Elena Bonet

The Metabolic Syndrome and Its Individual Components: Its Association with Venous Thromboembolism in a Mediterranean Population

INTRODUCTION The association of metabolic syndrome with venous thromboembolism (VTE) remains uncertain. Moreover, the relevance of abdominal obesity as an independent or related risk factor for VTE in the metabolic syndrome cluster is controversial. We aimed to evaluate the impact of metabolic syndrome and its individual components on VTE risk. METHODS We conducted a case-control study to investigate the presence of metaoblic syndrome in 150 healthy individuals (43 ± 13 years) and 146 patients with a first objectively confirmed episode of deep venous thrombosis or pulmonary embolism (44 ± 13 years) who underwent a thrombophilia work up. RESULTS Metabolic syndrome was present in 19% of cases and 8% of controls (P = 0.008). In the unadjusted analysis, metabolic syndrome was statistically associated with higher VTE risk [odds ratio (OR) = 2.7, 95% confidence interval (CI) 1.3-5.6]. However, hypertriglyceridemia (OR = 2.0, 95% CI 1.1-3.5), high glucose levels (OR = 2.0, 95% CI 1.2-3.5), and abdominal obesity (OR = 5.7, 95% CI 3.4-9.6) were also significantly associated with higher VTE risk. Abdominal obesity was the factor that had the highest OR. Moreover, after multivariate analysis in which each independent factor was adjusted for the others, only abdominal obesity remained statistically associated with higher VTE risk, revealing its relevance. Further adjustment for the presence of thrombophilia did not change the estimation. CONCLUSION We conclude that, in subjects with a mean age of 44 years, metabolic syndrome increases VTE risk, although abdominal obesity is the pivotal factor.

Association of the Thrombomodulin Gene c.1418C>T Polymorphism With Thrombomodulin Levels and With Venous Thrombosis Risk

Objective—To investigate the association of the THBD c.1418C>T polymorphism, which encodes for the replacement of Ala455 by Val in thrombomodulin (TM), with venous thromboembolism (VTE), plasma soluble TM, and activated protein C levels. In addition, human umbilical vein endothelial cells (HUVEC) isolated from 100 umbilical cords were used to analyze the relation between this polymorphism and THBD mRNA and TM protein expression. Approach and Results—The THBD c.1418C>T polymorphism was genotyped in 1173 patients with VTE and 1262 control subjects. Levels of soluble TM and activated protein C were measured in 414 patients with VTE (not on oral anticoagulants) and 451 controls. HUVECs were genotyped for the polymorphism and analyzed for THBD mRNA and TM protein expression and for the ability to enhance protein C activation by thrombin. The 1418T allele frequency was lower in patients than in controls (P<0.001), and its presence was associated with a reduced VTE risk, reduced soluble TM levels, and increased circulating activated protein C levels (P<0.001). In cultured HUVEC, the 1418T allele did not influence THBD expression but was associated with increased TM in cell lysates, increased rate of protein C activation, and reduced soluble TM levels in conditioned medium. Conclusions—The THBD 1418T allele is associated with lower soluble TM, both in plasma and in HUVEC-conditioned medium, and with an increase in functional membrane–bound TM in HUVEC, which could explain the increased activated protein C levels and the reduced VTE risk observed in individuals carrying this allele.

The endothelial cell protein C receptor: Its role in thrombosis

The protein C anticoagulant pathway plays a crucial role as a regulator of the blood clotting cascade. Protein C is activated on the vascular endothelial cell membrane by the thrombin-thrombomodulin complex. The endothelial protein C receptor binds protein C and further enhances protein C activation. Once formed, activated protein C down-regulates thrombin formation by inactivating factors Va and VIIIa and exerts cytoprotective effects through endothelial protein C receptor binding. An adequate generation of activated protein C depends on the precise assembly, on the surface of the endothelial cells, of thrombin, thrombomodulin, protein C, and endothelial protein C receptor. Therefore, any change in the efficiency of this assembly may cause a reduction or increase in activated protein C generation and modulate the risk of thrombosis. This review highlights the role of the endothelial protein C receptor in disease and discusses the association of its mutations with the risk of thrombosis.

Functional Analysis of Two Haplotypes of the Human Endothelial Protein C Receptor Gene

Objective— To confirm the effect of the endothelial protein receptor gene (PROCR) haplotypes H1 and H3 on venous thromboembolism (VTE), to study their effect on endothelial protein C receptor (EPCR) expression in human umbilical vein endothelial cells, and to investigate the functionality of H1 tagging single-nucleotide polymorphisms in an in vitro model. Approach and Results— Protein C (PC), activated PC, and soluble EPCR (sEPCR) levels were measured in 702 patients with VTE and 518 healthy individuals. All subjects were genotyped for PROCR H1 and H3. Human umbilical vein endothelial cells isolated from 111 umbilical cords were used to study the relation between PROCR haplotypes, PROCR mRNA, cellular distribution of EPCR, and rate of PC activation. Finally, the functionality of the intragenic PROCR H1 single-nucleotide polymorphisms was analyzed using a luciferase-based method. We confirmed that individuals carrying H1 have reduced VTE risk, increased plasma activated PC levels, and reduced plasma sEPCR levels and that individuals with the H3H3 genotype have an increased VTE risk and increased plasma sEPCR levels. In cultured human umbilical vein endothelial cells, H1 is associated with increased membrane-bound EPCR, increased rate of PC activation, and reduced sEPCR in conditioned medium, but does not significantly influence PROCR mRNA levels. In contrast, H3 is associated with reduced membrane-bound EPCR and increased sEPCR in human umbilical vein endothelial cell–conditioned medium, higher levels of a truncated mRNA isoform, and a lower rate of PC activation. Finally, we identified the g.2132T>C single-nucleotide polymorphism in intron 1 as an intragenic H1-specific functional single-nucleotide polymorphism. Conclusions— These results support a protective role of PROCR H1 against VTE and an increased risk of VTE associated with the H3 haplotype.

Hyperhomocysteinemia and the methylene tetrahydrofolate reductase C677T mutation in splanchnic vein thrombosis.

Introduction: The role that hyperhomocysteinemia (HH) and the C677T mutation in 5,10‐methylenetetrahydrofolate reductase (MTHFR) play in splanchnic vein thrombosis (SVT) remains unclear due to this unusual thrombotic location. Objective: To analyse the possible association of HH with the C677T mutation in the MTHFR gene in SVT. Material and methods: We determined homocysteine levels and the C677T MTHFR mutation, along with classical cardiovascular risk factors, in 48 patients with SVT (18 Budd‐Chiari syndrome, 11 mesenteric vein thrombosis, 19 portal vein thrombosis) and 84 controls. Results: In the univariate analysis, patients with SVT showed statistically higher homocysteine levels (P = 0.044). After adjusting for total cholesterol, differences disappeared (P = 0.256). However, no differences in homocysteine levels were observed when comparing the three SVT types (P = 0.199), even after adjusting for age and total cholesterol (P = 0.095). In addition, the prevalence of the TT genotype was no different when controls were compared with patients with SVT (P = 0.253) or with SVT subtypes (P = 0.885). No association was found between HH (>15 μm) and the TT genotype in cases (P = 0.404), controls (P = 0.178), or in the different SVT subtypes (P = 0.495). Conclusions: Our results suggest that HH and the homozygous genotype in the MTHFR C677T mutation do not seem to play a role in SVT development.

Haplotypes of the endothelial protein C receptor gene and Behçet's disease

INTRODUCTION Behçets disease is a vasculitis of unknown cause in which thrombosis occurs in about 25% of patients. Two haplotypes of the endothelial protein C receptor gene, H1 and H3, are associated with the risk of thrombosis. Thus, the objective of this study was to evaluate the influence of these haplotypes on the thrombosis risk in Behçets disease. MATERIAL AND METHODS We evaluated the H1 and H3 haplotypes in 87 patients with Behçets disease, 19 with and 68 without a history of thrombosis, and in 260 healthy individuals. We also measured protein C, activated protein C, and soluble endothelial protein C receptor levels in all individuals. RESULTS The presence of the H1 haplotype seemed to protect Behçets patients against thrombosis (odds ratio 0.21; 95% CI 0.1-0.8; p=0.023), whereas the frequency of the H3 haplotype was lower in patients than in control individuals (0.19; 0.1-0.5; p=0.006). Furthermore, the H1 haplotype was associated with increased levels of activated protein C, whereas the H3 haplotype was associated with the highest soluble endothelial protein C levels. Moreover, activated protein C levels were lower in patients with than in patients without posterior uveitis (p<0.001). CONCLUSIONS These findings indicate that the H1 haplotype protects Behçets patients from thrombosis, likely via increased levels of activated protein C, whereas individuals carrying the H3 haplotype seem to be protected from the clinical manifestations associated with Behçets disease, probably via increased soluble endothelial protein C levels.

Effects of oral anticoagulant therapy and haplotype 1 of the endothelial protein C receptor gene on activated protein C levels.

Oral anticoagulants (OACs) reduce activated protein C (APC) plasma levels less than those of protein C (PC) in lupus erythematosus and cardiac patients. Carriers of the H1 haplotype of the endothelial PC receptor gene (PROCR) have higher APC levels than non-carriers. We aimed to confirm these results in a large group of patients treated with OACs because of venous thromboembolism (VTE) and to assess whether the effect is influenced by the PROCR H1 haplotype. We evaluated APC, PC, and factor (F)II levels in 502 VTE patients (158 with and 344 without OACs) and in 322 healthy individuals. Mean APC, PC and FII levels were significantly lower in OAC patients than in patients not taking OACs. During anticoagulant therapy, the FII/PC ratios were independent of the PC values, whereas APC/FII and APC/PC ratios significantly increased when FII and PC levels decreased. Of the 22 OAC patients carrying the H1H1genotype, 11 (50%) showed APC/PCag ≥2.0 and 10 (45%) APC/FIIag ratios ≥2.0, whereas for the 49 OAC patients non-carrying the H1 haplotype these figures were 6 (12%) and 4 (8%), respectively (p<0.001). Barium citrate adsorption of plasma from OAC patients showed that most of the circulating free and complexed APC, but only part of PCag, is fully carboxylated. In conclusion, during anticoagulant therapy VT patients have APC levels disproportionately higher than the corresponding PC levels, mainly due to the presence of the PROCR H1 haplotype. Furthermore, a sufficiently carboxylated PC Gla-domain seems to be essential for PC activation in vivo.

A simplified assay for the quantification of circulating activated protein C

Available assays for circulating levels of activated protein C (APC) are either time-consuming or difficult to use in a routine laboratory, or have a detection limit above normal levels. We have developed a simplified assay that measures both the in vivo free APC and the in vivo APC complexed to PC inhibitor (PCI). We measured APC levels, with both assays, in 339 plasma samples, 165 from patients with venous thromboembolism (VTE) and 174 from healthy individuals. The mean APC level in the 339 samples was 0.038±0.010 nM, using a previous assay that measures only the in vivo APC level, and 0.041±0.010 nM with the present new assay. The coefficient of correlation between assays was r=0.954 (P<0.001). The mean APC level in VTE patients was 0.034±0.009 nM (previous assay) and 0.037±0.009 nM (new assay), significantly lower than those in controls (P<0.001). In both groups there was a significant correlation between the levels obtained by the two assays (P<0.001). These results show that both assays are equivalent, and confirm that the APC level is lower in VTE patients than in healthy individuals. Therefore, the new simplified assay, which measures the sum of circulating free APC and APC complexed to PCI, may be used to estimate the level of circulating APC, and will allow its use in routine laboratories.

Association Between the Metabolic Syndrome, Its Individual Components, and Unprovoked Venous ThromboembolismSignificance

Objective—The metabolic syndrome (MetS) may contribute to the pathogenesis of venous thromboembolism (VTE), but this association requires additional investigation. Approach and Results—We performed a patient-level meta-analysis of case–control and cohort studies that evaluated the role of MetS and risk of unprovoked VTE. For case–control studies, odds ratios and 95% confidence intervals were calculated using logistic regression analysis to estimate the influence of individual variables on the risk of VTE; &khgr;2 tests for trend were used to investigate the effect of increasing number of components of MetS on the risk of VTE and to explore the influence of abdominal obesity on this relationship. For cohort studies, hazard ratios and 95% confidence interval were calculated using multivariable Cox regression analysis. Six case–control studies were included (908 cases with unprovoked VTE and 1794 controls): in multivariate analysis, MetS was independently associated with VTE (odds ratio, 1.91; 95% confidence interval, 1.57–2.33), and both MetS and abdominal obesity were better predictors of unprovoked VTE than obesity defined by the body mass index. Two prospective cohort studies were included (26 531 subjects and 289 unprovoked VTE events): age, obesity, and abdominal obesity, but not MetS were associated with VTE. Conclusions—Case–control but not prospective cohort studies support an association between MetS and VTE. Abdominal adiposity is a strong risk factor for VTE.

C0034 A simplified assay for quantification of circulating activated protein C levels

Background: Activated protein C (APC) is an enzyme with anticoagulant and cytoprotective functions. Accumulative data suggest that the number of situations in which in vivo circulating APC will need to be measured will increase over the next few years. Therefore, the availability of a rapid, sensitive and simple assay for quantifying circulating APC is crucial. Available assays are difficult to apply in routine laboratory. Our previously reported assay required an excessive pre-analytical handling: drawing of blood into two citrate tubes; immediate addition of a fresh APC inhibitor to one tube in order to block complexation of circulating APC to protein C inhibitor (PCI), and of heparin to the other tube to force all circulating APC to bind to PCI, and measurement of APC:PCI complexes in both tubes by ELISA (method A). Here we describe a simplifiedmethod based on the measurement of APC:PCI in a unique blood tube anticoagulated with heparin (method B). Methods: We measured APC levels, with both methods, in 125 plasma samples, 69 from patients with venous thrombosis (VT) and 56 from healthy controls. Results: ThemeanAPC level in the 125 sampleswas 1.06±0.46 ng/ml (method A) and 2.21±0.85 ng/ml (method B). The coefficient of correlation between both methods was r=0.849 (Pb0.001). The mean APC level in the 69 VT patients wase 0.84±0.38 (method A) and 1.80± 0.67 (method B), significantly lower (as previously reported) than those in the 56 controls, 1.33±0.39 and 2.72±0.79, respectively, (Pb0.001). In both groups there was a significant correlation between the levels obtained by the two methods (VT, r=0.772; controls, r=0.777 (Pb0.001). Comment: These results show that both assays are equivalent, and confirm that the APC level is lower in VT patients than in healthy individuals. Therefore, the new simplified assay, which measures the sum of circulating free APC and APC complexed to PCI, may be used to estimate the level of circulating APC, and will allow its use in routine laboratories. (FIS PS09/00610, RECAVA RD06/0014/0004, Prometeo/ 2011/027, y Fundacion para la Investigacion Hospital La Fe; Pilar Medina is a Miguel Servet Researcher, ISCIII CP09/00065).