PubMed Health⌕ Search

Biomedical subjects

Rogier M Bertina

Publications and source records attributed to Rogier M Bertina.

At least 19 recordsLinked to original sources
PubMed2006

Interleukin-6 induction of protein s is regulated through signal transducer and activator of transcription 3.

OBJECTIVE: The protein C anticoagulant pathway is an essential process for attenuating thrombin generation by the membrane-bound procoagulant complexes tenase and prothrombinase. In this pathway, protein S (PS) serves as a cofactor for activated protein C. PS circulates in plasma both in a free form and in complex with complement component 4b-binding protein (C4BP). C4BP is a known acute phase reactant, thereby suggesting a relation between PS and the acute phase response. Interleukin (IL)-6 has been shown to increase both PS and C4BP gene expression. Our objective was to study the regulation of PS gene expression by IL-6 in detail. METHODS AND RESULTS: IL-6 upregulates both PS mRNA and protein levels in liver-derived HepG2 cells. The promoter of the PS gene (PROS1) was cloned upstream from a luciferase reporter gene. After transfection in HepG2 cells, the luciferase activity was shown to be stimulated by the addition of IL-6. IL-6 exerts its effect through Signal Transducer and Activator of Transcription 3 (STAT3) that interacts with the PROS1 promoter at a binding site in between nucleotides 229 to 207 upstream from the translational start. CONCLUSIONS: IL-6 induces PS expression via STAT3. A possible function for IL-6-induced PS expression in cell survival is discussed.

Cell Line, Tumor↗
PubMed2006

The constitutive expression of anticoagulant protein S is regulated through multiple binding sites for Sp1 and Sp3 transcription factors in the protein S gene promoter.

Protein S (PS) is a vitamin K-dependent plasma protein that inhibits blood coagulation by serving as a nonenzymatic cofactor for activated protein C in the protein C anticoagulant pathway. Low PS levels are a risk factor for the development of deep venous thrombosis. The regulation of PS levels through transcriptional regulation of the PS gene was investigated in this report. A minimal PS gene promoter 370 bp upstream from the translational initiation codon was sufficient for maximal promoter activity in transient transfections regardless of the cell type. A pivotal role for Sp1 in the constitutive expression of the PS gene was demonstrated through electrophoretic mobility shift assay experiments, transient expression of mutant PS promoter-reporter gene constructs, and chromatin immunoprecipitations in HepG2 cells. At least four Sp-binding sites were identified. The two sites most proximal to the translational start codon were found to be indispensable for PS promoter activity, whereas mutation of the two most distal Sp-binding sites had a negligible influence on basal promoter activity. In addition, all other major promoter-binding proteins that were found by electrophoretic mobility shift assay could be positively identified in supershift assays. We identified binding sites for the hepatocyte-specific forkhead transcription factor FOXA2, nuclear factor Y, and the cAMP-response element-binding protein/activating transcription factor family of transcription factors. Their relevance was investigated using site-directed mutagenesis.

Base Sequence↗
PubMed2006

Haplotypes encoding the factor VIII 1241 Glu variation, factor VIII levels and the risk of venous thrombosis.

Levels of factor VIII (FVIII) are associated with the risk of venous thrombosis. The FVIII variation D1241E has been reported to be associated with decreased levels of FVIII. Our objective was to study whether D1241E is associated with levels of FVIII and the risk of venous thrombosis and whether this association is caused by D1241E or another linked variation. We analyzed the association of three FVIII gene haplotypes encoding 1241E (further denoted as HT1, HT3, and HT5) with FVIII levels and thrombosis risk. This analysis was performed in the Leiden Thrombophilia Study (LETS). The control populations of two case-controls studies on arterial thrombosis in men and women, respectively, were used to confirm the effects observed on FVIII:C in the LETS. In men, HT1 was associated with a 6% reduction in FVIII:C and with a reduced risk of venous thrombosis [odds ratio 0.4 (CI95 0.2-0.8)]. Logistic regression showed that the risk reduction was only partially dependent of the reduction in FVIII levels. HT1 showed no effects in women on either FVIII:C or risk of thrombosis. The number of carriers of HT3 and HT5 was too low to make an accurate estimate of the risk of venous thrombosis. Neither HT3 nor HT5 showed effects on levels of FVIII:C. When we consider that all three haplotypes encoding 1241E show different effects on FVIII:C and thrombosis risk, it is possible that D1241E is not the functional variation. However, FVIII gene variations do contribute to both levels of FVIII and the risk of thrombosis.

Aged↗
PubMed2006

Differential effects of the loss of intrachain- versus interchain-disulfide bonds in the cystine-knot domain of von Willebrand factor on the clinical phenotype of von Willebrand disease.

Von Willebrand factor (VWF) contains a large number of cysteine residues, which all form disulfide bonds. Mutations of cysteines located in the cystine-knot (CK) domain of VWF have been identified in both qualitative type 2A (IID) and quantitative type 3 von Willebrand disease (VWD). Our objective was to test the hypothesis that the difference in phenotype is related to whether the mutated cysteine residue is involved in either interchain- or intrachain-disulfide-bond formation. The effects of three cysteine mutations which are all located in the CK-domain of VWF, C2773S (type 2A(IID)), C2739Y (type 3), and C2754W (type 3), were studied by transient expression in 293T cells. Cotransfection of wild-type (wt) and C2773S VWF constructs reproduced the plasma phenotype of heterozygous type 2A(IID) patients, with normal to high levels of VWF antigen (VWF:Ag), absence of high-molecular-weight multimers, and the presence of intervening bands between the normal multimers. In contrast, single transfections of C2739Y or C2754W resulted in a quantitative VWF defect with low VWF:Ag levels, and co-transfections of wt and mutant constructs resulted in a 50% reduction of VWF:Ag and only a minor effect on VWF multimerization. We demonstrated N-terminal dimerization of VWF-C2773S and both N- and C-terminal dimerization of VWF-C2754W. Our data suggest that loss of a single disulfide bond in the CK-domain of VWF leads to a recessive, quantitative VWF deficiency if an intrachain-disulfide bond is involved, and to a dominant-negative, qualitative defect of VWF if an interchain-disulfide bond is involved.

ADAM Proteins↗
PubMed2005

Genetic variation in the fibrinogen gamma gene increases the risk for deep venous thrombosis by reducing plasma fibrinogen gamma' levels.

We investigated the association between haplotypes of fibrinogen alpha (FGA), beta (FGB), and gamma (FGG), total fibrinogen levels, fibrinogen gamma' (gammaA/gamma' plus gamma'/gamma') levels, and risk for deep venous thrombosis. In a population-based case-control study, the Leiden Thrombophilia Study, we typed 15 haplotype-tagging single nucleotide polymorphisms (htSNPs) in this gene cluster. None of these haplotypes was associated with total fibrinogen levels. In each gene, one haplotype increased the thrombosis risk approximately 2-fold. After adjustment for linkage disequilibrium between the genes, only FGG-H2 homozygosity remained associated with risk (odds ratio [OR], 2.4; 95% confidence interval [95% CI], 1.5-3.9). FGG-H2 was also associated with reduced fibrinogen gamma' levels and reduced ratios of fibrinogen gamma' to total fibrinogen. Multivariate analysis showed that reduced fibrinogen gamma' levels and elevated total fibrinogen levels were both associated with an increased risk for thrombosis, even after adjustment for FGG-H2. A reduced fibrinogen gamma' to total fibrinogen ratio (less than 0.69) also increased the risk (OR, 2.4; 95% CI, 1.7-3.5). We propose that FGG-H2 influences thrombosis risk through htSNP 10034C/T [rs2066865] by strengthening the consensus of a CstF site and thus favoring the formation of gammaA chain above that of gamma' chain. Fibrinogen gamma' contains a unique high-affinity, nonsubstrate binding site for thrombin, which seems critical for the expression of the antithrombin activity that develops during fibrin formation (antithrombin 1).

Adolescent↗
PubMed2005

Influence of the 4600A/G and 4678G/C polymorphisms in the endothelial protein C receptor (EPCR) gene on the risk of venous thromboembolism in carriers of factor V Leiden.

Two polymorphisms in the endothelial protein C receptor (EPCR) gene, 4600A/G and 4678G/C, have been reported to influence the risk of venous thromboembolism (VTE). The objective of this study was to assess whether these polymorphisms modify the risk of VTE in carriers of factor V (FV) Leiden. We genotyped 295 carriers of FV Leiden for these polymorphisms: 100 unrelated patients with a history of VTE (propositi) and 195 relatives (14 of them symptomatic) of 81 of the propositi. Spontaneous VTE events occurred in 71% of propositi carrying the 4678GG genotype, 65% carrying the GC, and 43% with the CC genotype. The mean age at the first onset was significantly higher in propositi carrying the 4678CC than in those with the GC or GG genotype (p = 0.046). Among the 276 carriers of FV Leiden from the 81 families studied, the 95 symptomatic members had similar 4600G allele and 4600AG genotype frequencies but significantly lower 4678C allele (p = 0.002) and 4678CC genotype (p = 0.004) frequencies than the 181 asymptomatic members. The probability of being free of thrombosis at age 40 was significantly higher in the 66 carriers of the 4678CC genotype (94%) than in the 138 carrying the GC (72%) or in the 72 with the 4678GG genotype (60%) (p < 0.001). Multivariate analysis showed that the 4678CC genotype reduced the risk of thrombosis in carriers of FV Leiden (OR = 0.31;95% CI = 0.16-0.83). The incidence of VTE was higher in the 195 relatives with FV Leiden than in the 133 without FV Leiden (OR = 4.7; CI = 1.3-7.2). These results show that carriers of FV Leiden with the 4678CC genotype have a significantly reduced risk of VTE compared with those carrying the 4678GG or GC genotype, probably due to the higherAPC levels previously observed in individuals carrying the 4678CC genotype.

Alleles↗
PubMed2004

C-reactive protein does not directly induce tissue factor in human monocytes.

OBJECTIVE: It is generally assumed that C-reactive protein (CRP) induces synthesis of tissue factor (TF) in monocytic cells, thereby potentially initiating intravascular blood coagulation. We aimed to elucidate the mechanism of CRP-induced TF expression in monocytes and monocyte-derived macrophages (MDMs) in vitro. METHODS AND RESULTS: Monocytes were isolated from the blood of healthy donors and cultured with or without CRP or lipopolysaccharide (LPS) to study the time course of TF antigen and TF mRNA expression. Addition of 100 microg/mL CRP did not result in a significant increase in TF antigen (range: 9 to 163 pg/10(6) cells, n=11) and TF mRNA (relative number of TF transcripts; N(TF)=0.01 to 0.33), when compared with nonstimulated cells (TF antigen 7 to 46 pg/10(6) cells, N(TF)=0.01 to 0.13). Variation of CRP concentration and exposure time did not affect the TF response. Similar results were obtained in monocytes cultured in suspension and in MDMs. In contrast, TF was strongly induced by 10 microg/mL LPS (TF antigen 1125 to 6120 pg/10(6) cells, N(TF)=5.94 to 23.43). Cultured monocytes did express FcRgammaII, a putative CRP receptor, and addition of CRP induced a 7-fold increase in the production of monocyte chemoattractant protein-1 (MCP-1). Interestingly, CRP addition to peripheral blood mononuclear cells (PBMCs) did result in TF expression on monocytic cells. CONCLUSIONS: The absence of TF induction after incubation of purified monocytes with CRP indicates that CRP is unable to induce TF expression in monocytes and MDMs directly. The presence of CRP-induced TF expression in PBMCs suggests that CRP can induce TF indirectly, probably through cross-talk between cells.

Blood Cells↗
PubMed2004

Candidate gene approach in association studies: would the factor V Leiden mutation have been found by this approach?

A re-emerging strategy in the search for disease susceptibility genes is the evaluation of candidate genes, which are thought to play a role in disease pathogenesis. Candidate genes are screened for single nucleotide polymorphisms (SNPs) in a case-control study. The factor V Leiden (FVL) mutation (1691G --> A in the F5 gene) is an important risk factor for venous thrombosis. We asked ourselves whether the FVL mutation would have been found using the candidate gene approach in the absence of prior knowledge of the haplotype structure of the F5 gene. We typed four SNPs in the F5 gene in the Leiden Thrombophilia study, that is, promoter (99930G --> A), exon 13 (55907A --> G), exon 16 (42855A --> G), and intron 19 (37833T --> G). These SNPs were known to have different population frequencies, making their presence in distinct haplotypes likely. None of these SNPs has previously been associated with venous thrombotic risk. Subsequently we derived haplotypes. One haplotype was clearly more frequent in patients than controls (GAAT; 20 versus 9%), suggesting that a polymorphism in or near the F5 gene in this haplotype is associated with an increased thrombotic risk. If we had sequenced the F5 gene in patients homozygous for this haplotype, in order to locate the possible causal polymorphism, we would have found that 16 (76%) patients were homozygous or heterozygous for a missense mutation in exon 10 (1691G --> A), which predicts the replacement of Arg506 by Gln in one of the cleavage sites for activated protein C, a mutation that we now know as the FVL mutation.

Case-Control Studies↗
PubMed2004

C825T polymorphism in the human G protein beta 3 subunit gene and preeclampsia; a case control study.

OBJECTIVE: Recently, a polymorphism of the gene encoding for the G protein beta3-subunit (GNB3) has been described. The T allele of this polymorphism (825T) is associated with endothelium dysfunction. Endothelium dysfunction has been described in women with preeclampsia. We, therefore, tested the hypothesis that in women who have had preeclampsia the T allele is more prevalent than in controls. STUDY DESIGN: We conducted a case-control study of 157 women with preeclampsia during 1991-1996. Cases and controls were tested for the presence of 825T by genotyping. Logistic regression methods were used for data analysis. RESULTS: The frequency of the T allele of the GNB3 gene was similar in cases (0.30) and controls (0.27) (odds ratio 1.24, 95% confidence interval 0.88-1.75). Compared to controls, we found a high frequency of the T allele in patients with the hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome (odds ratio 4.25, 95% confidence interval 1.12-16.05). CONCLUSION: The frequency of the T allele of the GNB3 gene, which serves as a marker for endothelium dysfunction, was not different between women with preeclampsia and controls. Women with the HELLP syndrome had a high frequency of the T allele. This suggests that this polymorphism in the GNB3 gene does not contribute to endothelium dysfunction in women with preeclampsia while it does contribute in women with the HELLP syndrome.

Adult↗
PubMed2004

Contribution of polymorphisms in the endothelial protein C receptor gene to soluble endothelial protein C receptor and circulating activated protein C levels, and thrombotic risk.

Endothelial cell protein C receptor (EPCR) enhances the generation of activated protein C (APC) by the thrombin-thrombomodulin complex. A soluble form of EPCR (sEPCR), which is generated by metalloprotease activity, is present in plasma. The distribution of sEPCR levels in healthy populations is bimodal. Previously, we described two polymorphisms in exon 4 of the EPCR gene, 4600A/G that encodes the substitution of Ser219 by Gly in the transmembrane region of EPCR and 4678G/C in the 3'-UT region. The aim of this study was to investigate the relationship between these two polymorphisms and plasma sEPCR and APC levels and risk of venous thrombosis. We genotyped 401 healthy controls from the Spanish population and measured their plasma sEPCR and APC levels. Carriers of the 4600AG genotype had significantly higher sEPCR levels than those with the AA genotype, while the 4678CC genotype was associated, to a lesser extent, with elevated APC levels. To assess the effect of these polymorphisms on the risk of thrombosis, we genotyped 405 patients with venous thromboembolism. The frequency of the 4600AG genotype was very similar in patients and controls (p=0.975), whereas the 4678CC genotype was significantly more frequent in controls than in patients (p=0.008). In multivariate analysis, carriers of the 4678CC genotype had a decreased risk of thrombosis (OR=0.61, p=0.009). These data indicate that individuals carrying the 4600AG genotype have high sEPCR levels but do not have an increased risk of thrombosis, whereas individuals carrying the 4678CC genotype have higher APC levels and lower risk of venous thromboembolism.

Adult↗
PubMed2003

Factor Va is inactivated by activated protein C in the absence of cleavage sites at Arg-306, Arg-506, and Arg-679.

Activated protein C (APC) exerts its anticoagulant activity via proteolytic degradation of the heavy chains of activated factor VIII (FVIIIa) and activated factor V (FVa). So far, three APC cleavage sites have been identified in the heavy chain of FVa: Arg-306, Arg-506, and Arg-679. To obtain more insight in the structural and functional implications of each individual cleavage, recombinant factor V (rFV) mutants were constructed in which two or three of the APC cleavage sites were mutated. After expression in COS-1 cells, rFV mutants were purified, activated with thrombin, and inactivated by APC. During this study we observed that activated rFV-GQA (rFVa-GQA), in which the arginines at positions 306, 506, and 679 were replaced by glycine, glutamine, and alanine, respectively, was still inactivated by APC. Further analysis showed that the inactivation of rFVa-GQA by APC was phospholipid-dependent and sensitive to an inhibitory monoclonal antibody against protein C. Inactivation proceeded via a rapid phase (kx1=5.4 x 10(4) M(-1) s(-1)) and a slow phase (kx2=3.2 x 10(3) M(-1) s(-1)). Analysis of the inactivation curves showed that the rapid phase yielded a reaction intermediate that retained approximately 80% of the original FVa activity, whereas the slow cleavage resulted in formation of a completely inactive reaction product. Inactivation of rFVa-GQA was accelerated by protein S, most likely via stimulation of the slow phase. Immunoblot analysis using a monoclonal antibody recognizing an epitope between Arg-306 and Arg-506 indicated that during the rapid phase of inactivation a fragment of 80 kDa was generated that resulted from cleavage at a residue very close to Arg-506. The slow phase was associated with the formation of fragments resulting from cleavage at a residue 1.5-2 kDa carboxyl-terminal to Arg-306. Our observations may explain the unexpectedly mild APC resistance associated with mutations at Arg-306 (FV HongKong and FV Cambridge) in the heavy chain of FV.

Animals↗
PubMed2003

Low levels of tissue factor pathway inhibitor (TFPI) increase the risk of venous thrombosis.

There is now strong experimental evidence that tissue factor pathway inhibitor (TFPI) is a critical inhibitor to modulate tissue factor-induced coagulation, but the role of TFPI as a risk factor for thrombosis is yet to be to be determined. This study investigated the role of low TFPI levels for the development of deep-vein thrombosis (DVT). We determined TFPI activity and TFPI-free and total antigen levels in the subjects enrolled in the Leiden Thrombophilia Study, which is a large population-based case-control study of 474 patients and 474 controls. The odds ratio (OR) for DVT in subjects who had TFPI-free antigen levels below the 10th percentile, as compared with those who had TFPI-free antigen levels above this cutoff, was 1.7 (95% confidence interval [CI], 1.1-2.6). The ORs for low TFPI activity and low total antigen were also mildly increased. When the 5th percentile was used as a cutoff, the ORs were 2.1 (95% CI, 1.1-4.1) for both TFPI-free antigen and TFPI total antigen. Exogenous female hormones had a profound lowering effect on TFPI levels, with lower levels in oral contraceptive users than in premenopausal nonusers, who had lower levels than men and postmenopausal women. These results indicate that low levels of TFPI, especially low TFPI-free and total antigen in plasma, constitute a risk factor for DVT.

Adolescent↗
PubMed2003

Activated protein C resistance determined with a thrombin generation-based test predicts for venous thrombosis in men and women.

Activated protein C (APC) resistance, determined with a thrombin-generation-based APC resistance test, may explain risk differences of venous thrombosis in users of second- and third-generation oral contraceptives (OC). To clinically validate this test, we analysed the Leiden thrombophilia case-control study (474 patients with a first episode of deep vein thrombosis and 474 age- and sex-matched control subjects). Data for men and women were analysed separately. As hormonal status in women is known to strongly influence the APC sensitivity ratio (APCsr), additional strata (OC use and menopausal state) were defined. The APCsr was higher in all patients than in control subjects. Odds ratios (OR), using the 90th percentile of all control subjects (APCsr > 4.5) as cut-off, were: 7.5 [95% confidence interval (CI) 1.6-33.8] for men, 3.0 (95% CI 1.0-8.8) for premenopausal women not using OC, 4.8 (95% CI 1.6-14.7) for premenopausal women using OC and 4.7 (95% CI 1.4-15.6) for postmenopausal women. After excluding the carriers of factor V Leiden, the OR became infinite for men (no control had an APCsr > 4.5), 1.4 (95% CI 0.2-8.2) for premenopausal women not using OC, 3.4 (95% CI 1.1-10.8) for premenopausal women using OC and 3.6 (95% CI 0.6-20.5) for postmenopausal women. A high APCsr, determined with the thrombin-generation-based APC resistance test, predicts venous thrombotic risk, in populations with and without factor V Leiden. In addition, acquired APC resistance resulting from OC use predicts an increased risk for venous thrombosis independent of factor V Leiden.

Activated Protein C Resistance↗
PubMed2003

Frequency of the TAFI -438 G/A and factor XIIIA Val34Leu polymorphisms in patients with objectively proven pulmonary embolism.

Deep vein thrombosis (DVT) and pulmonary embolism (PE) are considered to be two forms of the same disease, however it is not fully understood what determines their clinical presentation. Proteins encoded by the FXIIIA and TAFI genes are involved in stabilizing the fibrin clot and in making it more lysis resistant. The FXIIIA 34Leu and TAFI -438A alleles might protect against DVT. Information on such an association with PE is either contradictory or missing. We hypothesized that both polymorphisms might influence the formation and fate of emboli and accordingly the risk of PE. We determined the frequencies of both polymorphisms in patients with objectively demonstrated PE. The frequency of FXIIIA Leu34Leu in PE patients and non-PE patients was 4.5% and 8.8%, [OR 0.5 (95% CI: 0.1 to 1.9)], respectively. For -438 A/A TAFI genotype the frequency was 1.5% and 8.1% [OR 0.1 (95% CI: 0.02 to 1.1)], respectively.

Adult↗
PubMed2003

The R2-haplotype associated Asp2194Gly mutation in the light chain of human factor V results in lower expression levels of FV, but has no influence on the glycosylation of Asn2181.

The R2 haplotype of the FV gene spans from exon 8 through 25 and comprises several strongly linked polymorphisms in the FV gene, including some missense mutations. Carriership of the R2-FV allele has been associated with reduced plasma FV levels, increased FV1/FV2 ratios and mild APC resistance. Some studies have reported that carriership of the R2-FV allele is associated with an increased risk of venous thombosis. At this moment, the individual contribution to the R2-associated phenotypes of the different mutations linked to the R2 haplotype of FV is unclear. The main objective of our study was to obtain insight in the influence of the R2-related Asp2194Gly mutation on FV expression, FV structure and FV function using Bdomainless rFV mutants. Replacing Asp at position 2194 by Gly resulted in a more than threefold reduction of rFV expression compared to rFV wild-type. Therefore, we propose that the R2-linked Asp2194Gly mutation is an important determinant of the association of the R2-FV allele with lower FV levels. Furthermore, the light chains from Asp2194Gly containing rFV mutants showed similar molecular weights as the light chains of the non-glycosylated rFVwt or the plasma FV2 isoform, indicating that glycosylation at Asn2181 is not stimulated by the presence of a glycine in position 2194. Finally, the apparent K(d) for dissociation of the FXaVa complex (K(1/2Xa)) was not higher in rFV mutants with the Asp2194Gly mutation than for rFVwt, suggesting that also the affinity for negatively charged phospholipids is not affected by substitution of Asp into Gly at position at 2194.

Alleles↗