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PubMed1981

Taipan viper venom and chromogenic substrate (chromozym Th). Prothrombin assay. No sensitivity to coumarin-induced prothrombin.

Prothrombin (factor II) was assayed in congenital or acquired prothrombin deficiencies and abnormalities using Taipan viper (Oxyuranus scutellatus) venom as activating agent and adsorbed normal plasma or a chromogenic compound (Chromozym-Th) as substrates. Prothrombin was found to be low, as expected in every instance, regardless of the substrate used. In coumarin treated patients the levels observed were similar to the prothrombin time percentile values and definitely lower than the immunological counterparts. Therefore, the Taipan viper venom appears suited for prothrombin assay even during anticoagulant therapy. Since the cost of the chromogenic substrate is about 20 times that of adsorbed normal plasma and since no special information is obtained by it, the use of the chromogenic method is not justified for routine purposes.

Blood Coagulation Disorders↗
PubMed1996

A common genetic variation in the 3'-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis.

We have examined the prothrombin gene as a candidate gene for venous thrombosis in selected patients with a documented familial history of venous thrombophilia. All the exons and the 5'- and 3'-UT region of the prothrombin gene were analyzed by polymerase chain reaction and direct sequencing in 28 probands. Except for known polymorphic sites, no deviations were found in the coding regions and the 5'-UT region. Only one nucleotide change (a G to A transition) at position 20210 was identified in the sequence of the 3'-UT region. Eighteen percent of the patients had the 20210 AG genotype, as compared with 1% of a group of healthy controls (100 subjects). In a population-based case-control study, the 20210 A allele was identified as a common allele (allele frequency, 1.2%; 95% confidence interval, 0.5% to 1.8%), which increased the risk of venous thrombosis almost threefold {odds ratio, 2.8; 95% confidence interval, 1.4 to 5.6}. The risk of thrombosis increased for all ages and both sexes. An association was found between the presence of the 20210 A allele and elevated prothrombin levels. Most individuals (87%) with the 20210 A allele are in the highest quartile of plasma prothrombin levels (> 1.15 U/mL). Elevated prothrombin itself also was found to be a risk factor for venous thrombosis.

Alleles↗
PubMed1992

Monitoring "mini-intensity" anticoagulation with warfarin: comparison of the prothrombin time using a sensitive thromboplastin with prothrombin fragment F1+2 levels.

Treatment with warfarin using a target International Normalized Ratio (INR) range of 1.7 to 2.5 is efficacious for many clinical indications, but the minimal intensity of anticoagulation required for antithrombotic protection has yet to be determined. To evaluate whether patients could be reliably monitored with a less intense regimen, we anticoagulated patients with warfarin for several months using a target INR range of 1.3 to 1.6 as determined by prothrombin time (PT) using a sensitive thromboplastin (Dade IS, International Sensitivity Index [ISI] = 1.3). Plasma measurements of F1+2, a marker of factor Xa action on prothrombin in vivo, were also obtained to determine the suppressive effect of warfarin on hemostatic system activity. Overall, 20 of 21 patients with a history of cerebrovascular events (mean age, 61 years) could be reliably regulated with warfarin in the target INR range. F1+2 levels were significantly suppressed from baseline in all patients, with a mean reduction of 49% (range, 28% to 78%). We found a significant relationship between the extent of suppression of prothrombin activation levels and the baseline measurements. A mean reduction of 65% was observed for those patients with baseline F1+2 greater than or equal to 1.5 nmol/L, but only 38% for baseline F1+2 less than or equal to 0.5 nmol/L. Overall, 68% of plasma samples obtained during stable anticoagulation were within the target INR range. PTs were also determined on all plasma samples with two thromboplastins of lower sensitivity (C+, ISI = 2.09; and automated simplastin, ISI = 2.10). Only 47% and 35% of PT determinations, respectively, were within the target range with these reagents. We conclude that prothrombin activation can be significantly suppressed in vivo with use of warfarin in an INR range of 1.3 to 1.6. This level of anticoagulation can be reliably achieved by monitoring PTs with a thromboplastin of high sensitivity.

Blood Coagulation↗
PubMed2002

Prospective study of the G20210A polymorphism in the prothrombin gene, plasma prothrombin concentration, and incidence of venous thromboembolism.

Case-control studies have indicated increased risk of venous thrombosis associated with the prothrombin gene G20210A polymorphism and with elevated plasma prothrombin levels. We sought to confirm these results in a prospective population-based study of 21,690 persons. We measured G20210A and prothrombin antigen on pre-event blood samples of 302 participants who developed venous thromboembolism (VTE) and 626 participants who remained free of VTE. Approximately 4.0% of cases and 2.4% of controls carried the G20210A polymorphism, but only one of 137 African Americans did. The odds ratio in whites was 1.87 (95% CI = 0.85, 4.11)--higher for those who reported a prior history of VTE (OR = 5.44) than those reporting no VTE history (OR = 1.41) and in those with idiopathic VTE (OR = 2.51) than those with secondary VTE (OR = 1.38). There was no association between venous thromboembolism and plasma prothrombin antigen level. We estimated that the G20210A polymorphism may account for approximately 2.5% of venous thromboembolism events in United States whites.

Age Factors↗
PubMed1987

Prothrombin "Mexico City," an asymptomatic autosomal dominant prothrombin variant.

A functionally normal but structurally abnormal prothrombin variant was found in a Mexican family. Immunoisoelectricfocusing studies revealed that this variant has a more acidic isoelectric point (4.01) than normal prothrombin (4.29), but it proved to have a normal molecular weight as assessed by sodium-dodecyl-sulphate polyacrylamide gel electrophoresis. Two-dimensional immunoelectrophoresis studies showed an abnormal cleavage of the prothrombin molecule by factor Xa and Echis carinatus venom as well, despite the fact that both yield functionally normal thrombin molecules. Finally, the ability of the molecule to bind calcium ions as well as its overall antigenic structure were investigated and found to be normal. These results taken together suggest a simple (substitution or translocation) mutation at the fragment 2 level. Since this prothrombin variant is different from others described previously, the name "Mexico City" is proposed to identify it.

Adult↗
PubMed1982

Partially carboxylated prothrombins. II. Effect of gamma-carboxyglutamyl residues on the properties of prothrombin fragment 1.

Purified prothrombin fragments 1 derived from normal (10-carboxyglutamyl) and dicoumarol-induced 7-, 5-, 2-, 1-, and 0-carboxyglutamyl prothrombins contained the same number of gamma-carboxyglutamyl residues as their respective parent molecules. The effect of gamma-carboxyglutamyl residues was more pronounced on the fragments 1 than on the prothrombins. Consequently, the pI values of the fragments 1 were very well differentiated, with normal fragment 1 focusing at pH 3.58, 7-carboxyglutamyl fragment 1 at 3.79, 5- at 3.97, and 2- at pH 4.29. Similarly, by agar gel electrophoresis, normal fragment 1 was the most mobile, followed by 7-, 5-, 2-, 1- and lastly 0-carboxyglutamyl fragment 1. Because of Ca2+ being bound to the carboxyglutamyl residues, the electrophoretic mobility of normal fragment 1, in the presence of Ca2+, was reduced the most, followed by 7-, 5- and then 2-carboxyglutamyl fragment 1, while the mobilities of the 1- and 0-carboxyglutamyl fragments 1 were not affected. In contrast to their parent molecules, all of the fragments 1 in the presence of EDTA gave negative immunoprecipitation reactions against antibodies produced against normal prothrombin. In the presence of Ca2+, conversely, the fragments 1 containing comparable amounts of antigenic activity all gave positive reactions. However, the intensity of the immunoprecipitates varied, as normal fragment 1 gave the most prominent immunoprecipitation reaction, consecutively followed by 7-, 5-, 2-, 1- and lastly 0-carboxyglutamyl fragment 1 where the precipitation was so faint that it was hardly visible.

1-Carboxyglutamic Acid↗
PubMed1987

Production of abnormal prothrombin (des-gamma-carboxy prothrombin) by hepatocellular carcinoma. A clinical and experimental study.

We measured plasma abnormal prothrombin (des-gamma-carboxy prothrombin; DCP) levels in normal subjects and in patients with hepatocellular carcinoma and other various diseases using the enzyme-linked immunosorbent assay developed by Motohara et al. (Pediatr Res 1985; 19: 354-357). Fifty-eight percent of 52 patients with hepatocellular carcinoma had elevated DCP levels; 24 of 28 patients with advanced or moderately advanced hepatocellular carcinoma were positive. By contrast, 50 normal controls, 13 pregnant women and 10 patients with acute hepatitis had normal levels. Three of 55 patients with chronic liver disease, and 6 of 32 patients with other malignancies, showed a slight increase. Thus, increased plasma DCP appears useful for the diagnosis of hepatocellular carcinoma. To elucidate the mechanism for the increase of DCP in hepatocellular carcinoma, we cultured a human hepatoma cell line, huH-2, and measured the levels of this abnormal prothrombin in the medium. The huH-2 cells produced large amounts of DCP in the medium without added vitamin K. It increased in a cell concentration- and time-dependent fashion. These cells produced no detectable amount of DCP in the medium with added vitamin K. Thus, human hepatoma cell line huH-2 produces DCP, and its production is dependent on the amount of vitamin K available in the medium. Des-gamma-carboxy prothrombin may be a useful tumor marker for the diagnosis of hepatocellular carcinoma.

Biomarkers↗
PubMed2002

Role of procoagulant lipids in human prothrombin activation. 1. Prothrombin activation by factor X(a) in the absence of factor V(a) and in the absence and presence of membranes.

Activation of prothrombin by factor X(a) requires proteolysis of two bonds and is commonly assumed to occur via by two parallel, sequential pathways. Hydrolysis of Arg(322)-Ile(323) produces meizothrombin (MzII(a)) as an intermediate, while hydrolysis of Arg(273)-Thr(274) produces prethrombin 2-fragment 1.2 (Pre2-F1.2). Activation by human factor X(a) of human prothrombin was examined in the absence of factor V(a) and in the absence and presence of bovine phosphatidylserine (PS)/palmitoyloleoylphosphatidylcholine (25:75) membranes. Four sets of data were collected: fluorescence of an active site probe (DAPA) was sensitive to thrombin, MzII(a), and Pre2-F1.2; a synthetic substrate (S-2238) detected thrombin or MzII(a) active site formation; and SDS-PAGE detected both intermediates and thrombin. The fluorescence data provided an internal check on the active site and SDS-PAGE measurements. Kinetic constants for conversion of intermediates to thrombin were measured directly in the absence of membranes. Both MzII(a) and Pre2-F1.2 were consumed rapidly in the presence of membranes, so kinetic constants for these reactions had to be estimated as adjustable parameters by fitting three data sets (thrombin and MzII(a) active site formation and Pre2 appearance) simultaneously to the parallel-sequential model. In the absence of membranes, this model successfully described the data and yielded a rate constant, 44 M(-1) s(-1), for the rate of MzII(a) formation. By contrast, the parallel-sequential model could not describe prothrombin activation in the presence of optimal concentrations of PS-containing membranes without assuming that a pathway existed for converting prothrombin directly to thrombin without release from the membrane-enzyme complex. The data suggest that PS membranes (1) regulate factor X(a), (2) alter the substrate specificity of factor X(a) to favor the meizothrombin intermediate, and (3) "channel" intermediate (MzII(a) or Pre2-F1.2) back to the active site of factor X(a) for rapid conversion to thrombin.

Animals↗
PubMed2000

The prothrombin Denver patient has two different prothrombin point mutations resulting in Glu-300-->Lys and Glu-309-->Lys substitutions.

Dysprothrombinaemia is a rare, congenital cause of bleeding. Fewer than 25 families who express a functional prothrombin (factor II) defect have been reported. The original patient with prothrombin Denver had a severe haemophilia-like bleeding disorder treated with weekly prophylactic factor replacement. Analysis of factor II activity and antigen in the patient showed a factor II activity of 5 units/dl and factor II antigen of 21 units/dl. Genomic DNA from the patient, mother and brother was obtained from peripheral blood white cells. Oligonucleotides were constructed, and prothrombin exons were amplified via polymerase chain reaction (PCR). The entire sequence of the thrombin portion of the molecule (exons VIII-XIV) and that of exons I-II and IV-VII was determined. This moderately severe dysprothrombinaemia was found to be associated with compound heterozygosity for two different Glu-->Lys point mutations, at amino acid positions 300 and 309. Assays of plasma from the prothrombin Denver proband suggested that the functional defect was in the activation of zymogen to enzyme.

Adolescent↗
PubMed2006

Prothrombin Saint-Denis: a natural variant with a point mutation resulting in Asp to Glu substitution at position 552 in prothrombin.

A new prothrombin variant, with a point mutation at nucleotide 20 029 resulting in Asp 552 to Glu substitution (prothrombin numbering), has been identified in a male newborn. Plasma prothrombin level was <3%, 16% and 60% when measured by clotting, chromogenic and immunological assays respectively. The substitution did not affect the rate of prothrombin conversion to thrombin but altered thrombin activity. Amino acid 552 has been reported to be involved in the allosteric transition, which is induced by sodium binding to thrombin. This is the first known amino acid substitution at this site to result in dysprothrombinaemia.

Base Sequence↗
PubMed2003

The APC-independent anticoagulant activity of protein S in plasma is decreased by elevated prothrombin levels due to the prothrombin G20210A mutation.

Protein S exhibits anticoagulant activity independent of activated protein C (APC). An automated factor Xa-based one-stage clotting assay was developed that enables quantification of the APC-independent activity of protein S in plasma from the ratio of clotting times (protein S ratio [pSR]) determined in the absence and presence of neutralizing antibodies against protein S. The pSR was 1.62 +/- 0.16 (mean +/- SD) in a healthy population (n = 60), independent of plasma levels of factors V, VIII, IX, and X; protein C; and antithrombin, and not affected by the presence of factor V Leiden. The pSR strongly correlates with the plasma level of protein S and is modulated by the plasma prothrombin concentration. In a group of 16 heterozygous protein S-deficient patients, the observed mean pSR (1.31 +/- 0.09) was significantly lower than the mean pSR of the healthy population, as was the pSR of plasma from carriers of the prothrombin G20210A mutation (1.47 +/- 0.21; n = 46). We propose that the decreased APC-independent anticoagulant activity of protein S in plasma with elevated prothrombin levels may contribute to the thrombotic risk associated with the prothrombin G20210A mutation.

Antibodies↗
PubMed1975

Plasma abnormal prothrombin and microsomal prothrombin precursor in various species (38492).

Abnormal, biologically inactive forms of prothrombin have previously been shown to appear in the plasma of cows or humans given coumarin anticoagulants. We have previously shown that a protein with similar properties increases in the liver of rats given these vitamin K antagonists, and have postulated that this protein represents the liver precursor to plasma prothrombin. Eight species, rats, mice, guinea pigs, hamsters, rabbits, calves, dogs, and chickens, have now been surveyed for both plasma abnormal prothrombin and liver precursor activity. Large amounts of plasma abnormal prothrombin were found in the bovine, substantial amounts were seen in the chick, and small amounts in rat and mouse plasma. With the exception of the bovine, all anticoagulant treated animals showed elevated levels of liver precursor activity in microsomal preparations. The relationship of these observations to the mechanism of action of vitamin K is discussed.

Animals↗
PubMed1975

Vitamin K and the biosynthesis of prothrombin. V. Gamma-carboxyglutamic acids, the vitamin K-dependent structures in prothrombin.

Tryptic peptides obtained from normal prothrombin have been compared with those obtained from prothrombin synthesized by cattle given the vitamin K antagonist dicumarol. Two peptides were found which contain vitamin K-dependent structures. These peptides contain residues 4 through 10 and residues 12 through 44, respectively. One of these (residues 4 through 10) has previously been shown to contain gamma-carboxyglutamic acid residues. Digestion of this peptide with aminopeptidase M and carboxypeptidase B yielded a tetrapeptide (residues 6 through 9). Mass spectra of this peptide showed that it has the structure Leu-Glu(CO2)-Glu(CO2)-Val. The structure of the peptide containing residues 12 through 44 was determined by automated degradation in a peptide sequenator. The modified glutamic acid residues were identified by mass spectrometric comparison with the thiohydantoin derivatives of synthetic gamma-carboxyglutamic acid. This approach unequivocally demonstrated that all of the first 10 glutamic acid residues in prothrombin are carboxylated to form gamma-carboxyglutamic acid residues. Evidence is also presented that indicates that these gamma-carboxyglutamic acid residues constitute the entire vitamin K-dependent modification of prothrombin.

Amino Acid Sequence↗
PubMed1999

Factor V G1691A and prothrombin G20210A in childhood spontaneous venous thrombosis--evidence of an age-dependent thrombotic onset in carriers of factor V G1691A and prothrombin G20210A mutation.

UNLABELLED: Risk factors for venous thrombosis in adults are the prothrombin (PT) G20210A, the factor (F) V G1691A mutations and hereditary deficiencies of protein C, protein S and antithrombin. However, data are limited on the relevance of these risk factors for thrombosis in children and adolescents. We therefore investigated 119 patients aged 0-18 with spontaneous venous thrombosis and controls (n = 100) for the presence of the factor V G1691A mutation and the prothrombin G20210A variant with respect to thrombotic onset and thrombosis location. The following frequencies (patients vs. controls), odds ratios (OR), 95%-confidence intervals (CI) and p-values were found: FV G1691A, 19.3% vs. 5%, OR/CI 4.55/1.66-12.5, p = 0.0038 and prothrombin G20210A, 8.4% vs. 3%, OR/CI 2.96/0.8-11, p = 0.17. A combination of the FV G1691A mutation with the PT G20210A variant was found in 3 children (2.5% of cases) but only once in the controls. With a median (range) age of 2 years (0-17), carriers of the FV mutation were significantly younger compared with patients carrying the PT variant (16 years: 0-18, p < 0.001). Vascular accidents in carriers of the FV mutation occurred in deep veins of the leg (n = 11), cerebral veins (n = 4), renal veins (n = 3) and portal veins (n = 2). Patients with the PT mutation showed spontaneous thrombosis in the majority of cases in the deep veins of the leg (n = 5) and in the central nervous system (n = 2). Combined defects were found in a neonate with renal venous thrombosis and in two adolescents with deep vein thrombosis. CONCLUSION: Data presented here suggest that the heterozygous FV mutation is the most commonly found prothrombotic risk factor responsible for spontaneous thrombosis during infancy and early childhood. In contrast, the PT G20210A variant is likely to be more important during puberty and adolescence.

Adolescent↗
PubMed1978

The role of blood clotting factor V in the conversion of prothrombin and a decarboxy prothrombin into thrombin.

Purified PIVKA-II exhibits some factor II (prothrombin) activity in the one-stage coagulation assay and this factor II activity does not come from residual amounts of factor II but originates from PIVKA-II itself. It is shown that PIVKA-II is converted by a normal prothrombinase complex (factor Va and factor Xa adsorbed onto a phospholipid interface) more readily than by phospholipids and factor Xa alone. This suggests that binding between PIVKA-II and factor Va is an essential feature in the formation of the enzyme . substrate complex and from this we infer that a direct interaction between factor Va and prothrombin plays a rôle in the prothrombinase . prothrombin complex.

Animals↗
PubMed1979

Decarboxylation of bovine prothrombin fragment 1 and prothrombin.

Bovine prothrombin fragment 1 and prothrombin undergo decarboxylation of their gamma-carboxyglutamic acid residues when the lyophilized proteins are heated in vacuo at 110 degrees C for several hours. The fully decarboxylated fragment 1 product has lost its barium-binding ability as well as the calcium-binding function which causes fluorescence quenching in the presence of 2 mM Ca2+. There is no sign of secondary structure alteration in solution upon analysis by fluorescence emission and circular dichroic spectroscopy. A family of partially decarboxylated fragment 1 species generated by heating for shorter periods shows that the initial decrease in calcium-binding ability occurs almost twice as rapidly as the loss of gamma-carboxyglutamic acid. This is consistent with the idea that differential functions can be ascribed to the 10 gamma-carboxyglutamic acid residues in fragment 1, including both high- and low-affinity metal ion binding sites. Prothrombin itself also undergoes total decarboxylation without any apparent alteration in secondary structure. However, in this case the latent thrombin activity is progressively diminished during the heating process in terms of both clotting activity and hydrolysis of the amide substrate H-D-Phe-Pip-Arg-pNA. The present results indicate that in vitro decarboxylation of gamma-carboxyglutamic acid in dried proteins is useful for analyzing the detailed calcium-binding proteins of vitamin K dependent coagulation factors.

1-Carboxyglutamic Acid↗
PubMed1978

Metal ion induced conformational transitions of prothrombin and prothrombin fragment 1.

Circular dichroism experiments indicate that prothrombin fragment 1 undergoes essentially the same secondary structural change whether in the presence of Ca(2+), Mg(2+), or Mn(2+). Titration with any of these metal ions results in a sigmoidal titration curve indicative of cooperative binding. Mg(2+) and Ca(2+) have nearly identical transition midpoints, while that for Mn(2+) is an order of magnitude less. These results correlate well with the results of previous metal ion intrinsic fluorescence quenching experiments. Fragment 1 has previously been shown to undergo a second transition corresponding to dimerization at high calcium concentrations. The present circular dichroism experiments show that this transition does not result in a gross alteration of secondary structure in the fragment 1 molecule. Studies with prothrombin, similar to those with fragment 1, indicate a similar metal ion dependent conformational change but of smaller magnitude. As apparently only the fragment 1 portion of the molecule undergoes the transition, it would appear that the covalently linked fragment 1 is constrained from attaining the same conformation as the purified entity. This suggests that caution must be used in interpreting the results of metal ion binding studies using fragment 1 as an analogue for prothrombin.

Animals↗
PubMed2002

Role of procoagulant lipids in human prothrombin activation. 2. Soluble phosphatidylserine upregulates and directs factor X(a) to appropriate peptide bonds in prothrombin.

Activation of human prothrombin to thrombin (II(a)) by factor X(a) during blood coagulation requires proteolysis of two bonds and thus involves two possible activation pathways (parallel-sequential activation model). Hydrolysis of Arg(322)-Ile(323) produces meizothrombin (MzII(a)) as an intermediate, while hydrolysis of Arg(273)-Thr(274) produces prethrombin 2-fragment 1.2 (Pre2-F1.2). A soluble lipid, dicaproylphosphatidylserine (C6PS), enhances activation by 60-fold [Koppaka et al. (1996) Biochemistry 35, 7482]. We report here that C6PS binding to factor X(a) not only enhances the rate of activation but also alters the pathway. Activation was monitored using a chromogenic substrate (S-2238) to detect both II(a) and MzII(a) active site formation and SDS-PAGE to detect Pre2-F1.2 as well as II(a) and MzII(a). Of the four kinetic constants needed to describe activation, two (MzII(a) and Pre2-F1.2 consumption) were measured directly, and two (MzII(a) and Pre2-F1.2 formation) were obtained by fitting the three time courses simultaneously to the parallel-sequential reaction model. The time courses of II(a), MzII(a), and Pre2-F1.2 formations were all well described below the C6PS critical micelle concentration (CMC) by this activation model. The rate of Arg(322)-Ile cleavage leading to MzII(a) formation increased by 150-fold, while the rate of Arg(273)-Thr cleavage leading to Pre2-F1.2 formation was inhibited slightly. At concentrations of water-soluble C6PS above its CMC, all four proteolytic reactions increased in rate by 2-5-fold at the C6PS CMC. We conclude that soluble C6PS differentially affects the rate of individual bond cleavages during prothrombin activation in solution such that activation occurs almost exclusively via MzII(a) formation. Finally, C6PS enhanced the rates of all proteolytic reactions to within a factor of 3 of the enhancement seen with PS-containing membranes. We conclude that PS-containing membranes regulate prothrombin activation by factor X(a) mainly via interaction of individual PS molecules with factor X(a).

Enzyme Activation↗