma from a patient with thrombosis [2]. The protein C assay included isolation of protein C by barium citrate adsorption, activation of the protein C by a thrombin–thrombomodulin complex and finally an APTT reaction to monitor the anticoagulant response of APC. In the analysis, it was observed that the patient sample and the standard did not yield parallel dose–response curves. This stimulated our curiosity and we performed a large number of experiments to understand the problem. During these studies it became apparent that the unexpected behavior was not related to the patient's protein C but that something was wrong with the patient's plasma—we found a poor anticoagulant response of the patient's plasma to added normal APC. This observation lead to the design of a new APTT-based assay in which normal APC was added to the patient's plasma and the anticoagulation response measured. Using this first version of the APC resistance test, we found that not only the original patient but also many of his family members responded poorly to APC. Stimulated by these observations, we and others investigated cohorts of thrombosis patients and it became obvious that APC resistance was a major risk factor for venous thrombosis [3-5]. Moreover, in a majority of the cases, the APC resistance was inherited and was due to the same genetic defect [4, 6]. The APTT-based APC-resistance test was instrumental in identifying factor (F)V as the causative molecule for the APC-resistance phenotype and laid the foundation for the subsequent identification of the responsible mutation in the FV gene [7]. Almost all patients with APC resistance were found to carry the same mutation, which replaces Arg506 in one of the APC-cleavage sites with a Gln (FV Leiden) [8-11]. As a consequence of this mutation, the APC-mediated degradation of FVa was impaired. This observation has provided great stimulus to investigations of the mechanisms of APC-mediated degradation of FVa and factor (F)VIIIa and the role of protein S. During the efforts to elucidate the molecular mechanisms of APC resistance, it was discovered that FV is a Janus-faced protein with a potential to express both pro- and anticoagulant activities [7, 12, 13]. The anticoagulant function of FV is that of a cofactor to APC in the degradation of FVIIIa. In APC resistance, this anticoagulant property of FV is lost due to the FV Leiden mutation. The first diagnostic assay for APC resistance, the ‘classical’ APC-resistance test, was modified based on the discovery of the involvement of FV in the pathogenesis of APC resistance. In the modified test, FV-deficient plasma was included in the clotting reaction [14]. The modified APC-resistance test has been found to be highly specific and sensitive for the FV Leiden mutation and is often used as a first screening method to identify individuals with APC resistance. One might reflect on what made the APTT reaction particularly suitable for research on APC resistance. A contributing factor is that the APTT is dependent on the activity of both FVa and FVIIIa. It is therefore sensitive to the effect of APC at two levels of the coagulation reaction. In contrast, the regular prothrombin time reaction is sensitive to changes only in FVa activity and unlike the APTT it is sensitive neither to loss of FVIIIa activity nor to loss of the anticoagulant function of FV. In conclusion, I wish to add to the praise of the APTT method. It was important not only for defining a new era in coagulation but also for research on anticoagulant pathways, for the discovery of APC resistance and the elucidation of pathogenic mechanisms of thrombophilia.

Prevalence of Activated Protein C Resistance Due To Factor V Leiden Mutation among South Asians (India and Pakistan) with Venous Thromboembolism.

Objective: To study the prevalence of Activated Protein C (APC) resistance due to Factor V Leiden (FV Leiden) mutation among the first generation immigrants from India and Pakistan with venous thromboembolism (VTE).Introduction: APC resistance due to the substitution of Arginine 506 by Glutamine in coagulation Factor V is caused by G1691A mutation in exon 10 of Factor V gene. This is the commonest cause of inherited thrombophilia in Caucasians, but the frequency of this mutation is low in non-Caucasians. Among subjects in the Physician Health Study, the frequency of FV Leiden was found to be 5.27% in Caucasian Americans vs. 0.45% in Asian Americans. Another study found no mutation in 191 Asian Americans tested. In non-Caucasians with VTE, it is generally considered not cost effective to screen for this mutation. However Asians are a heterogeneous group and the Leiden gene frequency varies among different ethnic populations. While the frequency of FV Leiden gene has been documented to be low in China, Korea, Japan, Thailand, Indonesia etc, the frequency in India and Pakistan is not well studied. Two studies found a carrier frequency of 2% (Rees et al) and 4.2 % (Gou et al) among the general population from India and Pakistan. This is similar to the frequency found in Middle Eastern and European population. We did not come across any study of FV Leiden gene frequency in patients with VTE from India and Pakistan.Patients and Methods: A retrospective chart review of patients of Indian or Pakistani origin seen at Coney Island Hospital, from July 1996 to June 2003, who had a work up for inherited thrombophilia after an episode of VTE. During the chart review age, sex, first or recurrent episode and any predisposing factors such as immobilization, malignancy, hormonal therapy, surgery, pregnancy, and the presence of SLE or MPD were noted. Thrombophilia work up included functional assays for Protein C, S and Antithrombin III, Lupus anticoagulant, ACA and Homocysteine levels. APC resistance was measured by a clotting assay using Factor V depleted plasma and all patients who were borderline or resistant were tested for the presence of FV Leiden mutation by PCR.Results: A total of 18 patients were studied. All had an episode of VTE documented by a Doppler ultrasonography or a Ventilation Perfusion lung scan or a CT angiogram. 3 out of 18 patients (16.6%) had APC resistance. All the three patients were confirmed to be heterozygous for FV Leiden mutation. Two were male and one was a female with a median age of 36 yrs (27, 36 and 57 yrs). The female patient had a recurrent episode, first one occurred during pregnancy, but the second episode had no precipitating events. One male patient had trauma to the leg and was immobilized at the time of the VTE, another male patient was a cab driver by occupation. None of the patients had any other concurrent inherited thrombophilic state.Conclusions: The prevalence of the FV Leiden mutation is significantly high among South Asians with VTE in our study. If the findings are confirmed by a larger study, screening for this mutation for thrombophilia would be relevant in patients of South Asian origin and screening recommendations for family members would be identical to Caucasian population. The high prevalance as in Caucasians suggests a founder effect and possible spread of the mutation by the migration of Neolithic farmers from the Middle East towards Europe and India, ten thousand years ago. This has been confirmed by haplotype analysis.

Prevalence of factor V Leiden and G6PD 1311 silent mutations in dalmatian population

Factor V Leiden has been described as a common genetic risk factor for venous thromboembolism. The geographic distribution of this abnormality varies greatly, being high in Europe and almost absent in Asia and Africa. Particularly high prevalence is observed in some Mediterranean countries, which suggests the Mediterranean origin of this mutation. Similarly, prevalence of silent mutation 1311 of the G6PD gene seems to be higher among Mediterranean populations. Since the Dalmatian population (of south Croatia) geographically belongs to the Mediterranean populations we analyzed the prevalence of FV-Leiden and silent mutation 1311 in this region. Furthermore, because the coincidence of G6PD deficiency and venous thromboembolism was described earlier, we tested a possible association of FV-Leiden and G6PD deficiency. One hundred sixty-eight healthy blood donors and 55 G6PD deficient individuals originating from the Dalmatian region were tested for the presence of FV-Leiden mutation and silent mutation 1311. Prevalence of FV-Leiden among blood donors was 2.4%, while among G6PD deficient individuals it was significantly higher, 11% (p=0.011). Prevalence of silent mutation 1311 among blood donors and G6PD deficient individuals was 21 and 15%, respectively. Observed allele frequencies among individuals originating from the Dalmatian region is similar to the neighboring European and Mediterranean populations. Interestingly, our results indicate the association of the FV-Leiden and G6PD deficiency and warrant further studies.

Familial thrombophilia and lifetime risk of venous thrombosis

We started a large multicenter prospective follow-up study to provide reliable risk estimates of venous thrombosis in families with various thrombophilic defects. This paper describes data collected at study entry on venous events experienced before study inclusion, i.e. the baseline data. All individuals (probands, relatives) registered in nine European thrombosis centers with the factor (F)V Leiden mutation, a deficiency of antithrombin, protein C or protein S, or a combination of these defects, were enrolled between March 1994 and September 1997. As control individuals, partners, friends or acquaintances of the thrombophilic participants were included. Incidence and relative risk of objectively confirmed venous thrombotic events (VTEs) prior to entry were calculated for the relatives with thrombophilia and the controls. Of the 846 relatives with thrombophilia (excluding probands), 139 (16%) had experienced a VTE with an incidence of 4.4 per 1000 person years. Of the controls, 15 of the 1212 (1%) controls had experienced a VTE with an incidence of 0.3 per 1000 person years. The risk of venous thrombosis associated with familial thrombophilia was 15.7 (95% CI 9.2-26.8) and remained similar after adjustment for regional and sex-effects (16.4; 95% CI 9.6-28.0). The highest incidence per 1000 person years was found in relatives with combined defects (8.4; 95% CI 5.6-12.2), and the lowest incidence was found in those with the FV Leiden mutation (1.5; 95% CI 0.8-2.6). Considerable differences in the lifetime risk of VTE were observed among individuals with different thrombophilia defects.

Molecular basis of inherited antithrombin deficiency in Portuguese families: identification of genetic alterations and screening for additional thrombotic risk factors.

Antithrombin (AT), the most important coagulation serine proteases inhibitor, plays an important role in maintaining the hemostatic balance. Inherited AT deficiency, mainly characterized by predisposition to recurrent venous thromboembolism, is transmitted in an autosomal dominant manner. In this study, we analyzed the underlying genetic alterations in 12 unrelated Portuguese thrombophilic families with AT deficiency. At the same time, the modulating effect of the FV Leiden mutation, PT 20210A, PAI-1 4G, and MTHFR 677T allelic variants, on the thrombotic risk of AT deficient patients was also evaluated. Three novel frameshift alterations, a 4-bp deletion in exon 4 and two 1-bp insertions in exon 6, were identified in six unrelated type I AT deficient families. A novel missense mutation in exon 3a, which changes the highly conserved F147 residue, and a novel splice site mutation in the invariant acceptor AG dinucleotide of intron 2 were also identified in unrelated type I AT deficient families. In addition to these, two previously reported missense mutations changing the AT reactive site bond (R393-S394) and leading to type II-RS deficiency, and a previously reported cryptic splice site mutation (IVS4-14G-->A), were also identified. In these families, increased thrombotic risk associated with co-inheritance of the FV Leiden mutation and of the PAI-1 4G variant was also observed. In conclusion, we present the first data regarding the underlying genetic alterations in Portuguese thrombophilic families with AT deficiency, and confirm that the FV Leiden mutation and probably the PAI-1 4G variant represent additional thrombotic risk factors in these families.

Venous thrombosis among patients with AIDS.

Thrombosis has been considered an uncommon complication in patients with AIDS. In a 42-month period, 28 adult male homosexuals with AIDS experienced 34 thrombotic events. All but three received HAART regimen, two a successful round of double nucleoside analog therapy, and one patient received no treatment. Median age of group was 38.5 years (range, 24 to 56 years). Median time from HIV infection to thrombosis was 40.5 months (range, 3 to 108 months). No patient had previous thrombosis, family history of thrombosis, or prothrombotic conditions. There were 31 deep vein thromboses, two pulmonary thromboembolisms, and one renal vein thrombosis. Six patients had two thrombotic events. The rate of thrombosis during the 42-month study period was 1.52% (cumulative incidence = 0.30%/year), while the rate of thrombosis in 600 patients before the era of protease inhibitor therapy was 0.33% (cumulative incidence approximately 0.055%/year) (p < 0.001). Due to high incidence of thrombotic recurrences and hemorrhagic complications while using oral anticoagulants, acetylsalicylic acid was initiated; no thrombotic episodes were recorded while using this drug. Protein C and protein S deficiency were found in nine and two patients, respectively. Two patients had lupus anticoagulant and two activated protein C resistance (APCR) without FV Leiden mutation (APCR test was negative after initial screening). Fifteen patients had no thrombophilic abnormalities. These data suggest that protease inhibitors could be a risk factor for venous thrombosis not due to thrombophilic abnormalities but likely related to abnormalities in platelets or endothelium.