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.

Genome-wide linkage scan in affected sibling pairs identifies novel susceptibility region for venous thromboembolism: Genetics In Familial Thrombosis study.

BACKGROUND: Venous thromboembolism (VTE) is a multicausal disorder involving environmental and genetic risk factors. In many thrombophilic families the clustering of thrombotic events cannot be explained by known genetic risk factors, indicating that some remain to be discovered. OBJECTIVES: We aimed to identify novel thrombosis susceptibility alleles in a large panel of small thrombophilic families: the Genetics In Familial Thrombosis (GIFT) study. PATIENTS/METHODS: In the GIFT study, 201 families were recruited consisting of 438 siblings with an objectively confirmed VTE at a young age. Multipoint linkage analysis (402 SSR markers) and fine mapping were performed, followed by genotyping of tagging SNPs in positional candidate genes. RESULTS: Established genetic risk factors such as factor V Leiden, ABO blood group non-O, prothrombin 20210A, fibrinogen gamma 10034T and deficiencies of antithrombin, protein C and protein S were more frequent in GIFT patients than in unselected VTE patients. Linkage supported the presence of novel thrombosis susceptibility loci on 7p21.3-22.2 (LOD score = 3.23) and Xq24-27.3 (LOD score = 1.95). Simulation analysis showed that the chr7 signal was genome-wide statistically significant (P = 0.022). Tagging SNPs (n = 157) in eight positional candidate genes (LOD drop 1.5 regions) were genotyped in GIFT patients and 332 healthy controls. Five chr7 SNPs associated with VTE. SNP THSD7A rs2074597 was responsible for part of the chr7 signal. CONCLUSIONS: The GIFT panel is rich in established genetic risk factors for VTE, but genetic factors remain unidentified in many families. Genome-wide linkage failed to identify the previously established genetic risk factors for VTE, but identified a novel VTE susceptibility locus on chr7.

Sex hormone-binding globulin levels are not causally related to venous thrombosis risk in women not using hormonal contraceptives.

BACKGROUND: Oral contraceptive use increases the risk of venous thrombosis as well as sex hormone-binding globulin (SHBG) levels. Furthermore, increased SHBG levels are positively associated with activated protein C (APC) resistance and thrombotic risk in oral contraceptive users. OBJECTIVES: To determine whether increased SHBG levels are causally related to venous thrombosis in women not using hormonal contraceptives. METHODS: Premenopausal women were selected from a case-control study on venous thrombosis, the Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis (MEGA) study (23 patients; 258 controls). Women using hormonal contraceptives were excluded. First, the risk of venous thrombosis with SHBG levels above the normal reference range (70 nm) was determined. Second, because multiple regulatory factors affect SHBG levels and residual confounding may remain, we determined six single-nucleotide polymorphisms (SNPs) in the SHBG gene and assessed the risk of venous thrombosis in a different case-control study, the Leiden Thrombophilia Study (LETS) (20 patients; 74 controls), and in the MEGA study. Finally, the association between SHBG levels and the normalized activated partial thromboplastin time-based APC resistance (an intermediate endpoint for venous thrombosis) was determined. RESULTS: Elevated SHBG levels (> 70.0 nm) were associated with venous thrombosis (odds ratio 1.92; 95% confidence interval [CI] 0.74-5.00). However, this finding can be explained by residual confounding. Two SNPs in the SHBG gene affected SHBG levels, but not venous thrombosis risk. Furthermore, SHBG levels in controls were not associated with APC resistance (SHBG level, > 70.0 vs. ≤ 70.0 nm: mean difference in normalized APC sensitivity ratio, 0.03; 95% CI -0.05 to 0.10). Exclusion of women with FV Leiden did not materially change these results. CONCLUSIONS: Increased SHBG levels are not causally related to the risk of venous thrombosis.

The minor allele of GP6 T13254C is associated with decreased platelet activation and a reduced risk of recurrent cardiovascular events and mortality: results from the SMILE-Platelets project.

BACKGROUND: Contradictory results have been published on the effects of T13254C (rs1613662), which distinguishes the two major isoforms of GP6, the gene encoding the platelet receptor glycoprotein VI, on platelet function and the risk of cardiovascular disease. METHODS: We performed a population-based case-control study, the Study of Myocardial Infarctions in Leiden, among 547 male patients with a first myocardial infarction (MI) and 646 control subjects, as well as a prospective cohort study in which the same MI patients were followed for recurrent events (fatal and non-fatal MI and unstable angina) and mortality (median follow-up of 12 years). P-selectin expression by platelets induced by crosslinked collagen-related peptide (CRP-XL) was measured by whole blood flow cytometry in 274 MI patients. RESULTS: T13254C was not associated with a first MI, but seemed to be associated with a reduced incidence of recurrent events [per-allele hazard ratio 0.77, 95% confidence interval (CI) 0.56-1.06] and mortality (hazard ratio 0.57, 95% CI 0.37-0.89). Pooling with the Heart and Estrogen/Progestin Replacement Study revealed hazard ratios of 0.81 (95% CI 0.66-0.99) and 0.73 (95% CI 0.55-0.96). The minor C-allele was also strongly associated with a reduced percentage of P-selectin-expressing platelets. The reduction per C-allele was 23% (95% CI 18-28%). In an independent study of 219 healthy volunteers, the per-allele reduction of CRP-XL-induced aggregation was 10% (95% CI 2-18%). CONCLUSION: The minor allele of GP6 T13254C that reduced platelet activation and aggregation also seemed to be associated with a reduced incidence of recurrent cardiovascular events and mortality, but was not associated with first MI.

Functional variation in the arginine vasopressin 2 receptor as a modifier of human plasma von Willebrand factor levels.

SUMMARY OBJECTIVES: Stimulation of arginine vasopressin 2 receptor (V2R) with arginine vasopressin (AVP) results in a rise in von Willebrand factor (VWF) and factor VIII plasma levels. We hypothesized that gain-of-function variations in the V2R gene (AVPR2) would lead to higher plasma levels of VWF and FVIII. METHODS AND RESULTS: We genotyped the control populations of two population-based studies for four AVPR2 variations: a-245c, G12E, L309L, and S331S. Rare alleles of a-245c, G12E, and S331S, which were in linkage disequilibrium, were associated with higher VWF propeptide, VWF and FVIII levels. The functionality of the G12E variant was studied in stably transfected MDCKII cells, expressing constructs of either 12G-V2R or 12E-V2R. Both V2R variants were fully glycosylated and expressed on the basolateral membrane. The binding affinity of V2R for AVP was increased three-fold in 12E-V2R-green fluorescent protein (GFP) cells, which is in accordance with increased levels of VWF propeptide associated with the 12E variant. The dissociation constant (K(D)) was 4.5 nm [95% confidence interval (CI) 3.6-5.4] for 12E-V2R-GFP and 16.5 nm (95% CI 10.1-22.9) for 12G-V2R-GFP. AVP-induced cAMP generation was enhanced in 12E-V2R-GFP cells. CONCLUSIONS: The 12E-V2R variant has increased binding affinity for AVP, resulting in increased signal transduction, and is associated with increased levels of VWF propeptide, VWF, and FVIII.

Prothrombotic gene variation and new vascular events after cerebral ischemia of arterial origin.

BACKGROUND: Several genetic variants involved in hemostasis have been associated with ischemic stroke or myocardial infarction (MI). Stroke patients who carry a prothrombotic genotype may also be at increased risk for subsequent vascular events. PATIENTS AND METHODS: We included 887 patients with non-disabling cerebral ischemia of arterial origin, who were referred to the University Medical Center Utrecht in the Netherlands between 1995 and 2005 and followed them for the occurrence of ischemic stroke, MI or death. The primary outcome was a composite of death from all vascular causes, non-fatal ischemic stroke, non-fatal MI, whichever happened first. We selected 22 prothrombotic variants in 14 genes that were previously associated with ischemic stroke or MI or had evidence of functionality. RESULTS: During a 4.6-year mean follow-up period new vascular events occurred in 135 patients (annual event rate 3.3%). None of the 22 variants was associated with the occurrence of new vascular events. Eight additional analyses with secondary outcomes or among subgroups revealed four associations that were likely to be false positive after accounting for multiple testing. CONCLUSIONS: In this cohort, prothrombotic genetic variants do not affect the risk of new vascular events after cerebral ischemia of arterial origin. This study does not support the use of prothrombotic genetic variants to identify stroke patients at increased risk for new vascular events or to guide antithrombotic treatment.

Selectin haplotypes and the risk of venous thrombosis: influence of linkage disequilibrium with the factor V Leiden mutation.

BACKGROUND: Selectins (E-, L- and P-selectin) and their most important counter-receptor P-selectin glycoprotein ligand (SELPLG) facilitate the interaction of platelets, leukocytes and endothelial cells at inflammatory sites. Selectin polymorphisms/haplotypes have been associated with cardiovascular disease. OBJECTIVES: We investigated the association between haplotypes (H) of these four genes and deep venous thrombosis (DVT) risk. We additionally explored the effect of linkage disequilibrium (LD) with the nearby Factor V Leiden mutation (FVL). Furthermore, interactions between SELPLG polymorphisms and selectin polymorphisms were investigated. PATIENTS/METHODS: Leiden Thrombophilia Study (LETS) subjects were genotyped for 24 polymorphisms by TaqMan or PCR-RFLP, detecting all common haplotypes in four blocks. P-selectin was analyzed in two blocks, upstream (SELPup) and downstream (SELPdown) of the recombination hotspot. RESULTS: In E- and L-selectin, none of the haplotypes was associated with DVT risk. In SELPup, H2-carriers had a 1.3-fold increased risk (95% CI, 1.0-1.7), whereas H4-carriers had a 1.4-fold decreased risk (95% CI, 0.5-1.0). In SELPdown, H2-carriers had a 1.3-fold increased risk (95% CI, 1.0-1.7). Because of LD with FVL, we subsequently excluded all FVL-carriers and all risks disappeared. Mutual adjustment within a logistic regression model resulted in disappearance of the risks for the SELP haplotypes, whereas FVL risk remained. CONCLUSIONS: After adjustment for LD with FVL, none of the selectin haplotypes was associated with DVT risk, showing that the increased risks of the selectin haplotypes were a reflection of the effect of FVL on thrombosis risk.

Polymorphism 10034C>T is located in a region regulating polyadenylation of FGG transcripts and influences the fibrinogen gamma'/gammaA mRNA ratio.

BACKGROUND: Fibrinogen gamma haplotype 2 (FGG-H2) is associated with reduced fibrinogen gamma' levels and fibrinogen gamma'/total fibrinogen ratios and with an increased deep-venous thrombosis (DVT) risk. Two FGG-H2 tagging single nucleotide polymorphisms (SNPs), 9615C>T and 10034C>T, are located in the region of alternative FGG pre-mRNA processing. 10034C>T is located in a GT-rich downstream sequence element (DSE) that comprises a putative cleavage stimulation factor (CstF) binding site. OBJECTIVES: To investigate the functionality of SNPs 9615C>T and 10034C>T, and the importance of the DSE containing 10034C>T. METHODS: Different minigene constructs containing FGG exon 9, intron 9, exon 10 and the 3' region were transiently transfected into HepG2 cells and quantitative real-time polymerase chain reaction was used to measure relative polyadenylation (pA) signal usage (pA1/pA2 ratio). RESULTS: Compared with the reference construct CC (9615C-10034C; FGG-H1; pA1/pA2 ratio set at 100%), the pA1/pA2 ratio of construct TT (9615T-10034T; FGG-H2) was 1.4-fold decreased (71.5%, P = 0.015). The pA1/pA2 ratio of construct CT (9615C-10034T) was almost 1.2-fold decreased (85.3%, P = 0.001), whereas the pA1/pA2 ratio of construct TC (9615T-10034C) did not differ significantly from the reference construct (101.6%, P = 0.890). Functionality of the putative CstF binding site was confirmed using constructs in which this site was deleted or its sequence altered by point mutations. CONCLUSIONS: SNP 10034C>T is located in a GT-rich DSE involved in regulating the usage of the pA2 signal of FGG, which may represent a CstF binding site. We propose that the 10034C>T change is the functional variation in FGG-H2 that is responsible for the reduction in the fibrinogen gamma'/total fibrinogen ratio and the increased DVT risk.

The association of prothrombin A19911G polymorphism with plasma prothrombin activity and venous thrombosis: results of the MEGA study, a large population-based case-control study.

BACKGROUND: Prothrombin (FII) G20210A mutation and elevated plasma prothrombin activity are known risk factors for venous thrombosis. The risk of venous thrombosis among 19911G carriers of the prothrombin A19911G polymorphism has not been extensively investigated. OBJECTIVES AND METHODS: We assessed prothrombin activity, FIIG20210A, and FIIA19911G polymorphisms in a large population-based case-control study, the Multiple Environmental and Genetic Assessment (MEGA) study of risk factors for venous thrombosis. Four thousand three hundred and sixty-five consecutive patients with a first episode of deep vein thrombosis of the leg or pulmonary embolism were included. The control group (n = 4779) consisted of partners of patients or persons gathered using a random-digit dialing method. We studied the effect of FIIA19911G polymorphism on prothrombin activity and thrombosis risk, also in combination with factor V Leiden. RESULTS: Among FII20210-GG control subjects, FII19911-GG carriers had 7.1% [95% confidence interval (CI): 5.7-8.5] higher mean prothrombin activity than FII19911-AA carriers and the risk for GG carriers was 1.43-fold increased compared to AA carriers [odds ratio (OR) 1.43; 95% CI: 1.27-1.61]. Among FII20210-GA control carriers, the mean prothrombin activity in both FII19911-AA and -AG carriers was nearly equivalent [131.7% and 133.4%; mean difference (95% CI) = 1.7% (-7.2-10.7)]. Because of genetic linkage, FII19911-GG carriers were very rare on a FII20210-GA background, as only one FII20210A carrier had FII19911-GG. In FII20210-GA carriers, the OR increased from 3.05 (95% CI: 2.17-4.27) in subjects with FII19911-AA to 3.33 (2.28-4.85) in subjects with FII19911-AG, compared to those with FII20210-GG and FII19911-AA. CONCLUSIONS: The FIIA19911G polymorphism is associated with mildly elevated prothrombin activity and is a risk factor for venous thrombosis.

Homozygous C2362F von Willebrand factor induces intracellular retention of mutant von Willebrand factor resulting in autosomal recessive severe von Willebrand disease.

The missense mutation of cysteine 2362 to a phenylalanine in von Willebrand factor (VWF) has been detected in several Italian families with autosomal recessive, severe von Willebrand disease. We investigated how this amino acid change in VWF may lead to a predominantly quantitative defect. This mutation was studied in vitro by transient expression of the full-length mutant VWF-C2362F protein and in vivo by analysis of plasma VWF after infusion of 1-deamino-8-d-arginine vasopressin (DDAVP) in a patient homozygous for this mutation. Single transfections of pSVHVWF-C2362F and co-transfections of mutant and wild-type constructs resulted in 8% and 50% VWF antigen, respectively, in conditioned medium. These reduced levels are in accordance with observations in homozygous and heterozygous carriers of the mutation. In addition, VWF-C2362F was retained intracellularly. Similar results were obtained for C2362F and C2362A. After infusion of DDAVP in a homozygous patient, a twofold decrease in half-life of plasma VWF-C2362F was observed. This was not explained by increased susceptibility of recombinant VWF-C2362F to ADAMTS13. It was concluded that VWF-C2362F causes reduced VWF plasma levels due to impaired secretion and intracellular retention. Furthermore, it is the loss of cysteine 2362 rather than the introduction of the bulky amino acid side chain that causes these effects.

Inherited defects of coagulation Factor V: the thrombotic side.

DNA variations in the Factor V gene have played a major role in thrombosis research ever since the discovery of Factor V Leiden. Here, all relatively common DNA variations in the coding regions of the Factor V gene are discussed. Many of them have been associated with venous thrombosis or related diseases. However, most variations have been studied separately, without taking the presence of other variations in the same gene into account. This means that their association with disease should be interpreted with caution, as it may reflect linkage with another variation. An approach in which a haplotype-based analysis of the Factor V gene is combined with in vitro assays of recombinant proteins is advocated. Finally, a possible reason for the relatively polymorphic nature of the Factor V protein is discussed.

Cysteine-mutations in von Willebrand factor associated with increased clearance.

BACKGROUND: von Willebrand disease (VWD) is a bleeding disorder caused by the decrease of functional von Willebrand factor (VWF). Low levels of VWF can result from decreased synthesis, impaired secretion, increased clearance or combinations thereof. Several mutations lead to impaired synthesis or secretion of VWF, however, little is known about the survival of VWF in the circulation. OBJECTIVES: To evaluate the effect of several VWF mutations on VWF clearance. PATIENTS/METHODS: The effect of three cysteine-mutations (C1130F, C1149R or C2671Y) on the in vivo survival of VWF was studied in patients carrying these mutations and in a VWF-deficient mice model. RESULTS: In patients carrying these mutations, we observed increased propeptide/mature VWF ratios and rapid disappearance of VWF from the circulation after desmopressin treatment. Detailed analysis of in vivo clearance of recombinant VWF in a VWF-deficient mice model revealed a fourfold increased clearance rate of the mutants. The mutations C1130F, C1149R and C2671Y are each associated with reduced survival of VWF in the circulation. Detailed analysis of the recombinant mutant VWF demonstrated that increased clearance was not due to increased proteolysis by ADAMTS-13. We did not identify functional or structural characteristics that the mutant proteins have in common and could be associated with the phenomenon of increased clearance. CONCLUSIONS: Cysteine-mutations in VWF may result in reduced in vivo survival. The observation that various mutations are associated with increased in vivo clearance may have major implications for the therapeutic strategies that rely on the rise of endogenous VWF after desmopressin administration.