Large-scale genomic studies reveal central role of ABO in sP-selectin and sICAM-1 levels.

P-selectin and intercellular adhesion molecule-1 (ICAM-1) participate in inflammatory processes by promoting adhesion of leukocytes to vascular wall endothelium. Their soluble levels have been associated with adverse cardiovascular events. To identify loci affecting soluble levels of P-selectin (sP-selectin) and ICAM-1 (sICAM-1), we performed a genome-wide association study in a sample of 4115 (sP-selectin) and 9813 (sICAM-1) individuals of European ancestry as a part of The Cohorts for Heart and Aging Research in Genome Epidemiology consortium. The most significant SNP association for sP-selectin was within the SELP gene (rs6136, P = 4.05 x 10(-61)) and for sICAM-1 levels within the ICAM-1 gene (rs3093030, P = 3.53 x 10(-23)). Both sP-selectin and sICAM-1 were associated with ABO gene variants (rs579459, P = 1.86 x 10(-41) and rs649129, P = 1.22 x 10(-15), respectively) and in both cases the observed associations could be accounted for by the A1 allele of the ABO blood group. The absence of an association between ABO blood group and platelet-bound P-selectin levels in an independent subsample (N = 1088) from the ARIC study, suggests that the ABO blood group may influence cleavage of the P-selectin protein from the cell surface or clearance from the circulation, rather than its production and cellular presentation. These results provide new insights into adhesion molecule biology.

Genome-wide association study identifies novel loci for plasma levels of protein C: the ARIC study.

Protein C is an important endogenous anticoagulant in hemostasis. Deficiencies of protein C due to genetic mutations or a low level of circulating protein C increase the risk of venous thromboembolism. We performed a genome-wide association scan for plasma protein C antigen concentration with approximately 2.5 million single-nucleotide polymorphisms in 8048 individuals of European ancestry and a replication analysis in a separate sample of 1376 individuals in the Atherosclerosis Risk in Communities Study. Four independent loci from 3 regions were identified with genome-wide significance: 2p23 (GCKR, best SNP rs1260326, P = 2.04 × 10(-17)), 2q13-q14 (PROC, rs1158867, P = 3.77 × 10(-36)), 20q11 (near and within PROCR, rs8119351, P = 2.68 × 10(-203)), and 20q11.22 (EDEM2, rs6120849, P = 7.19 × 10(-37) and 5.23 × 10(-17) before and after conditional analysis, respectively). All 4 loci replicated in the independent sample. Furthermore, pooling the discovery and replication sets yielded an additional locus at chromosome 7q11.23 (BAZ1B, rs17145713, P = 2.83 × 10(-8)). The regions marked by GCKR, EDEM2, and BAZ1B are novel loci that have not been previously reported for association with protein C concentration. In summary, this first genome-wide scan for circulating protein C concentration identified both new and known loci in the general population. These findings may improve the understanding of physiologic mechanisms in protein C regulation.

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.

Genetic variants in TLR2 and TLR4 are associated with markers of monocyte activation: the Atherosclerosis Risk in Communities MRI Study.

Markers of monocyte activation play a critical role in atherosclerosis, but little is known about the genetic influences on cellular levels. Therefore, we investigated the influence of genetic variants in monocyte differentiation antigen (CD14), toll-like receptor-4 (TLR4), toll-like receptor-2 (TLR2), and myeloperoxidase (MPO) on monocyte surface receptor levels. The study sample consisted of 1,817 members of a biracial cohort of adults from the Atherosclerosis Risk in Communities Carotid MRI Study. Monocyte receptors were measured using flow cytometry on fasting whole blood samples. TLR2 rs1816702 genotype was significantly associated with CD14+/TLR2+ percent of positive cells (%) and median fluorescence intensity (MFI) in whites but not in blacks (p < 0.001). Specifically, the presence of the minor T-allele was associated with increased receptor levels. In blacks, TLR4 rs5030719 was significantly associated with CD14+/TLR4+ monocytes (MFI) with mean ± SE intensities of 16.7 ± 0.05 and 16.0 ± 0.14 for GG and GT/TT genotypes, respectively (p < 0.001). Variants in TLR2 and TLR4 were associated with monocyte receptor levels of TLR2 and TLR4, respectively, in a biracial cohort of adults. To our knowledge, this is the first study to look at associations between variants in the toll-like receptor family and toll-like receptor levels on monocytes.

Novel hemostatic factor levels and risk of ischemic stroke: the Atherosclerosis Risk in Communities (ARIC) Study.

BACKGROUND AND OBJECTIVE: The role of hemostatic factor levels in cerebral infarction remains uncertain. We studied the association of levels of several under-studied hemostatic factors with ischemic stroke in a population-based cohort. METHODS: The Atherosclerosis Risk in Communities (ARIC) study includes 15,792 individuals aged 45-54 years at intake. Hemostatic factors II, V, IX, X, XI, XII, plasminogen and alpha(2)-antiplasmin were measured on frozen citrate plasma samples from 1990 to 1992. A case-cohort design was used, including all incident ischemic strokes (n = 89) over a median of 7.5 years and a stratified cohort random sample (n = 412). To determine the association of hemostatic factors with incident ischemic stroke, we computed hazard ratios (HRs) using multivariate proportional hazard regression analyses adjusted for demographic and other cardiovascular risk factors. RESULTS: The cohort random sample had a mean age (SD) of 56.9 (5.4) years and 42% were men. The age-, sex- and race-adjusted HRs for highest versus lowest quartiles were: factor XI (2.74, 95% CI 1.42-5.29), factor IX (1.92, 95% CI 0.99-3.73), and alpha(2)-antiplasmin (2.24, 95% CI 1.16-4.33). Correspondingly, the HRs of ischemic stroke per SD increment of factors XI, IX, and alpha(2)-antiplasmin were 1.64, 1.46 and 1.52, respectively (all p < 0.05). After multivariate adjustment including other clinical variables, the standardized HR remained significant for factor XI (1.50, 95% CI 1.10-2.05), but no other factor. CONCLUSION: A greater level of factor XI was associated with an increased risk of ischemic stroke. Higher factor XI levels might help identify patients at elevated ischemic stroke risk.

Atherosclerosis Risk in Communities (ARIC) Carotid MRI flow cytometry study of monocyte and platelet markers: intraindividual variability and reliability.

BACKGROUND: Cellular markers help identify different components of a pathological process and may contribute to the diagnosis, prognostic assessment, and management of patients with suspected syndromes. Flow cytometry can be used to accurately assess markers of platelet and leukocyte activation and cellular aggregation in whole blood. To use cell markers as predictors of disease requires that they be measured reliably and show modest within-individual, day-to-day variation. METHODS: We used whole blood flow cytometry to analyze monocyte and platelet markers in the Atherosclerosis Risk in Communities (ARIC) Carotid MRI study. We estimated laboratory variability using 20 split samples, process variation using replicate blood tubes taken from 112 subjects, and biologic plus process variation using replicate blood samples taken 4-8 weeks apart from 55 people. RESULTS: For most analytes, the laboratory CV was <10% (mean 3.6%, range 0%-14.5%) and reliability was excellent (75% of analytes had R > 0.90). Reliability coefficients based on repeat-visit data indicated substantial to high repeatability (R > 0.60) for CD14, Toll-like receptor (TLR)-2, CD162, CD61, CD41, CD62P, CD154, and platelet-leukocyte aggregates. In contrast, TLR-4, CD45, myeloperoxidase (MPO), and cyclooxygenase (COX)-2 had slight to moderate repeat visit reliability. CONCLUSIONS: The high repeatability results for selected platelet and monocyte markers indicate that they can be reliably measured in multicenter studies with delayed sample processing, provided that rigorous standardization of sample collection, shipping, and flow cytometry procedures is applied.

Interaction between soluble thrombomodulin and intercellular adhesion molecule-1 in predicting risk of coronary heart disease.

BACKGROUND: Results from previous ARIC (Atherosclerosis Risk In Communities) analyses indicate that soluble intercellular adhesive molecule-1 (sICAM) and soluble thrombomodulin (sTM) levels are associated with risk of coronary heart disease (CHD) in an opposite direction. A high sICAM level increases the risk of CHD, whereas a high level of sTM has a lower risk of CHD. It was unclear whether there was an interaction between sTM and sICAM. METHODS AND RESULTS: Using a nested case-cohort design, we measured sTM and sICAM in 317 incident CHD cases and 726 non-cases from the ARIC participants. Consistent with our previous reports, sICAM values in the upper versus the lower tertile increased the risk of CHD event by approximately 2-fold (95% confidence interval [CI], 1.46 to 2.87) whereas sTM values in the lower versus the upper tertile increased CHD risk by approximately 4-fold (95% CI, 2.80 to 5.74). Interaction between these 2 parameters was determined by weighted Cox proportional hazard regression. A significant interaction (P=0.038) was noted. Combinatorial analysis shows a significant increase in CHD risk ratio (RR) (4.66, 95% CI, 1.89 to 11.46) of the lower sTM/upper sICAM group versus the upper sTM/lower sICAM group. Individuals whose sTM values were in the upper tertile had a RR below 1, even when sICAM were in the upper tertile. The RR of lower tertile sTM was increased by sICAM in a tertile-dependent manner. CONCLUSIONS: Weighted Cox proportional hazard analysis shows a significant interaction between sTM and sICAM in predicting risk of CHD event. Combinatorial analysis reveals that an upper tertile sICAM had a significant increase in the risk of a CHD event only when sTM was in the lower tertile.

Cyclooxygenase-2 inhibitor treatment improves left ventricular function and mortality in a murine model of doxorubicin-induced heart failure.

BACKGROUND: Progression of heart failure after initial myocardial injury is mediated in part by various redundant inflammatory mediators, including the widely expressed cyclooxygenase-2 (COX-2). Because COX-2 inhibitors are useful in treating many inflammation-mediated diseases, we asked whether COX-2 inhibition can attenuate heart failure progression. METHODS AND RESULTS: Heart failure was experimentally induced in 100 mice by administration of doxorubicin (4 mg. kg(-1). wk(-1) for 6 weeks). Beginning at day 42, mice were fed daily with either COX-2 inhibitor-containing mice chow (n=50) or plain mice chow (controls; n=50). Left ventricular ejection fraction was evaluated as a measure of heart failure by a novel method of transthoracic echocardiography (with intravascular ultrasound catheters) at baseline and on days 42, 56, and 70. From baseline to study termination, left ventricular ejection fraction in COX-2 inhibitor-treated mice decreased significantly less than in control mice (9% versus 29%, P<0.01). Mortality was significantly lower for COX-2 inhibitor-treated mice than for control mice (18% versus 38%, P<0.01). These results were confirmed in a revalidation study in COX-2 inhibitor-treated mice (n=25) and controls (n=25). That study revealed that the hearts from control mice weighed roughly the same as hearts from COX-2 inhibitor-treated mice but showed more extensive signs of cardiomyopathy (as determined by pathological analysis by an independent, blinded observer) and higher levels of COX-2 proteins (as determined by immunoblotting [6442+/-1635 versus 4300+/-2408 arbitrary units, P<0.022]). CONCLUSIONS: COX-2 inhibitors can attenuate the progression of heart failure in a murine model of doxorubicin-induced heart failure.

Protein C, antithrombin, and venous thromboembolism incidence: a prospective population-based study.

Although deficiencies of protein C and antithrombin, 2 natural plasma anticoagulants, are known risk factors for venous thrombosis, population-based prospective incidence data on these associations are lacking. Venous thromboembolic events have been identified in adults in 2 longitudinal cohort studies, the Atherosclerosis Risk in Communities (ARIC) Study and the Cardiovascular Health Study (CHS). Incidence was examined in relation to prediagnostic plasma levels of protein C (ARIC Study only) and antithrombin. Over a mean of 8.1 years of follow-up, there were 130 incident venous thromboembolic events that were not due to cancer in the ARIC Study. The age-adjusted incidence was elevated 3.36-fold (95% CI 1.24 to 9.11) in the 1.1% of subjects with protein C values <2.0 mg/L compared with subjects with higher values. In contrast, in the ARIC Study and the CHS, there was no association between low plasma antithrombin and venous thromboembolism. In conclusion, in this population-based study, a low protein C, but not antithrombin, level has been determined to be associated with an increased incidence of venous thromboembolism. Attributable risk estimates suggest that low protein C levels account for approximately 2.5% of venous thromboembolic events in the ARIC population.

Factor XIIIA Val34Leu polymorphism does not predict risk of coronary heart disease: The Atherosclerosis Risk in Communities (ARIC) Study.

Factor XIII catalyzes the cross-linking of fibrin. Cross-sectional studies have suggested that a common polymorphism site at residue 34 of the A subunit of factor XIII (FXIIIA) with a substitution of Leu for Val (FXIIIA Val34Leu) was associated with reduced coronary heart disease (CHD). This association has not been examined in prospective studies. Healthy subjects (n=15 792) were recruited from 4 US communities into the Atherosclerosis Risk in Communities (ARIC) study from 1987 to 1989. They were followed, and incident CHD events were identified and verified. For the present study, we included 423 patients with CHD as cases and 479 noncases. FXIIIA Val34Leu polymorphism was determined by the single-strand conformational polymorphism method. There were no significant differences in the genotype frequencies between cases and noncases. The genotypes showed little association with known CHD risk factors. A weighted proportional hazards regression analysis adjusting for other risk factors confirmed no association of the genotypes with risk of CHD. This prospective study did not provide evidence of a reduced CHD risk for the FXIIIA 34Leu allele.

Human fallopian tubes express prostacyclin (PGI) synthase and cyclooxygenases and synthesize abundant PGI.

Animal studies unequivocally support the indispensable role of prostaglandin (PG) and cyclooxygenase (COX) in ovulation and implantation. Available data also suggest that PG and COX may be important in the transport of embryos. The effects of PGE(2) and PGF(2alpha) on the contractility of human tubal muscle have been studied extensively; the expression of COX in human fallopian tubes was also reported. Despite all these, two fundamentally important questions remained to be answered: 1) which PGs are produced by human fallopian tubes; and 2) which COX isoform(s) is expressed by the fallopian tubes. We used reverse-phase HPLC to study the metabolism of [1-(14)C] arachidonic acid by the fallopian tubes. We found that 6 keto-PGF(1alpha), a stable metabolite of prostacyclin (PGI), and PGE(2) constituted 56% +/- 10% and 35% +/- 10% (mean +/- SEM, four samples), respectively, of total eicosanoids synthesized. Western blot analysis revealed the expression of both COX isoforms. Immunohistochemistry study showed that both COX-1 and -2 were localized to nonciliated epithelia and tubal smooth muscle. In addition, COX-2 was also expressed in ciliated epithelial cells. Western blot analysis revealed the expression of PGI synthase (PGIS) and PGI receptor by fallopian tubes. Immunohistochemistry confirmed the expression of PGIS by luminal epithelia, tubal smooth muscle, vascular endothelial cells, and vascular smooth muscle cells. Iloprost, a PGI analog, inhibited the activities of circular and longitudinal muscles of the fallopian tube. Thus, the fallopian tube expresses both COX isoforms and PGIS. Furthermore, it is a source and a target of PGI. PGI and COX may be important to gamete function, embryo transport, and embryo development.

Coagulation factors, inflammation markers, and venous thromboembolism: the longitudinal investigation of thromboembolism etiology (LITE).

PURPOSE: We sought to assess prospectively whether higher levels of blood coagulation factors and inflammation markers are risk factors for venous thromboembolism. SUBJECTS AND METHODS: In two pooled population-based cohort studies, we measured levels of factor VII, factor VIII, von Willebrand factor, fibrinogen, and C-reactive protein, and white blood cell count, in samples obtained from 19,237 adults with no baseline history of venous thromboembolism, cancer, or warfarin use. The endpoint was validated venous thromboembolism during follow-up (median, 7.8 years). RESULTS: A total of 159 venous thromboembolism events occurred. Factor VIII and von Willebrand factor were linearly associated with increased risk of venous thromboembolism (P for trend <0.0001). As compared with those in the lowest quartile, the multivariate-adjusted hazard ratio (HR) of venous thromboembolism was 2.6 (95% confidence interval [CI]: 1.6 to 4.3) for factor VIII levels in the highest quartile and 3.8 (95% CI: 2.0 to 7.2) for the highest fifth percentile. For von Willebrand factor, the hazard ratios in middle-aged subjects were 4.6 (95% CI: 2.2 to 9.2) for the highest quartile and 7.6 (95% CI: 3.1 to 18) for the highest fifth percentile. Factor VII levels above the 95th percentile, as compared with the lowest quartile, also conveyed a higher risk of venous thromboembolism (HR = 2.4; 95% CI: 1.2 to 4.8). In contrast, there was no association of venous thromboembolism with fibrinogen or C-reactive protein levels, or white cell count. CONCLUSIONS: In this prospective study, elevated factor VIII and von Willebrand factor levels were common, independent, and dose-dependent risk factors for venous thromboembolism, and an elevated factor VII level was a possible risk factor. Venous thromboembolism, unlike arterial disease, was not related to inflammatory markers.

C-reactive protein and incident coronary heart disease in the Atherosclerosis Risk In Communities (ARIC) study.

BACKGROUND: Recent evidence implicates inflammation in the pathogenesis of coronary heart disease (CHD). C-reactive protein, a plasma marker of inflammation, is a marker of CHD risk but has been studied in few prospective investigations of the general population. METHODS AND RESULTS: We prospectively examined the association of CRP with incident CHD among middle-aged adults in the Atherosclerosis Risk In Communities (ARIC) study. With the use of a nested case-cohort approach, we measured CRP in stored, baseline blood samples of 2 groups of subjects in whom CHD developed during follow-up (242 incident cases from 1987 to 1993 and 373 from 1990 to 1995) and, for comparison, 2 stratified random samples of noncases. In analyses adjusted for demographic variables and traditional CHD risk factors, the relative risk of CHD across quintiles of CRP was 1.0, 0.8, 1.6, 1.9, and 1.5 for events from 1987 to 1995 (P for trend =.01). As expected, inclusion of fibrinogen, intracellular adhesion molecule-1, and white blood cell count (other potential markers of the inflammatory reaction) attenuated the association of CRP with CHD incidence. In a supplemental cross-sectional analysis, CRP was not associated with carotid intima-media thickness after adjustment for major risk factors. CONCLUSIONS: C-reactive protein is a moderately strong marker of risk of CHD in this cohort of middle-aged adults, consistent with the role of inflammation in the pathogenesis of CHD events. The association was not specific to CRP because other markers of inflammation could largely account for the finding.

Prospective study of fibrinolytic markers and venous thromboembolism.

Prior research has conflicted on whether increased levels of plasma fibrinolytic factors may identify patients at risk of venous thromboembolism (VTE). We therefore performed a nested case-control study of VTE within two prospective population-based studies. In 308 participants who developed VTE and 640 controls, we measured PAI-1 antigen, tPA/PAI-1 complex, plasmin-alpha 2-antiplasmin (PAP), and the PAI-1 -675 4G/5G promoter polymorphism on pre-event blood samples. There was no overall association between any of these fibrinolytic variables and VTE, after adjustment for age or for multiple VTE risk factors. There was weak evidence for an interaction of PAP with elevated factor VIIIc and elevated D-dimer in augmenting VTE risk. We conclude that, for the most part, these fibrinolytic markers do not identify healthy subjects at risk for VTE.

Coagulation factors II, V, IX, X, XI, and XII, plasminogen, and alpha-2 antiplasmin and risk of coronary heart disease.

AIM: To examine whether plasma levels of coagulation factors II, V, IX, X, XI, and XII, plasminogen, and alpha-2 antiplasmin are associated with coronary heart disease (CHD) in a prospective case-cohort study. METHODS: This case-cohort sample consisted of 368 African-Americans or whites with incident CHD that occurred between 1990-92 and 1998, from the Atherosclerosis Risk in Communities (ARIC) study, and a cohort random sample of n=412. Hemostatic factors were measured in the case-cohort sample using plasma stored at -70 degrees C since 1990-92. RESULTS: After adjustments for age, sex and race, coagulation factors IX and XI, and alpha-2 antiplasmin were associated positively with risk of CHD: The hazard ratio [95% confidence interval] for the highest vs lowest quartiles was 1.52 [1.01-2.27] for factor IX; 2.26 [1.47-3.48] for factor XI; and 1.64 [1.05-2.57] for alpha-2 antiplasmin. However, as these hemostatic factors were correlated with classical risk factors, their association with CHD was attenuated and no longer statistically significant after multivariable adjustments. No association was observed between CHD and factor II, V, X, or XII, or plasminogen. CONCLUSIONS: Positive associations of factors IX and XI, and alpha-2 antiplasmin with incident CHD were not strong and accounted for by classical coronary risk factors.

Risk factor correlates of platelet and leukocyte markers assessed by flow cytometry in a population-based sample.

BACKGROUND: Platelet and leukocyte products are involved in atherothrombosis. However, the determinants of platelet and leukocyte markers assessed by flow cytometry have not been documented in a population-based sample. METHODS AND RESULTS: We performed flow cytometry on blood from participants (n=1894) in the Atherosclerosis Risk in Communities (ARIC) Carotid MRI Study. Cellular aggregates and multiple platelet and leukocyte markers, such as myeloperoxidase in granulocytes and toll-like receptor-4, CD14, and CD45 in monocytes, were quantified. Their cross-sectional associations with demographic and risk factors were assessed using multiple linear regression. Mean values of most cellular markers and aggregates were considerably higher in blacks than whites (p<0.01). There were some differences in cellular markers between men and women, but little association with age. LDL-cholesterol was associated positively with several markers (toll-like receptor-4 and myeloperoxidase in granulocytes and CD162 in lymphocytes). Cholesterol-lowering therapy tended to show opposite associations. Smokers had much higher granulocyte myeloperoxidase than nonsmokers. However, most other correlations between risk factors and cellular markers were nonsignificant. CONCLUSIONS: Race/ethnicity, sex, and to a lesser degree LDL-cholesterol and cholesterol-lowering therapy, but few other risk factors, were correlated with markers of cellular activation in this population-based study.

Assessment of coronary heart disease risk by combined analysis of coagulation factors.

OBJECTIVE: Prospective studies have reported a positive association of coagulation factors with risk of coronary heart disease (CHD). It is unclear whether these coagulation factors interact. METHODS AND RESULTS: Using a prospective case-cohort design, we analyzed by Cox proportional hazard regression interactions between soluble thrombomodulin (sTM) and fibrinogen, factor VIII (FVIII), FVII, or plasminogen activator inhibitor-1 (PAI-1) in 410 CHD cases and 721 non-cases from the Atherosclerosis Risk in Communities (ARIC). There was a significant interaction between sTM and fibrinogen (p=0.027). We next assessed risk ratios (RR) by combined tertile analysis. Combined analysis revealed that being in the upper sTM tertile counteracted the CHD risk imposed by higher fibrinogen whereas being in the lower sTM tertile amplified the CHD risk of higher fibrinogen. sTM and fibrinogen mutually influenced CHD incidence in a concentration-dependent manner. When analyzed as single factors by tertiles, FVIII, FVII and PAI-1 were not associated with CHD. However, when analyzed together with sTM, FVIII and PAI-1 were both positively associated with CHD for those in the lower sTM tertile. CONCLUSION: There is a complex interaction between sTM and prothrombotic coagulation factors. Combined analysis improves CHD risk assessment.

Prostacyclin inhibits endothelial cell XIAP ubiquitination and degradation.

To understand the role of prostacyclin (PGI(2)) in protecting endothelial cells from apoptosis, we evaluated the effects of carbaprostacyclin (cPGI(2)) on H(2)O(2)-induced human umbilical vein endothelial cell (HUVEC) apoptosis. cPGI(2) suppressed H(2)O(2)-induced annexin V-positive cells in a concentration- and time-dependent manner. Pre-treatment of HUVEC with 50 microM cPGI(2) for 4 h produced the maximal anti-apoptotic effect. Authentic PGI(2) generated by adenoviral transfer of PGI(2) synthetic genes exerted a similar protective effect. cPGI(2) inhibited Smac/DIABLO release from mitochondria, caspase 3 activation, focal adhesion protein degradation, and cell detachment. cPGI(2) selectively protected X-linked inhibitor of apoptosis protein (X-linked IAP, XIAP) from H(2)O(2)-induced ubiquitination, and preserved XIAP protein levels. PD-98059 but not H-89 abrogated the protective action of cPGI(2). cPGI(2) increased ERK phosphorylation which was blocked by PD-98059. HUVEC stably transfected with dominant negative Ras abrogated XIAP preservation by cPGI(2) while constitutive active Ras increased ERK phosphorylation and protected XIAP from degradation. Our results demonstrate for the first time that PGI(2) inhibits XIAP ubiquitination and degradation via the Ras/MEK-1/ERK signaling pathway. Preservation of XIAP proteins represents a key mechanism by which PGI(2) protects endothelial cells from oxidant-induced apoptosis.

Molecular aspects of thrombosis and antithrombotic drugs.

There have been major advances in our understanding of thrombosis and antithrombotic drugs. This review focuses on the molecular aspects of thrombus formation and antithrombotic therapy. Molecules involved in arterial thrombosis are derived from inflammatory cells in the atherosclerotic plaque and blood platelets. These molecules work in concert to promote plaque instability and thrombogenicity. Thrombus formation on the ruptured plaque is mediated by platelet and coagulation activation. By contrast, molecules involved in venous thrombosis are derived from the activated coagulation cascade. Platelets appear to play a secondary role. The antithrombotic drugs are classified according to their targeted constituents: antiplatelet agents and anticoagulants; the latter are further divided into non-specific anticoagulants, such as vitamin K antagonists and heparin, and direct thrombin inhibitors, including hirudin and argatroban. Currently available antiplatelet agents target glycoprotein IIbIIIa (abciximab, tirofiban, eptifibatide), cyclooxygenase-1 (aspirin) or adenosine diphosphate receptor, P2Y12 (clopidogrel).

Mitochondrial localization of cyclooxygenase-2 and calcium-independent phospholipase A2 in human cancer cells: implication in apoptosis resistance.

Cyclooxygenase-2 (COX-2) is inducible by myriad stimuli. The inducible COX-2 in primary cultured human cells has been reported to localize to nuclear envelope, endoplasmic reticulum, nucleus and caveolae. As COX-2 plays an important role in tumor growth, we were interested in its subcellular location in cancer cells. We examined COX-2 localization in several cancer cell lines by confocal microscopy. A majority of COX-2 was colocalized with heat shock protein 60, a mitochondrial protein, in colon cancer (HT-29, HCT-15 and DLD-1), breast cancer (MCF7), hepatocellular cancer (HepG2) and lung cancer cells (A549) with a similar distribution pattern. By contrast, COX-2 was not localized to mitochondria in human foreskin fibroblasts or endothelial cells. Immunoblot analysis of COX-2 in mitochondrial and cytosolic fractions confirmed localization of COX-2 to mitochondria in HT-29 and DLD-1 cells but not in fibroblasts. Calcium-independent phospholipase A2 was colocalized with heat shock protein 60 to mitochondria not only in cancer cells (HT-29 and DLD-1) but also in fibroblasts. HT-29 which expressed more abundant mitochondrial COX-2 than DLD-1 was highly resistant to arachidonic acid and H2O2-induced apoptosis whereas DLD-1 was less resistant and human fibroblasts were highly susceptible. Treatment of HT-29 cells with sulindac or SC-236, a selective COX-2 inhibitor, resulted in loss of resistance to apoptosis. These results suggest that mitochondrial COX-2 in cancer cells confer resistance to apoptosis by reducing the proapoptotic arachidonic acid.