Montserrat Borrell

Genetic Susceptibility to Thrombosis and Its Relationship to Physiological Risk Factors: The GAIT Study

Although there are a number of well-characterized genetic defects that lead to increased risk of thrombosis, little information is available on the relative importance of genetic factors in thrombosis risk in the general population. We performed a family-based study of the genetics of thrombosis in the Spanish population to assess the heritability of thrombosis and to identify the joint actions of genes on thrombosis risk and related quantitative hemostasis phenotypes. We examined 398 individuals in 21 extended pedigrees. Twelve pedigrees were ascertained through a proband with idiopathic thrombosis, and the remaining pedigrees were randomly ascertained. The heritability of thrombosis liability and the genetic correlations between thrombosis and each of the quantitative risk factors were estimated by means of a novel variance component method that used a multivariate threshold model. More than 60% of the variation in susceptibility to common thrombosis is attributable to genetic factors. Several quantitative risk factors exhibited significant genetic correlations with thrombosis, indicating that some of the genes that influence quantitative variation in these physiological correlates also influence the risk of thrombosis. Traits that exhibited significant genetic correlations with thrombosis included levels of several coagulation factors (factors VII, VIII, IX, XI, XII, and von Willebrand), tissue plasminogen activator, homocysteine, and the activated protein C ratio. This is the first study that quantifies the genetic component of susceptibility to common thrombosis. The high heritability of thrombosis risk and the significant genetic correlations between thrombosis and related risk factors suggest that the exploitation of correlated quantitative phenotypes will aid the search for susceptibility genes.

A quantitative-trait locus in the human factor XII gene influences both plasma factor XII levels and susceptibility to thrombotic disease.

One approach to the identification of genetic loci that influence complex diseases is through the study of quantitative risk factors correlated with disease susceptibility. Factor XII (FXII) plasma levels, a related phenotype correlated with thrombosis, is such a risk factor. We conducted the first genomewide linkage screen to localize genes that influence variation in FXII levels. Two loci were detected: one on chromosome 5 and another on chromosome 10 (LOD scores 4.73 and 3.53, respectively). On chromosome 5, the peak LOD score occurred in the 5q33-5ter region, near the FXII gene. Addition of a 46C/T mutation in the FXII gene increased the multipoint LOD score to 10.21 (P=3.6 x 10(-12)). A bivariate linkage analysis of FXII activity and thrombosis further improved the linkage signal (LOD = 11.73) and provided strong evidence that this quantitative-trait locus (QTL) has a pleiotropic effect on the risk of thrombosis (P=.004). Linkage analysis conditional on 46C/T indicated that this mutation alone cannot explain the chromosome 5 signal, implying that other functional sites must exist. These results represent the first direct genetic evidence that a QTL in or near the FXII gene influences both FXII activity and susceptibility to thrombosis and suggest the presence of one or more still unknown functional variants in FXII.

Risk of Ischemic Stroke Associated With Functional Thrombin-Activatable Fibrinolysis Inhibitor Plasma Levels

Background and Purpose— Recently, a novel procarboxypeptidase B–like proenzyme, called thrombin-activatable fibrinolysis inhibitor (TAFI), has been described. It plays an important role in the delicate balance between coagulation and fibrinolysis. TAFI leads to potent inhibition of tissue plasminogen activator–induced fibrinolysis. The relevance of TAFI in thromboembolic disease is unclear. We have investigated the risk of ischemic stroke (IS) in relation to plasma levels of functional TAFI. Methods— In a case-control study, we enrolled 264 individuals; 114 had IS, and 150 were recruited as controls who were age and sex matched and had no history of arterial disease. The individuals supplied information on their personal and family histories of cardiovascular diseases and conventional cardiovascular risk factors. Functional TAFI assays were performed by use of a method based on the activation of TAFI with thrombin-thrombomodulin and the measure of the TAFI activity generated. Other hemostatic parameters assayed were factor VIIIc, anti-phospholipid antibodies,fibrinogen, factor V Leiden, and the prothrombin gene G20210A mutations (PT20210A). Results— Functional TAFI levels were significantly higher in patients with IS (113.7±25%; range, 57% to 209%) than in controls (102.6±19%). The odds ratio for IS in patients with functional TAFI levels >120% was 5.7 (95% confidence interval, 2.3 to 14.1). Conclusions— We found that functional TAFI levels in plasma (>120%) increased the risk of IS ≈6-fold. Further studies should elucidate the physiological role of TAFI in arterial disease and possibly provide clues to therapeutic approaches.

Association after linkage analysis indicates that homozygosity for the 46C-->T polymorphism in the F12 gene is a genetic risk factor for venous thrombosis.

In a family-based study called GAIT (Genetic Analysis of Idiopathic Thrombophilia) that included a genome-wide scan we demonstrated that a polymorphism (46C-->T) in the F12 locus jointly influences variability of plasma (Factor XII) FXII levels and susceptibility to thrombotic disease. It then became germane to determine the prevalence of the 46C-->T polymorphism and its relative risk of thrombotic disease. We followed up evidence for genetic linkage with a case-control study, including 250 unrelated consecutive Spanish patients suffering from venous thrombotic disease and 250 Spanish subjects matched for sex and age as a controls. We measured FXII levels and genotyped the 46C-->T polymorphism, as well as a number of classical risk factors for thrombotic disease. We confirmed that individuals with different genotypes for this polymorphism showed significant differences in their FXII levels. Most importantly, the mutated T allele in the homozygous state (genotype T/T) was associated with an increased risk of thrombosis (adjusted OR of 4.82; 95% CI 1.5-15.6), suggesting that the polymorphism itself is an independent risk factor for venous thromboembolism. This study confirms that the 46C-->T polymorphism is a genetic risk factor for venous thrombosis in the Spanish population. In addition, our results confirm that a genome-wide scan coupled with a classical case-control association study is an extremely valuable approach to identify DNA variants that affect complex diseases.

Pretreatment hemostatic markers of symptomatic intracerebral hemorrhage in patients treated with tissue plasminogen activator.

Background and Purpose— Symptomatic intracerebral hemorrhage (ICH) is a major complication of thrombolysis in patients with acute ischemic stroke. We analyzed whether baseline hemostatic markers could predict symptomatic ICH (SICH). Methods— In a multicenter study of patients treated with intravenous tissue plasminogen activator (t-PA) within 3 hours of stroke onset, we analyzed the following variables: demographic data, vascular risk factors, blood glucose at admission, time from the onset of symptoms to t-PA infusion, blood pressure, neurological deficit measured by the National Institutes of Health Stroke Scale (NIHSS) score, early signs of ischemia on the baseline computed tomography (CT) scan, and protocol deviations. In blood samples, the following markers of coagulation/fibrinolysis were measured before treatment: fibrinogen, prothrombin fragments 1+2, Factor XIII, Factor VII, &agr;2 antiplasmin, plasminogen activator inhibitor-1 (PAI-1), and thrombin-activatable fibrinolysis inhibitor. ICH was classified according to the European Cooperative Acute Stroke Study (ECASS) II criteria. SICH was defined as a parenchymal hematoma-1 (PH1) or PH2 type, associated with an increase in ≥4 points on the NIHSS score appearing within 36 hours after infusion. Results— We studied 114 patients. Mean age was 68.4±12.7 years, and 61% were men. The median baseline NIHSS score was 14. Mean time to treatment was 153±33 minutes. Eight patients had SICH (7%), and 18 patients (15.7%) had asymptomatic ICH. None of the baseline markers of coagulation/fibrinolysis were associated with SICH. In the multivariate analysis, only NIHSS on admission was an independent risk factor for SICH. Conclusions— None of the hemostatic markers analyzed in our study predicted symptomatic cerebral hemorrhage in patients with ischemic stroke treated with t-PA.

Hemostatic markers of recanalization in patients with ischemic stroke treated with rt-PA.

Objective: To determine whether pretreatment markers of coagulation and fibrinolysis are related to recanalization and functional outcome. Methods: The authors included patients treated with IV rt-PA with occlusion on baseline transcranial Doppler (Thrombolysis in Brain Ischemia [TIBI] criteria) in whom recanalization within 6 hours was monitored. At baseline, the authors recorded data about demographics, vascular risk factors, the NIH Stroke Scale (NIHSS) score, early CT signs, etiology, blood glucose, and time to rt-PA. The authors also measured plasmatic markers of coagulation (fibrinogen, prothrombin fragments 1 + 2, Factor XIII, Factor VII) and fibrinolysis (α2-antiplasmin, Plasminogen Activator Inhibitor, Functional Thrombin Activatable Fibrinolysis Inhibitor [fTAFI]). A favorable outcome was defined as a modified Rankin score < 2 at 3 months. Results: The authors studied 63 patients with a mean age of 67.3 ± 12.5 years. The median NIHSS score was 16. Patients who recanalized had lower concentrations of α2-antiplasmin (87.5 ± 18% vs 96.5 ± 12.5%, p = 0.023) and fTAFI (91.7 ± 26.7% vs 104.4 ± 21%, p = 0.039). A multivariant logistic regression analysis showed that the level of α2-antiplasmin was the only predictive variable of recanalization (OR 0.95, 95% CI 0.91, 0.99, p = 0.038), while the NIHSS score was the only predictive variable of functional outcome (OR 0.81, 95% CI 0.72, 0.92, p = 0.001). Conclusion: Baseline levels of α2-antiplasmin were predictive of recanalization but were not related to the long-term outcome in patients treated with rt-PA within the first 3 hours.

Fibrinolysis changes in normal pregnancy.

The aim of this study was to evaluate the changes in fibrinolysis parameters during pregnancy. Normal pregnant women (n = 60) formed the study population. Blood samples were taken in the first, second and third trimester, during delivery and three days after delivery. Fibrinolysis parameters were estimated using commercial tests. Tissue plasminogen activator, D-dimer and plasminogen activator inhibitors (PAI-1 and PAI-2) were determined. Tissue plasminogen activator and D-dimer increased after the first trimester and reached maximum levels during delivery. Plasminogen activator inhibitors type 1 and type 2 were also higher, in particular PAI-2, and reached maximum levels in the third trimester. On the third day after delivery, fibrinolysis activity recovered, but D-dimer and PAI-2 levels remained above the normal non-pregnant range.

Optimization of a simple and rapid single-strand conformation analysis for detection of mutations in the PROS1 gene: identification of seven novel mutations and three novel, apparently neutral, variants.

Anticoagulant protein S (PS) deficiency is a known risk factor for thrombophilia. The structure and high allelic heterogeneity of the PS gene (PROS1), together with the presence of a 97% homologous pseudogene, complicates PROS1 analysis. We have optimized a simple, fast, and non‐isotopic Single‐Strand Conformation Analysis (SSCA or SSCP) method for PROS1 mutation detection. This is accomplished through the analysis of the single‐stranded and heteroduplex DNA fragments corresponding to 15 PCR segments that include part of the 5′‐upstream region and the 15 PROS1 exons with their intron boundaries. To standardize the method, 13 known PROS1 mutations or allele variants in 10 different fragments were analyzed under different electrophoretic conditions. The results indicated that, using a combination of two different electrophoretic settings, all the allele variants could be detected as a single‐strand band shift and/or by the presence of a heteroduplex. This method was used to analyze the PROS1 gene in 31 propositi with different types of PS deficiency and thrombosis. Ten different cosegregating mutations, seven of which are novel (143C‐>G, L‐27H, G96X, M599T, P626L, 1418delA, and 1877delT), were identified in the five families suffering from type I or quantitative PS deficiency and in four of the nine families with coexistence of type I and type III phenotypes. No clearly co‐segregating PROS1 mutations were identified in any of the 17 type III propositi analyzed, although eight of them were heterozygotes for the uncommon P460 allele of the S/P460 variant. Furthermore, five apparently neutral allelic variants, three of which are novel (‐296C‐>T, 182G‐>C and T57S), were identified in a normal control, two type I/III and two type III PS‐deficient pedigrees. Hum Mutat 15:463–473, 2000.

Hemostatic Proteins and Their Association With Hematoma Growth in Patients With Acute Intracerebral Hemorrhage

Background and Purpose— We tested the hypothesis that proteins of hemostasia could be associated with hematoma growth (HG) in patients with acute intracerebral hemorrhage. Methods— We prospectively studied patients with spontaneous supratentorial intracerebral hemorrhage within the first 6 hours after the onset of symptoms. HG was defined as an increase >33% in the volume of hematoma on CT obtained 24 to 72 hours after the onset of symptoms in comparison with the CT obtained at admission. We collected admission and follow-up blood samples. We measured fibrinogen, factor XIII, thrombin activatable fibrinolysis inhibitor, plasminogen activator inhibitor, plasminogen, &agr;;2-antiplasmin, tissue plasminogen activator, d-dimer, thrombomodulin, thrombin–antithrombin complex, and plasmin–antiplasmin complex. Results— We included 90 patients with a mean age of 71±10.8 years; 61% were men. HG was observed in 35 (39%) of the patients. Mean baseline and follow-up protein measurements showed no difference between the groups with and without HG. The analysis of variance showed that factor XIII activity decreased in the non-HG group in the 24 to 72 hours sample, whereas it increased in the HG group (P=0.001). Conclusion— Factor XIII was the only measured protein related to HG. The levels at the follow-up sample decreased in the non-HG group and increased in the HG group. Further studies are needed to confirm this association.

Antiphospholipid antibodies in inflammatory bowel disease

To The Editor: The presence of antiphospholipid antibodies (APA) is frequently associated with thromboembolic phenomena by as yet incompletely understood mechanisms (1). Inflammatory bowel disease (IBD) patients have a high incidence of thrombotic disease, and recent investigations suggest that microthrombi formation in bowel capillaries could be a determinant factor in IBD pathogenesis (2). Recently, Chamouard et al have reported in your journal a prevalence of anticardiolipin antibodies of about 22% in a sample of 50 patients with Crohns disease (3). They detected IgG isotype in five patients, IgM in three, and IgA in three. None of them had lupus anticoagulant. Other antiphospholipid antibodies were not investigated. We have measured serum levels of IgM and IgG anticardiolipin (ACL) and antiphosphatidilserine (APS) in 101 patients with IBD (75 ulcerative colitis, 26 Crohns disease; 30 in active phase, 71 in inactive). APA detection was performed essentially as described by Gharavi et al (4) using as antigens cardiolipin and phosphatidilserine from Sigma (St. Louis, Missouri). In our method, the cutoff levels of positivity (based on 97.5 percentile from 100 controls) were: <2.54 binding index (BI) for IgG ACL, <4.64 BI for IgM ACL, <2 BI for IgG APS, and <3.77 BI for IgM APS. We found only positive APA in five cases. One patient who had both anticardiolipinand antiphosphatidilserine-positive antibodies suffered from rheumatoid arthritis and had an ulcerative colitis in active phase. The remaining four cases had weak antiphosphatidilserine antibodies (<4.5 BI). All were inactive ulcerative colitis patients. None of them had history of thromboembolic events. No patient with Crohns disease had positive APA. In immunological disorders, such as rheumatoid arthritis, there is a known high prevalence of APA, so if we exclude the patient with rheumatoid arthritis, prevalence of positive APA in our sample of IBD patients was 3.9% (95% confidence interval 0.1-9.8), almost equal to that observed in the healthy population (5). Hudson et al also found an absence of lupus anticoagulant--an antibody closely related with APA--in this disease (6), and in the study by Webberly et al, anticardiolipin antibodies were absent in 103 of 104 patients (7). However, these results disagree with those of Reicht et al (8), who found a significantly higher prevalence of anticardiolipin antibodies in their sample of 121 patients compared to healthy controls; indeed they found that prevalence in Crohns disease was at least 25%. In our opinion, these contradictory results could be explained by either methodological differences or by distinct positivity thresholds for each antibody. Thus, new data are necessary to resolve these discrepancies because our study (and others) almost discards the implication of APA in thromboembolic alterations in IBD patients, due both to the low observed prevalence and to their low titers in the few positive cases, but results from Chamouard et al and Reicht et al may indicate a higher risk for APAassociated disorders in IBD and may imply a pathogenic role of APA in the genesis of IBD, especially in Crohns disease.