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.

Inhibition of thrombin-mediated factor V activation contributes to the anticoagulant activity of fibrinogen γ'.

BACKGROUND: Besides its role in blood clotting, fibrinogen exerts a poorly understood anticoagulant function by binding thrombin and modulating its activity. In particular, the γA/γ' fibrinogen isoform binds with high affinity to thrombin exosite II through the anionic carboxyl-terminal end of the γ' chain. This interaction down-regulates thrombin-mediated factor VIII (FVIII) activation, but its effect on FV activation is unknown. OBJECTIVES: To investigate the overall anticoagulant activity of fibrinogen and particularly of fibrinogen γ' in plasma, and to verify whether the fibrinogen γ' carboxyl-terminal peptide affects thrombin-mediated FV activation. METHODS: Thrombin generation was measured by calibrated automated thrombography in whole and defibrinated plasma and in plasma supplemented with the (sulfated) fibrinogen γ' carboxyl-terminal peptide (0-500 µmol L(-1) ). The effect of the peptide on thrombin-mediated FV activation was studied in model systems and in plasma. RESULTS: Total fibrinogen prolonged the lag time of thrombin generation at low tissue factor (TF) concentrations. The fibrinogen γ' peptide dose-dependently prolonged the lag time and decreased the peak height of thrombin generation at low TF, whereas a scrambled control peptide was ineffective. These effects persisted in the presence of an anti-FVIII antibody, suggesting that the peptide may also inhibit thrombin-mediated activation of FV. This was confirmed in model systems and in plasma. CONCLUSIONS: Total fibrinogen and the fibrinogen γ' peptide have an overall anticoagulant effect on thrombin generation determined at low TF. Inhibition of thrombin-mediated FV activation by the fibrinogen γ' peptide is a novel mechanism of the anticoagulant activity of fibrinogen γ'.

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.

Microparticle-associated tissue factor activity, venous thromboembolism and mortality in pancreatic, gastric, colorectal and brain cancer patients.

BACKGROUND: Tissue factor (TF) expression by tumors contributes to tumor growth. Release of TF-positive microparticles (MPs) may contribute to venous thromboembolism (VTE). OBJECTIVES: To conduct a prospective cohort study to determine whether elevated MP-associated TF (MP-TF) activity is predictive of VTE and mortality in four cancer types. PATIENTS/METHODS: We determined MP-TF activity in pancreatic, gastric, colorectal and brain cancer patients. We used a chromogenic endpoint assay for all patients and also a chromogenic kinetic assay for patients with pancreatic and brain cancer. RESULTS: During follow-up, 12/60 (20%) pancreatic, 6/43 (14%) gastric, 12/126 (10%) colorectal and 19/119 (16%) brain cancer patients developed VTE; 46/60 (77%), 30/43 (70%), 47/126 (37%) and 67/119 (56%), respectively, died. MP-TF activity levels were highest in pancreatic cancer. We did not find a statistically significant association of MP-TF activity with the risk of VTE in any of the four cancer types by using two statistical methods. An association of MP-TF activity with the risk of mortality was detected in pancreatic cancer with the endpoint assay (hazard ratio [HR] 1.8 and 95% confidence interval [CI] 1.4-2.3 per doubling of activity, P < 0.001) and the kinetic assay (HR 1.2, 95% CI 1.1-1.4, P < 0.001); adjustment for type of treatment was not performed. In pancreatic cancer, MP-TF activity correlated with D-dimer level (endpoint assay, r = 0.51; chromogenic assay, r = 0.48), and a correlation between assays (r = 0.61) was found. CONCLUSION: MP-TF activity was not associated with future VTE in pancreatic, gastric, colorectal and brain cancer. However, we found a strong association of MP-TF activity with mortality in pancreatic cancer. MP-TF activity might be reflective of an aggressive pancreatic cancer phenotype.

Determination of the size distribution of blood microparticles directly in plasma using atomic force microscopy and microfluidics.

Microparticles, also known as microvesicles, found in blood plasma, urine, and most other body fluids, may serve as valuable biomarkers of diseases such as cardiovascular diseases, systemic inflammatory disease, thrombosis, and cancer. Unfortunately, the detection and quantification of microparticles are hampered by the microscopic size of these particles and their relatively low abundance in blood plasma. The use of a combination of microfluidics and atomic force microscopy to detect microparticles in blood plasma circumvents both problems. In this study, capture of a specific subset of microparticles directly from blood plasma on antibody-coated mica surface is demonstrated. The described method excludes isolation and washing steps to prepare microparticles, improves the detection sensitivity, and yields the size distribution of the captured particles. The majority of the captured particles have a size ranging from 30 to 90 nm, which is in good agreement with prior results obtained with microparticles immediately isolated from fresh plasma. Furthermore, the qualitative shape of the size distribution of microparticles is shown not to be affected by high-speed centrifugation or the use of the microfluidic circuit, demonstrating the relative stable nature of microparticles ex vivo.

Plasma levels of microparticle-associated tissue factor activity in patients with clinically suspected pulmonary embolism.

INTRODUCTION: Microparticles (MPs) carrying active tissue factor (TF) have been detected in the plasma of cancer patients in particular in those presenting with acute deep vein thrombosis (DVT) or pulmonary embolism (PE). Experimental studies in mice have revealed that circulating MPs carrying TF contribute to thrombus formation. AIM: To study whether unselected patients with an acute confirmed PE have elevated TF activity in the MP fraction (MP-TF activity). MATERIALS AND METHODS: Plasma MP-TF activity was measured in 159 non-selected patients with clinically suspected PE and in 48 healthy controls as previously described. Blood was collected at time of inclusion. The diagnosis of acute PE was confirmed in 54 patients and excluded in 105 patients. RESULTS: Median MP-TF activity in 159 patients with clinically suspected PE was 72 fM Xa/min [range 32-6657] fM Xa/min and higher than in healthy controls (66 [range 28-183] fM Xa/min; P<0.05). There was no significant difference (P=0.169) in MP-TF activity between patients with confirmed PE (median 84.5 fM Xa/min [range 36-2149]) and patients without PE (72 fM Xa/min [range 32-6657]) fM Xa/min). In the 159 patients with clinically suspected PE we observed in an exploratory analysis higher MP-TF activity levels in patients with active cancer (median 137 fM Xa/min [range 36-6657]) and cardiovascular disease (median 131.5 fM Xa/min [range 45-2149]) than in patients without these disorders (P=0.0004 and P=0.014, respectively). CONCLUSION: In patients presenting with clinically suspected PE plasma MP-TF activity was not associated with confirmed PE.

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.

Atomic force microscopy: a novel approach to the detection of nanosized blood microparticles.

BACKGROUND: Microparticles (MPs) are small vesicles released from cells of different origin, bearing surface antigens from parental cells. Elevated numbers of blood MPs have been reported in (cardio)vascular disorders and cancer. Most of these MPs are derived from platelets. OBJECTIVES: To investigate whether atomic force microscopy (AFM) can be used to detect platelet-derived MPs and to define their size distribution. METHODS: Blood MPs isolated from seven blood donors and three cancer patients were immobilized on a modified mica surface coated with an antibody against CD41 prior to AFM imaging. AFM was performed in liquid-tapping mode to detect CD41-positive MPs. In parallel, numbers of CD41-positive MPs were measured using flow cytometry. Mouse IgG1 isotype control was used as a negative control. RESULTS: AFM topography measurements of the number of CD41-positive MPs were reproducible (coefficient of variation=16%). Assuming a spherical shape of unbound MPs, the calculated diameter of CD41-positive MPs (dsph) ranged from 10 to 475 nm (mean: 67.5+/-26.5 nm) and from 5 to 204 nm (mean: 51.4+/-14.9 nm) in blood donors and cancer patients, respectively. Numbers of CD41-positive MPs were 1000-fold higher than those measured by flow cytometry (3-702x10(9) L(-1) plasma vs. 11-626x10(6) L(-1) plasma). After filtration of isolated MPs through a 0.22-microm filter, CD41-positive MPs were still detectable in the filtrate by AFM (mean dsph: 37.2+/-11.6 nm), but not by flow cytometry. CONCLUSIONS: AFM provides a novel method for the sensitive detection of defined subsets of MPs in the nanosize range, far below the lower limit of what can be measured by conventional flow cytometry.

The -589C>T polymorphism in the interleukin-4 gene (IL-4) is associated with a reduced risk of myocardial infarction in young individuals.

BACKGROUND: Inflammatory reactions contribute to the development of arterial disease. We investigated the role of interleukin-4 (IL-4) in the development of myocardial infarction (MI) by genotyping patients with MI and control subjects for the -589C>T (rs2243250) single nucleotide polymorphism (SNP), which tags a functional haplotype of IL-4. METHODS AND RESULTS: Study of Myocardial Infarctions Leiden (SMILE) included 560 men with a first MI and 646 control subjects. The Valencia study included 305 patients with MI at T genotype was found [odds ratio (OR) 0.84; 95% CI 0.37-1.95 for -589TT and 0.82; 95% CI 0.62-1.07 for -589CT compared with -589CC]. In patients younger than 50 years, carriership of one or two -589T alleles was associated with a reduced risk of MI (OR 0.57: 95% CI 0.34-0.95). This result was replicated in the Valencia study, where carriers of one or two -589T alleles had a reduced risk of MI (OR 0.67: 95% CI 0.47-0.95), with a strong protective effect of the -598T allele in homozygous -589T (OR 0.33: 95% CI 0.10-1.05). In the control subjects of the Valencia study, the -589T allele was associated with reduced levels of F1+2. CONCLUSION: Our data indicate that the IL-4 haplotype tagged by the -589T allele reduces the risk of MI in young individuals.

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.

Different effects of oral contraceptives containing different progestogens on protein S and tissue factor pathway inhibitor.

BACKGROUND: Oral contraceptives (OC) containing different types of progestogens induce different sensitivities to activated protein C (APC) measured with the thrombin generation-based APC-resistance test. These differences in APC resistance may be the biological explanation for the differences in thrombotic risk of the various pills. The mechanistic basis of APC resistance observed in OC users is unknown. Our objective was to study the effect of OC on the two main determinants of the APC-resistance test, free protein S and free tissue factor pathway inhibitor (TFPI). PATIENTS/METHODS: We measured free protein S and free TFPI in 156 users of various types of OC. RESULTS: Users of desogestrel-containing OC, known to double the risk of thrombosis compared with levonorgestrel-containing OC, had lower free protein S (24 vs. 33 U dL(-1)) and TFPI free antigen (2.9 vs. 3.6 ng mL(-1)) levels than users of OC containing levonorgestrel. Women using cyproterone acetate-containing OC, known to confer a high thrombotic risk, had the lowest free protein S (19 U dL(-1)) and free TPFI antigen (2.5 ng mL(-1)) levels. Users of OC containing drospirenone had lower free protein S (23 U dL(-1)) and TFPI antigen levels (3.2 ng mL(-1)) than users of levonorgestrel-containing OC. Low free protein S and low free TFPI antigen levels were associated with an increased resistance to APC, an established risk factor for thrombosis. CONCLUSIONS: This study observed that the differences in APC resistance induced by OC containing different progestogens can at least in part be explained by different effects of OC on free protein S and TFPI.

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.

Plasma fibrinogen gamma' chain content in the thrombotic microangiopathy syndrome.

BACKGROUND: Human fibrinogen gamma chain variants, termed gamma' chains, contain a unique 20-residue sequence after gamma chain residue 407 that ends at gamma'427, and is designated gamma'(427L). Full-length (FL) gamma'(427L) chains are constituents of a fibrin-dependent thrombin inhibitory system known as antithrombin I, whereas a gamma' chain processed in vivo, termed gamma'(423P), lacks the C-terminal tetrapeptide EDDL, and does not bind thrombin. Together, the gamma'(423P) and gamma'(427L) chains comprise the total plasma fibrinogen gamma' chain content. OBJECTIVES: Lowered plasma gamma' chain content (i.e. gamma' chain-containing fibrinogen/total fibrinogen ratio) has been shown to correlate with susceptibility to venous thrombosis, thus prompting this study on the total and FL gamma' chain content in 45 subjects with thrombotic microangiopathy (TMA), a disorder characterized by microvascular thrombosis. METHODS: We measured by enzyme-linked immunosorbent assay the total gamma' chain-containing fibrinogen/total fibrinogen (Total gamma'-fgn/Total fgn) ratio and the FL gamma' chain-containing fibrinogen/total fibrinogen (FL gamma'-fgn/Total fgn) ratio in these plasmas and in healthy subjects (n = 87). RESULTS: In healthy subjects, the mean Total gamma'-fgn/Total fgn ratio was 0.127, whereas the FL gamma'-fgn/Total fgn ratio was somewhat lower at 0.099 (P < 0.0001), a difference reflecting the presence of gamma'(423P) chains. In TMA plasmas, both the Total gamma'-fgn and FL gamma'-fgn/Total fgn ratios (0.099 and 0.084, respectively) were lower than those of their healthy subject counterparts (P < 0.0001). CONCLUSIONS: These findings in TMA suggest that reductions in the gamma' chain content indicate reduced antithrombin I activity that may contribute to microvascular thrombosis in TMA.

Microparticle-associated tissue factor activity: a link between cancer and thrombosis?

BACKGROUND: Cancer, in particular mucinous adenocarcinoma, is associated with venous thromboembolism (VTE). Tissue factor (TF), initiator of coagulation, plays a central role in the paradigm that clotting and tumor growth form a vicious circle, in which hypercoagulability facilitates the aggressive biology of cancer and vice versa. Expression of TF in tumors is associated with poor differentiation and poor prognosis. PATIENT/METHODS: We investigated the association between clinically manifest VTE and procoagulant properties of circulating microparticles (MP) isolated from blood of unselected pancreatic and breast adenocarcinoma patients' consecutive subjects, who presented with ultrasound or CT-scan confirmed VTE, and healthy subjects. RESULTS: Patients with disseminated breast and pancreatic cancer had significantly increased levels of MP-associated TF activity compared with healthy controls, subjects with idiopathic acute VTE and non-metastatic cancer patients. Patients with both high MP-associated TF-activity and MP-associated epithelial mucin (MUC1) had a lower survival rate at 3-9 months follow-up than those with low TF-activity and no MUC1 expression: the likelihood of survival was 0.42 (95% CI: 0.19- 0.94) for an individual with these two predictor variables present, after adjustment for other factors (age cohort, type of cancer, VTE) in the Cox proportional hazards model. CONCLUSIONS: Our results suggest an important role for MP-associated TF and MUC1 in the pathogenesis of thrombosis in disseminated mucinous adenocarcinoma patients. Future studies should reveal the mechanism underlying the observed associations.

Matrix metalloproteinase-9 gene -1562C/T polymorphism mitigates preeclampsia.

Although the aetiology of preeclampsia is unknown, there is substantial evidence that it finds its roots in abnormal placentation. Prerequisites for successful placentation include trophoblast invasion, degradation and remodelling of the uterine decidual extracellular matrix, and apoptosis without thrombosis. We tested this hypothesis by analysing the effect of functional polymorphisms in the genes coding for MMP9, MMP3 and annexin A5 on the risk of preeclampsia using a case-control design. In 163 women with preeclampsia and 163 controls we studied the association with polymorphisms in the MMP9 (-1562 C/T), MMP3 (-1612 5A/6A) and annexin A5 (-1 C/T) genes using logistic regression analysis. A lower prevalence of the rare T allele of the MMP9 (-1562 C/T) polymorphism in women with preeclampsia was found (odds ratio 0.48, 95% confidence interval 0.25-0.90). The distribution of the MMP3 (-1612 5A/6A) and annexin A5 (-1 C/T) gene polymorphisms were similar in cases and controls. Our results suggest that the MMP9-1562T allele is associated with a reduced risk of preeclampsia and therefore may protect against maladaptation of the spiral arteries and decreased decidual degradation. The elevated MMP9 concentrations reported to be associated with the -1562T allele might be essential for the development of an adequate maternal-fetal interface early in pregnancy by facilitating trophoblast apoptosis and degradation.

von Willebrand factor and its propeptide: the influence of secretion and clearance on protein levels and the risk of venous thrombosis.

BACKGROUND AND OBJECTIVES: Elevated levels of factor (F)VIII are associated with an increased risk of thrombosis. FVIII levels are determined mainly by von Willebrand factor (VWF). We have investigated the contribution of secretion and clearance rates to the elevated VWF antigen (VWF:Ag) and to the risk of thrombosis. VWF is secreted in equimolar amounts with its propeptide, which has a shorter half-life. VWF propeptide can be used as a measure of VWF secretion and allows estimation of the VWF half-life. METHODS AND RESULTS: We have measured VWF propeptide, VWF:Ag, FVIII:Ag and FVIII activity (FVIII:C) in the Leiden Thrombophilia Study. In controls, high VWF propeptide was associated with high VWF:Ag, FVIII:Ag and FVIII:C. In contrast to mature VWF:Ag, VWF propeptide was not influenced by blood groups. Using an ELISA-based assay we have shown that VWF propeptide lacks ABO antigens. Levels were higher in men and increased with age. A long VWF half-life was also associated with high VWF:Ag, FVIII:Ag and FVIII:C. The VWF half-life was influenced by blood group (10 h in O vs. 12 h in non-O individuals), but not by sex, and only slightly by age. VWF propeptide was higher in thrombosis patients than in controls. The VWF half-life was similar in patients and controls (11.4 and 11.1 h, respectively). CONCLUSIONS: Both secretion and clearance rates are important determinants of VWF and FVIII levels. However, mainly high VWF and FVIII levels caused by increased secretion seem to be associated with thrombosis. ABO blood group influences the clearance rates of VWF rather than VWF secretion rates.