Genetic variants associated with protein C levels.

BACKGROUND: Normal protein C (PC) plasma levels range widely in the general population. Factors influencing normal PC levels are thought to influence the risk of venous thrombosis. Little is known about the underlying genetic variants. OBJECTIVES: We performed a genome scan of normal PC levels to identify genes that regulate normal PC levels. PATIENTS/METHODS: We performed a variance components linkage analysis for normal PC levels in 275 individuals from a single, large family. We then sequenced candidate genes under the identified linkage peak in eight family members: four with high and four with low, but normal, PC levels. For variants showing a difference in carriers between those with high and low PC levels, we re-evaluated linkage in the 275 family members conditional on the measured genotype effect. Genotype-specific mean PC levels were determined using likelihood analysis. Findings were replicated in the Leiden Thrombophilia Study (LETS). RESULTS: We identified a quantitative trait locus at chromosome 5q14.1 affecting normal PC plasma level variability. Next-generation sequencing of 113 candidate genes under the linkage peak revealed four SNPs in BHMT2, ACOT12, SSBP2 and XRCC4, which significantly increased PC levels in our thrombophilic family, but not in LETS. CONCLUSIONS: We identified four genes at chromosome 5q14.1 that might influence normal PC levels. BHMT2 seems the most likely candidate to regulate PC levels via homocysteine, a competitive inhibitor to thrombin. Failure to replicate our findings in LETS might be due to differences between the studies in genetic background and linkage disequilibrium patterns.

Genome scan of clot lysis time and its association with thrombosis in a protein C-deficient kindred.

BACKGROUND: Previously, we found increased clot-lysis time (CLT), as measured with a plasma-based assay, to increase the risk of venous thrombosis in two population-based case-control studies. The genes influencing CLT are as yet unknown. PATIENTS/METHODS: We tested CLT as risk factor for venous thrombosis in Kindred Vermont II (n = 346), a pedigree suffering from a high thrombosis risk, partially attributable to a type I protein C deficiency. Furthermore, we tested for quantitative trait loci (QTLs) for CLT, using variance component linkage analysis. RESULTS: Protein C-deficient family members had shorter CLTs than non-deficient members (median CLT 67 min vs. 75 min). One standard deviation increase in CLT increased the risk of venous thrombosis 2.4-fold in non-deficient family members. Protein C deficiency without elevated CLT increased the risk 6.9-fold. Combining both risk factors yielded a 27.8-fold increased risk. The heritability of CLT was 42-52%. We found suggestive evidence of linkage on chromosome 11 (62 cM), partly explained by the prothrombin 20210A mutation, and on chromosome 13 (52 cM). Thrombin-activatable fibrinolysis inhibitor genotypes did not explain the variation in CLT. CONCLUSION: Hypofibrinolysis appears to increase thrombosis risk in this family, especially in combination with protein C deficiency. Protein C deficiency is associated with short CLT. CLT is partly genetically regulated. Suggestive QTLs were found on chromosomes 11 and 13.

The endothelial microenvironment in the venous valvular sinus: thromboresistance trends and inter-individual variation.

The valve sinuses of the deep venous system are frequent sites of venous thrombus initiation. We previously reported that, in comparison with the non-valvular lumenal endothelium, the valve sinus endothelium had decreased expression of von Willebrand factor (vWF) and increased expression of endothelial protein C receptor (EPCR) and thrombomodulin (TM), suggesting alteration in the procoagulant/anticoagulant balance. We hypothesized that increased stasis in the deeper recesses of the venous valves would be associated with a gradient of increased thromboresistance. Expression of EPCR, TM, and vWF was analyzed via quantitative confocal immunofluorescence in residual saphenous veins collected following coronary artery bypass procedures. In agreement with our hypothesis, endothelial expression of vWF in the valve sinus decreased from the uppermost to the deepest region of the valve sinus. In contrast to our hypothesis, EPCR expression decreased from the uppermost to the deepest region of the valve sinus (p < 0.001) and TM expression remained unchanged throughout the valve sinus. Comparison of the non-valvular lumenal endothelium with the valve sinus endothelium demonstrated significantly decreased vWF expression (p < 0.001) in the valvular sinus consistent with our previous report; however, we did not observe statistically significant differences in EPCR or TM expression in this comparison. In addition, remarkable inter-individual variation in expression of these three proteins was also observed. These findings suggest that the genesis of these observations is more complex than predicted by our initial hypothesis, likely due, at least in part, to the complex rheology of the valvular sinus microenvironment.

Risk factors for peripheral venous disease resemble those for venous thrombosis: the San Diego Population Study.

BACKGROUND: Clinically silent deep vein thrombosis (DVT) is common and may cause chronic venous disease that resembles post-thrombotic syndrome. OBJECTIVE: We evaluated whether peripheral venous disease in a general population shares risk factors with DVT. METHODS: In an established cohort of 2404 men and women, the San Diego Population Study, peripheral venous disease was evaluated using physical examination, symptom assessment and venous ultrasound. We performed a case-control study including 308 cases in four hierarchical groups by severity and 346 controls without venous abnormalities, frequency matched to cases by 10-year age group, race and gender. Cases and controls had no prior history of venous thrombosis. Hemostatic risk factors were measured in cases and controls. RESULTS: Accounting for age, obesity and family history of leg ulcer, odds ratios (ORs) of venous disease for elevated factor VIII, von Willebrand factor (VWF), D-dimer and for factor V Leiden were 1.4 (95% CI 0.9-2.1), 1.5 (CI 1.0-2.3), 1.7 (CI 1.1-2.8) and 1.1 (CI 0.5-2.4), respectively. These associations were larger for the two most severe case groups; ORs 2.0 (CI 1.0-3.8), 1.7 (CI 0.9-3.3), 2.7 (CI 1.2-6.1) and 2.3 (CI 0.8-7.1). Each hemostatic factor was also associated with severity of venous disease, for example elevated D-dimer was associated with a 2.2-fold increased odds of being in one higher severity case group. Prothrombin 20210A was not associated with venous disease. CONCLUSIONS: DVT risk factors are associated with presence and severity of peripheral venous disease. Results support a hypothesis that peripheral venous disease may sometimes be post-thrombotic syndrome as a result of a previous unrecognized DVT.

A genetic basis for the interrelation of coagulation factors.

BACKGROUND: Evidence found in the literature for a strong correlation between coagulation factors suggests that single genes might influence the plasma concentrations of multiple coagulation factors (i.e. pleiotropically acting genes). OBJECTIVE: To determine whether there is a genetic basis for the correlation among coagulation factors by assessing the heritability of interrelated coagulation factors. PATIENTS/METHODS: We performed principal components analysis, and subsequently variance components analysis, to estimate the heritability of principal components of coagulation factors in family members of a large French-Canadian kindred. RESULTS: Four clusters were identified by principal components analysis in 200 family members who did not carry the protein C 3363C mutation. Cluster 1 consisted of prothrombin, factor VII (FVII), FIX, FX and protein S; cluster 2 consisted of FV, FIX, protein C and tissue factor pathway inhibitor; cluster 3 consisted of FVIII and von Willebrand factor; and cluster 4 consisted of antithrombin, protein C and FVII. The heritability of the principal components estimated by variance components analysis was, respectively, 37%, 100%, 37%, and 37%. CONCLUSION: Our findings support the hypothesis that genes can influence plasma levels of interrelated coagulation factors.

Attitudes toward genetic testing for thrombophilia in asymptomatic members of a large family with heritable protein C deficiency.

BACKGROUND: Little research has been performed regarding the psychological consequences of knowing that one is at an increased risk for venous thrombosis. OBJECTIVES: The aim of this study was to explore attitudes toward genetic testing for protein C deficiency. METHODS: Questionnaires about genetic testing attitudes, dispositional anxiety, risk perception, and thrombosis-related worry were completed by 168 asymptomatic members of a North-American kindred with a high incidence of heritable protein C deficiency conferring a high lifetime risk of venous thrombosis. A total of 76 subjects (45%) had not been tested for protein C deficiency before participating in our study whereas the other 92 subjects (55%) had been tested prior to filling in the questionnaire, of whom 34 people had protein C deficiency, while 58 did not. RESULTS: Family members with protein C deficiency perceived a higher risk of suffering venous thrombosis and scored higher on thrombosis-related worry than family members without protein C deficiency. Participants who had not been tested did not report excessive thrombosis-related worry. Participants with protein C deficiency reported a belief in the psychological and health benefits of testing, and felt that they experienced low psychological distress following the genetic test. High psychological distress following the test was related to dispositional anxiety and thrombosis-related worry. Participants without protein C deficiency were relieved after finding out that they did not have the deficiency. CONCLUSION: There seem to be few negative psychological consequences of knowing that one is at an increased risk for venous thrombosis, except in vulnerable individuals.

Chronic venous abnormalities in symptomatic and asymptomatic protein C deficiency.

BACKGROUND: Thrombophilia is a frequent medical condition associated with symptomatic deep vein thrombosis (DVT). Unlike other clinical risk factors associated with DVT, such as surgery, thrombophilia has not been demonstrated to be associated with asymptomatic venous thrombotic events. Our aim was to search for asymptomatic sequelae of DVT in a protein C (PC)-deficient family. METHODS: We studied 228 individuals from a large kindred with PC deficiency and performed a systematic ultrasound examination. RESULTS: Among the 203 patients without a known history of venous thrombosis we found seven patients with abnormalities indicative of prior asymptomatic thrombosis: six (7.4%) in the PC-deficient group (n = 81) and only one (0.8%) in the non-deficient group (n = 122). The relative risk for these sequelae associated with PC deficiency was 9.0 (95% CI: 1.1-73.7). CONCLUSIONS: These data suggest that chronic venous abnormalities are frequently present and that thrombotic events in asymptomatic individuals with familial PC deficiency may be underestimated.

First experience with direct, selective factor Xa inhibition in patients with non-ST-elevation acute coronary syndromes: results of the XaNADU-ACS Trial.

BACKGROUND: Unfractionated heparin is widely used in patients with non-ST-elevation acute coronary syndromes but has important limitations. Anticoagulants with predictable kinetics and anticoagulant effects, better efficacy, and greater safety are needed. OBJECTIVE: To investigate the efficacy and safety of a direct, selective factor Xa inhibitor, DX-9065a (Daiichi Pharmaceuticals LTD, Inc.) compared with heparin, in patients with non-ST-elevation acute coronary syndromes. PATIENTS AND METHODS: Patients (n = 402) from the USA, Canada, and Japan were randomized to blinded, weight-adjusted heparin, low-dose DX-9065a, or high-dose DX-9065a. RESULTS: The primary efficacy endpoint of death, myocardial infarction, urgent revascularization, or ischemia on continuous ST-segment monitoring occurred in 33.6%, 34.3%, and 31.3% of patients assigned to heparin, low-dose DX-9065a, and high-dose DX-9065a (P = 0.91 for heparin vs. combined DX-9065a). The composite of death, myocardial infarction, or urgent revascularization occurred in 19.5%, 19.3%, and 11.9% (P = 0.125 for heparin vs. high-dose DX-9065a) of patients; major or minor bleeding occurred in 7.7%, 4.2%, and 7.0% of patients; and major bleeding in 3.3%, 0.8%, and 0.9% of patients. Higher concentrations of DX-9065a were associated with a lower likelihood of ischemic events (P = 0.03) and a non-significant tendency toward a higher likelihood of major bleeding (P = 0.32). CONCLUSIONS: In this small phase II trial, there was a non-significant tendency toward a reduction in ischemic events and bleeding with DX-9065a compared with heparin in patients with acute coronary syndromes. The absence of an effect on ST-monitor ischemia warrants further investigation. These data provide the rationale for adequately powered studies of DX-9065a in acute coronary syndromes or percutaneous intervention.

Genome scan of venous thrombosis in a pedigree with protein C deficiency.

Kindred Vermont II has a high frequency of venous thrombosis, occurring primarily in pedigree members with type I protein C deficiency due to a 3363 inserted (Ins) C mutation in exon 6 of the protein C gene. However, only a subset of 3363 InsC carriers have suffered thrombotic episodes, suggesting that the increased risk of thrombosis results upon the co-occurrence of 3363 InsC with a second, unknown, thrombophilic mutation that segregates independently within the pedigree. To test this hypothesis and to localize the co-occurring gene, we performed a genome scan of venous thrombosis in Kindred Vermont II. Non-parametric linkage statistics identified three potential gene locations, on chromosomes 11q23 (nominal P < 0.0001), 18p11.2-q11.2 (P < 0.0007), and 10p12 (P < 0.0003), supporting the presence of at least one additional thrombophilic mutation in the pedigree. Identification of the unknown mutation(s) promises to reveal a new genetic risk factor for thrombophilia, contribute to our understanding of the blood clotting mechanism, and expand our knowledge of the diversity of oligogenic disease.

Initial experience with factor-Xa inhibition in percutaneous coronary intervention: the XaNADU-PCI Pilot.

BACKGROUND: Direct factor (F)Xa inhibition is an attractive method to limit thrombotic complications during percutaneous coronary intervention (PCI). OBJECTIVES: To investigate drug levels achieved, effect on coagulation markers, and preliminary efficacy and safety of several doses of DX-9065a, an intravenous, small molecule, direct, reversible FXa inhibitor during PCI. PATIENTS AND METHODS: Patients undergoing elective, native-vessel PCI (n = 175) were randomized 4 : 1 to open-label DX-9065a or heparin in one of four sequential stages. DX-9065a regimens in stages I-III were designed to achieve concentrations of > 100 ng mL-1, > 75 ng mL-1, and > 150 ng mL-1. Stage IV used the stage III regimen but included patients recently given heparin. RESULTS: At 15 min median (minimum) DX-9065a plasma levels were 192 (176), 122 (117), 334 (221), and 429 (231) ng mL-1 in stages I-IV, respectively. Median whole-blood international normalized ratios (INRs) were 2.6 (interquartile range 2.5, 2.7), 1.9 (1.8, 2.0), 3.2 (3.0, 4.1), and 3.8 (3.4, 4.6), and anti-FXa levels were 0.36 (0.32, 0.38), 0.33 (0.26, 0.39), 0.45 (0.41, 0.51), and 0.62 (0.52, 0.65) U mL-1, respectively. Stage II enrollment was stopped (n = 7) after one serious thrombotic event. Ischemic and bleeding events were rare and, in this small population, showed no clear relation to DX-9065a dose. CONCLUSIONS: Elective PCI is feasible using a direct FXa inhibitor for anticoagulation. Predictable plasma drug levels can be rapidly obtained with double-bolus and infusion DX-9065a dosing. Monitoring of DX-9065a may be possible using whole-blood INR. Direct FXa inhibition is a novel and potentially promising approach to anticoagulation during PCI that deserves further study.

Heritability of plasma concentrations of clotting factors and measures of a prethrombotic state in a protein C-deficient family.

BACKGROUND: Earlier studies found strong support for a genetic basis for regulation of coagulation factor levels and measures of a prethrombotic state (d-dimer, prothrombin fragment 1.2). OBJECTIVES: Estimation of how much of the variation in the levels of coagulation factors and measures of a prethrombotic state, including measures of protein C activation and inactivation, could be attributed to heritability and household effect. PATIENTS AND METHODS: Blood samples were collected from 330 members of a large kindred of French-Canadian origin with type I protein C deficiency. Heritability and common household effect were estimated for plasma concentrations of prothrombin, factor (F)V, factor VIII, factor (F)IX, fibrinogen, von Willebrand factor (VWF), antithrombin, protein C, protein S, protein Z, protein Z-dependent protease inhibitor (ZPI), fibrinopeptide A (FPA), protein C activation peptide (PCP), activated protein C-protein C inhibitor complex (APC-PCI), activated protein C-alpha1-antitrypsin complex (APC-alpha1AT), prothrombin fragment 1.2 (F1.2) and d-dimer, using the variance component method in sequential oligo-genic linkage analysis routines (SOLAR). RESULTS: The highest heritability was found for measures of thrombin activity (PCP and FPA). High estimates were also found for prothrombin, FV, FIX, protein C, protein Z, ZPI, APC-PCI and APC-alpha1AT. An important influence of shared household effect on phenotypic variation was found for VWF, antithrombin, protein S and F1.2. CONCLUSIONS: We found strong evidence for the heritability of single coagulation factors and measures of a prethrombotic state. Hemostatic markers with statistically significant heritability constitute potential targets for the identification of novel genes involved in the control of quantitative trait loci.

Characterization of the human prostaglandin H synthase 1 gene (PTGS1): exclusion by genetic linkage analysis as a second modifier gene in familial thrombosis.

Genetic evidence from a large Vermont kindred indicates that an unknown gene promotes thrombosis when inherited in conjunction with type I protein C deficiency. Cyclooxygenase-1 [prostaglandin H synthase 1 gene (PTGS1)] was tested as a plausible candidate for the unknown gene because of its role in primary hemostasis. The complete sequence of PTGS1 (25 638 nucleotides) was determined from a 37 kb human genomic cosmid clone to characterize intronic regions and subsequently to allow the search for mutations by direct sequencing of genomic DNA. Northern blot analysis confirms usage of a newly described distal poly-adenylation signal. Short tandem repeat (STR) sequences found in intron 2 and the 3' flanking region were developed as new genetic markers for PTGS1. The position of PTGS1 was refined on the CHLC chromosome 9 linkage map using the new markers scored in four Centre d'Etude du Polymorphisme Humain families and multipoint linkage analysis. Direct sequencing of DNA from members of the Vermont kindred led to the discovery of two new single nucleotide polymorphisms (SNPs) that give rise to non-conservative amino acid changes in the signal peptide (Arg(8) to Trp and Pro(17) to Leu) of cyclooxygenase-1. Linkage analysis of the SNP and STR markers indicated that PTGS1 is not the interacting gene associated with an increased incidence of thrombosis in the Vermont kindred.

Evidence of a founder effect for the protein C gene 3363 inserted C mutation in thrombophilic pedigrees of French origin.

We have previously reported that the 3363 inserted (Ins) C mutation in exon 6 of the protein C gene was present in four unrelated French patients and in four French Canadian families with type I protein C deficiency as well as in a large Vermont protein C deficient kindred of French Canadian origin. The present study was designed to investigate the likelihood of the existence of a founder effect for this mutation in protein C deficient individuals of French origin living in France, Quebec and Vermont. In order to demonstrate a possible founder effect for the 3363 InsC mutation, we have previously constructed a high-resolution genetic map to locate several highly polymorphic markers close to the protein C locus. Thereafter, the markers D2S347, D2S2339, D2S383, D2S2271 and D2S2215 were genotyped in 117 heterozygotes from France (n = 7), Quebec (n = 36) or Vermont (n = 74). The allelic frequency distribution of these five markers was also determined in fifty control French Canadian subjects and thirty-two unaffected members of the Vermont kindred with normal protein C levels and compared with their frequency in our cohort of heterozygotes. Our data suggest that patients from Quebec and Vermont carry a common haplotype at the protein C locus. Moreover, in order to study the evolutionary history of the 3363 InsC mutation, we traced back the ascending genealogy of one proband in each of the families with this mutation. These results showed that the 3363 InsC mutation was most probably introduced in North America by a couple of French settlers who established themselves in 1669 on Isle d'Orleans located near Quebec City. All heterozygotes for the 3363 InsC mutation living in North America are related to these founders within 10 generations. Thus, these families afford a unique opportunity to evaluate the role of the protein C system in thrombophilia due to the high degree of linkage disequilibrium at the protein C gene, which in essence holds that variable more constant than in a more heterogeneous population.

The association of vitamin K status with warfarin sensitivity at the onset of treatment.

We investigated the association of vitamin K status with warfarin sensitivity among 40 orthopaedic patients beginning perioperative algorithm-dosed warfarin. Baseline vitamin K status was assessed using plasma vitamin K-1 and vitamin K-1 2,3 epoxide concentrations, and a questionnaire-based estimation of usual vitamin K intake. Warfarin sensitivity was assessed as the increase in the International Normalized Ratio (INR) after two doses of 5 mg of warfarin and as the 4-d accumulation of under-gamma-carboxylated prothrombin (PIVKA-II), adjusted for warfarin dose requirement. Multivariate models were used to assess vitamin K variables as predictors of warfarin sensitivity. The mean INR increase was 0.53 U and the mean PIVKA-II increase was 771 ng/ml/mg warfarin. Demographic factors were not associated with warfarin response. For each 1 standard deviation (SD) lower value of plasma vitamin K-1, but not the other vitamin K variables, the INR rose 0.24 U (P < or = 0.01). A higher usual vitamin K intake and plasma vitamin K-1, and lower plasma vitamin K-1 2,3 epoxide, were all associated with a lower PIVKA-II increase over 4 d. Respective differences in PIVKA-II accumulation per SD increase of each variable were -165, -218 and 236 ng/ml/mg warfarin (all P < or = 0.05). We concluded that dietary and biochemical measures of vitamin K status were associated with early warfarin sensitivity.

Genetic screening of candidate genes for a prothrombotic interaction with type I protein C deficiency in a large kindred.

The incomplete penetrance of thrombosis in familial protein C deficiency suggests disease occurs when this deficit is combined with additional abnormalities in the hemostatic system. The pattern of inherited thrombophilia in the Vermont II kindred, which is affected by a clinically dominant type I protein C deficiency, provides strong evidence for a second unidentified gene that segregates independently of protein C deficiency and increases susceptibility to thrombosis. To test the second gene hypothesis, thirty-four candidate genes for proteins involved in hemostasis or inflammation were tested as the unknown defect, using highly polymorphic short tandem repeat (STR) markers in an informative subset (n = 31) of the kindred. The genes considered are; alpha-fibrinogen, beta-fibrinogen, gamma-fibrinogen, prothrombin, tissue factor, factor V, protein S, complement component 4 binding protein, factor XI, factor XII, factor XIIIa, factor XIIIb, histidine rich glycoprotein, high molecular weight kininogen, kallikrein, von Willebrands factor, platelet factor 4, thrombospondin, antithrombin III, alpha-1-antitrypsin, thrombomodulin, plasminogen, tissue plasminogen activator, urokinase plasminogen activator, plasminogen activator inhibitor-1, plasminogen activator inhibitor-2, protein C inhibitor, alpha-2-plasmin inhibitor, kallistatin, lipoprotein a, interleukin 6, interleukin 1, cystathionine-beta-synthase, and methylenetetrahydrofolate reductase. Mutations in many of these genes have been previously established as independent risk factors for thrombosis. However, linkage analysis provided no evidence to implicate any of the candidate genes as the second inherited factor that promotes thrombophilia in this kindred.

Severe acquired vitamin K deficiency: a hypothesis for rapid response to therapy.

The potential mechanism underlying the rapid response to vitamin K replacement in acquired deficiency states is incompletely understood. To examine vitamin K metabolism, a 10-year-old boy with autoimmune enteropathy on oral vitamin K supplementation, who presented with profuse nosebleeds and calf tenderness, was evaluated. Laboratory analyses were consistent with severe vitamin K deficiency: vitamin K dependent protein (VKDP) levels < 5%, normal vitamin K epoxide level and depressed total prothrombin antigen (carboxylated and undercarboxyated forms). Intramuscular vitamin K (10 mg) was administered. Nine hours following therapy, VKDP levels corrected completely. Total prothrombin antigen increased indicating new prothrombin synthesis. However, the increase in the prothrombin-clotting assay far exceeded the increase in total prothrombin, supporting storage of undercarboxylated prothrombin in vitamin K deficiency states, with carboxylation and secretion after vitamin K replacement. Although this mechanism is known to occur in rodents, it has not been reported in humans. Our findings suggest a new potential mechanism of prothrombin metabolism in humans.