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PubMed1993

Thrombin exosite for fibrinogen recognition is partially accessible in prothrombin.

The procoagulant alpha-thrombin is produced by the proteolytic cleavages of a minimum of two peptide bonds Arg274-Thr275 and Arg323-Ile324 in prothrombin. The Arg323-Ile324 cleavage is required for the expression of the active site of thrombin (Morita, T., Iwanaga, S. Suzuki, T. (1976) J. Biochem. (Tokyo) 79, 1089-1108; Hibbard, L. S., Nesheim, M. E., and Mann, K. G. (1982) Biochemistry 21, 2285-2292). It is not yet clear to what extent the proteolytic events are responsible for exposing protein recognition exosites on thrombin. We employed high resolution NMR spectroscopy to examine interactions of prothrombin and thrombin with synthetic hirudin peptides targeted toward the fibrinogen recognition exosite of thrombin. The hirudin tail synthetic analogues (acetyl-Asp55-Phe-Glu-Glu-Ile-Pro-Glu-Glu-Tyr-Leu-Gln65/G ly65-OH) exhibited similar NMR relaxation enhancements (line broadening patterns and transferred nuclear Overhauser effects) with human prothrombin as with human alpha-thrombin, indicating that both proteins bind the peptide in a similar manner. The protein-induced relaxation enhancements are specific to the interaction of the hirudin peptides with the fibrinogen recognition exosite of thrombin since no significant effects were observed with either human serum albumin or with human gamma-thrombin, which has an impaired recognition exosite. The binding affinities were determined from NMR relaxation time measurements, which gave approximate Kd values of 500 microM and < 100 microM for prothrombin and alpha-thrombin, respectively. Since the hirudin tail fragment binds specifically to the fibrinogen recognition exosite in alpha-thrombin, this exosite appears to be partially accessible in prothrombin in a proenzyme form.

Amino Acid Sequence↗
PubMed1993

Vitamin K1 metabolism and the production of des-carboxy prothrombin and protein C in the term and premature neonate.

This study investigated the incidences of undercarboxylated (protein induced by vitamin K absence: PIVKA) prothrombin and protein C in 496 neonates across a wide range of gestational ages. These findings are related to vitamin K1 levels (an indicator of cofactor availability) and vitamin K1 epoxide levels (a measure of the efficiency of the hepatic vitamin K cycle). PIVKA protein C was present in at least trace amounts in 27% of infants; whereas, PIVKA prothrombin was present in 7% of infants. PIVKA prothrombin and protein C were present at high plasma concentrations in 2% to 3% of term and preterm neonates and both PIVKA protein C and prothrombin increased with gestational age. Despite elevated plasma concentrations of PIVKA protein C and diminished levels of normally carboxylated protein C, clinical thrombosis was not observed. The mean (+/- SD) vitamin K1 level in the study population was 0.009 +/- 0.02 nmol/L (adult reference interval: 0.3 to 2.6 nmol/L) with no clear relationship between vitamin K1 levels and production of PIVKA protein C or prothrombin. By comparison with adults, the epoxide form of the vitamin comprised an abnormally high proportion of total vitamin K1; this suggests possible inefficiencies in hepatic reductase cycling.

Biomarkers↗
PubMed1993

The effect of activated protein C on fibrinolysis in cell-free plasma can be attributed specifically to attenuation of prothrombin activation.

The effect of human activated protein C (APC) on tissue plasminogen activator (tPA)-induced fibrinolysis was studied in cell free plasma and in a system of purified components. Clots were produced by adding plasma or a solution of fibrinogen and plasminogen to the wells of a microtiter plate containing small separated aliquots of Ca2+, thrombin, and tPA, plus and minus APC. Initial clotting and subsequent fibrinolysis were monitored continuously by turbidity. The lysis time of dialyzed normal human plasma (NHP) was longer than that of dialyzed barium citrate-adsorbed plasma (BAP). APC had no effect on the lysis time of BAP but shortened the lysis time of NHP to that of BAP. Two fractions were produced from material eluted from the barium citrate pellet by precipitation of selective components with polyethylene glycol 8000 (PEG). One fraction comprised materials which precipitated at 5% PEG (5% PF) and the other materials which precipitated between 5 and 40% PEG (5-40% PF). Both fractions together, but neither alone, prolonged the lysis time of BAP, an effect which could be reversed by APC. Fractionation of the 5% PF showed that the component with the required activity has properties of the procoagulant surface and can be replaced with vesicles of phosphatidylcholine/phosphatidylserine (PCPS). In addition, the 5-40% PF can be replaced with either the combination of purified coagulation Factors II, IX, and X or Factor II plus the prothrombin activator Factor Xa. When Factor Xa was used as the activator in BAP plus PSPC vesicles, a dose-dependent saturable increase in lysis time was observed with a half-maximal increase occurring at 32 pM Factor Xa. This effect was eliminated by APC. In a system of purified components comprising PCPS vesicles, Factors V and II, protein S, plasminogen and fibrinogen; the prothrombin activators Factor Xa and ecarin both induced a prolongation of the lysis time. APC prevented prolongation by Factor Xa but not by ecarin. The time courses of the generation of thrombin and plasmin during fibrinolysis of clots produced from systems of purified components in the presence and absence of APC, and with Factor Xa as the prothrombin activator, were determined by standardized activity assays using chromogenic substrates. In the absence of APC the lysis time was 145 min, and prothrombin was quantitatively converted to thrombin. In the presence of APC, however, the lysis time was reduced to 100 min with no evidence for the activation of prothrombin.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell-Free System↗
PubMedDate not supplied

[Determination of the general prothrombin level and detection of its functionally inactive forms using the enzyme ecamulin purified from Echis multisquamatus venom].

The method for determination of the general prothrombin level and detection of its inactive forms (prothrombin-1, decarboxylated prothrombin, etc.) has been worked out. Enzyme ecamulin was purified from the venom of Echis multisquamatus. Ecamulin test was used for plasma of practically healthy people and patients with hypertension. The results obtained proved that this test is informative and precise. Comparison of two tests, prothrombin and ecamulin ones, permits characterizing the state of blood coagulation and finding functionally inactive prothrombin forms which are the markers of some pathological states.

Adult↗
PubMed1996

[Transitions of each inhibitor in a patient with lupus anticoagulant and anti-prothrombin antibody].

In a 2-year-old girl showing purpura on her legs after administration of antibiotics, marked prolongation of activated partial thromboplastin time(APTT) and prothrombin time was noted. Circulating anticoagulants were demonstrated by the failure to correct APTT on neutralization test. A lupus anticoagulant(LA), one of the circulating anticoagulants, was detected by rabbit brain phospholipid neutralization procedure and platelet neutralization procedure. On crossed immunoelectrophoresis, the abnormal prothrombin peaks with reduced electrophoretic mobilities were considered prothrombin/prothrombin-antibody complexes because pretreatment with anti-human IgG serum caused their disappearance. This prothrombin-antibody seemed to be another circulating anticoagulant. The anti-prothrombin-antibody reduced prothrombin activities in the circulating blood of the patient to 20 approximately 30% of normal and the condition persisted for two weeks resulting in the purpura which occurred during that period. After two months, APTT was restored to normal following the disappearance of LA.

Animals↗
PubMed1995

Reference materials and reference measurement systems in laboratory medicine. Prothrombin time standardization: the problem of the control plasma.

The prothrombin time of fresh normal pooled plasma and the mean normal prothrombin time are currently recommended as the denominator term in the expression of prothrombin time ratios and International Normalised Ratio (INR) values. Fresh normal pooled plasma is also required for correct extrapolation of percent prothrombin time activity values. To avoid collection and measurement of a relevant number of normal individual samples for calculation of the mean normal prothrombin time or the necessity for a fresh plasma pool, lyophilised normal control plasma is made available by the manufacturers of thromboplastin reagents. The Verband der Diagnostica- und Diagnostikgerätehersteller (VDGH) has adopted a lyophilised normal pooled plasma (R82A) which was calibrated in two studies against fresh normal plasma pools and fresh individual normal plasmas, using a variety of plain and combined thromboplastins. Both studies concluded that plasma R82A could be used as a substitute for normal plasma, but a correction should be made for plain thromboplastin reagents. These data have been confirmed in an IFCC collaborative international study organised by the IFCC Working Group Standardisation of Coagulation Tests. In the latter study, in addition to plain and combined thromboplastin reagents, the recently introduced recombinant thromboplastins were evaluated and they showed a significant deviation from the sensitivity observed with extracted thromboplastins. Recombinant thromboplastins are under consideration as future international reference thromboplastins; the results of the IFCC collaborative study call for additional experimentation before this is accomplished.

Blood↗
PubMed1996

[Isolation and characteristics of ekamulin--a prothrombin activator from multiscaled viper (Echis multisquamatus) venom].

Ecamulin, a novel prothrombin activating enzyme, has been isolated and purified 63-fold with a 57% yield from the venom of the Middle-Asian sand viper Echis multisquamatus using three-step ion-exchange chromatography. The enzyme was shown to activate prothrombin similarly to Ecarin, a prothrombin-converting enzyme from Echis carinatus venom, however, differing from the latter by structural and physico-chemical properties. The enzyme is a Zn-proteinase: it contains 1 mol Zn per 1 mol of protein. The molecular mass of the enzyme as determined by Sephacryl S-200 chromatography is 93 +/- 2 kDa. Upon SDS-PAAG electrophoresis ecamulin produces two bands with Mr of 67 and 27 kDa under non-reducing conditions, and three bands with Mr of 67, 14 and 13 kDa in the presence of DTT. During native PAGE without SDS, the activator yields one slow mobility band: two bands are observed after addition of DTT or EDTA. Carbohydrates containing N-acetyl-alpha-D-glucosamine residues are localized in the 67 kDa chain. Ecamulin has two isoforms, S2 and S3, that are distinguished by the charge and partial coagulation activities: form S2 has 250 NIH units/mg, while the S3 form has 524 NIH units/mg. The amino acid sequences of the both isoforms are similar but the more active S3 form has 4 times higher content of Gln and 4 times less of Gly than the S2 form. The isoelectric point is 4.3-4.5; E280 of 1% solution is 10.2. Forms S2 and S3 of ecamulin hydrolyze chromogenic substrates of plasma kallikrein S2302 and glandular kallikrein 2266. Ecamulin does not hydrolyze BAEE, TAME, LEE, thrombin substrates Chromozym TH and S2160, factor Xa-S2222, protein Ca-Chromozym PCa and Plasmin S2251. The amidase activity is nonreversibly inhibited by EDTA, o-phenanthroline (the activity is recovered by addition of Zn2+), Cys or DTT, EGTA, DFP, PMSF or pCMB do not inhibit the enzyme activity. Ecamulin converts prothrombin to alpha-thrombin passing by a shunt via the meizothrombin stage. The reaction of prothrombin activation does not require Ca2+, phospholipids of factor Va. Part of this work was presented at the International Conference "Fibrinogen and fibrinolysis", Yalta, September 23-28, 1995.

Amino Acids↗
PubMed1976

Clinical usefulness of measuring prothrombin time as a routine admission test.

A questionnaire indicating the presence of a history or physical findings consistent with liver desease or bleeding disorders was completed by house officers on 301 admissions to a Veterans Administration medical service. Each patient had prothrombin time determined on admission. Only two of 107 patients for whom the prothrombin time was measured as a screening test had an abnormal results and one of these was normal when repeated. Of 73 patients with a history of alcoholism but no other pertient case history or physical examination results, only one had an abnormal prothrombin time. Of the remaining 121 patients, who had a pertinent history or physical examination, 41 had an abnormal admission prothrombin time. We conclude that measurement of prothrombin time adds little additional information to that obtained by history and physical examination in screening for liver disease and coagulation defects unless the patient has specific clinical evidence of liver disease, anticoagulation, or other conditions predisposing to bleeding disorders.

Alcoholism↗
PubMed1998

Prothrombin kringle-2 domain has a growth inhibitory activity against basic fibroblast growth factor-stimulated capillary endothelial cells.

Recently, O'Reilly et al. (O'Reilly, M. S., Holmgren, L., Shing, Y., Chen, C., Rosenthal, R. A., Moses, M., Lane, W. S., Cao, Y., Sage, E. H., and Folkman, J. (1994) Cell 79, 315-328; O'Reilly, M. S., Boehm, T., Shing, Y., Fukai, N., Vasios, G., Lane, W. S., Flynn, E., Birkhead, J. R., Olsen, B. R., and Folkman, J. (1997) Cell 88, 277-285) developed a simple in vitro angiogenesis assay system using bovine capillary endothelial cell proliferation and purified potent angiogenic inhibitors, including angiostatin and endostatin. Using a simple in vitro assay for angiogenesis, we purified a protein molecule that showed anti-endothelial cell proliferative activity from the serum of New Zealand White rabbits, which was stimulated by lipopolysaccharide. The purified protein showed only bovine capillary endothelial cell growth inhibition and not any cytotoxicity. This molecule was identified as a prothrombin kringle-2 domain (fragment-2) using Edman degradation and the amino acid sequence deduced from the cloned cDNA. Both the prothrombin kringle-2 domain released from prothrombin by factor Xa cleavage and the angiogenic inhibitor purified from rabbit sera exhibited anti-endothelial cell proliferative activity. The recombinant rabbit prothrombin kringle-2 domain showed potent inhibitory activity with half-maximal concentrations (ED50) of 2 microg/ml media. As in angiostatin, the recombinant rabbit prothrombin kringle-2 domain also inhibited angiogenesis in the chorioallantoic membrane of chick embryos.

Amino Acid Sequence↗
PubMed1987

Long-term results of the treatment of children with factor VIII inhibitors with prothrombin-complex concentrates.

Twelve boys with haemophilia and factor VIII inhibitors have received initial treatment for each episode of bleeding with prothrombin-complex concentrate. The follow-up period for these boys has ranged from two months to 14 years, and a total of 732 bleeding episodes were reviewed. Each boy underwent a full clinical and radiological assessment of knee-joint function, and this was compared with knee-joint function in 29 patients with severe haemophilia but without factor VIII antibodies. A change of treatment from prothrombin-complex concentrate to some other form of therapy was considered to be required in only 4% of the bleeding episodes. Of 10 severe bleeding episodes, five episodes clearly were controlled with prothrombin-complex concentrate, but five episodes necessitated alternative treatment. Four of the 12 families considered prothrombin-complex concentrate as relatively ineffective. All boys who were older than 10 years of age showed grade-1 or grade-2 impairment of knee-joint mobility, as well as radiological evidence of moderate-to-severe knee-joint changes. In the group of haemophiliac patients who were older than 10 years of age and did not have factor VIII antibodies, only 10 of the 58 knee-joints that were examined showed grade-1 or grade-2 functional impairment. Although prothrombin-complex concentrates were effective in the control of the majority of minor bleeding episodes, they were effective in only 50% of severe episodes of bleeding and did not prevent the early development of haemophilic arthropathy.

Blood Coagulation Factors↗
PubMed1986

Prothrombin Perija: a new congenital dysprothrombinemia in an Indian family.

A Western Venezuelan indigenous Yukpa(Irapa) family with moderate bleeding and discrepancy in the prothrombin values by biological and immunological methods is described. The propositus presented biological activity of 2% by the methods in one-stage including Staphylocoagulase and Echis carinatus. Electrophoretic migration of the prothrombin in plasma was normal, while the serum only showed one precipitation arc instead of the three of normal serum. The propositus appeared to be homozygous for the abnormal prothrombin which we have denominated Prothrombin Perijá.

Adolescent↗
PubMed1997

Performance of prothrombin-proconvertin time as a monitoring test of oral anticoagulation therapy.

Outcome and anticoagulation intensity was evaluated during 121 patient years of oral anticoagulant therapy monitored with the prothrombin-proconvertin clotting time (PP, also known as P&P). The PP-based international normalized ratio (INR; PP-INR) correlated well with the INR calculated from the prothrombin clotting time (PT; r = 0.92), and results were almost identical over a wide range after linear conversion (1/INR). When the PP-INR was 4.5 or less, the risk of major bleeding was 1 for every 118 treatment years, but it was 1 for every 73 days when the INR was 6 or more. The 1/PP-INR correlated better with factor II coagulant activity (r = 0.85) than did the 1/PT-INR (r = 0.78). The 1/PP-INR also correlated better with the native prothrombin antigen (r = 0.76) than did the 1/PT-INR (r = 0.68). The PP and PT results correlated better with factor II coagulant activity than with native prothrombin antigen. Thus, the PP clotting time results can be accurately converted to INR. The results also suggest that the PP may have advantages over the PT as an indicator of anticoagulation intensity during oral anticoagulation.

Administration, Oral↗
PubMed1975

Statistical comparison of the fibrometer and the Electra 600 for prothrombin time determination.

Determinations of prothrombin time with the semiautomatic fibrometer and with a more automated machine (Electra 600) were compared in regard to reproducibility, accuracy, and speed. Prothrombin times determined for replicate samples with the two machines showed a correlation coefficient of 0.98, but the Electra 600 sensed the clot 0.5 seconds before the fibrometer. The overall coefficients of variation for multiple tests in the normal therapeutic and elevated ranges of prothrombin time were 3.5% for the fibrometer and 2.0% for the Electra 600. The average technician working time was 60% shorter with the fully automated machine than with fibrometer. It is concluded that the automated machine was more accurate and more rapid in determining prothrombin time.

Automation↗
PubMed1976

Antihemophilic factor inhibitors. Management with prothrombin complex concentrates.

Prothrombin complex concentrates have been evaluated in the treatment of acute bleeding episodes occurring in 11 hemophilic and two nonhemophilic patients with factor VIII inhibitors. In several of the hemophilic patients, a continued fall in inhibitor levels has been observed coincident with continued use of the concentrate Proplex, and one of these patients is now responsive to factor VIII therapy. The therapeutic effectiveness of prothrombin complex products appears to be related to the presence of small amounts of activated factors or to the continued formation of factor Xa in vivo as a result of the increased levels of prothrombin and factors VII and X. Until high-potency activated products are available, prothrombin concentrates offer an effective means of treating patients with factor VIII inhibitor.

Acute Disease↗
PubMed2002

Prevalence of factor V G1691A (factor V-Leiden) and prothrombin G20210A gene mutations in a recurrent miscarriage population.

Factor V G1691A (FV-Leiden) and prothrombin G20210A mutations are major inherited risk factors for venous thrombosis. Recently, it was suggested that both mutations, through stimulation of venous and placental thrombosis events, were strongly associated with recurrent idiopathic miscarriages, although other studies disputed such a link. The aim of this study was to determine the prevalence of prothrombin G20210A and factor V G1691A (R506Q, FV-Leiden) mutations in women with recurrent idiopathic abortions and to recommend management for high-risk mutation carriers. One hundred ten women with two or more consecutive unexplained first-trimester miscarriages (mean age +/- SD, 32.3 +/- 5.3) were compared to 67 parous women with uncomplicated pregnancies (mean age +/- SD, 33.9 +/-7.3) (P = 0.134) from the same ethnic background. The presence or absence of the prothrombin G20210A and FV-Leiden mutations was assessed by PCR and RFLP analysis, using HindIII and MnlI digestion, respectively. In women with primary habitual abortion, 45 (40.91%) carried the FV-Leiden mutation, of whom 7 were in the homozygote and 38 were in the heterozygote states, and 15 (13.64%) carried the prothrombin G20210A mutation all as heterozygotes, compared to 16.42% and 2.99% carrier rates among controls, respectively, all of whom were heterozygote carriers. Of the other risk factors analyzed, smoking (OR 1.76; 95% CI = 0.79-3.94) was more prevalent in habitual aborters compared to controls. Both FV-Leiden and factor II G20210A mutations are major inherited risk factor associated with primary recurrent miscarriages. Women with a family or personal history of thrombosis should be screened before or early in the pregnancy for FV-Leiden and factor II G20210A mutations.

Abortion, Habitual↗
PubMed2001

A functional prothrombin gene product is synthesized by human kidney cells.

gamma-carboxylated polypeptides were detected in the human kidney by immunohistochemistry with a monoclonal antibody (M3B) specific for gamma-carboxyglutamyl residues. An approximately 70-kDa gamma-carboxylated protein, subsequently identified as prothrombin, was isolated from the intracellular compartment of cultured human embryonic kidney (HEK293) cells by immunoaffinity chromatography on M3B-coupled resin. Immunohistochemical analyses demonstrated that prothrombin and another vitamin K-dependent protein, the growth arrest-specific protein 6, were detectable in human kidney. As in the liver, the kidney synthesizes prothrombin as a zymogen that can be cleaved by ecarin to an amidolytically active serine protease that is inhibited by hirudin. This demonstrates for the first time the de novo synthesis of a full-length, gamma-carboxylated, and functional prothrombin gene product by human kidney cells.

Antibodies, Monoclonal↗
PubMed1996

Prothrombin fragment F1+2 and thrombin-antithrombin III complex (TAT) plasma levels in patients with gynecological cancer.

The pretreatment plasma levels of prothrombin fragment F1+2 and thrombin-antithrombin III complex (TAT) were measured in 56 consecutive patients with gynecological cancer and in 33 apparently healthy volunteer nonpregnant women as control. Prothrombin fragment F1+2 concentrations were significantly elevated in the 18 patients with ovarian cancer (median, 3.2 nmol/ liter; range, 0.9-17.0 nmol/liter, P < 0.0001), in the 24 patients with cervical cancer (median, 1.7 nmol/liter; range, 0.6-15.0 nmol/ liters, P < 0.0001), and in the 14 patients with endometrial cancer (median, 1.5 nmol/liter; range, 0.6-3.0 nmol/liter, P = 0.036) when compared to controls (median, 1.0 nmol/liter; range, 0. 1 -2.1 nmol/ liter). Similarly, TAT levels were significantly higher in patients with ovarian cancer (median, 5.3 microgram /ml; range, 1.3-65.0 microgram/ml , P < 0.0001), cervical cancer (median, 3.8 microgram/ml; range, 2.1 - 11.0 microgram / ml, P < 0.0001), and endometrial cancer (median, 2.7 microgram/ml; range, 1.9-19.0 microgram /ml, P = 0.008) when compared to controls (median, 2.2 microgram/ml; range, 1.0-5.0 microgram/ml). Prothrombin fragment F1+2 levels correlated with TAT levels (r = 0.659, P < 0.0001). Among ovarian cancer patients, prothrombin fragment F1+2 and TAT concentrations correlated with FIGO stage (III-IV versus 1, P = 0.01 and P = 0.003, respectively) and CA 125 levels (P 0.02 and P < 0.0001, respectively). The present data confirmed the occurrence of hemostasis activation in patients with gynecological cancer, and in particular in those with ovarian cancer.

Adenocarcinoma↗
PubMed1975

Breakdown of prothrombin and proconvertin in rats with experimental extrahepatic cholestasis.

Changes in the half-life of prothrombin and proconvertin were studied in rats with experimental extraphepatic cholestasis induced by ligation of the common bile duct. By the end of the second week of cholestasis the prothrombin activity was reduced almost by half and the proconvertin activity by more than two-thirds compared with the control. Meanwhile the half-life of prothrombin after cholestatsis for 14 days was increased by 1.f h and that of proconvertin by 4.7 h compared with the control. The slower breakdown of prothrombin and proconvertin during cholestasis is regarded as a compensatory reation.

Animals↗