A genome search for genetic determinants of markers of protein C activation.
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Adolescent↗
Biomedical subjects
E G Bovill
Publications and source records attributed to E G Bovill.
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.
Acute Disease↗
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.
Adolescent↗
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.
Adolescent↗
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.
Adolescent↗
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.
Adolescent↗
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.
Aged↗
Heritability of clotting factors and the revival of the prothrombotic state.
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Blood Coagulation↗
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.
Base Sequence↗
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.
Aged↗
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.
Blood Coagulation Factors↗
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.
Consanguinity↗
The G20210A prothrombin polymorphism is not associated with increased thromboembolic risk in a large protein C deficient kindred.
Likelihood analysis was used to test the effect of the G20210A prothrombin mutation and the His107Pro protein C mutation (resulting from a C insertion) on thrombosis status and prothrombin level in a large kindred of French Canadian descent with type I protein C deficiency. Genotypes were available on 279 pedigree members or their spouses. Of this total, 36 pedigree members were heterozygous for the G20210A variant and one pedigree member was homozygous for G20210A, while 64 were heterozygous for the His107Pro protein C mutation. The factor V Leiden mutation (Arg506Gln) was observed in only one of 181 tested family members. Objectively verified thrombosis was present in 26 of the 279 pedigree members. Thrombosis was suspected in an additional 19 pedigree members. The transmission disequilibrium test of Spielman, 1996, as extended to pedigrees, was used to test for excess transmission of G20210A or His107Pro to thrombosis cases, with transmission of 0.5 specifying no effect. Although the His107Pro mutation was over transmitted (0.837 +/- 0.075 p <0.001) to thrombosis cases in this pedigree, the G20210A variant was not (0.491 +/- 0.130 NS). Measured genotype analysis was used to examine a total of 184 individuals for the relationship between prothrombin level and both the G20210A variant and thrombosis. The G20210A variant increased prothrombin level from 97 +/- 2% to 124 +/- 4% (p <0.0001), but thrombosis status was not associated with any additional increase in prothrombin level. Thus, in a large thrombophilic, protein C deficient kindred, with the G20210A variant present in a proportion (13%) far higher than the general Caucasian population (approximately 2%), neither the presence of the variant nor the plasma concentration of prothrombin were associated with increased risk for thrombosis. These findings contrast with those of others who have established the G20210A variant as a thrombophilic risk factor; and emphasize the complex nature of the multigenic pathogenesis of thrombophilia.
Female↗
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.
Blood Coagulation↗
Quality assessment of atomic force microscopy probes by scanning electron microscopy: correlation of tip structure with rendered images.
While image quality from instruments such as electron microscopes, light microscopes, and confocal laser scanning microscopes is mostly influenced by the alignment of optical train components, the atomic force microscope differs in that image quality is highly dependent upon a consumable component, the scanning probe. Although many types of scanning probes are commercially available, specific configurations and styles are generally recommended for specific applications. For instance, in our area of interest, tapping mode imaging of biological constituents in fluid, double ended, oxide-sharpened pyramidal silicon nitride probes are most often employed. These cantilevers contain four differently sized probes; thick- and thin-legged 100 microm long and thick- and thin-legged 200 microm long, with only one probe used per cantilever. In a recent investigation [Taatjes et al. (1997) Cell Biol. Int. 21:715-726], we used the scanning electron microscope to modify the oxide-sharpened pyramidal probe by creating an electron beam deposited tip with a higher aspect ratio than unmodified tips. Placing the probes in the scanning electron microscope for modification prompted us to begin to examine the probes for defects both before and after use with the atomic force microscope. The most frequently encountered defect was a mis-centered probe, or a probe hanging off the end of the cantilever. If we had difficulty imaging with a probe, we would examine the probe in the scanning electron microscope to determine if any defects were present, or if the tip had become contaminated during scanning. Moreover, we observed that electron beam deposited tips were blunted by the act of scanning a hard specimen, such as colloidal gold with the atomic force microscope. We also present a mathematical geometric model for deducing the interaction between an electron beam deposited tip and either a spherical or elliptical specimen. Examination of probes in the scanning electron microscope may assist in interpreting images generated by the atomic force microscope.
DNA↗
Imaging of collagen type III in fluid by atomic force microscopy.
Type III collagen is a component of the basement membrane of endothelial cells, and may play a role in the interaction between hemostatic system proteins and the basement membrane of blood vessels. To begin to investigate these structural interactions, we have imaged type III collagen in solution by atomic force microscopy. A 20 microg/ml solution of type III collagen in bicarbonate buffer (pH 9.5) from calf skin was deposited onto a freshly cleaved mica substrate. Atomic force microscopy images were acquired using a fluid cell and tapping mode with oxide-sharpened silicon nitride probes 2, 3, and 4 hours after deposition of the collagen onto the mica. Two-hour preparations displayed fibrillar networks with well-defined sites of nucleation and lateral growth. At 3 and 4 hour polymerizations, more mature fibrils of increasing lengths, diameters, and complexity were observed. Fibrils appeared to be aligning and twisting (helical formation) to form a mature fibril with a higher mass per unit area. Interestingly, the mature fibrils appeared larger centrally with tapered ends displaying declining slopes. These observations compare favorably with those previously published on collagen type I assembly [Gale et al. (1995) Biophys. J. 68:2124-2128]. High resolution atomic force microscopy images of type III collagen in solution should provide a template for observation of the interactions between basement membrane components and hemostatic system proteins present in cardiovascular disease.
Animals↗
Stroke risk in an elderly population with atrial fibrillation.
Patients with nonvalvular atrial fibrillation (AF) have an increased risk of stroke, but the absolute rate of stroke varies widely depending on coexistent vascular disease. We assessed the stroke rate and predictive value of two published schemes for stroke risk stratification in a population-derived cohort of 259 elderly people with nonvalvular AF followed for a median of 5.3 years. The rate of ischemic stroke was 2.8% per year (95% confidence interval [CI] 1.9, 3.9). Thirty-one percent were predicted to be at low risk, and their stroke rate was 1.7% per year (95% CI 0.6, 3.8). Many people with AF in this population-derived cohort had relatively low rates of stroke. Further studies to reliably stratify stroke risk in patients with AF are needed.
Aged↗
Clinically significant differences in the International Normalized Ratio measured with reagents of different sensitivities. SPAF Investigators. Stroke Prevention in Atrial Fibrillation.
The International Normalized Ratio (INR) system was introduced a decade ago as a way of standardizing the results of prothrombin time testing for patients taking oral anticoagulants. A strong emphasis has been placed upon using thromboplastin reagents that are very sensitive to the effects of oral anticoagulants upon the prothrombin time [i.e. reagents with low International Sensitivity Index (ISI)]. In order to assess how well the INR system functions as currently used in clinical laboratories, we compared the INRs determined using thromboplastins of differing ISIs in samples collected during a large clinical trial of oral anticoagulation for atrial fibrillation (Stroke Prevention in Atrial Fibrillation III trial). Frozen plasma was subjected to prothrombin time testing using thromboplastins with ISIs ranging from 0.97 to 2.49. INRs were calculated using machine-specific ISIs and Westgard's rules were followed to maintain quality control. An unanticipated coagulometer failure allowed a determination of the effect of machine recalibration upon the INR of control plasmas. The correlation between each pair of INRs obtained from 1181 plasmas was high (> 0.9), but the differences between reagents were statistically different from zero (P<0.001 for pairwise comparisons). Plasmas had INRs within the therapeutic range (2.0-3.0) with one reagent but not with another in an average of 20% of instances. Among the 20% discordant pairings, 43% (8.5% of the total tested) showed a difference in INR of more than 0.2 INR units above or below the target range. Low ISI thromboplastins did not perform better in this pairwise comparison than other reagents or the locally determined INR. Recalibration of a coagulometer resulted in a significant change in the INRs obtained from control plasmas (P<0.0001), which confirms and extends the observations of other authors concerning the sensitivity of the INR to coagulometer-related variables. There was a clinically significant difference in the INRs obtained with different thromboplastins, and low ISI reagents did not perform better than others. Since the risk of thrombosis rises sharply below the lower limit of the currently recommended target ranges, consideration should be given to narrowing the recommended range, or advising clinicians to aim for its mid-point. These findings illustrate the difficulties in imposing standardization upon coagulation testing after a test is in widespread use.
Administration, Oral↗