Endothelial protein C receptor polymorphisms and risk of myocardial infarction

Haplotypes A1 and A3 in the endothelial protein C receptor gene are tagged by the 4678G/C and 4600A/G polymorphisms, respectively, and have been reported to influence the risk of venous thromboembolism. This study shows that A1 and A3 haplotype carriers have a reduced risk of myocardial infarction.  Haplotypes A1 and A3 in the endothelial protein C receptor gene are tagged by the 4678G/C and 4600A/G polymorphisms, respectively, and have been reported to influence the risk of venous thromboembolism. We assessed whether these haplotypes modify the risk of premature myocardial infarction.  We genotyped these polymorphisms in 689 patients with premature myocardial infarction and 697 control subjects. Activated protein C and soluble endothelial protein C receptor levels were also measured.  After adjustment for other cardiovascular risk factors, A1 and A3 haplotypes protected against premature myocardial infarction (odds ratio 0.7, 95% CI 0.4–0.8, p=0.044 and 0.5, 0.3–0.6, p<0.001, respectively). Moreover, the protective role of these haplotypes seemed to be additive, as carriers of both the A1 and A3 haplotypes had adjusted odds ratios of 0.3 (0.2–0.5, p<0.001) and 0.4 (0.2–0.8, p=0.006) compared to those carrying only the A1 or A3 haplotype, respectively. The presence of the A1 haplotype was associated with increased levels of activated protein C whereas individuals carrying the A3 haplotype showed the highest soluble endothelial protein C receptor levels.  These results show that A1 haplotype carriers have a reduced risk of premature myocardial infarction via the association of this haplotype with increased activated protein C plasma levels. The study also shows that carriers of the A3 haplotype have a reduced risk of myocardial infarction, only in part due to increased soluble endothelial protein C levels.

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title: Elevated factor XIII level and the risk of myocardial infarction in women.
abstract: Factor XIII (FXIII) activity and antigen levels were determined in 955 patients investigated by coronary angiography. Patients were sub-grouped according to the presence or absence of coronary sclerosis (CS+, CS-) and a positive history of myocardial infarction (MI+, MI-). In females, but not in males, adjusted FXIII activity and antigen levels were significantly elevated in the CS+MI+ group compared to in the CS+MI- group. FXIII levels in the upper tertile were associated with significantly increased risk of MI in females, but not in males.

title: A polymorphism of a platelet glycoprotein receptor as an inherited risk factor for coronary thrombosis.
abstract: Platelet glycoprotein IIb/IIIa is a membrane receptor for fibrinogen and von Willebrand factor, and it has an important role in platelet aggregation. It is known to be involved in the pathogenesis of acute coronary syndromes. Previously, we found a high frequency of a particular polymorphism, PlA2, of the gene encoding glycoprotein IIIa in kindreds with a high prevalence of premature myocardial infarction.



To investigate the relation between the PlA2 polymorphism and acute coronary syndromes, we conducted a case-control study of 71 case patients with myocardial infarction or unstable angina and 68 inpatient controls without known heart disease. The groups were matched for age, race, and sex. We used two methods to determine the PlA genotype: reverse dot blot hybridization and allele-specific restriction digestion.



The prevalence of PlA2 was 2.1 times higher among the case patients than among the controls (39.4 percent vs. 19.1 percent, P=0.01). In a subgroup of patients whose disease began before the age of 60 years, the prevalence of PlA2 was 50 percent, a value that was 3.6 times that among control subjects under 60 years of age (13.9 percent, P=0.002). Among subjects with the PlA2 polymorphism, the odds ratio for having a coronary event was 2.8 (95 percent confidence interval, 1.2 to 6.4). In the patients less than 60 years of age at the onset of disease, the odds ratio was 6.2 (95 percent confidence interval, 1.8 to 22.4).



We observed a strong association between the PlA2 polymorphism of the glycoprotein IIIa gene and acute coronary thrombosis, and this association was strongest in patients who had had coronary events before the age of 60 years.

title: Role of hemostatic gene polymorphisms in venous and arterial thrombotic disease.
abstract: Venous thromboembolic disorders form a spectrum of conditions characterized by in situ thrombus formation and the variable presence of embolic manifestations. The clinical presentation depends to a large extent on the site and extent of thrombus formation and on the underlying cause. In the absence of genetic deficiencies, thrombosis occurs in the older population, largely in the context of marked environmental influences such as surgery, obesity, and underlying malignancy. In contrast, at the other extreme, familial thrombosis associated with mutations in genes for protein S, protein C, or antithrombin is associated with younger age of onset, lesser environmental stimulus, and thrombus formation often in unusual sites. There is an increasing awareness of the importance of hemostatic mechanisms in the pathogenesis of venous thrombosis. The hemostatic process comprises coagulation and fibrinolytic systems with activators, zymogens, cofactors, surfaces (such as platelets), and inhibitors. The function and regulation of these composite systems and factors have been extensively studied. Understanding their functional integration in vivo, however, still presents a major challenge. A starting point for a simplified model of integration envisages a balance between procoagulant (clot-promoting) and anticoagulant (clot-preventing) mechanisms. This balance serves to prevent an explosive generation of thrombin when coagulation is triggered. The balance is determined by the (functional) levels of all coagulation and fibrinolytic factors, with increases in the levels of coagulation factors and decreases in the levels of both fibrinolytic factors and natural inhibitors being procoagulant. The role of inhibitory mechanisms in preventing venous occlusion was established after the identification of the link between inherited deficiencies of antithrombin, protein C, protein S, and venous thrombosis during the 1960s and the 1970s. The general importance of the anticoagulant pathway involving protein C and protein S was established by the widespread prevalence among white populations and the clinical consequences of the phenotype of activated protein C resistance (APCR) and its main causative gene mutation, factor V G1691A (factor V Arg506Gln, factor V Leiden). There has been slower appreciation of the procoagulant effect of increased levels of coagulation factors in venous disease, but recent work has highlighted the likely importance of prothrombin and factor VIII levels in this regard.

title: Myocardial infarction in young adults: angiographic characterization, risk factors and prognosis (Coronary Artery Surgery Study Registry).
abstract: This study examined the angiographic characteristics, coronary risk factors and prognosis in young men and women with a history of myocardial infarction compared with that in older patients.



There are few data regarding myocardial infarction in young adults. It is undetermined whether the development of myocardial infarction at a young age represents a form of coronary heart disease with an adverse prognosis.



Of the 8,839 patients with a history of myocardial infarction in the Coronary Artery Surgery Study (CASS), there were 294 men < or = 35 years old and 210 women < or = 45 years old. Coronary anatomy, baseline characteristics and prognosis were compared in younger and older patients.



Young men and women more often had angiographically normal coronary arteries, nonobstructive disease < 70% stenosis and single-vessel disease than older patients (p < 0.0001). Current smoking was more frequent in young patients (p < 0.0001). Hypertension and diabetes were more frequent in both older men and women, whereas a positive family history of premature coronary disease was significantly more prevalent only in young men. The survival rate at 7 years was improved for young men compared with that in older men (84% vs. 75%, p = 0.0094) and for young women compared with that in older women (90% vs. 77%, p = 0.0004). When multivariate analysis was applied to the data, the survival advantage for young patients remained after adjustment.



Young patients with a myocardial infarction have a favorable prognosis compared with that in older patients.

title: Myocardial infarction in young people with normal coronary arteries
abstract: Myocardial infarction occurring in young people with angiographically normal coronary arteries is well described but the pathophysiology of this condition remains unknown. Coronary artery spasm in association with thrombus formation and minimal atheromatous disease or spontaneous coronary artery dissection are possible causes. Two young men presented with severe chest pain after acute alcohol intoxication and each sustained an extensive anterior myocardial infarction. Investigations including intravascular ultrasound showed no evidence of atherosclerotic coronary artery disease. Coronary artery spasm associated with acute alcohol intoxication as well as a prothrombotic state and endothelial damage related to cigarette smoking may be mechanisms leading to acute myocardial infarction in these cases. Acute myocardial infarction occurs in young persons with normal coronary arteries and the diagnosis should be considered in young patients presenting with severe chest pain, particularly those abusing cocaine or alcohol, so that reperfusion therapy can be initiated promptly.

title: Inflammation, sepsis, and coagulation.
abstract: The molecular links between inflammation and coagulation are unquestioned. Inflammation promotes coagulation by leading to intravascular tissue factor expression, eliciting the expression of leukocyte adhesion molecules on the intravascular cell surfaces, and down regulating the fibrinolytic and protein C anticoagulant pathways. Thrombin, in turn, can promote inflammatory responses. This creates a cycle that logically progresses to vascular injury as occurs in septic shock. Most complex systems are regulated by product inhibition. This inflammation-coagulation cycle seems to follow this same principle with the protein C pathway serving as the regulatory mechanism. The molecular basis by which the protein C pathway functions as an anticoagulant is relatively well established compared to the mechanisms involved in regulating inflammation. As one approach to identifying the mechanisms involved in regulating inflammation, we set out to identify novel receptors that could modulate the specificity of APC in a manner analogous to the mechanisms by which thrombomodulin modulates thrombin specificity. This approach led to the identification of an endothelial cell protein C receptor (EPCR). To understand the mechanism, we obtained a crystal structure of APC (lacking the Gla domain). The crystal structure reveals a deep groove in a location analogous to anion binding exosite 1 of thrombin, the location of interaction for thrombomodulin, platelet thrombin receptor and fibrinogen. Thrombomodulin blocks the activation of platelets and fibrinogen without blocking reactivity with chromogenic substrates or inhibitors. Similarly, in solution, EPCR blocks factor Va inactivation without modulating reactivity with protease inhibitors. Thus, these endothelial cell receptors for the protein C system share many properties in common including the ability to be modulated by inflammatory cytokines. Current studies seek to identify the substrate for the APC-EPCR complex as the next step in elucidating the mechanisms by which the protein C pathway modulates the response to injury and inflammation.

title: The multifunctional protein C system.
abstract: The protein C pathway is a major regulator of blood coagulation, since it controls the conversion of prothrombin to thrombin through a feedback inhibition mechanism. Protein C circulates in plasma as an inactive zymogen and is activated on the surface of endothelial cells by the thrombin-thrombomodulin complex, a process that can be further enhanced when protein C binds to its membrane receptor, the endothelial-cell protein C receptor. Activated protein C (APC) is then released from the complex, binds protein S and inhibits thrombin formation by inactivating coagulation factors Va and VIIIa. The importance of the protein C anticoagulant pathway is emphasized by the increased risk of venous thromboembolism (VTE) associated with protein C and protein S deficiencies, the factor V Leiden mutation, and reduced circulating APC levels. The protein C pathway also plays a significant role in inflammatory processes, since it prevents the lethal effects of E. coli-associated sepsis in animal models and improves the outcome of patients with severe sepsis. APC seems to display anti-apoptotic and neuroprotective activities. Thus, it reduces organ damage in animal models of sepsis, ischemic injury, endothelial cell injury, or stroke. Further research will hopefully widen the current therapeutic perspectives in all these illnesses, where these effects might play a crucial role in their treatment. This review will summarize the mechanisms that contribute to these biological activities of the protein C pathway.

title: Regulation and functions of the protein C anticoagulant pathway.
abstract: The protein C pathway plays a critical role in the negative regulation of the blood clotting process. We recently identified an endothelial cell receptor for protein C/activated protein C (APC). The receptor is localized almost exclusively on endothelial cells of large vessels and is present at only trace levels or indeed absent from capillaries in most tissues. Patients with sepsis or lupus erythematosus exhibit elevated levels of plasma EPCR which migrates on gels as a single band and is fully capable of binding protein C/APC. There is no correlation with thrombomodulin levels, probably due to different vascular localizations and/or cellular release mechanisms. To understand the mechanisms by which EPCR plasma levels are elevated, we examined EPCR mRNA expression in a rat endotoxin shock model. The EPCR mRNA gene exhibited an early immediate gene response to endotoxin with the mRNA levels increasing nearly 4 fold in the first 3-6 hrs, before returning toward baseline. Plasma levels of EPCR also rose about 4 fold with little change in tissue EPCR levels. Both processes were markedly attenuated by hirudin suggesting that thrombin was responsible for increases in mRNA and plasma EPCR levels. At the level of mRNA, the induction is mediated by a thrombin response element in the 5' flanking region of the gene. Direct thrombin infusion and cell culture experiments support this contention. On endothelium, thrombin is capable of releasing cell surface EPCR and this process is blocked by the metalloproteinase inhibitor orthophenanthroline. Taken together these studies indicate that elevation in soluble plasma EPCR reflects endothelial cell activation in the larger vessels and is likely to be an indication of local thrombin generation near these vessel surfaces.

title: Reconstitution of the Human Endothelial Cell Protein C Receptor with Thrombomodulin in Phosphatidylcholine Vesicles Enhances Protein C Activation*
abstract: Blocking protein C binding to the endothelial cell protein C receptor (EPCR) on the endothelium is known to reduce protein C activation rates. Now we isolate human EPCR and thrombomodulin (TM) and reconstitute them into phosphatidylcholine vesicles. The EPCR increases protein C activation rates in a concentration-dependent fashion that does not saturate at 14 EPCR molecules/TM. Without EPCR, the protein C concentration dependence fits a single class of sites (K m = 2.17 ± 0.13 μm). With EPCR, two classes of sites are apparent (K m = 20 ± 15 nm andK m = 3.2 ± 1.7 μm). Increasing the EPCR concentration at a constant TM concentration increases the percentage of high affinity sites. EPCR ratio constant while decreasing the density of these proteins results in a decrease in the EPCR enhancement of protein C activation, suggesting that there is little affinity of the EPCR for TM. Negatively charged phospholipids also enhance protein C activation. EPCR acceleration of protein C activation is blocked by anti-EPCR antibodies, but not by annexin V, whereas the reverse is true with negatively charged phospholipids. Human umbilical cord endothelium expresses approximately 7 times more EPCR than TM. Anti-EPCR antibody reduces protein C activation rates 7-fold over these cells, whereas annexin V is ineffective, indicating that EPCR rather than negatively charged phospholipid provide the surface for protein C activation. EPCR expression varies dramatically among vascular beds. The present results indicate that the EPCR concentration will determine the effectiveness of the protein C activation complex.

title: Autoantibodies against endothelial protein C receptor and the risk of a first deep vein thrombosis
abstract: The endothelial protein C receptor (EPCR) binds protein C and enhances its activation. Anti‐EPCR autoantibodies are found in patients with antiphospholipid syndrome and may explain the increased risk of thrombosis in these patients. Anti‐EPCR autoantibodies have been associated with fetal death and myocardial infarction in young women.  To determine whether anti‐EPCR autoantibodies are associated with deep vein thrombosis (DVT).  We measured plasma anti‐EPCR autoantibody levels in the Leiden Thrombophilia Study (LETS), a population‐based case–control study consisting of 474 patients with a first DVT and 474 control subjects.  The estimated risk of DVT was increased approximately 2‐fold in the presence of elevated IgA, IgG or IgM anti‐EPCR autoantibodies (i.e. levels above the 90th percentile as measured in the control subjects). The risk conferred by  increased in a dose‐dependent manner for IgA and IgG. When anti‐EPCR autoantibodies were considered in the co‐presence of lupus anticoagulant (LAC) the odds ratio (OR) was 6.1 [95% CI 1.3–27.9]. Anti‐EPCR without LAC remained associated with DVT (OR 1.6; 95% CI 1.2–2.1). Anti‐EPCR autoantibodies were associated with high levels of D‐dimer and soluble EPCR in controls, suggestive of a prothrombotic status induced by the autoantibodies.  This study demonstrates that the presence of anti‐EPCR autoantibodies is a moderate risk factor for DVT in the general population.

title: Autoantibodies against the endothelial receptor of protein C are associated with acute myocardial infarction in young women
abstract: Acute myocardial infarction (AMI) is rare among young women. The search for unknown risk factors is warranted. Endothelial protein C receptor (EPCR) is largely present at the endothelial surface of large arteries. No studies about association of anti‐EPCR autoantibodies (anti‐EPCR) with AMI are available.  Plasma IgA, IgM and IgG anti‐EPCR levels were measured by enzyme‐linked immunosorbent assay in 165 women younger than 45 years who survived a first AMI and 165 healthy women, matched by age and geographical origin.  Using the 90th percentile of IgA anti‐EPCR in the control group, IgA anti‐EPCR were independently associated with AMI after adjustment for cardiovascular risk factors (OR 5.1; 95% CI 1.7–15.6; P = 0.004). The risk apparently conferred by IgA anti‐EPCR increased dose‐dependently (P for trend =0.0002). IgM anti‐EPCR were less consistently associated with AMI: a significant increase in the risk was found when women above the 90th percentile were compared with those in the lowest quartile (OR 3.6; 95% CI 1.2–11.5; P = 0.03). IgG anti‐EPCR were similar in patients and controls. A total of 145 patients underwent coronary arteriography. IgA or IgM anti‐EPCR were not different among patients with different degrees of atherosclerotic lesion (anova, P = 0.77 and 0.24, respectively).  High levels of IgA and, to a lesser extent, IgM anti‐EPCR, are associated with AMI in young women.

title: A haplotype of the EPCR gene is associated with increased plasma levels of sEPCR and is a candidate risk factor for thrombosis.
abstract: The endothelial cell protein C (PC) receptor (EPCR) facilitates PC activation by the thrombin-thrombomodulin complex. A soluble form of this receptor (sEPCR) found in plasma inhibits both activated PC (aPC) activity and PC activation by competing for PC with membrane-associated EPCR. Elevated sEPCR levels are found in approximately 20% of healthy subjects, but the mechanisms underlying this interindividual variability are unknown. We measured sEPCR levels in 100 healthy male volunteers, and observed 2 phenotypic groups of subjects. The temporal stability of sEPCR levels suggested genetic control. Extensive analysis of the EPCR gene in these subjects revealed 13 polymorphisms in complete linkage disequilibrium; these defined 3 haplotypes, 1 of which (A3) was strongly associated with high sEPCR levels. The high constitutive sEPCR levels observed in A3 haplotype carriers might reduce the efficiency of the PC system and predispose these subjects to venous thrombosis. By studying 338 patients with venous thrombosis and 338 age- and sex-matched healthy subjects, we found that the A3 haplotype was overrepresented in the patients. In multivariate analysis, subjects carrying the A3 haplotype had an increased risk of thrombosis (odds ratio [OR] = 1.8; P =.004). Thus, the A3 haplotype, which is associated with elevated plasma sEPCR levels, is a candidate risk factor for venous thrombosis.

title: Contribution of polymorphisms in the endothelial protein C receptor gene to soluble endothelial protein C receptor and circulating activated protein C levels, and thrombotic risk.
abstract: Endothelial cell protein C receptor (EPCR) enhances the generation of activated protein C (APC) by the thrombin-thrombomodulin complex. A soluble form of EPCR (sEPCR), which is generated by metalloprotease activity, is present in plasma. The distribution of sEPCR levels in healthy populations is bimodal. Previously, we described two polymorphisms in exon 4 of the EPCR gene, 4600A/G that encodes the substitution of Ser219 by Gly in the transmembrane region of EPCR and 4678G/C in the 3'-UT region. The aim of this study was to investigate the relationship between these two polymorphisms and plasma sEPCR and APC levels and risk of venous thrombosis. We genotyped 401 healthy controls from the Spanish population and measured their plasma sEPCR and APC levels. Carriers of the 4600AG genotype had significantly higher sEPCR levels than those with the AA genotype, while the 4678CC genotype was associated, to a lesser extent, with elevated APC levels. To assess the effect of these polymorphisms on the risk of thrombosis, we genotyped 405 patients with venous thromboembolism. The frequency of the 4600AG genotype was very similar in patients and controls (p=0.975), whereas the 4678CC genotype was significantly more frequent in controls than in patients (p=0.008). In multivariate analysis, carriers of the 4678CC genotype had a decreased risk of thrombosis (OR=0.61, p=0.009). These data indicate that individuals carrying the 4600AG genotype have high sEPCR levels but do not have an increased risk of thrombosis, whereas individuals carrying the 4678CC genotype have higher APC levels and lower risk of venous thromboembolism.

title: Haplotypes of the EPCR gene, plasma sEPCR levels and the risk of deep venous thrombosis
abstract: Binding of protein C (PC) to the endothelial cell PC receptor (EPCR) stimulates PC activation by increasing the affinity of PC for the thrombin‐thrombomodulin complex. A soluble form of this receptor (sEPCR) circulates in plasma and inhibits both PC activation and APC anticoagulant activity.  The aim of this study was to investigate whether variations in the EPCR gene or plasma sEPCR levels are risk factors for deep venous thrombosis (DVT).  In a large case‐control study, the Leiden Thrombophilia Study (LETS), sEPCR levels were measured by ELISA. All subjects were genotyped for three haplotype‐tagging SNPs, enabling us to detect all four common haplotypes of the EPCR gene.  The distribution of sEPCR levels in the control population was trimodal and was genetically controlled by haplotype 3 (H3). This haplotype explained 86.5% of the variation in sEPCR levels. Carriers of two H3 alleles had higher sEPCR levels (439 ng mL−1) than carriers of one H3 allele (258 ng mL−1), which had higher levels than non‐H3 carriers (94 ng mL−1). Haplotype 4 was associated with a slightly increased risk (OR = 1.4, 95%CI:1.0–2.2). The risk of subjects with sEPCR levels in the top quartile (≥ 137 ng mL−1) was increased compared to that of subjects in the first quartile (< 81 ng mL−1), but since there was no dose–response effect, it is most likely that low sEPCR levels reduce the risk of DVT.  Our data do not suggest a strong association between EPCR haplotypes and thrombosis risk, but low sEPCR levels appear to reduce the risk of DVT.

title: EPCR Ser219Gly: elevated sEPCR, prothrombin F1+2, risk for coronary heart disease, and increased sEPCR shedding in vitro.
abstract: We have progressively analysed three studies of coronary heart disease (CHD) for a variant in EPCR (Ser219Gly). Initially, in a prospective study, NPHSII, while no overall CHD-risk was identified in heterozygotes, homozygotes for 219Gly exhibited a three-fold elevated risk (HR 3.3, CI 1.22-8.96). In diabetics within NPHSII, there was a suggestion that 219Gly+ was associated with elevated CHD-risk (HR 1.89, CI 0.39-9.06) although numbers were small. To further assess the effect of the variant in diabetes, a case-control study of MI, HIFMECH, was used, in which previous analysis had defined a group with metabolic syndrome, by factor analysis. A significant CHD-risk interaction was identified between genotype and the 'metabolic syndrome' factor (interaction p=0.009). To further assess CHD-risk for this variant in type-2 diabetes and to assess the effect of the variant upon thrombin generation and plasma levels of soluble EPCR, a cross-sectional study of type-2 diabetes was used. A significant CHD-risk was identified for European Whites (OR 2.84, CI 1.38-5.85) and Indian Asians in this study (OR 1.6, CI 1.00-2.57) and the frequency of 219Gly was two-fold higher in Indian Asians. Soluble EPCR levels were strongly associated with genotype, with homozygotes for 219Gly having four-fold higher levels (p<0.0001). In vitro studies of EPCR-transfected cells suggested increased basal release of sEPCR from cells expressing the 219Gly EPCR phenotype. Furthermore, in base-line samples from NPHSII and in the diabetic study, a significant increase in prothrombin F1+2 level was observed for 219Gly. The increased CHD-risk and thrombin generation appears to be acting through increased shedding of the Gly allele from the cell surface.

title: Haplotypes of the EPCR gene, prothrombin levels, and the risk of venous thrombosis in carriers of the prothrombin G20210A mutation
abstract: Haplotypes A1 and A3 in the endothelial protein C receptor (EPCR) gene are tagged by 4678G/C and 4600A/G respectively. We assessed whether these haplotypes modify the risk of venous thromboembolism in carriers of the prothrombin 20210A allele.  We genotyped 4678G/C and 4600A/G in 246 20210A carriers: 84 venous thromboembolism propositi and 162 relatives (13 symptomatic), and in 140 relatives not carrying the 20210A variant. Prothrombin and soluble EPCR (sEPCR) levels were also measured.  Among propositi, the mean age at first onset was lower in carriers (35±8 years) than non-carriers of the 4600G allele (44±14 years) (p=0.004). The probability of being free of thrombosis at age 40 was lower in 20210A carriers with the EPCR 4600G allele (p=0.015). The frequency of the 4600G allele (p=0.002) and the levels of prothrombin antigen (p=0.002) and sEPCR (p<0.001) were higher in the propositi than in their asymptomatic relatives. Multivariate analyses showed that the presence of the 4600G allele (OR=2.5, 95% confidence interval 1.3–5.0), sEPCR >147 ng/mL (2.8, 1.5–5.2) and prothrombin >129% (3.8, 1.8–8.3) all increased the thrombotic risk. In bivariate analysis, including the 4600G allele and sEPCR>147 ng/mL, only the latter remained associated with risk.  These results show that in 20210A carriers the venous thromboembolism risk is influenced both by the actual prothrombin levels and by the EPCR A3 haplotype, via its effect on sEPCR levels.

title: The Ser219–>Gly dimorphism of the endothelial protein C receptor contributes to the higher soluble protein levels observed in individuals with the A3 haplotype
abstract: The endothelial cell protein C receptor (EPCR) plays an important role in regulating blood coagulation and in activated protein C‐mediated anti‐inflammatory and antiapoptotic processes. Recent studies reported that there are polymorphisms in the human EPCR gene. One of the polymorphisms (haplotype A3) results in substitution of the Ser at residue 219 with Gly in the transmembrane domain. This haplotype is associated with increased plasma levels of soluble EPCR and is a candidate risk factor for thrombosis. We established stable cell lines expressing either the EPCR A1 (Ser at residue 219) or A3 (Gly at residue 219) haplotype. Both constitutive and PMA‐stimulated shedding are five‐ to sevenfold higher in the A3 cell line than the A1 cell line. We also isolated human umbilical vein endothelial cells (HUVEC) from A1/A1 or A1/A3 origins. PMA‐stimulated shedding is fourfold higher in HUVEC derived from A1/A3 origin than from A1/A1 origin. After PMA treatment, the rate of human protein C activation decreased 36% in HUVEC derived from A1/A3 origin, while it only decreased 18% in HUVEC derived from A1/A1 origin. These results indicate that the A3 haplotype does promote cellular shedding in either 293 or endothelial cells and therefore is likely directly contributory to the higher soluble EPCR levels seen in patients carrying this haplotype.

title: Regulated endothelial protein C receptor shedding is mediated by tumor necrosis factor‐α converting enzyme/ADAM17
abstract: Endothelial protein C receptor (EPCR) plays an important role in the protein C anticoagulation pathway. Previously, we have reported that EPCR can be shed from the cell surface, and that this is mediated by an unidentified metalloproteinase. In this study, we demonstrate that tumor necrosis factor‐α converting enzyme/ADAM17 (TACE) is responsible for EPCR shedding. Phorbol‐12‐myristate 13‐acetate (PMA)‐stimulated EPCR shedding is reduced by approximately 50% in HEK293 cells transfected with human EPCR cDNA and by 60% in human umbilical vein endothelial cells after transfection of TACE small interfering RNA (siRNA) into these cells. PMA‐stimulated EPCR shedding is completely blocked in fibroblasts from TACE‐deficient mice transfected with human EPCR cDNA, and restored by transfection of TACE cDNA into this cell line. To characterize the EPCR sequence requirement for shedding, we generated several mutants of EPCR. Replacing amino acids from residue 193 to residue 200 with the FLAGTM sequence (DYKDDDDK) completely blocks EPCR shedding, whereas a single amino acid substitution in this region has less effect on EPCR shedding.

title: Endothelial Cell Protein C Receptor Acts as a Cellular Receptor for Factor VIIa on Endothelium*
abstract: Although factor VII/factor VIIa (FVII/FVIIa) is known to interact with many non-vascular cells, activated monocytes, and endothelial cells via its binding to tissue factor (TF), the interaction of FVII/FVIIa with unperturbed endothelium and the role of this interaction in clearing FVII/FVIIa from the circulation are unknown. To investigate this, in the present study we examined the binding of radiolabeled FVIIa to endothelial cells and its subsequent internalization. 125I-FVIIa bound to non-stimulated human umbilical vein endothelial cells (HUVEC) in time- and dose-dependent manner. The binding is specific and independent of TF and negatively charged phospholipids. Protein C and monoclonal antibodies to endothelial cell protein C receptor (EPCR) blocked effectively 125I-FVIIa binding to HUVEC. FVIIa binding to EPCR is confirmed by demonstrating a marked increase in 125I-FVIIa binding to CHO cells that had been stably transfected with EPCR compared with the wild-type. Binding analysis revealed that FVII, FVIIa, protein C, and activated protein C (APC) bound to EPCR with similar affinity. FVIIa binding to EPCR failed to accelerate FVIIa activation of factor X or protease-activated receptors. FVIIa binding to EPCR was shown to facilitate FVIIa endocytosis. Pharmacological concentrations of FVIIa were found to impair partly the EPCR-dependent protein C activation and APC-mediated cell signaling. Overall, the present data provide convincing evidence that EPCR serves as a cellular binding site for FVII/FVIIa. Further studies are needed to evaluate the pathophysiological consequences and relevance of FVIIa binding to EPCR.

title: Binding of factor VIIa to the endothelial cell protein C receptor reduces its coagulant activity
abstract: Endothelial cell protein C receptor (EPCR) binds protein C through its γ‐carboxyglutamic acid (Gla) domain and enhances its thrombin–thrombomodulin complex‐dependent activation. So far, only protein C/activated protein C has been shown to interact with EPCR. Factor VII (FVII), the coagulation trigger upon tissue factor (TF) interaction, is a serine protease whose Gla domain is highly homologous to the Gla domain of protein C. To characterize the binding of FVII/FVIIa to EPCR and its functional consequences. We demonstrated by surface plasmon resonance (SPR) that FVII/FVIIa binds to EPCR through its Gla domain. At therapeutic concentrations, FVIIa reduced the activation of protein C by 40%. Soluble EPCR (sEPCR) was also able to prolong dose‐dependently the clotting time induced by the FVIIa–TF complex. SPR and amidolytic experiments showed that FVIIa is able to interact simultaneously with TF and EPCR, thus ruling out the possibility that the effect of EPCR on clotting time was due to the inhibition of the binding between FVIIa and TF. sEPCR inhibited dose‐dependently the activation of FX by the FVIIa–TF complex. Notably, blocking the binding site of EPCR on the endothelial surface increased the generation of FXa 2‐fold.  EPCR binds to FVII/FVIIa and inhibits the procoagulant activity of the FVIIa–TF complex.

title: Influence of the 4600A/G and 4678G/C polymorphisms in the endothelial protein C receptor (EPCR) gene on the risk of venous thromboembolism in carriers of factor V Leiden.
abstract: Two polymorphisms in the endothelial protein C receptor (EPCR) gene, 4600A/G and 4678G/C, have been reported to influence the risk of venous thromboembolism (VTE). The objective of this study was to assess whether these polymorphisms modify the risk of VTE in carriers of factor V (FV) Leiden. We genotyped 295 carriers of FV Leiden for these polymorphisms: 100 unrelated patients with a history of VTE (propositi) and 195 relatives (14 of them symptomatic) of 81 of the propositi. Spontaneous VTE events occurred in 71% of propositi carrying the 4678GG genotype, 65% carrying the GC, and 43% with the CC genotype. The mean age at the first onset was significantly higher in propositi carrying the 4678CC than in those with the GC or GG genotype (p = 0.046). Among the 276 carriers of FV Leiden from the 81 families studied, the 95 symptomatic members had similar 4600G allele and 4600AG genotype frequencies but significantly lower 4678C allele (p = 0.002) and 4678CC genotype (p = 0.004) frequencies than the 181 asymptomatic members. The probability of being free of thrombosis at age 40 was significantly higher in the 66 carriers of the 4678CC genotype (94%) than in the 138 carrying the GC (72%) or in the 72 with the 4678GG genotype (60%) (p < 0.001). Multivariate analysis showed that the 4678CC genotype reduced the risk of thrombosis in carriers of FV Leiden (OR = 0.31;95% CI = 0.16-0.83). The incidence of VTE was higher in the 195 relatives with FV Leiden than in the 133 without FV Leiden (OR = 4.7; CI = 1.3-7.2). These results show that carriers of FV Leiden with the 4678CC genotype have a significantly reduced risk of VTE compared with those carrying the 4678GG or GC genotype, probably due to the higherAPC levels previously observed in individuals carrying the 4678CC genotype.

title: Myocardial infarction redefined--a consensus document of The Joint European Society of Cardiology/American College of Cardiology Committee for the redefinition of myocardial infarction.
abstract: This document was developed by a consensus conference initiated by Kristian Thygesen, MD, and Joseph S. Alpert, MD, after formal approval by Lars Rydén, MD, President of the European Society of Cardiology (ESC), and Arthur Garson, MD, President of the American College of Cardiology (ACC). All of the participants were selected for their expertise in the field they represented, with approximately one-half of the participants selected from each organization. Participants were instructed to review the scientific evidence in their area of expertise and to attend the consensus conference with prepared remarks. The first draft of the document was prepared during the consensus conference itself. Sources of funding appear in Appendix A. The recommendations made in this document represent the attitudes and opinions of the participants at the time of the conference, and these recommendations were revised subsequently. The conclusions reached will undoubtedly need to be revised as new scientific evidence becomes available. This document has been reviewed by members of the ESC Committee for Scientific and Clinical Initiatives and by members of the Board of the ESC who approved the document on April 15, 2000.

title: Circulating activated protein C is reduced in young survivors of myocardial infarction and inversely correlates with the severity of coronary lesions 1
abstract: Cardiovascular risk factors for myocardial infarction (MI) are less frequent in younger than in older MI survivors. Therefore, the thrombotic component of MI may play a more important role at a young age. As activated protein C (APC) provides systemic anticoagulant and anti‐inflammatory protection, a low plasma APC level may be an arterial thrombotic risk factor.  To determine whether there is an association between reduced APC levels and early MI and severe coronary lesions.  APC was measured in 231 young MI survivors and 231 controls.  Low APC levels were significantly associated with MI. Compared with the fourth quartile, the odds ratio (OR) for APC values in the first quartile was 3.7 [95% confidence interval (CI) = 2.1–6.4], and 3.2 (1.5–7.0) after adjustment for cardiovascular risk factors. Moreover, each decrease of 0.43 ng mL−1 (1 SD) in APC increased the OR 1.7 times (1.4–2.2), and 1.5 times (1.2–1.9) after adjustment for cardiovascular risk factors. Low APC levels were also associated with the number of coronary arteries affected and with the severity of coronary lesions (P < 0.001).  There is a significant association between low circulating APC levels and both early MI and the extent and severity of coronary atherosclerosis, which might be related to the anticoagulant and anti‐inflammatory properties of APC.

title: Quantification of circulating activated protein C in human plasma by immunoassays--enzyme levels are proportional to total protein C levels.
abstract: We have developed a simple assay that measures the circulating activated protein C (APC) in plasma. The assay requires collection of duplicate blood samples, one in citrate plus heparin and the other in citrate plus inhibitors of the enzyme. In the heparin tube, APC reacts completely and irreversibly with its major plasma inhibitors, protein C inhibitor (PCI) and alpha 1-antitrypsin (alpha 1AT), and the complexes formed are measured by ELISAs. The amount of circulating APC is calculated from the difference between the total amount of complexed APC (sample in citrate plus heparin) and the amount of APC complexed in vivo (sample in citrate plus inhibitor). Over 95% of the APC added to blood collected with heparin was recovered in the assay. The assay can easily be performed in four hours, and had a detection limit of 0.1 ng/ml APC. The mean APC level in 18 protein C heterozygous members from seven kindreds was significantly lower (0.6 +/- 0.3 ng/ml) than in 20 healthy controls (1.1 +/- 0.3 ng/ml) (p < 0.001), whereas the mean level in 10 non-affected members from the kindreds studied was 1.5 +/- 0.3 ng/ml. In the group of 12 nonanticoagulated heterozygous protein C-deficient individuals, the three patients with a history of venous thrombosis had a mean APC level significantly lower than the nine asymptomatic members (p < 0.01), both subgroups showing similar protein C levels. There was a significant correlation in all groups between the levels of APC and the levels of protein C antigen (r = 0.758, p < 0.0001) and activity (r = 0.745, p < 0.0001), which means that APC circulating levels are proportional to protein C levels and suggests that the protein C level is the limiting factor in the rate of protein C activation in vivo.

title: Activation of Endothelial Cell Protease Activated Receptor 1 by the Protein C Pathway
abstract: The coagulant and inflammatory exacerbation in sepsis is counterbalanced by the protective protein C (PC) pathway. Activated PC (APC) was shown to use the endothelial cell PC receptor (EPCR) as a coreceptor for cleavage of protease activated receptor 1 (PAR1) on endothelial cells. Gene profiling demonstrated that PAR1 signaling could account for all APC-induced protective genes, including the immunomodulatory monocyte chemoattractant protein-1 (MCP-1), which was selectively induced by activation of PAR1, but not PAR2. Thus, the prototypical thrombin receptor is the target for EPCR-dependent APC signaling, suggesting a role for this receptor cascade in protection from sepsis.

title: Overexpressing endothelial cell protein C receptor alters the hemostatic balance and protects mice from endotoxin
abstract: Previous studies have shown that blocking endothelial protein C receptor (EPCR)‐protein C interaction results in about an 88% decrease in circulating activated protein C (APC) levels generated in response to thrombin infusion and exacerbates the response to Escherichia coli. To determine whether higher levels of EPCR expression on endothelial cells might further enhance the activation of protein C and protect the host during septicemia, we generated a transgenic mouse (Tie2‐EPCR) line which placed the expression of EPCR under the control of the Tie2 promoter. The mice express abundant EPCR on endothelial cells not only on large vessels, but also on capillaries where EPCR is generally low. Tie2‐EPCR mice show higher levels of circulating APC after thrombin infusion. Upon infusion with factor Xa and phospholipids, Tie2‐EPCR mice generate more APC, less thrombin and are protected from fibrin/ogen deposition compared with wild type controls. The Tie2‐EPCR animals also generate more APC upon lipopolysaccharide (LPS) challenge and have a survival advantage. These results reveal that overexpression of EPCR can protect animals against thrombotic or septic challenge.

title: Recombinant human activated protein C attenuates the inflammatory response in endothelium and monocytes by modulating nuclear factor-&kgr;B
abstract: ObjectiveTo review the anti-inflammatory and anti-apoptotic properties of drotrecogin alfa (activated) (recombinant human activated protein C), emphasizing its modulatory effects on endothelial nuclear factor-&kgr;B. We propose a broad anti-inflammatory effect of drotrecogin alfa (activated), acting on both endothelium and monocytes. Data SourcesA selected review of the published literature on nuclear factor-&kgr;B, severe sepsis, and the use of drotrecogin alfa (activated) in clinical and preclinical models, together with data derived from preclinical gene profiling of model systems.  Extraction and SynthesisData from the PROWESS trial support the preclinical evidence of an antithrombotic effect of drotrecogin alfa (activated). Anti-inflammatory effects through reduction of thrombin generation and through thrombin-independent mechanisms in mononuclear and endothelial cells are reviewed. Inhibition of apoptosis is used as an example of the protective effect of drotrecogin alfa (activated) on endothelial and mononuclear cell dysfunction. ConclusionsDrotrecogin alfa (activated) acts as a modulator of nuclear factor-&kgr;B to aid in the host immune response in endothelium and monocytes. Extrapolation of gene array findings to explain apoptosis in endothelium and monocytes, coupled with emerging preclinical reports, provides evidence to support the role of drotrecogin alfa (activated) in modulating nuclear factor-&kgr;B.

title: Activated protein C and inflammation in human myocardium after heart surgery
abstract: To assess possible interactions between inflammation and activation of the anticoagulant protein C system during post‐ischemic reperfusion protein C, APC, neutrophil L‐selectin expression and myocardial myeloperoxidase activity (MPO) were measured in 19 patients undergoing cardiopulmonary bypass. After reperfusion for 10 min, APC to protein C ratio (APC/PC) increased from pre‐reperfusion value 1.43 ± 0.12 (mean ± SEM) to 2.25 ± 0.29, p = 0.015. Negative correlations were observed between APC/PC and MPO activity (Spearman r −0.64, p = 0.007) and APC/PC and neutrophil L‐selectin expression (r = −0.7, p = 0.007, demonstrating that post‐ishemic protein C activation was associated with decreased neutrophil tissue sequestration. Thus, physiological protein C activation may be involved in regulation of the inflammatory injury during reperfusion of human ischemic coronary circulation.  2001.