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At least 973 records · Page 54Linked to original sources
PubMed2001

The omega-loop region of the human prothrombin gamma-carboxyglutamic acid domain penetrates anionic phospholipid membranes.

The hydrophobic omega-loop within the prothrombin gamma-carboxyglutamic acid-rich (Gla) domain is important in membrane binding. The role of this region in membrane binding was investigated using a synthetic peptide, PT-(1-46)F4W, which includes the N-terminal 46 residues of human prothrombin with Phe-4 replaced by Trp providing a fluorescent probe. PT-(1-46)F4W and PT-(1-46) bind calcium ions and phospholipid membranes, and inhibit the prothrombinase complex. PT-(1-46)F4W, but not PT-(1-46), exhibits a blue shift (5 nm) and red-edge excitation shift (28 nm) in the presence of phosphatidylserine (PS)-containing vesicles, suggesting Trp-4 is located within the motionally restricted membrane interfacial region. PS-containing vesicles protect PT-(1-46)F4W, but not PT-(1-46), fluorescence from potassium iodide-induced quenching. Stern-Volmer analysis of the quenching of PT-(1-46)F4W in the presence and absence of 80% phosphatidylcholine/20% PS vesicles suggested that Trp-4 is positioned within the membrane and protected from aqueous quenching agents whereas Trp-41 remains solvent-accessible in the presence of PS-containing vesicles. Fluorescence quenching of membrane-bound PT-(1-46)F4W is optimal with 7- and 10-doxyl-labeled lipids, indicating that Trp-4 is inserted 5 to 7 A into the bilayer. This report demonstrates that the omega-loop region of prothrombin specifically interacts with PS-containing membranes within the interfacial membrane region.

1-Carboxyglutamic Acid↗
PubMed2003

Proexosite-1 on prothrombin is a factor Va-dependent recognition site for the prothrombinase complex.

Although the contribution of basic residues of exosite-1 to the catalytic function of thrombin has been studied extensively, their role in the specificity of prothrombin recognition by factor Xa in the prothrombinase complex (factor Xa, factor Va, phosphatidylcholine/phosphatidylserine vesicles, and Ca2+) has not been examined. In this study, we prepared several mutants of prethrombin-1 (prothrombin lacking Gla and Kringle-1 domains) in which basic residues of this site (Arg35, Lys36, Arg67, Lys70, Arg73, Arg75, and Arg77 in chymotrypsinogen numbering) were individually substituted with a Glu. Following expression in mammalian cells and purification to homogeneity, these mutants were characterized with respect to their ability to function as zymogens for both factor Xa and the prothrombinase complex. Factor Xa by itself exhibited similar catalytic activity toward both the wild type and mutant substrates; however, its activity in the prothrombinase complex toward most of mutants was severely impaired. Further kinetic studies in the presence of Tyr63-sulfated hirudin-(54-65) peptide suggested that although the peptide inhibits the prothrombinase activation of the wild type zymogen with a KD of 0.5-0.7 microm, it is ineffective in inhibiting the activation of mutant zymogens (KD = 2-30 microm). These results suggest that basic residues of proexosite-1 on prothrombin are factor Va-dependent recognition sites for factor Xa in the prothrombinase complex.

Binding Sites↗
PubMed2004

Proexosite-1-dependent recognition and activation of prothrombin by taipan venom.

An activator complex from the venom of Oxyuranus scutellatus scutellatus (taipan venom) is known to rapidly activate prothrombin to thrombin. To determine whether, similar to prothrombinase, taipan venom utilizes proexosite-1 on prothrombin for a productive complex assembly, the activation of proexosite-1 mutants of prethrombin-1 by the partially purified venom was studied. It was discovered that basic residues of this site (Arg(35), Lys(36), Arg(67), Lys(70), Arg(73), Arg(75), and Arg(77)) are also crucial for recognition and rapid activation of the substrate by taipan venom. This was evidenced by the observation that the K(m) and k(cat) values for the activation of the charge reversal mutants of prethrombin-1 (in particular K36E, R67E, and K70E) were markedly impaired. Competitive kinetic studies with the Tyr(63)-sulfated hirudin(54-65) peptide revealed that although the peptide inhibits the activation of the wild type zymogen by taipan venom with a K(D) of approximately 2 microm, it is ineffective in inhibiting the activation of mutant zymogens (K(D) > 4-30 microm). Interestingly, an approximately 50-kDa activator, isolated from the taipan venom complex, catalyzed the activation of prothrombin in a factor Va-dependent manner and exhibited identical activation kinetics toward the substrate in the presence of the hirudin peptide. These results suggest that, similar to prothrombinase, proexosite-1 is a cofactor-dependent recognition site for taipan venom.

Antithrombins↗
PubMed2002

Prothrombin activation in eastern tiger snake bite.

AIMS: To perform ex vivo studies in eastern tiger snake envenomation which define the haemostatic events associated with prothrombin activation. METHOD: Serial studies were performed on plasma from six individuals with evidence of eastern tiger snake envenomation. These analyses were particularly directed to fibrinogen levels, F1 + 2, TAT and evidence of fibrinolysis. RESULTS: There was a substantial rise in F1 + 2 and thrombin-antithrombin (TAT) complexes in all cases, even with minimal evenomation. In some cases the molar ratio of F1 + 2 and TAT was reduced from the relationship normally seen in vitro and ex vivo in clinical thrombosis. There was a dramatic fall in factor V and VIII levels which occurred 4-6 hours before the decline in prothrombin and AT3. This related in time to a fall in alpha2AP and plasminogen. Protein C levels also declined dramatically but many hours after presentation. CONCLUSIONS: F1 + 2 and TAT are sensitive markers of tiger snake evenomation. In some patients with massive prothrombin activation, the common mechanism for TAT clearance may be altered or overwhelmed. Conversely, the renal clearance of the smaller F1 + 2 may be enhanced. In the absence of thrombocytopaenia, which is a very sensitive marker of DIC, the fall in labile factors with tiger snake envenomation is significantly contributed to by proteolytic digestion of clotting factors.

Adolescent↗
PubMed1990

Prothrombin time as an index of mortality in kwashiorkor.

Prothrombin time, serum albumin, aminotransferases and liver size were evaluated in 40 consecutive cases of kwashiorkor. Eleven (27.5%) of the 40 patients died. Eight out of the 11 patients who died had a prolonged prothrombin time of more than 3 s above the control compared to only 4 out of the 29 who survived (p = 0.005). Mean serum aspartate aminotransferase (AST), alanine aminotransferase (ALT) albumin, globulin and liver size were abnormal but similar in both groups. These results may indicate a predictive mortality value of prothrombin time in kwashiorkor.

Bacterial Infections↗
PubMed2001

Low prevalence of large intraventricular haemorrhage in very low birthweight infants carrying the factor V Leiden or prothrombin G20210A mutation.

UNLABELLED: The influence of genetic factors that increase coagulation on the extension of intraventricular haemorrhage (IVH) in very low birthweight infants has not been studied previously. This study investigated the frequency and effect of the factor V Leiden and prothrombin G20210A mutations in a population-based cohort of 305 preterm infants with a birthweight below 1500 g. The overall prevalence of IVH was similar in infants with (n = 43) and without (n = 262) prothrombotic mutations (18.6% vs 16.4%, respectively). However, infants with prothrombotic mutations had a significantly reduced risk of developing extension to IVH grade II or more [p = 0.023, odds ratio (OR) 0.11, 95% confidence interval (CI) 0.02-0.5]. The carrier state of a factor V Leiden or prothrombin G20210A mutation was still predictive for a low rate of IVH grade II-IV if possible confounding variables were included in a multivariate regression model (OR 0.12; 95%CI: 0.017-0.86). CONCLUSION: The data suggest that the factor V Leiden and prothrombin G20210A mutations lead to improved control of intraventricular bleeding in very low birthweight infants.

DNA Mutational Analysis↗
PubMed2002

Mutations in the factor V, prothrombin and MTHFR genes are not risk factors for recurrent fetal loss.

OBJECTIVE: Recurrent fetal loss, defined as the occurrence of three or more consecutive spontaneous abortions regardless of previous live birth, is a condition that affects about 2% of all reproductive-aged women. The role of gene mutations in recurrent pregnancy loss is not fully understood. The present research examined the relationship between factor V Leiden, factor V HR2, prothrombin G20210A and MTHFR and recurrent fetal loss in a case-control study. METHODS: Women aged 22-45 with a history of three or more fetal losses, being seen at a perinatal medicine clinic in New Jersey or Georgia, were eligible as cases. Overall, the study consisted of 60 women with three or more fetal losses and 92 women with at least one successful pregnancy. RESULTS: Factor V HR2 and MTHFR were not related to recurrent fetal loss. The prothrombin G20210A mutation appeared to confer an elevation in risk but the association was based upon small numbers and was not statistically significant (OR 4.8, 95% CI 0.50-47.2). Cases were 90% less likely to have the factor V Leiden mutation than controls (OR 0.10, 95% CI 0.01-0.81). CONCLUSIONS: Our study did not demonstrate that women who are carriers of the factor V, prothrombin, or MTHFR mutations are at higher risk of recurrent fetal loss than women without these mutations. In regards to factor V Leiden, the prevalence in our cases (1.7%) was not statistically different from the known population prevalence of 5%. However, the high prevalence in our controls (14%) was unusual. Factor V Leiden may protect against bleeding in early pregnancy.

Abortion, Habitual↗
PubMed1951

Fractionation of plasma globulin for prothrombin, thrombokinase, and accessory thromboplastin.

1. Crude globulin from more than 1,000 liters of citrated bovine plasma has been used in developing a procedure for moderately large scale separation of clotting factors. Fraction A, prothrombin, kinase, and thrombin fractions were prepared. Fraction A contained both kinase and accessory thromboplastin, the latter predominating when fraction A was diluted. 2. When prothrombin was activated by kinase, the rate of thrombin production was enhanced by the addition of platelets, or brain lipid, or dilute fraction A. These accessory thromboplastins caused this acceleration only when calcium chloride was added. Even with calcium, they were not effective unless kinase was present. 3. In contrast, the action of kinase was not entirely dependent on either ionic calcium or accessory thromboplastin. The concentrated kinase fraction activated prothrombin in the presence of excess oxalate. Although kinase often contaminates highly purified thrombins, it is probably distinct from thrombin. The ratio of kinase to thrombin was 100 times as great in the kinase fraction as in the thrombin fraction. 4. The kinase fraction, diluted 45,000-fold, to protein-nitrogen concentrations as low as 0.02 microgram per ml., accelerated the conversion of crude prokinase in three-stage tests. 5. The findings are consistent with the following concept of the basic enzymatic mechanism: See PDF for Structure It is now added that calcium and accessory thromboplastin exert their effects by impinging on the basic mechanism, in a chemically secondary or indirect manner.

Animals↗
PubMed2004

Multiplex PCR-RFLP assay for detection of factor V Leiden and prothrombin G20210A.

Factor V Leiden and prothrombin G20210A are clinically relevant genetic risk factors for venous thrombosis. Molecular diagnostic testing for factor V Leiden and prothrombin G20210A is widespread, and laboratories use a variety of technical approaches. Here we introduce a multiplex polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) based on single (Mn/l) restriction endonuclease digestion. The assay was shown to simultaneously and accurately detect factor V Leiden and prothrombin G20210A mutations.

Activated Protein C Resistance↗
PubMed1996

Prothrombin time: one tube or two.

This study compares the prothrombin times (PTs) and calculated international normalized ratios (INRs) from first and second evacuation blood tubes to determine the clinical importance of using a second tube specimen for protime coagulation studies. The National Committee for Clinical Laboratory Standards (NCCLS) currently recommends that all coagulation studies be done on a second or later drawn blood tube. For patients on long-term anticoagulation therapy, this often requires that first blood tubes be drawn and discarded at each prothrombin evaluation. In this prospective study, a first and second evacuation blood tube was drawn from 343 outpatients who had a physician-ordered prothrombin time test performed. There was no statistically significant difference in the paired PT or calculated INR from any of the first and second tubes. The average difference in the INR from tube 1 to tube 2 was 2% (standard deviation [SD] 1.1%). In this sample of outpatients, the use of a second tube for PT testing was not clinically justified.

Ambulatory Care↗
PubMed1998

A cross-Canada survey of prothrombin time testing: Does the establishment of local ISI values improve the accuracy of international normalized ratio reporting? Thrombosis Interest Group of Canada.

The international normalized ratio (INR) was established as a means of standardizing the prothrombin time regardless of the thromboplastin used in the individual laboratories. The INR is the prothrombin time ratio of the sample raised to the power of the International Sensitivity Index (ISI). Traditionally, the ISI is determined by using a manual clotting technique by comparing the test thromboplastin with a World Health Organization international reference thromboplastin with results from 60 patient samples standardized on warfarin, and 20 samples from normal volunteers. Most laboratories no longer perform prothrombin time testing by a manual technique but instead automate the procedure by using a variety of coagulation instruments. Thromboplastin ISI values have the potential to be modified considerably by instrumentation, and as a consequence, they, may result in INRs that are unreliable when compared to those obtained by the traditional manual method. It has been proposed that the use of calibrant plasmas tested with the laboratory's thromboplastin and instrument may overcome this problem and a local ISI value could be established. Our study objective was to determine whether a local ISI calibration with the plasma calibrants could reduce the variation of the INR over a wide range of thromboplastin/ instrument combinations. A total of 58 laboratories from across Canada, including community hospitals, private laboratories, and large tertiary care units, participated. The findings indicate that the use of calibrant plasmas to calculate the local ISI does improve the accuracy of INR reporting in the majority of thromboplastin/instrument combinations.

Autoanalysis↗
PubMed1979

Prothrombin time after heparin removal. Application to monitoring simultaneous anticoagulation with heparin and coumarins.

Coumarin-anticoagulated plasmas from 31 subjects comprising two study groups were passed through epichlorohydrin triethanolamine cellulose (ECTEOLA) chromatography columns. Group I plasmas, which were run with nothing added, had mean prothrombin times of 21.6 +/- 5.4 sec prior to and 22.4 +/- 5.6 sec following exposure to these columns (r = 0.9449; t = 1.8307, P less than 0.100). The mean prothrombin time for Group II, 18.2 +/- 4.9 sec, lengthened with 5 u/ml heparin to 35.5 +/- 16.2 sec, and returned to 19.2 +/- 5.0 (r = 0.9763) after chromatography. Therefore, it appears that coumarin anticoagulation had no significant influence upon the capacity of ECTEOLA effectively to remove heparin in therapeutic doses. This means that virtually all prothrombin time reagent systems can be employed to monitor concurrent heparin and coumarin anticoagulation. In addition, quality control of the combined technic is simpler, and the technic more sensitive to low levels of fibrinogen and factor V.

Cellulose↗
PubMed1982

A rapid chromogenic method for the determination of prothrombin precursor in plasma.

A method was described for the quantitation of precursor prothrombin using a chromogenic substrate S2238 and Dispholidus typus venom. Prothrombin precursor was detected in the adsorbed plasma of all 65 patients on coumadin therapy tested, but was absent in 20 controls. The technic offered a rapid and sensitive method for determining if a prolonged prothrombin time is due to vitamin K antagonism or deficiency.

Adsorption↗
PubMed1985

Inhibition of cold-promoted activation of the prothrombin time studies of new siliconized borosilicate collection tubes in normals and patients receiving warfarin.

Studies of the prothrombin time in normals and patients receiving oral anticoagulant therapy revealed a partial inhibition of the in vitro cold-promoted activation of Factor VII and shortening of the prothrombin time when blood was collected in a new commercial siliconized borosilicate tube (Becton-Dickinson, Rutherford, NJ, 6418 and 6419). The data collected with normal blood indicated that the cold-promoted activation was almost totally inhibited, while the patients had only a mean reduction of 50% of the cold-promoted activation. In pregnant patients there was no inhibition of the cold-promoted activation of the prothrombin time or Factor VII using the new siliconized tubes.

Blood Specimen Collection↗
PubMed2004

Factor V Leiden and G20210A prothrombin mutation and the risk of subclavian vein thrombosis in patients with breast cancer and a central venous catheter.

BACKGROUND: To analyze the influence of the prothrombotic gene mutation factor V G1691A (factor V Leiden) and prothrombin G20210A on the risk of a first episode of catheter-related deep venous thrombosis (DVT) in a group of patients with breast cancer treated with chemotherapy. PATIENTS AND METHODS: Between January 1999 and February 2001, the occurrence of a first symptomatic DVT was investigated in a cohort of 300 consecutive patients with locally advanced or metastatic breast cancer treated at a single institution with fluorouracil-based chemotherapy, administered continuously through a totally implanted access port. A nested case-control study included 25 women (cases) with catheter-related DVT and 50 controls without DVT matched with cases for age, identical chemotherapy, stage of disease and prognostic features. The G1691A factor V and G20210A prothrombin mutation genotypes were analyzed. RESULTS: Five cases [20%; 95% confidence interval (CI) 9% to 39%)] and two controls (4%; 95% CI 1% to 14%) were heterozygous carriers of G1691A factor V (P = 0.04). The age-adjusted odds ratio for catheter-related DVT was 6.1 (95% CI 1.1-34.3). Only one patient (case) had the G20210A prothrombin gene mutation. Time from start of chemotherapy infusion to DVT was not significantly different between patients with (median 31 days) and without (median 43 days) G1691A factor V mutation (P = 0.6). CONCLUSIONS: Factor V Leiden carriers with locally advanced or metastatic breast cancer have an increased risk of developing catheter-related DVT during chemotherapy.

Adult↗
PubMed1985

A method for systematic purification from bovine plasma of six vitamin K-dependent coagulation factors: prothrombin, factor X, factor IX, protein S, protein C, and protein Z.

A systematic purification scheme is presented for the isolation of six vitamin K-dependent coagulation factors from bovine plasma in a functionally and biochemically pure state. The vitamin K-dependent proteins concentrated by the ordinary barium citrate adsorption were first separated into four fractions, fractions A, B, C, and D, by DEAE-Sephadex A-50 chromatography. From the pooled fraction A, protein S, factor IX, and prothrombin were purified by column chromatography on Blue-Sepharose CL-6B. Heparin-Sepharose chromatography of the pooled fraction B provided mainly pure factor IX, in addition to homogeneous prothrombin. A high degree of resolution of protein C and prothrombin from the pooled fraction C was obtained with a Blue-Sepharose column. This dye-ligand chromatographic procedure was also very effective for the separation of protein Z and factor X contained in the pooled fraction D. Thus, these preparative procedures allowed high recovery of milligram and gram quantities of six vitamin K-dependent proteins from 15 liters of plasma in only two chromatographic steps, except for protein S, which required three (the third step was rechromatography on Blue-Sepharose CL-6B).

Amino Acids↗
PubMed1992

Reduction of plasma levels of prothrombin fragments 1 and 2 during thromboprophylaxis with a low-molecular-weight heparin.

In a randomized double-blind placebo-controlled study, plasma levels of prothrombin fragment 1 and 2 and total fibrin/fibrinogen degradation products were measured preoperatively and on days 1, 3, 5 and 7 postoperatively in 131 patients undergoing total hip replacement. Patients received a subcutaneous injection of either a low molecular weight heparin (Logiparin) or placebo once daily. Postoperative deep vein thrombosis was diagnosed by bilateral phlebography 7 to 10 days after operation. In the placebo group postoperative levels of prothrombin fragments 1 and 2 and total fibrin/fibrinogen degradation products were significantly higher in patients with postoperative thromboembolic complications, whereas in the low-molecular-weight heparin group no statistical differences were observed. Compared with placebo the administration of a low-molecular-weight heparin was associated with a significant reduction in the levels of prothrombin fragments 1 and 2 and total degradation products. Our observations suggest that the postoperative thrombin generation is moderated by thromboprophylaxis with Logiparin.

Adult↗
PubMed1994

Prothrombin Padua I: incomplete activation due to an amino acid substitution at a factor Xa cleavage site.

An individual and an affected brother previously identified as having the variant prothrombin Padua I were studied in order to identify underlying genetic defects. A heterozygous mutation in the prothrombin gene exon 8 was identified as substitution of A for G at nucleotide position 7,312 (Arg271 (CGT) to His (CAT)). An abolished RsaI restriction site was used to confirm heterozygosity for the defect. Lack of the requisite cleavage of the His271-Thr272 bond in prothrombin Padua I could prevent release of fragment 2 and block the conversion of the intermediate meizothrombin des fragment 1 to alpha-thrombin, providing an explanation of reduced potential for clotting activity and for the observed mild bleeding tendency.

Amino Acid Sequence↗