Moniek P.M. de Maat

Elevated levels of von Willebrand Factor in cirrhosis support platelet adhesion despite reduced functional capacity.

Cirrhosis of the liver is frequently accompanied by complex alterations in the hemostatic system, resulting in a bleeding tendency. Although many hemostatic changes in liver disease promote bleeding, compensatory mechanisms also are found, including high levels of the platelet adhesive protein von Willebrand Factor (VWF). However, conflicting reports on the functional properties of VWF in cirrhosis have appeared in literature. We have measured a panel of VWF parameters in plasma from patients with cirrhosis of varying severity and causes. Furthermore, we assessed the contribution of VWF to platelet adhesion, by measuring the ability of plasma from patients with cirrhosis to support adhesion of normal or patient platelets under flow conditions. VWF antigen levels were strongly increased in patients with cirrhosis. In contrast, the relative collagen binding activity, as well as the relative ristocetin cofactor activity, was significantly lower in patients as compared with controls, indicating loss of function. Accordingly, patients had a reduced fraction of high‐molecular‐weight VWF multimers. Both strongly elevated and reduced activity and antigen levels of the VWF cleaving protease ADAMTS13 were found in individual patients. Adhesion of either normal or patient platelets to a collagen surface was substantially increased when these platelets were resuspended in plasma of patients with cirrhosis, as compared with control plasma. In conclusion, highly elevated levels of VWF in patients with cirrhosis contribute to the induction of primary hemostasis despite reduced functional properties of the molecule. This phenomenon might compensate for defects in platelet number and function in patients with cirrhosis. (HEPATOLOGY 2006;44:53–61.)

Evaluation of newer risk markers for coronary heart disease risk classification: a cohort study.

BACKGROUND Whether newer risk markers for coronary heart disease (CHD) improve CHD risk prediction remains unclear. OBJECTIVE To assess whether newer risk markers for CHD risk prediction and stratification improve Framingham risk score (FRS) predictions. DESIGN Prospective population-based study. SETTING The Rotterdam Study, Rotterdam, the Netherlands. PARTICIPANTS 5933 asymptomatic, community-dwelling participants (mean age, 69.1 years [SD, 8.5]). MEASUREMENTS Traditional CHD risk factors used in the FRS (age, sex, systolic blood pressure, treatment of hypertension, total and high-density lipoprotein cholesterol levels, smoking, and diabetes) and newer CHD risk factors (N-terminal fragment of prohormone B-type natriuretic peptide levels, von Willebrand factor antigen levels, fibrinogen levels, chronic kidney disease, leukocyte count, C-reactive protein levels, homocysteine levels, uric acid levels, coronary artery calcium [CAC] scores, carotid intima-media thickness, peripheral arterial disease, and pulse wave velocity). RESULTS Adding CAC scores to the FRS improved the accuracy of risk predictions (c-statistic increase, 0.05 [95% CI, 0.02 to 0.06]; net reclassification index, 19.3% overall [39.3% in those at intermediate risk, by FRS]). Levels of N-terminal fragment of prohormone B-type natriuretic peptide also improved risk predictions but to a lesser extent (c-statistic increase, 0.02 [CI, 0.01 to 0.04]; net reclassification index, 7.6% overall [33.0% in those at intermediate risk, by FRS]). Improvements in predictions with other newer markers were marginal. LIMITATION The findings may not be generalizable to younger or nonwhite populations. CONCLUSION Among 12 CHD risk markers, improvements in FRS predictions were most statistically and clinically significant with the addition of CAC scores. Further investigation is needed to assess whether risk refinements using CAC scores lead to a meaningful change in clinical outcome. Whether to use CAC score screening as a more routine test for risk prediction requires full consideration of the financial and clinical costs of performing versus not performing the test for both persons and health systems. PRIMARY FUNDING SOURCE Netherlands Organization for Health Research and Development (ZonMw).

Novel Associations of Multiple Genetic Loci With Plasma Levels of Factor VII, Factor VIII, and von Willebrand Factor The CHARGE (Cohorts for Heart and Aging Research in Genome Epidemiology) Consortium

A complex cascade of coagulation factors underlies hemostasis and prevents life-threatening blood loss from damaged blood vessels. The hemostatic factors VII and VIII, both produced in the liver, play central roles in the initiation and propagation, respectively, of fibrin formation. In the tissue-factor pathway, blood coagulation factor VII (FVII), once activated, serves as a catalyst for factor X (FX) activation, which converts prothrombin to thrombin. During propagation, activated factor VIII (FVIII) activates FX in the presence of activated factor IX. Von Willebrand factor (vWF), produced by endothelial cells and megakaryocytes, has multiple roles in hemostasis. Its primary role is to serve as an adhesion molecule that anchors platelets to exposed collagen after endothelial cell damage. The factor also acts as a carrier protein of FVIII, thereby prolonging the half-life of FVIII. Elevated circulating levels of FVIII and vWF are risk factors for venous thrombosis but the data supporting an association of FVII levels with arterial thrombosis are less consistent.1-5 Hemorrhagic complications are associated with deficiency in FVII and vWF (von Willebrand disease), as well as X-linked deficiency in FVIII (Hemophilia A).6-9 Plasma levels of these proteins are affected by environmental factors but they also are genetically influenced.10-13 Heritability estimates range from 0.53-0.63 for FVII, 0.40-0.61 for FVIII, and 0.31-0.75 for vWF.12, 13 To date, our understanding of genetic variation influencing plasma levels has been focused primarily on cis-acting variation in the genes encoding each protein product (F7, F8, and VWF, respectively). A large-scale genome-wide investigation of the genomic correlates of plasma levels has not been previously published. Using data from 23,608 adults, we investigated genome-wide associations between common genetic variation and plasma levels of FVII, FVIII, and vWF.Background— Plasma levels of coagulation factors VII (FVII), VIII (FVIII), and von Willebrand factor (vWF) influence risk of hemorrhage and thrombosis. We conducted genome-wide association studies to identify new loci associated with plasma levels. Methods and Results— The setting of the study included 5 community-based studies for discovery comprising 23 608 European-ancestry participants: Atherosclerosis Risk In Communities Study, Cardiovascular Health Study, British 1958 Birth Cohort, Framingham Heart Study, and Rotterdam Study. All subjects had genome-wide single-nucleotide polymorphism (SNP) scans and at least 1 phenotype measured: FVII activity/antigen, FVIII activity, and vWF antigen. Each study used its genotype data to impute to HapMap SNPs and independently conducted association analyses of hemostasis measures using an additive genetic model. Study findings were combined by meta-analysis. Replication was conducted in 7604 participants not in the discovery cohort. For FVII, 305 SNPs exceeded the genome-wide significance threshold of 5.0×10−8 and comprised 5 loci on 5 chromosomes: 2p23 (smallest P value 6.2×10−24), 4q25 (3.6×10−12), 11q12 (2.0×10−10), 13q34 (9.0×10−259), and 20q11.2 (5.7×10−37). Loci were within or near genes, including 4 new candidate genes and F7 (13q34). For vWF, 400 SNPs exceeded the threshold and marked 8 loci on 6 chromosomes: 6q24 (1.2×10−22), 8p21 (1.3×10−16), 9q34 (<5.0×10−324), 12p13 (1.7×10−32), 12q23 (7.3×10−10), 12q24.3 (3.8×10−11), 14q32 (2.3×10−10), and 19p13.2 (1.3×10−9). All loci were within genes, including 6 new candidate genes, as well as ABO (9q34) and VWF (12p13). For FVIII, 5 loci were identified and overlapped vWF findings. Nine of the 10 new findings were replicated. Conclusions— New genetic associations were discovered outside previously known biological pathways and may point to novel prevention and treatment targets of hemostasis disorders.

C-Reactive Protein Predicts Progression of Atherosclerosis Measured at Various Sites in the Arterial Tree The Rotterdam Study

Background and Purpose— C-reactive protein (CRP) predicts myocardial infarction and stroke. Its role as a predictor of the progression of subclinical atherosclerosis is not yet known. We investigated whether CRP predicts progression of atherosclerosis measured at various sites in the arterial tree. Methods— CRP levels were measured in a random sample of 773 subjects ≥55 years of age who were participating in the Rotterdam Study. Subclinical atherosclerosis was assessed at various sites at 2 points in time, with a mean duration between measurements of 6.5 years. Results— After adjustment for age, sex, and smoking habits, odds ratios (ORs) associated with CRP levels in the highest compared with the lowest quartile were increased for progression of carotid (OR, 1.9; 95% CI, 1.1 to 3.3), aortic (OR, 1.7; 95% CI, 1.0 to 3.0), iliac (OR, 2.0; 95% CI, 1.2 to 3.3), and lower extremity (OR, 1.9; 95% CI, 1.0 to 3.7) atherosclerosis. The OR for generalized progression of atherosclerosis as indicated by a composite progression score was 4.5 (95% CI, 2.3 to 8.5). Except for aortic atherosclerosis, these estimates hardly changed after additional adjustment for multiple cardiovascular risk factors. In addition, ORs for progression of atherosclerosis associated with high CRP levels were as high as those associated with the traditional cardiovascular risk factors high cholesterol, hypertension, and smoking. Geometric mean levels of CRP increased with the total number of sites showing progression of atherosclerosis (P =0.002 for trend). Conclusions— CRP predicts progression of atherosclerosis measured at various sites in the arterial tree.

Genetic Variation, C-Reactive Protein Levels, and Incidence of Diabetes

C-reactive protein (CRP) has been shown to be associated with type 2 diabetes, but whether CRP has a causal role is not yet clear. We examined the association in the Rotterdam Study, a population-based prospective cohort study. The association of baseline serum CRP and incident diabetes during follow-up was investigated, and a meta-analysis was conducted on the BMI-adjusted relation of CRP and diabetes. Furthermore, the association of CRP haplotypes with serum CRP and risk of diabetes was assessed. The age- and sex-adjusted hazard ratio for diabetes was 1.41 (95% CI 1.29–1.54) per 1 SD increase in natural logarithm of CRP, and it was 1.88, 2.16, and 2.83 for the second, third, and fourth quartiles of CRP, respectively, compared with the first quartile. The risk estimates attenuated but remained statistically significant after additional adjustment for obesity indexes, which agreed with the results of the meta-analysis. The most common genetic haplotype was associated with a significantly lower CRP level compared with the three other haplotypes. The risk of diabetes was significantly higher in the haplotype with the highest serum CRP level compared with the most common haplotype (OR 1.45, 95% CI 1.08–1.96). These findings support the hypothesis that serum CRP enhances the development of diabetes.

High von Willebrand Factor Levels Increase the Risk of First Ischemic Stroke Influence of ADAMTS13, Inflammation, and Genetic Variability

Background and Purpose— Elevated von Willebrand factor (vWF) concentrations are associated with an increased risk of ischemic heart disease. Several factors influence vWF antigen levels and activity, including blood group, genetic variability, acute-phase response, and proteolysis by A Disintegrin and Metalloprotease with ThromboSpondin motif (ADAMTS13), a determinant of proteolytic cleavage of vWF. We assessed how these factors affect the relation between vWF and the occurrence of stroke to understand the underlying mechanism. Methods— In a case-control study of 124 first-ever ischemic stroke patients and 125 age- and sex-matched controls, we studied vWF antigen (vWF:Ag), vWF ristocetin cofactor activity (vWF:RCo), ADAMTS13 activity, the −1793C/G polymorphism in the vWF gene, and C-reactive protein. Results— vWF antigen and activity levels were significantly higher in cases than in controls. The relative risk of ischemic stroke was highest in individuals in the upper quartile of vWF:Ag (odds ratio, 3.2; 95% CI, 1.4 to 7.5) and vWF:RCo (odds ratio, 2.1; 95% CI, 0.9 to 4.8) compared with individuals in the lowest quartiles. In individuals with ADAMTS13 in the lowest quartile, the relative risk of stroke was 1.7 (95% CI, 0.7 to 3.9) compared with the highest quartile. C-reactive protein, ADAMTS13, and genetic variation did not affect the association between vWF and the relative risk of stroke, whereas blood group did affect the association. Conclusions— vWF antigen and activity are associated with the occurrence of acute ischemic stroke. This relation is unaffected by the severity of the acute-phase response or by genetic variation or degradation.

Inflammatory Mediators and Cell Adhesion Molecules as Indicators of Severity of Atherosclerosis The Rotterdam Study

Inflammatory mediators and soluble cell adhesion molecules predict cardiovascular events. It is not clear whether they reflect the severity of underlying atherosclerotic disease. Within the Rotterdam Study, we investigated the associations of C-reactive protein (CRP), interleukin-6 (IL-6), soluble intercellular adhesion molecule-1, and soluble vascular cell adhesion molecule-1 with noninvasive measures of atherosclerosis. Levels of CRP were assessed in a random sample of 1317 participants, and levels of IL-6 and soluble cell adhesion molecules were assessed in a subsample of 714 participants. In multivariate analyses, logarithmically transformed CRP (regression coefficient [&bgr;]=−0.023, 95% CI −0.033 to −0.012) and IL-6 (&bgr;=−0.025, 95% CI −0.049 to −0.001) were inversely associated with the ankle-arm index. Only CRP was associated with carotid intima-media thickness (&bgr;=0.018, 95% CI 0.010 to 0.027). Compared with the lowest tertile, the odds ratio for moderate to severe carotid plaques associated with levels of CRP in the highest tertile was 2.0 (95% CI 1.3 to 3.0). Soluble intercellular adhesion molecule-1 levels were strongly associated with carotid plaques (odds ratio 2.5, 95% CI 1.5 to 4.4 [highest versus lowest tertile]). Soluble vascular cell adhesion molecule-1 was not significantly associated with any of the measures of atherosclerosis. This study indicates that CRP is associated with the severity of atherosclerosis measured at various sites. Associations of the other markers with atherosclerosis were less consistent.

Association of plasma fibrinogen levels with coronary artery disease, smoking and inflammatory markers

The plasma level of fibrinogen is associated with the risk of ischaemic heart disease (IHD) and the severity of atherosclerosis. It has been suggested that an increased plasma level of fibrinogen is a coronary risk indicator because it reflects the inflammatory condition of the vascular wall. An inflamed vascular wall may increase the production of the cytokines interleukin 6 (IL6), interleukin 1-beta (IL1-beta), and tumour necrosis factor alpha(TNF-alpha), which have a major role in the regulation of synthesis in the liver of acute phase proteins, including fibrinogen. Smoking has also been reported to increase the levels of fibrinogen and C-reactive protein (CRP). This may indicate that smoking induces an inflammatory reaction, probably of the pulmonary bronchi and alveolae. Therefore, we anticipated that with both types of inflammation the levels of acute phase proteins and cytokines would be related. We have investigated the contribution of inflammation to the plasma levels of fibrinogen in 34 patients with severe coronary artery disease (CAD) and 30 healthy controls comparable for age and smoking habits. We did not find a parallel in the effects of smoking and ischaemic heart disease on the plasma levels of fibrinogen, CRP, IL6, IL1-beta and TNF-alpha. Cardiovascular disease had its most important effect on the plasma fibrinogen level, while smoking appeared to increase the CRP levels, while both CAD and smoking seemed to affect the IL6 levels. Our results indicate that both smoking and CAD induce an inflammatory condition but that the increase of plasma levels of different inflammatory markers is complex. Although the acute phase reaction is the main regulatory mechanism of fibrinogen, the increase of fibrinogen in our group of CAD patients could not be fully explained by increased inflammation.

Genetic Influence on Inflammation Variables in the Elderly

Background—Inflammation variables (C-reactive protein [CRP], fibrinogen, and soluble intercellular adhesion molecule-1 [sICAM-1]) have been identified as risk factors for cardiovascular disease. It is still not known how much the regulation of inflammatory risk factors is determined by genetic factors, and the aim of this study was to determine the heritability of these inflammation variables and of the acute phase regulating cytokines interleukin-6 (IL-6) and tumor necrosis factor-&agr; (TNF-&agr;) at older ages. Methods and Results—The heritability of CRP, fibrinogen, sICAM-1, IL-6, and TNF-&agr; was determined in a twin study consisting of 129 monozygotic twin pairs and 153 dizygotic same-sex twins aged 73 to 94 years who participated in the Longitudinal Study of Aging of Danish Twins. Furthermore, we determined the influence of selected genetic polymorphisms on the plasma level variations. Genetic factors accounted for 20% to 55% of the variation in plasma levels of the inflammation variables. The highest heritability was found for sICAM-1. The genetic polymorphisms we studied explained only a small, insignificant part of the heritability. Conclusions—This study in elderly twins provides evidence for a substantial genetic component of inflammatory cardiovascular risk factors among the elderly.

High von Willebrand Factor Levels Increase the Risk of Stroke The Rotterdam Study

Background and Purpose— Many studies have investigated the role of plasma von Willebrand factor level in coronary heart disease, but few have investigated its role in stroke. The aim of this study was to determine if von Willebrand factor levels are associated with the risk of stroke. Methods— The study was part of the Rotterdam Study, a large population-based cohort study among subjects aged ≥55 years. We included 6 250 participants who were free from stroke at baseline (1997 to 2001) and for whom blood samples were available. Follow-up for incident stroke was complete up to January 1, 2005. Data were analyzed with Cox proportional hazards models adjusted for age and sex and additionally with models adjusted for other potential confounders including ABO blood group. A subgroup analysis was performed in participants without atrial fibrillation. Effect modification by sex was tested on a multiplicative and on an additive scale. Results— During an average follow-up time of 5.0 years, 290 first-ever strokes occurred, of which 197 were classified as ischemic. The risk of stroke increased with increasing von Willebrand factor levels (age- and sex-adjusted hazard ratios per SD increase in von Willebrand factor level: 1.12 [95% CI, 1.01 to 1.25] for stroke, 1.13 [95% CI, 0.99 to 1.29] for ischemic stroke). Adjustments for additional confounders slightly attenuated the association. The association was also present in subjects without atrial fibrillation and did not differ between sexes. Conclusion— High von Willebrand factor levels are associated with stroke risk in the general population.