Several missense mutations in the von Willebrand Factor (VWF) gene of von Willebrand disease (VWD) patients have been shown to cause impaired constitutive secretion and intracellular retention of ...VWF. However, the effects of those mutations on the intracellular storage in Weibel-Palade bodies (WPBs) of endothelial cells and regulated secretion of VWF remain unknown. We demonstrate, by expression of quantitative VWF mutants in HEK293 cells, that four missense mutations in the D3 and CK-domain of VWF diminished the storage in pseudo-WPBs, and led to retention of VWF within the endoplasmic reticulum (ER). Immunofluorescence and electron microscopy data showed that the pseudo-WPBs formed by missense mutant C1060Y are indistinguishable from those formed by normal VWF. C1149R, C2739Y, and C2754W formed relatively few pseudo-WPBs, which were often short and sometimes round rather than cigar-shaped. The regulated secretion of VWF was impaired slightly for C1060Y but severely for C1149R, C2739Y, and C2754W. Upon co-transfection with wild-type VWF, both intracellular storage and regulated secretion of all mutants were (partly) corrected. In conclusion, defects in the intracellular storage and regulated secretion of VWF following ER retention may be a common mechanism underlying VWD with a quantitative deficiency of VWF.
Mutations of cysteine residues in von Willebrand factor are known to reduce the storage and secretion of this factor, thus leading to reduced antigen levels. However, one cysteine mutation, ...p.Cys2773Ser, has been found in patients with type 2A(IID) von Willebrand's disease who have normal plasma levels of von Willebrand factor. We hypothesize that disruption of either intra- or interchain disulfide bonds by cysteine mutations in von Willebrand factor has different effects on the biogenesis of Weibel-Palade bodies.
The effect of specific cysteine mutations that either disrupt intrachain (p.Cys1130Phe and p.Cys2671Tyr) or interchain (p.Cys2773Ser) disulfide bonds on storage and secretion of von Willebrand factor was studied by transient transfection of human embryonic kidney cell line 293. Upon expression of von Willebrand factor these cells formed endothelial Weibel-Palade body-like organelles called pseudo-Weibel-Palade bodies. Storage of von Willebrand factor was analyzed with both confocal immunofluorescence and electron microscopy. Regulated secretion of von Willebrand factor was induced by phorbol 12-myristate 13-acetate.
p.Cys1130Phe and p.Cys2671Tyr reduced the storage of von Willebrand factor into pseudo-Weibel-Palade bodies with notable retention of von Willebrand factor in the endoplasmic reticulum, whereas p.Cys2773Ser-von Willebrand factor was stored normally. As expected, wild-type von Willebrand factor formed proteinaceous tubules that were seen under electron microscopy as longitudinal striations in pseudo-Weibel-Palade bodies. p.Cys2773Ser caused severe defects in von Willebrand factor multimerization but the factor formed normal tubules. Furthermore, the basal and regulated secretion of von Willebrand factor was drastically impaired by p.Cys1130Phe and p.Cys2671Tyr, but not by p.Cys2773Ser.
We postulate that natural mutations of cysteines involved in the formation of interchain disulfide bonds do not affect either the storage in Weibel-Palade bodies or secretion of von Willebrand factor, whereas mutations of cysteines forming intrachain disulfide bonds lead to reduced von Willebrand factor storage and secretion because the von Willebrand factor is retained in the endoplasmic reticulum.
In collateral development (i.e. arteriogenesis), mononuclear cells are important and exist as a heterogeneous population consisting of pro-inflammatory and anti-inflammatory/repair-associated cells. ...Protease-activated receptor (PAR)1 and PAR2 are G-protein-coupled receptors that are both expressed by mononuclear cells and are involved in pro-inflammatory reactions, while PAR2 also plays a role in repair-associated responses. Here, we investigated the physiological role of PAR1 and PAR2 in arteriogenesis in a murine hind limb ischemia model.
PAR1-deficient (PAR1-/-), PAR2-deficient (PAR2-/-) and wild-type (WT) mice underwent femoral artery ligation. Laser Doppler measurements revealed reduced post-ischemic blood flow recovery in PAR2-/- hind limbs when compared to WT, while PAR1-/- mice were not affected. Upon ischemia, reduced numbers of smooth muscle actin (SMA)-positive collaterals and CD31-positive capillaries were found in PAR2-/- mice when compared to WT mice, whereas these parameters in PAR1-/- mice did not differ from WT mice. The pool of circulating repair-associated (Ly6C-low) monocytes and the number of repair-associated (CD206-positive) macrophages surrounding collaterals in the hind limbs were increased in WT and PAR1-/- mice, but unaffected in PAR2-/- mice. The number of repair-associated macrophages in PAR2-/- hind limbs correlated with CD11b- and CD115-expression on the circulating monocytes in these animals, suggesting that monocyte extravasation and M-CSF-dependent differentiation into repair-associated cells are hampered.
PAR2, but not PAR1, is involved in arteriogenesis and promotes the repair-associated response in ischemic tissues. Therefore, PAR2 potentially forms a new pro-arteriogenic target in coronary artery disease (CAD) patients.
Venous thrombosis, whose main clinical presentations include deep vein thrombosis and pulmonary embolism, represents a major health problem worldwide. Numerous conditions are known to predispose to ...venous thrombosis and these conditions are commonly referred to as risk indicators or risk factors. Generally accepted or "classically" acquired risk factors for venous thromboembolism include advanced age, prolonged immobilisation, surgery, fractures, use of oral contraceptives and hormone replacement therapy, pregnancy, puerperium, cancer and antiphospholipid syndrome. In addition to these well-established risk factors for venous thrombosis, several lines of evidence that have emerged over the past few decades indicate a role of novel genetic risk factors, mainly related to the haemostatic system, in influencing thrombotic risk. The most significant breakthrough has been the confirmation of the concept that inherited hypercoagulable conditions are present in a large proportion of patients with venous thromboembolic disease. These include mutations in the genes that encode antithrombin, protein C and protein S, and the factor V Leiden and factor II G20210 A mutations. Moreover, plasmatic risk indicators, such as hyperhomocysteinemia and elevated concentrations of factors II, VIII, IX, XI and fibrinogen, have also been documented. This extensive list of genetic and acquired factors serves to illustrate that a single cause of venous thrombosis does not exist and that this condition should be considered as a complex or multifactorial trait. Complex traits can be understood by assuming an interaction between different mutations in candidate susceptibility genes. The risk that is associated with each genetic defect may be relatively low in isolation but the simultaneous presence of several mutations may dramatically increase disease susceptibility. Moreover, environmental factors may interact with one or more genetic variations to add further to the risk. The analysis of genetic risk factors and plasmatic factors, together with private life style and environmental factors, has contributed significantly to our understanding of the genetic predisposition to venous thrombosis.
In case-control studies, elevated levels of interleukins 6 and 8 have been found to be associated with an increased risk of venous thrombosis (VT). Because of the design of these studies, it remained ...uncertain whether these alterations were a cause or a result of the VT. In order to distinguish between the two, we set out to measure the levels of six inflammatory markers prior to thrombosis in a population-based cohort using a nested case-cohort design.
Between August 1995 and June 1997, blood was collected from 66,140 people in the second Norwegian Health Study of Nord-Trøndelag (HUNT2). We identified venous thrombotic events occurring between entry and 1 January 2002. By this date we had registered 506 cases with a first VT; an age- and sex-stratified random sample of 1,464 controls without previous VT was drawn from the original cohort. Levels of interleukins 1beta, 6, 8, 10, 12p70, and tumour necrosis factor-alpha were measured in the baseline sample that was taken 2 d to 75 mo before the event (median 33 mo). Cut-off points for levels were the 80th, 90th, and 95th percentile in the control group. With odds ratios ranging from 0.9 (95% CI: 0.6-1.5) to 1.1 (95% CI: 0.7-1.8), we did not find evidence for a relationship between VT and an altered inflammatory profile.
The results from this population sample show that an altered inflammatory profile is more likely to be a result rather than a cause of VT, although short-term effects of transiently elevated levels cannot be ruled out.
Abstract Objectives Selection bias in case–control studies occurs when control selection is inappropriate. However, selection bias due to improper case sampling is less well recognized. We describe ...how to recognize survivor bias (i.e., selection on exposed cases) and illustrate this with an example study. Study Design and Setting A case–control study was used to analyze the effect of statins on major bleedings during treatment with vitamin K antagonists. A total of 110 patients who experienced such bleedings were included 18–1,018 days after the bleeding complication and matched to 220 controls. Results A protective association of major bleeding for exposure to statins (odds ratio OR: 0.56; 95% confidence interval: 0.29–1.08) was found, which did not become stronger after adjustment for confounding factors. These observations lead us to suspect survivor bias. To identify this bias, results were stratified on time between bleeding event and inclusion, and repeated for a negative control (an exposure not related to survival): blood group non-O. The ORs for exposure to statins increased gradually to 1.37 with shorter time between outcome and inclusion, whereas ORs for the negative control remained constant, confirming our hypothesis. Conclusion We recommend the presented method to check for overoptimistic results, that is, survivor bias in case–control studies.
The protein C pathway plays an important role in the maintenance of endothelial barrier function and in the inflammatory and coagulant processes that are characteristic of patients on dialysis. We ...investigated whether common single nucleotide variants (SNV) in genes encoding protein C pathway components were associated with all-cause 5 years mortality risk in dialysis patients.
Single nucleotides variants in the factor V gene (F5 rs6025; factor V Leiden), the thrombomodulin gene (THBD rs1042580), the protein C gene (PROC rs1799808 and 1799809) and the endothelial protein C receptor gene (PROCR rs867186, rs2069951, and rs2069952) were genotyped in 1070 dialysis patients from the NEtherlands COoperative Study on the Adequacy of Dialysis (NECOSAD) cohort) and in 1243 dialysis patients from the German 4D cohort.
Factor V Leiden was associated with a 1.5-fold (95% CI 1.1-1.9) increased 5-year all-cause mortality risk and carriers of the AG/GG genotypes of the PROC rs1799809 had a 1.2-fold (95% CI 1.0-1.4) increased 5-year all-cause mortality risk. The other SNVs in THBD, PROC, and PROCR were not associated with 5-years mortality.
Our study suggests that factor V Leiden and PROC rs1799809 contributes to an increased mortality risk in dialysis patients.
Coagulation proteases may act as cell signaling molecules via protease-activated receptor (PAR) cleavage, subsequently affecting cellular and inflammatory responses. Activation of PARs in the setting ...of systemic inflammation and disseminated intravascular coagulation (DIC) might thus exacerbate the inflammatory response contributing to tissue and organ damage. To investigate the role of PAR-4 in these processes, we subjected mice to a model of systemic inflammation and DIC (Shwartzman reaction) in the absence or presence of a cell-penetrating pepducin antagonist of PAR-4 (P4pal-10). P4pal-10 dose-dependently diminished the severity of endotoxemia and preserved liver, kidney, as well as lung function. Moreover, systemic inflammation and local (neutrophilic) inflammatory responses were attenuated. In vitro migration assays and P4pal-10 treatment in neutropenic mice suggest an essential role for neutrophils in PAR-4–mediated pathology. P4pal-10 treatment of thrombocytopenic mice excluded the involvement of platelets in this phenomenon. These results uncover an important role for PAR-4 in the Shwartzman reaction and suggest that inhibition of PAR-4 signaling in neutrophils could be protective in systemic inflammation and DIC.
Summary
Protein C is an important inhibitor of blood coagulation. We investigated the effect of two polymorphisms within the promoter region of the protein C gene (C/T at position 2405 and A/G at ...position 2418) on risk of venous thrombosis and on plasma protein C levels. In addition the combined effect of the two polymorphisms with factor V Leiden and oral contraceptive use was investigated. Previous studies on these polymorphisms were small and were not able to investigate synergistic effects. In the Multiple Environmental and Genetic Assessment of risk factors for venous thrombosis (MEGA study), protein C levels were determined in 2,043 patients with venous thrombosis and 2,857 control subjects, and the two polymorphisms in 4,285 patients and 4,863 control subjects. The CC/GG genotype was associated with the lowest protein C levels. Compared to carriers of the TT/AA genotype – a genotype associated with higher protein C levels – the risk of venous thrombosis in CC/GG carriers was 1.3-fold increased (95% confidence interval 1.09–1.48). The combination of factor V Leiden with the CC/GG genotype led to a 4.7-fold increased risk, compared to non-carriers with the TT/AA genotype. Oral contraceptive use together with the CC/ GG genotype resulted in a 5.2-fold increased risk. In conclusion, the CC/GG genotype is associated with lower levels of protein C and an elevated risk of venous thrombosis compared to the TT/AA genotype. There is no clear synergistic effect of the CC/ GG genotype with factor V Leiden or oral contraceptive use on thrombotic risk.
Murine atherosclerosis models do not spontaneously develop atherothrombotic complications. We investigated whether disruption of natural anticoagulation allows preexisting atherosclerotic plaques to ...progress toward an atherothrombotic phenotype.
On lowering of plasma protein C levels with small interfering RNA (si
) in 8-week Western-type diet-fed atherosclerotic apolipoprotein E-deficient mice, 1 out of 4 mice displayed a large, organized, and fibrin- and leukocyte-rich thrombus on top of an advanced atherosclerotic plaque located in the aortic root. Although again at low incidence (3 in 25), comparable thrombi at the same location were observed during a second independent experiment in 9-week Western-type diet-fed apolipoprotein E-deficient mice. Mice with thrombi on their atherosclerotic plaques did not show other abnormalities and had equally lowered plasma protein C levels as si
-treated apolipoprotein E-deficient mice without thrombi. Fibrinogen and thrombin-antithrombin concentrations and blood platelet numbers were also comparable, and plaques in si
mice with thrombi had a similar composition and size as plaques in si
mice without thrombi. Seven out of 25 si
mice featured clots in the left atrium of the heart.
Our findings indicate that small interfering RNA-mediated silencing of protein C in apolipoprotein E-deficient mice creates a condition that allows the occurrence of spontaneous atherothrombosis, albeit at a low incidence. Lowering natural anticoagulation in atherosclerosis models may help to discover factors that increase atherothrombotic complications.
PDF
