Joan Carles Souto

Prothrombotic state and signs of endothelial lesion in plasma of patients with inflammatory bowel disease

Recent investigations suggest that microthrombi formation in bowel capillaries could be a determinant factor in inflammatory bowel disease (IBD) pathogenesis. To evaluate the implication of the hemostatic system during these thrombotic events, we analyzed plasmatic values of prothrombotic state markers, physiologic inhibitors of coagulation, and endothelial lesion markers in 112 IBD patients. We found an increase in thrombin-antithrombin complexes and a decrease in antithrombin III, probably due to consumption, demonstrating an increase in thrombin generation. High levels ofd-dimer reflect increased fibrin formation, but there is no correlation between thrombin generation markers andd-dimer, possibly suggesting the presence of inadequate fibrinolysis. Levels of tissue factor pathway inhibitor were higher in patients than in controls. Nine patients with Crohns disease (35% of our sample) had levels of this marker under 70% (range 37–69%). Von Willebrand factor values were increased and those of thrombomodulin only in active patients. Most of the changes were detected in patients with inflammatory activity, and there were no differences between ulcerative colitis and Crohns disease. In conclusion, these results support the hypothesis that there is an endothelial lesion with sustained coagulation activation in IBD patients.

Vitamin B12 deficiency, hyperhomocysteinemia and thrombosis: a case and control study

This study aimed at assessing the relationship between thrombosis, hyperhomocysteinemia and vitamin B12 deficiency using a case–control study carried out in 326 patients with thrombosis (case group) and 351 patients from the same hospital (control group). Apart from the classic risk factors, a number of hematological variables were evaluated, including serum vitamin B12 (B12), red cell folate (RCF), and serum homocysteine (Hcy). An evaluation of serum methylmalonic acid (MMA) and a clinical study were carried out to investigate B12 pathology. Results of univariate analysis demonstrated decreased B12 levels in thrombosis (Student’s t test, p < 0.0001). Vitamin B12 below 200 pmol/l (LB200) or below 150 pmol/l (LB150), and red cell folate below 600 nmol/l were found in 17.2, 8.6, and 2.2% of cases with thromboembolism, respectively. An increase in Hcy was detected in 86 cases with thrombosis (26.3%). An abnormality in vitamin B12 and/or renal function was found in 80% of cases with hyperHcy and thrombosis. The MMA increase demonstrated that vitamin B12 deficiency was present in these patients with low levels of vitamin B12 in serum, and the MMA levels were in concordance with Hcy levels. The clinical study revealed B12 malabsorption in most cases with LB200. Multivariate analysis showed that serum vitamin B12 (RR 0.998, CI 0.997–0.999) was moderately related to thromboembolism. The results indicated that vitamin B12 deficiency was common among patients with hyperhomocysteinemia and thrombosis. Moreover, HyperHcy was caused by vitamin B12 deficiency and/or chronic renal failure in most patients with thrombosis. As the main cause of vitamin B12 deficiency was vitamin malabsorption, parenteral vitamin B12 with or without folic acid should be administered for the treatment of this condition. However, it remains to be demonstrated whether this treatment approach prevents recurrent thromboses in patients with vitamin B12 deficiency and thrombosis, as suggested by some case reports.

Protein S gene analysis reveals the presence of a cosegregating mutation in most pedigrees with type I but not type III PS deficiency.

DNA sequence analysis of the protein S gene (PROS1) in 22 Spanish probands with type I or III PS deficiency, has allowed the identification of 10 different mutations and 2 new sequence variants in 15 probands. Nine of the mutations, 8 of which are novel, cosegregate with type I or quantitative PS deficiency in 12 of the 13 pedigrees analyzed. One of these mutations (Q238X) also cosegregates with both type I and III PS‐deficient phenotypes coexisting in a type I/III pedigree. Another mutation identified in a pedigree with these two PS phenotypes is the missense mutation R520G, present in the homozygous form in the type I propositus and in the heterozygous form in his type III relatives. By contrast, no cosegregating PROS1 mutation has been found in any of the six families with only type III phenotypes. Three of these families, as well as the two families with type I and I/III phenotypes where no other PROS1 mutation has been identified, segregate the P allele of the S460P variant, although this allele does not always cosegregate with the deficient phenotype. From these results we conclude that while mutations in PROS1 are the main cause of type I PS deficiency, the molecular basis of the type III phenotype is probably more complex, with many cases not being explained by a PROS1 mutation. Hum Mutat 14:30–39, 1999.

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.

Influence of Age, Gender and Lifestyle in Lymphocyte Subsets: Report from the Spanish Gait-2 Study

Introduction: Flow cytometry analysis of lymphocyte subsets in peripheral blood is a common technique in diagnostic laboratories. Abnormal values have been identified in prevalent infections, autoimmune disorders and neoplastic diseases. Reference ranges for lymphocyte subsets of a healthy population from Spain are scarce. Methods: The study was performed on 319 healthy subjects, aged 4–88 years, from 709 individuals enrolled in the GAIT-2 Project (Genetic Analysis of Idiopathic Thrombophilia). Health status, age, sex, fertility, BMI and lifestyle (physical activity, cigarette smoking and ethanol intake) were assessed using standardized criteria. The percentage of lymphocyte subsets was determined using flow cytometry (Lymphogram™). Percentages of CD3+, CD4+, CD8+, CD19+, CD3–CD56+, CD3+CD4–CD8– double-negative (DN) T cells, CD3+CD4+CD8+ double-positive T cells and the CD4+/CD8+ ratio were recorded for each case. Results: Children had a significantly higher percentage of CD19+ and DN cells than adults. Women had a significantly higher percentage of CD3+ and CD4+ and a lower percentage of natural killer cells than men. Increases in BMI were inversely associated with the percentage of DN cells. Physical activity increased the percentage of lymphocytes and DN cells. Alcohol consumers had a lower percentage of CD19+ and DN cells, and a higher percentage of CD4+. Conclusion: This study provides reference ranges for lymphocyte subsets of healthy children and adults in a Mediterranean population (Spain) and determines the influence of lifestyle factors on these values.

Genetic determinants of variation in the plasma levels of the C4b-binding protein (C4BP) in Spanish families

The C4b-binding protein (C4BP) is a plasma glycoprotein implicated in the homeostasis of the complement and coagulation systems. It is composed of two polypeptides (α and β), which form three plasma oligomers with different subunit compositions (α7β1, α7β0, and α6β1). The β chain-containing C4BP isoforms (C4BPβ+ isoforms) bind and inactivate protein S (PS), downregulating the activated protein C (APC)-dependent anticoagulatory pathway. Because PS deficiency is associated with recurrent thrombosis, it has been suggested that increased levels of C4BPβ+ isoforms might diminish the free PS plasma level, affecting the risk of developing thromboembolism. Previous work has tested this hypothesis, but no definitive conclusions were reached, mostly because nothing is known about the factors influencing the high variability in C4BP plasma levels in humans. As a part of the GAIT project, using variance component analysis, this work provides the first estimation of the relative contributions of genetic and environmental influences on the plasma levels of total C4BP and C4BPβ+ isoforms. Plasma levels of total C4BP and C4BPβ+ isoforms showed strong evidence of genetic regulation (heritability 37.7% and 42.5%, respectively). They were also affected by age, smoking, and exogenous sex hormones. Our results constitute the first step in localizing and evaluating potential quantitative trait loci that affect the plasma levels of C4BP and C4BPβ+. Furthermore, analysis of phenotypic and genetic correlations between C4BPβ+ plasma levels and the components of the APC anticoagulatory pathway (total PS, free PS, functional PS, and functional PC) suggests a genetic co-regulation of the proteins. These observations might have important implications in the individual susceptibility to thrombotic disease.

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.

Activated protein C resistance assay when applied in the general population

We estimated the predictive values of activated protein C resistance in the diagnosis of women who are carriers of factor V Leiden. When the prevalence of factor V Leiden is 2%, the positive predictive value is 44% and the cost of preventing one thromboembolic death is

Genetic correlation between plasma levels of C4BP isoforms containing beta chains and susceptibility to thrombosis.

44,180,000. Thus the activated protein C resistance assay is not cost-effective in ruling out factor V Leiden in this population.

Lack of Effect of Influenza Vaccine on Anticoagulation by Acenocoumarol

Thrombosis is a complex trait with both genetic and environmental components.1 In general, disease status is a qualitative trait where individuals are diagnosed as affected or unaffected, but it is now accepted that underlying the disease there is a continuum trait termed liability, susceptibility, or risk. Liability cannot be measured directly, but it can be modelled and estimated. Disease results when an individual’s liability is above a critical value or threshold, whereas liability values below the threshold correspond to healthy individuals. These threshold models of an underlying continuous scale of risk allow inferences that are compatible with current models of gene action.2 Quantitative plasma phenotypes, such as those related to haemostasis, are also complex traits, whose regulation depends on multiple genetic and environmental factors.3 Classical statistical methods are unable to quantify or partition the genetic and environmental factors determining the variability in such complex traits. However, variance component methods have been developed which allow the examination of sources of correlation between quantitative physiological measures and disease outcomes.4 These statistical genetic methods also permit the localisation and evaluation of the relative effects of the genes involved.5 We have applied these methods to the Genetic Analysis of Idiopathic Thrombophilia Project (GAIT Project) to characterise the genetic determinants responsible for idiopathic thrombophilia. Using a family based approach, we estimated that idiopathic thrombophilia has a heritability of 0.61, indicating that 61% of variation in liability to thrombosis at the population level can be attributed to genetic factors.6 Phenotypic correlations were also evaluated between 27 plasma phenotypes related to haemostasis and thrombosis liability.6 It was found that genetic factors were mainly responsible for these phenotypic correlations,6 indicating that some of the genes that regulate quantitative variation in these plasma phenotypes also affect the risk of thrombosis. These …