H Ireland

Factor V Leiden Gene Mutation and Thrombin Generation in Relation to the Development of Acute Stroke

To determine the prevalence of the factor V Leiden gene mutation in relation to the phenotypes of cerebral infarction and cerebral hemorrhage, we studied 386 randomly selected cases of acute stroke and 247 control subjects. Factor V genotype was determined by amplification of a 267-bp sequence of exon/intron 10 of the factor V gene. Levels of prothrombin fragment F(1 + 2), a marker of thrombin generation, were determined in both acute and convalescent stroke and related to factor V genotype. Prothrombin fragment F(1 + 2) was assessed by using an enzyme-linked immunosorbent assay. Sixteen stroke cases (4.1%) were identified as having the mutation compared with 14 (5.6%) control subjects. Prothrombin fragment F(1 + 2) levels were estimated in 191 cases and found to be elevated both acutely and after 3 months, but they were not related to factor V genotype. Prothrombin fragment F(1 + 2) is elevated in acute stroke and requires further evaluation in relation to cerebrovascular disease. These results suggest that the factor V Leiden gene mutation is not a risk factor for arterial thrombosis causing stroke.

Clarification of the Risk for Venous Thrombosis Associated with Hereditary Protein S Deficiency by Investigation of a Large Kindred with a Characterized Gene Defect

Vitamin K-dependent protein S plays an important role in the regulation of the coagulation cascade [1]. This protein increases the rate of degradation of activated factors V and VIII by acting as a cofactor to activated protein C, thereby limiting thrombin production. The presence of factor V, with which protein S acts in synergy, amplifies the function of protein S as a cofactor to activated protein C in factor VIII proteolysis. Protein S has recently been found to have anticoagulant functions independent of activated protein C: It directly inhibits procoagulant enzyme complexes [2, 3]. The relative importance of these different functions in maintaining intravascular fluidity is still unknown. However, heterozygous deficiency of protein S was described as a cause of venous thrombosis in 1984 [4] and has subsequently been identified in numerous clinically affected families, in which it is inherited as an autosomal dominant trait. This deficiency has been found in 1.5% to 7% of selected groups of thrombophilic patients [5-8], although an estimate of the prevalence in the general population awaits a sufficiently large study. Homozygous protein S deficiency is an extremely rare and life-threatening disorder associated with severe neonatal purpura fulminans [9]. Unlike other coagulation inhibitors, protein S has some functions that are affected by C4b-binding protein, a component of the complement cascade, to which 60% to 70% of protein S is bound in vivo [10]. Once bound, protein S can no longer act as a cofactor to activated protein C but retains some of its inhibitory properties. Because of the difference in function between the bound and unbound forms of protein S and uncertainty over which function is the most important, levels of both total and free protein S antigen are usually measured in patient plasma samples. The function of protein S as a cofactor to activated protein C may also be assessed. Protein S deficiency is diagnosed if one or more of these measurements is found to be below the lower limit of a laboratory reference range. Several problems are encountered in the diagnosis of protein S deficiency, including the large overlap in antigen levels between normal and heterozygous persons [11]. This overlap may be due to the effect of sex and hormones on total protein S levels [12, 13]. We recently described an age-related increase in total protein S antigen, independent of the influence of sex, in both normal and protein S-deficient persons [14]. This phenomenon also complicates diagnosis made on the basis of total protein S measurement and causes phenotypic variation within the same kindred. Furthermore, some assays for protein S activity are influenced by a mutation in the gene for factor V (Arg506Gln), which causes resistance to activated protein C and is a common, if relatively mild, risk factor for thrombosis. Heterozygosity for this mutation can result in apparent reduction of protein S cofactor activity to activated protein C in the laboratory assessment of normal persons [15]. Despite these problems with diagnosis, some studies have attempted to compute the risk for thrombosis associated with phenotypic protein S deficiency [11, 16-18]. In deficient families, the probability that affected family members remain thrombosis-free at 45 years of age has been reported to be 0.35 to 0.50 [11, 16]. However, the incidence of thrombosis varies among different families; this suggests problems with precise diagnosis or the presence of other genetic risk factors. Of note, no study has examined the risk associated with genetically confirmed protein S deficiency, which would remove the diagnostic uncertainties. The identification of gene mutations that cause protein S deficiency is complicated by the size of the gene (>80 kilobase-pairs) that encodes protein S [19-21] and by the presence of a pseudogene. However, an increasing number of studies have identified such mutations in probands or small family groups. The first database of protein S gene mutations was recently published by the Scientific and Standardisation Committee of the International Society on Thrombosis and Haemostasis [22]. Mutations in the coding region or at intron-exon boundaries have generally been identified in approximately 50% of protein S-deficient probands. We recently identified a single causative mutation (which results in a Gly295Val substitution) in a large protein S-deficient kindred [14]. This mutation is not thought to be common in the general population. The availability of comprehensive phenotypic, genotypic, and clinical data has enabled the interrelations among these data to be investigated and has thereby provided quantitative information on the risk for thrombosis associated with a mutation in the protein S gene and the value of different assays in predicting clinical events. Methods Participants The manifestations of thrombosis in the 122-member family under investigation (most family members live in northern Sweden) were attributed to protein S deficiency in 1993 [23]. This kindred has also been part of a larger study that involved 18 families with phenotypic protein S deficiency [16, 24] and provided an explanation for phenotypic variation in familial protein S deficiency [14]. All participants gave informed consent, and the medical ethics committee at the University of Lund approved all of these studies, including the present one. Study participants answered a questionnaire about their medical history, with emphasis on manifestations of deep venous thrombosis, pulmonary embolism, superficial thrombophlebitis, and arterial thrombosis. Symptomatic family members were also interviewed by a physician or had their medical records reviewed. The term deep venous thrombosis includes deep venous thrombosis of the leg and thrombosis in such unusual locations as the axillary, mesenteric, and cerebral veins. Thrombotic event refers to deep venous thrombosis, pulmonary embolism, or superficial thrombophlebitis diagnosed by a physician on the basis of physical examination. Laboratory Methods Blood sampling and routine coagulation were performed as described elsewhere [25]. Total and free protein S antigen levels were measured by doing radioimmunoassay [26]. Protein S levels were compared with laboratory reference ranges for levels of both free (reference range, 56 to 182 nmol/L) and total (reference range, 219 to 407 nmol/L) antigen. Persons receiving anticoagulation were compared with an anticoagulated control group. The control groups that we used have been described elsewhere [24]. Of the 122 family members, 44 had free protein S antigen levels below the lower limit of the reference range; 13 of the 44 were receiving oral anticoagulants at the time of sampling. Molecular Genetic Investigation The methods used to identify and detect the novel protein S gene mutation, Gly295Val, in 122 genomic DNA samples have been reported elsewhere [14, 27]. All 44 family members with reduced free protein S antigen levels were heterozygous for the mutation; the remaining 78 relatives who had normal free protein S antigen levels were normal at this site. This finding confirmed that the Gly295Val mutation was the cause of protein S deficiency in this family. A single asymptomatic family member with normal free protein S antigen levels had previously been found to be heterozygous for the factor V Arg506Gln mutation. Statistical Analysis Thrombosis-free survival curves were constructed according to the method of Kaplan and Meier [28]. Two curves were compared by using the log-rank test, which results in a test statistic with chi-squared distribution and one degree of freedom [29]. This analysis was performed by using Statistica software (Statsoft, Inc., Tulsa, Oklahoma). Univariate and multivariate Cox regression analyses [30] were performed by using Statistica software or SPS (SPS, Inc., Chicago, Illinois). All 122 family members were included in the analysis for risk for thrombosis. Results Demographic and Clinical Data Samples of plasma and genomic DNA were available for 122 germline family members (60 men and 62 women; mean age SD, 36 17 years [range, 7 to 82 years]) spanning five generations. The distribution of patient samples was 1, 8, 41, 57, and 15 from the first, second, third, fourth, and fifth generations, respectively. A histogram of the current ages of the study participants is shown in Figure 1. Twenty-five (57%) of the 44 family members with the Gly295Val mutation had one or more venous thrombotic events (mean age at first event, 31 years [range, 11 to 71 years]) compared with 5 (6%) of the 78 family members who lacked the mutation (mean age at first event, 29 years [range, 16 to 43 years]). The clinical manifestations in symptomatic family members are summarized in Table 1. First thrombotic events were associated with one or more circumstantial risk factors in 12 symptomatic relatives (48%) with the Gly295Val mutation and 2 family members (40%) who lacked the mutation (Table 2). Three carriers of the mutation (6.8%) and none of the normal family members had arterial thrombotic events (postoperative bilateral arterial thrombosis requiring bilateral above-the-knee amputation, myocardial infarction, and embolization requiring below-the-knee amputation). Figure 1. Histogram of the current ages of the investigated persons in the study family. Table 1. Clinical Manifestations of Venous Thrombosis in Symptomatic Family Members with and without the Gly295Val Mutation Table 2. Circumstantial Risk Factors Associated with First Thrombotic Episodes in 25 Symptomatic Relatives with the Gly295Val Mutation and 5 Symptomatic Relatives without the Mutation According to Kaplan-Meier analysis of thrombosis-free survival, the probability that a family member who carries the Gly295Val mutation would remain free of venous thrombosis at 30 years of age is 0.5 (95% CI, 0.33 to 0.66) compared with 0.97 (CI, 0.93 to 1.0) for normal family members (Figure 2)

Methylenetetrahydrofolate Reductase 677 C→T Mutation and Coronary Heart Disease Risk in UK Indian Asians

Plasma homocysteine concentrations are elevated in UK Indian Asians and may contribute to twice as many coronary heart disease (CHD) deaths in this group compared with European whites. The mechanisms underlying elevated homocysteine concentrations among Indian Asians are not well understood. In this study, we have investigated the extent to which the methylenetetrahydrofolate reductase (MTHFR) 677 C →T mutation accounts for elevated plasma homocysteine and increased CHD risk in Indian Asians compared with European whites. We investigated 454 male cases (with myocardial infarction or angiographically proven CHD: 224 Indian Asians, 230 European whites) and 805 healthy male controls (381 Indian Asians, 424 European whites). Fasting homocysteine concentrations, MTHFR 677 C →T genotype, and conventional CHD risk factors were measured. The prevalence of homozygous MTHFR 677 T in Indian Asian controls was less than one third that in European white controls (3.1% versus 9.7%, P <0.001). In Indian Asians, the TT MTHFR genotype was not associated with homocysteine concentrations and was not present in any of the Asian controls with hyperhomocysteinemia (>15 &mgr;mol/L). In contrast, among European whites, the TT MTHFR genotype was strongly related to elevated plasma homocysteine concentrations and was found in 27% of the European controls with hyperhomocysteinemia. Elevated homocysteine in Indian Asian compared with European white controls was accounted for by their reduced levels of B vitamins but not by the MTHFR 677 T genotype. However, neither the TT MTHFR genotype nor B vitamin levels explained the elevated homocysteine concentrations in CHD cases compared with controls. TT MTHFR was not a risk factor for early-onset CHD in Indian Asians (odds ratio, 0.5; 95% confidence interval, 0.1 to 2.4;P =0.39), unlike in European whites (odds ratio, 2.1; 95% confidence interval, 1.1 to 4.1;P =0.02). We conclude that the MTHFR 677 T mutation does not contribute to elevated plasma homocysteine concentrations or increased CHD risk in Indian Asians compared with European whites. Our results suggest that novel genetic defects and/or environmental factors influence homocysteine metabolism in Indian Asians residing in the United Kingdom.

ORAL CONTRACEPTIVES ENHANCE THE RISK OF CLINICAL MANIFESTATION OF VENOUS THROMBOSIS AT A YOUNG AGE IN FEMALES HOMOZYGOUS FOR FACTOR V LEIDEN

In 29 patients (17 females) homozygous Arg 506 Gln mutation (FV Leiden) was identified. 25 had been investigated because of venous thromboembolism (VTE); four asymptomatic patients were found during family studies.

The factor VII activating protease G511E (Marburg) variant and cardiovascular risk

A previous study had shown a strong relationship between a variant in factor VII activating protease (FSAP G511E) and advanced carotid atheroma. In-vitro, the variant has reduced fibrinolytic but normal pro-coagulant activity, which may constitute a prothrombotic state. The current study has addressed risk for coronary heart disease in a prospective study of cardiovascular disorders (Northwick Park Heart Study II). An interactive effect upon risk was found between the 511E allele and elevated levels of cholesterol and triglyceride. Fibrinogen could substitute for triglyceride levels in this risk-interaction analysis. The findings support the proposal that the FSAP 511E allele exacerbates atherosclerosis or its clinical sequelae.

Plasma concentrations of fibrinopeptide a, fibrinogen fragment bβ1-42 and β-thromboglobulin following total hip replacement

Abstract Plasma concentrations of thrombin sensitive peptide fibrinopeptide A (FpA), plasmin sensitive fibrinogen fragment Bβ1-42 and the platelet release product β-thromboglobulin (βTG) have been measured in 36 patients before and after total hip replacement. Statistically significant elevations of all three activation products were observed in the days following operation. There were small differences in plasma concentrations of FpA, Bβ1-42 and βTG in patients who did (n = 13) and did not (n = 23) develop post operative deep vein thrombosis, as assessed by ascending venography on post operative day 10, but these differences were not statistically significant. It is concluded that coagulation and fibrinolytic systems and also blood platelets are activated following total hip replacement operations. However, the formation of post operative deep vein thrombosis can not be effectively monitored by measurement of the activation products.

Antithrombin III Northwick Park: demonstration of an inactive high MW complex with increased affinity for heparin.

Summary. It has been shown previously that antithrombin III Northwick Park has reduced ability to inactivate thrombin and is characterized by an additional anodal component on crossed immunoelectrophoresis (Howarth et al. 1985). We have applied plasma from an affected family member to heparin‐Sepharose and eluted the antithrombin III with a salt gradient. Evidence is presented that a variant component has slightly higher affinity for heparin than normal antithrombin III. Furthermore, this variant component is present in plasma as an ∼ 120000 MW inactive antithrombin III complex that can be reduced with dithiothreitol to MW ∼ 60 000, indicating disulphide bridging. Using ionexchange chromatography, the inactive complex has been isolated and shown to migrate in the same position as the anodal peak on crossed immunoelectrophoresis.

Antithrombin III Glasgow: a variant with increased heparin affinity and reduced ability to inactivate thrombin, associated with familial thrombosis.

A functional antithrombin III (AT III) deficiency has been identified in two generations of a family with a high incidence of thrombosis. The deficiency presented as 50% reduction in heparin cofactor activity compared to its antigen concentration. No abnormality was detected by crossed immunoelectrophoresis in the presence or absence of heparin. Plasma from the propositus was precipitated with dextran sulphate, applied to heparin‐Sepharose and the AT III stepwise eluted with NaCl. The AT III had a reduced ability to inactivate thrombin, when this was monitored by substrate hydrolysis or by SDS polyacrylamide gel electrophoresis. Its mobility was normal by the latter technique using 10–20% gradient gels under reducing and non‐reducing conditions. AT III from the patient was reapplied to heparin‐Sepharose and eluted with a NaCl gradient. An active pool eluted in the same NaCl concentration range used to purify normal AT III, while predominantly inactive AT III eluted at higher NaCl concentrations. It is concluded that this variant, designated AT III Glasgow, has increased affinity for heparin but reduced ability to inactivate thrombin.

Novel point mutations leading to type 1 antithrombin deficiency and thrombosis

Summary. Direct sequencing of antithrombin III (AT) gene fragments specifically amplified by the polymerase chain reaction was utilized to identify the molecular basis of type 1 AT deficiency in two unrelated kindreds, both with thrombotic disease. Two novel point mutations were identified, deletion of a T from the second position of codon 81 in one propositus and insertion of a G in codon 424 in the second kindred. The AT 81(‐T) frameshift mutation leads to a premature stop signal in codon 89, while the AT 424(+G) allele has a premature stop only one codon short of the normal gene. The latter mutation changes the eight carboxy terminal residues of AT, including 429Cys. and increases the proportion of polar amino acids in this region. We suggest that altered folding of the mutant protein may explain the AT deficiency.

Relationship between ex vivo anti-proteinase (factor Xa and thrombin) assays and in vivo anticoagulant effect of very low molecular weight heparin, CY222

There is uncertainty as to which activities of unfractionated heparin (UFH) and low MW heparin are responsible for their anticoagulant and antithrombotic properties. We have sought to answer this question by examining plasma samples taken during a recently conducted dose‐finding study of the low MW heparin, CY222, in haemodialysis for chronic renal failure. In this study, in vivo anticoagulant effect was assessed by measurement of plasma FPA levels. UFH was administered as a dose of 5000 iu bolus + 1500 iu/h maintenance infusion, while the effects of three doses of CY222 were studied (10000, 15000 and 20000 Institute Choay anti‐factor Xa u bolus, all with 1500 Institute Choay anti‐factor Xa u/h maintenance infusion). Anti‐factor Xa levels were determined by chromogenic substrate assay. Anti‐thrombin levels were determined by chromogenic substrate assay and by quantitation of catalysed thrombin–inhibitor complexes (using autoradiography). Analysis of the results indicate that plasma fibrinopeptide A (FPA) levels correlate with anti‐factor Xa (r= 0.45) and anti‐thrombin (substrate) (r= ‐0.63) levels of UFH, but only with the anti‐factor Xa levels (r= ‐0.41) of CY222. These results suggest that the anti‐factor Xa assay is currently the most suitable assay for monitoring low MW heparins such as CY222 in humans.