Genetic variants in Cell Adhesion Molecule 1 (CADM1): a validation study of a novel endothelial cell venous thrombosis risk factor.

INTRODUCTION: In a protein C deficient family, we recently identified a candidate gene, CADM1, which interacted with protein C deficiency in increasing the risk of venous thrombosis (VT). This study aimed to determine whether CADM1 variants also interact with protein C pathway abnormalities in increasing VT risk outside this family. MATERIALS AND METHODS: We genotyped over 300 CADM1 variants in the population-based MEGA case-control study. We compared VT risks between cases with low protein C activity (n=194), low protein S levels (n=23), high factor VIII activity (n=165) or factor V Leiden carriers (n=580), and all 4004 controls. Positive associations were repeated in all 3496 cases and 4004 controls. RESULTS: We found 22 variants which were associated with VT in one of the protein C pathway risk groups. After mutual adjustment, six variants remained associated with VT. The strongest evidence was found for rs220842 and rs11608105. For rs220842, the odds ratio (OR) for VT was 3.2 (95% CI 1.2-9.0) for cases with high factor VIII activity compared with controls. In addition, this variant was associated with an increased risk of VT in the overall study population (OR: 1.5, 95% CI 1.0-2.2). The other variant, rs11608105, was not associated with VT in the overall study population (OR: 1.0, 95% CI 0.8-1.1), but showed a strong effect on VT risk (OR: 21, 95% CI 5.1-88) when combined with low protein C or S levels. CONCLUSIONS: In a population-based association study, we confirm a role for CADM1 variants in increasing the risk of VT by interaction with protein C pathway abnormalities.

A genome-wide association study for venous thromboembolism: the extended cohorts for heart and aging research in genomic epidemiology (CHARGE) consortium.

Venous thromboembolism (VTE) is a common, heritable disease resulting in high rates of hospitalization and mortality. Yet few associations between VTE and genetic variants, all in the coagulation pathway, have been established. To identify additional genetic determinants of VTE, we conducted a two-stage genome-wide association study (GWAS) among individuals of European ancestry in the extended cohorts for heart and aging research in genomic epidemiology (CHARGE) VTE consortium. The discovery GWAS comprised 1,618 incident VTE cases out of 44,499 participants from six community-based studies. Genotypes for genome-wide single-nucleotide polymorphisms (SNPs) were imputed to approximately 2.5 million SNPs in HapMap and association with VTE assessed using study-design appropriate regression methods. Meta-analysis of these results identified two known loci, in F5 and ABO. Top 1,047 tag SNPs (P ≤ 0.0016) from the discovery GWAS were tested for association in an additional 3,231 cases and 3,536 controls from three case-control studies. In the combined data from these two stages, additional genome-wide significant associations were observed on 4q35 at F11 (top SNP rs4253399, intronic to F11) and on 4q28 at FGG (rs6536024, 9.7 kb from FGG; P < 5.0 × 10(-13) for both). The associations at the FGG locus were not completely explained by previously reported variants. Loci at or near SUSD1 and OTUD7A showed borderline yet novel associations (P < 5.0 × 10(-6) ) and constitute new candidate genes. In conclusion, this large GWAS replicated key genetic associations in F5 and ABO, and confirmed the importance of F11 and FGG loci for VTE. Future studies are warranted to better characterize the associations with F11 and FGG and to replicate the new candidate associations.

Multifluorescence confocal microscopy: application for a quantitative analysis of hemostatic proteins in human venous valves.

Confocal laser scanning microscopy is commonly used to visualize and quantify protein expression. Visualization of the expression of multiple proteins in the same region via multifluorescence allows for the analysis of differential protein expression. The defining step of multifluorescence labeling is the selection of primary antibodies from different host species. In addition, species-appropriate secondary antibodies must also be conjugated to different fluorophores so that each protein can be visualized in separate channels. Quantitative analysis of proteins labeled via multifluorescence can be used to compare relative changes in protein expression. Multifluoresecence labeling and analysis of fluorescence intensity within and among human venous specimens, for example, allowed us to determine that the anticoagulant phenotype of the venous valve is defined not by increased anticoagulant expression, but instead by significantly decreased procoagulant protein expression (Blood 114:1276-1279, 2009 and Histochem Cell Biol 135:141-152, 2011).

Cell adhesion molecule 1 (CADM1) is ubiquitously present in the endothelium and smooth muscle cells of the human macro- and micro-vasculature.

Cell adhesion molecule 1 (CADM1) is a member of the immunoglobulin cell adhesion molecule family. Recently, we identified CADM1 to be a novel risk factor for venous thrombosis in a large, protein C deficient, thrombophilic family and showed, for the first time, the expression of CADM1 in endothelial cells (Hasstedt et al. in Blood 114:3084-3091, 2009). To further investigate its role in venous thrombosis, as well as other vasculopathies, we undertook a systematic confocal microscopic investigation for the presence of CADM1 in the vasculature of 28 different human tissues. Paraffin embedded tissue sections were dual immunostained with an antibody against CADM1, together with an antibody against either von Willebrand factor (to identify endothelial cells), or α-smooth muscle actin (to identify smooth muscle cells). The results showed that CADM1 was ubiquitously present in endothelial cells and smooth muscle cells in the vasculature from all 28 tissues, though its representation in the various classes of vessels was tissue dependent.

The prothrombotic phenotypes in familial protein C deficiency are differentiated by computational modeling of thrombin generation.

The underlying cause of thrombosis in a large protein C (PC) deficient Vermont kindred appears to be multicausal and not explained by PC deficiency alone. We evaluated the contribution of coagulation factors to thrombin generation in this population utilizing a mathematical model that incorporates a mechanistic description of the PC pathway. Thrombin generation profiles for each individual were generated with and without the contribution of the PC pathway. Parameters that describe thrombin generation: maximum level (MaxL) and rate (MaxR), their respective times (TMaxL, TMaxR), area under the curve (AUC) and clotting time (CT) were examined in individuals ± PC mutation, ± prothrombin G20210A polymorphism and ± thrombosis history (DVT or PE). This family (n = 364) is shifted towards greater thrombin generation relative to the mean physiologic control. When this family was analyzed with the PC pathway, our results showed that: carriers of the PC mutation (n = 81) had higher MaxL and MaxR and greater AUC (all p<0.001) than non-carriers (n = 283); and individuals with a DVT and/or PE history (n = 13) had higher MaxL (p = 0.005) and greater AUC (p<0.001) than individuals without a thrombosis history (n = 351). These differences were further stratified by gender, with women in all categories generating more thrombin than males. These results show that all individuals within this family with or without PC deficiency have an increased baseline procoagulant potential reflective of increased thrombin generation. In addition, variations within the plasma composition of each individual can further segregate out increased procoagulant phenotypes, with gender-associated plasma compositional differences playing a large role.

The effects of aging on the intimal region of the human saphenous vein: insights from multimodal microscopy and quantitative image analysis.

We hypothesized that structural remodeling associated with advancing age occurs in human saphenous veins. To address this hypothesis, we have identified structural remodeling in human saphenous veins by applying histochemistry, fluorescence staining and quantitative image analysis to specifically assess intimal area, intimal cellularity and intimal collagen content and organization. Saphenous veins were collected from patients undergoing coronary artery bypass graft surgery. Area measurements and cellularity were quantified using the image analysis software Stereo Investigator, employing planimetry and counting frames, respectively. Collagen content and organization were quantified in MetaMorph image analysis software based on measurements of color (hue, saturation, and intensity) from polarized light images. Intimal area and cellularity showed no statistically significant increases with age; in contrast, total collagen content showed a significant decrease with advancing age. Furthermore, collagen fiber types also demonstrated a statistically significant alteration with age; increases in age resulted in decreases in larger collagen fibers. No significant changes in small collagen fibers were identified. These results raise the possibility that age-associated structural alterations in total collagen content, specifically collagen fiber size, could be a factor in the etiology of age-associated venous diseases.

Effect of ageing on the murine venous circulation.

The effect of ageing on the morphology of veins, venous valves and arteries was investigated in male wild-type mice using an adapted procedure with injection of a silicone polymer Microfil(®) that preserves morphology of the vasculature. Throughout the hind limb the arterial, but not the venous, lumen area and wall thickness were significantly greater in 24-month as compared to 10-week-old C57BL/6 mice. Venous valves were most frequently located at the sapheno-femoral vein junction in the lower extremities, and appeared thicker at the base supported by structurally intact collagen fibers, and thinner towards the proximal end of the valve leaflet, with less organized collagen. Overall, valves were less supported by structurally intact collagen at 24 months as compared to 10 weeks. Endothelial expression of CD31, endothelial protein C receptor or von Willebrand factor (VWF) was not affected by age, while thrombomodulin expression was lower in aged versus young arteries. At both ages, expression of VWF was lower at venous valves versus veins. Evaluation of the blood coagulation profile revealed that aged mice had shortened prothrombin time, elevated plasma levels of factor (F)VII, FVIII and VWF and increased neutrophil and platelet counts. Thus, our data indicate that in mice with ageing, venous valves become more fragile, in association with a procoagulant and inflammatory blood phenotype. Taken together, we found that the procoagulant state in ageing, is accompanied by mild vascular changes.

Genetic predictors of fibrin D-dimer levels in healthy adults.

BACKGROUND: Fibrin fragment D-dimer, one of several peptides produced when crosslinked fibrin is degraded by plasmin, is the most widely used clinical marker of activated blood coagulation. To identity genetic loci influencing D-dimer levels, we performed the first large-scale, genome-wide association search. METHODS AND RESULTS: A genome-wide investigation of the genomic correlates of plasma D-dimer levels was conducted among 21 052 European-ancestry adults. Plasma levels of D-dimer were measured independently in each of 13 cohorts. Each study analyzed the association between ≈2.6 million genotyped and imputed variants across the 22 autosomal chromosomes and natural-log­transformed D-dimer levels using linear regression in additive genetic models adjusted for age and sex. Among all variants, 74 exceeded the genome-wide significance threshold and marked 3 regions. At 1p22, rs12029080 (P=6.4×10(-52)) was 46.0 kb upstream from F3, coagulation factor III (tissue factor). At 1q24, rs6687813 (P=2.4×10(-14)) was 79.7 kb downstream of F5, coagulation factor V. At 4q32, rs13109457 (P=2.9×10(-18)) was located between 2 fibrinogen genes: 10.4 kb downstream from FGG and 3.0 kb upstream from FGA. Variants were associated with a 0.099-, 0.096-, and 0.061-unit difference, respectively, in natural-log­transformed D-dimer and together accounted for 1.8% of the total variance. When adjusted for nonsynonymous substitutions in F5 and FGA loci known to be associated with D-dimer levels, there was no evidence of an additional association at either locus. CONCLUSIONS: Three genes were associated with fibrin D-dimer levels. Of these 3, the F3 association was the strongest, and has not been previously reported.

Venous valvular stasis-associated hypoxia and thrombosis: what is the link?

This review focuses on the role of the venous valves in the genesis of thrombus formation in venous thromboembolic disease (VTE). Clinical VTE and the evidence for the valvular origin of venous thrombosis are reviewed. Virchow's triad is then used as a framework for discussion to approach the question posed regarding the link between venous valvular stasis-associated hypoxia and thrombosis. Thus, the effects of blood flow stasis, hypercoagulability of blood, and the characteristics of the vessel wall within the venous valvular sinus are assessed in turn.

Genetic variation associated with plasma von Willebrand factor levels and the risk of incident venous thrombosis.

In a recent genome-wide association study, variants in 8 genes were associated with VWF level, a risk factor for venous thrombosis (VT). In an independent, population-based, case-control study of incident VT, we tested hypotheses that variants in these genes would be associated with risk. Cases were 656 women who experienced an incident VT, and controls comprised 710 women without a history of VT. DNA was obtained from whole blood. Logistic regression was used to test associations between incident VT and single nucleotide polymorphisms (SNPs) in 7 genes not previously shown to be associated with VT. Associations with P < .05 were candidates for replication in an independent case-control study of VT in both sexes. Two of the 7 SNPs tested yielded P < .05: rs1039084 (P = .005) in STXBP5, a novel candidate gene for VT, and rs1063856 (P = .04) in VWF, a gene whose protein level is associated with VT risk. Association results for the remaining 5 variants in SCARA5, STAB2, STX2, TC2N, and CLEC4M were not significant. Both STXBP5 and VWF findings were replicated successfully. Variation in genes associated with VWF levels in the genome-wide association study was found to be independently associated with incident VT.

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.

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 5.0x10(-8) and comprised 5 loci on 5 chromosomes: 2p23 (smallest P value 6.2x10(-24)), 4q25 (3.6x10(-12)), 11q12 (2.0x10(-10)), 13q34 (9.0x10(-259)), and 20q11.2 (5.7x10(-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 (1.2x10(-22)), 8p21 (1.3x10(-16)), 9q34 (<5.0x10(-324)), 12p13 (1.7x10(-32)), 12q23 (7.3x10(-10)), 12q24.3 (3.8x10(-11)), 14q32 (2.3x10(-10)), and 19p13.2 (1.3x10(-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.

Cell adhesion molecule 1: a novel risk factor for venous thrombosis.

Protein C (PC) deficiency increases the risk of venous thrombosis (VT) among members of Kindred Vermont II but fails to fully account for the inheritance pattern. A genome scan of the pedigree supported the presence of a prothrombotic gene on chromosome 11q23 (nominal P < .0001), with weaker support on chromosomes 10p12 (P < .0003) and 18p11.2-q11 (P < .0007). Resequencing of 109 genes in the linkage regions identified 5030 variants in a sample of 20 kindred members. Of 16 single nucleotide polymorphisms in 6 genes tested in the larger family set, only single nucleotide polymorphisms in cell adhesion molecule 1 (CADM1) associated with VT. Among the 8 CADM1 single nucleotide polymorphisms genotyped in the complete sample, rs6589488 was most strongly supported (P < .000007), but the association was limited to the PC-deficient subset of the sample (P < .000001). Haplotype analysis narrowed the region containing the causative variant to the coding region of the CADM1 gene. CADM1 gene expression analyzed in blood outgrowth endothelial cells cultured from family members was decreased compared with control subjects, lending phenotypic support to this conclusion. Finally, we have for the first time demonstrated CADM1 in endothelial cells, where it appears to be selectively involved in endothelial cell migration, suggesting a role in endothelial barrier repair.

Valves of the deep venous system: an overlooked risk factor.

Deep venous valves are frequent sites of deep venous thrombosis initiation. However, the possible contribution of the valvular sinus endothelium has received little attention in studies of thrombosis risk. We hypothesized that the endothelium of valve sinus differs from that of vein lumen with up-regulation of anticoagulant and down-regulation of procoagulant activities in response to the local environment. In pursuit of this hypothesis, we quantified endothelial protein C receptor (EPCR), thrombomodulin (TM), and von Willebrand factor (VWF) by immunofluorescence in great saphenous veins harvested at cardiac bypass surgery. We found significantly increased expression of EPCR and TM in the valvular sinus endothelium as opposed to the vein lumenal endothelium, and the opposite pattern with VWF (paired t test for TM and EPCR, each P < .001; for VWF, P = .01). These data support our hypothesis and suggest that variation in valvular sinus thromboresistance may be an important factor in venous thrombogenesis.

Imaging aspects of cardiovascular disease at the cell and molecular level.

Cell and molecular imaging has a long and distinguished history. Erythrocytes were visualized microscopically by van Leeuwenhoek in 1674, and microscope technology has evolved mightily since the first single-lens instruments, and now incorporates many types that do not use photons of light for image formation. The combination of these instruments with preparations stained with histochemical and immunohistochemical markers has revolutionized imaging by allowing the biochemical identification of components at subcellular resolution. The field of cardiovascular disease has benefited greatly from these advances for the characterization of disease etiologies. In this review, we will highlight and summarize the use of microscopy imaging systems, including light microscopy, electron microscopy, confocal scanning laser microscopy, laser scanning cytometry, laser microdissection, and atomic force microscopy in conjunction with a variety of histochemical techniques in studies aimed at understanding mechanisms underlying cardiovascular diseases at the cell and molecular level.

Lipoprotein-associated phospholipase A2 and risk of venous thrombosis in older adults.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) is an enzyme involved in inflammation and platelet function. Inherited deficiency and elevated levels are associated with atherosclerosis. Given potential common etiologies of atherosclerosis and venous thrombosis (VT), we hypothesized that low and high Lp-PLA2 would be associated with VT risk. Lp-PLA(2) mass and activity were measured in baseline samples of Cardiovascular Health Study participants (5,888 men and women age > or =65), excluding 354 reporting pre-baseline VT. The study endpoint was VT unrelated to cancer after 11.6 years follow-up. Hazard ratios were estimated using Cox proportional hazard models, adjusting for age, race, sex, and body-mass index. With 129 cases of VT, there was no association of Lp-PLA2 activity with risk. Adjusted hazard ratios were 1.19 (CI 0.62, 2.29) and 0.87 (CI 0.43, 1.76) for the lowest and highest decile, respectively, compared to the 10-25th percentile. Corresponding hazard ratios for Lp-PLA2 mass were 1.63 (CI 0.79, 3.34) and 1.33 (CI 0.61, 2.87). Results were robust to several definitions of low or high Lp-PLA2. While the association of Lp-PLA(2) levels with arterial disease events implies a role for this enzyme in atherogenesis, our findings suggest that it is not prothrombotic.

Novel isoforms of intracellular platelet activating factor acetylhydrolase (PAFAH1b2) in human testis; encoded by alternatively spliced mRNAs.

Platelet activating factor acetylhydrolase (paf-ah), a potent regulator of platelet activating factor activity, plays an important role in various physiological and pathophysiological functions including development, reproduction, inflammation, hemostasis, and apoptosis. Intracellular paf-ah (paf-ah-Ib) is composed of a regulatory subunit, Pafah1b1, and two highly conserved but non-identical catalytic subunits, Pafah1b2 and Pafah1b3. The present study identifies new splice variants of the Pafah1b2 gene transcript. The splice variants retain exons 1-5 and replace exon 6 with alternative exons derived from genomic sequence 3' to exon 6. Splice variants encode two proteins with different novel carboxy termini. One of the isoforms is expressed exclusively in testis. These new isoforms of pafah1b2 retain the ability to form higher order complexes while replacing known key catalytic residues, which raises the possibility that they may alter the subunit composition and catalytic function of paf-ah-Ib.

Exclusion of the alpha2 subunit of platelet-activating factor acetylhydrolase 1b (PAFAH1B2) as a prothrombotic gene in a protein C-deficient kindred and population-based case-control sample.

Protein C deficiency increases the risk of venous thromboembolic disease among members of Kindred Vermont II, but fails to fully account for the inheritance pattern. A genome scan of the pedigree supported the presence of a prothrombotic gene on chromosome 11q23 (107-119 Mb, nominal P < 0.0001), with weaker support on chromosomes 10p12 (11-25 Mb, P < 0.0003) and 18p11.2-q11 (12-24 Mb, P < 0.0007). The 11q23 region contains the alpha(2) subunit (gene name PAFAH1B2) of platelet-activating factor acetylhydrolase 1b, a candidate prothrombotic gene. Re-sequencing of the PAFAH1B2 regulatory region in 137 pedigree members, including 25 thrombosis cases, revealed 12 variants; eight were present in only 0-2 affected individuals; the other four assorted into three haplotypes and included three variants predicted to destroy transcription factor-binding sites. More extensive re-sequencing of the PAFAH1B2 gene in 11 affected and five unaffected pedigree members revealed an additional 13 variants that assorted into the same three haplotypes. We rejected as thrombosis risk factors each of the three presumed destructive variants as well as each of the three haplotypes. We also rejected (odds ratio = 1.31 CI: 0.91-1.88) one of the three variants in 469 cases and 472 controls from the Leiden Thrombophilia Study (LETS). Therefore, PAFAH1B2 is not the gene responsible for the linkage evidence on chromosome 11q23.

SELDI-TOF plasma profiles distinguish individuals in a protein C-deficient family with thrombotic episodes occurring before age 40.

We tested the hypothesis that differences in the low-molecular-weight (500-20,000 Da) proteomic profile of plasma may be detectable between members of a protein C-deficient family who have suffered thrombotic events before age 40 compared to family members without a history of venous thrombosis. Unfractionated plasma samples from members of a previously described large thrombophilic kindred with type I protein C deficiency were applied to ProteinChip weak cation exchange interaction arrays (WCX2; Ciphergen Biosystems, Fremont, CA, USA) and subjected to SELDI-TOF (surface-enhanced laser desorption/ionization time-of-flight) mass spectrometry using the Ciphergen PBSII ProteinChip System (Ciphergen Biosystems). Profiles were analyzed by a boosted decision-tree algorithm. When individuals who had presented with deep venous thrombosis (DVT) before the age of 40 (n = 21) were compared to age-matched, healthy family members (n = 50), the proteomic patterns defined by the decision-tree analysis could classify the entity of DVT before age 40 with 67% sensitivity, at a specificity of 86%. When a small group of cases with history of superficial venous thrombosis (n = 6) was added to the case group, the sensitivity was 87.5% at a specificity of 80%. These data support the hypothesis that members of the protein C deficient Vermont kindred II who suffer a thrombotic event before age 40 display significant differences in low-molecular-weight proteomics profile compared to those who remain disease-free. This is the first study to apply SELDI-TOF technology in conjunction with a bioinformatics tool to analyze low-molecular-weight proteomic patterns in patients with venous thrombosis.

Effect of the novel direct factor Xa inhibitor DX-9065a on thrombin generation and inhibition among patients with stable atherosclerotic coronary artery disease.

INTRODUCTION: Thrombin, a pluripotential effector enzyme with prothrombotic, proinflammatory, and mitogenic properties, plays a pivotal role in the pathobiology and clinical expression of atherothrombotic coronary artery disease. Existing anticoagulant drugs have not been shown to attenuate thrombin generation or activity consistently. We sought to investigate the effect of DX-9065a on thrombin generation and inhibition in patients with stable CAD. DX-9065a is a small-molecule, synthetic, direct inhibitor of factor Xa. MATERIALS AND METHODS: Peripheral venous blood samples were collected serially during and after administration of either placebo or 1 of 4 weight-adjusted regimens of DX-9065a, in 73 patients with stable CAD participating in the XaNADU-1B study. RESULTS AND CONCLUSIONS: At baseline, the median (25th, 75th) prothrombin activation fragment 1.2 (F1.2) level was 2.56 (2.05, 3.20) nmol/L, and the median d-dimer level was 0.26 (0.19, 0.38) microg FEU/L. There were significant relationships between measured plasma DX-9065a concentrations and both F1.2 (4.9% decrease for each doubling of DX-9065a) (P<0.0001) and d-dimer (5.5% decrease for each doubling of DX-9065a) (P=0.001). F1.2 was suppressed (below baseline) at 96 h after administration of DX-9065a. Coronary thrombotic events did not occur during or after study drug administration. DX-9065a, the first in a class of small-molecule, direct, selective and reversible factor Xa inhibitors, reduces thrombin generation and fibrin formation among patients with stable CAD. The effect is concentration-dependent and persists for at least 96 h following drug cessation, without biochemical or clinical evidence of rebound.