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PubMed2004

A proteomic analysis of changes in prothrombin and plasma proteins associated with the G20210A mutation.

The G-->A mutation at position 20210 of the prothrombin gene, localized in the 3'-polyadenylation untranslated region of the mRNA, is a recognized genetic risk factor for venous thromboembolism. The mechanism by which this base change confers an increased risk of thrombosis compared to noncarriers is undefined. Studies on the mRNA suggest enhanced cleavage site recognition and a change in the location of the 3'-cleavage/polyadenylation reaction, but no defined model has been proposed. The present study, based on proteomic investigation by two-dimensional gel electrophoresis and electrospray ionization (ESI) tandem mass spectrometry (MS/MS) protein identification, suggests that the G20210A mutation is associated with increased glycosylation of prothrombin, which confers greater stability to the protein. Additionally, proteomic investigation of pooled plasma showed that expression levels of six spots, three of them identified by ESI MS/MS, were altered in subjects carrying the mutation, suggesting a possible cooperative effect in the thrombotic risk increment induced by the mutation.

Blood Proteins↗
PubMed1995

Homology modeling and molecular dynamics simulation of human prothrombin fragment 1.

The crystallographic structure of bovine prothrombin fragment 1 bound with calcium ions was used to construct the corresponding human prothrombin structure (hf1/Ca). The model structure was refined by molecular dynamics to estimate the average solution structure. Accommodation of long-range ionic forces was essential to reach a stable solution structure. The gamma-carboxyglutamic acid (Gla) domain and the kringle domain of hf1/Ca independently equilibrated. Likewise, the hydrogen bond network and the calcium ion coordinations were well preserved. A discussion of the phospholipid binding of the vitamin K-dependent coagulation proteins in the context of the structure and mutational data of the Gla domain is presented.

1-Carboxyglutamic Acid↗
PubMed1997

Evidence for prothrombin production and thrombin expression by phorbol ester-treated THP-1 cells.

With the addition of fibrinogen, fibrin clots form in serum-free culture medium recovered from phorbol ester-treated THP-1 cells. We attribute this coagulant activity to thrombin generated as a consequence of cell stimulation because the coagulant activity exists in serum-free culture medium from treated cells only, and it is inhibited by hirudin. The thrombin does not derive from a prothrombin/thrombin contaminant since no detectable prothrombin/thrombin preexists in either the serum-free culture medium or the fibrinogen preparations used for our experiments. We hypothesized that the thrombin is synthesized by the cells themselves. In support of this hypothesis, we found that prothrombin mRNA is expressed in THP-1 cells following their treatment with phorbol ester. Accompanying expression of this mRNA, prothrombin antigen becomes detectable in lysates of PMA-treated THP-1 cells, and thrombin antigen and activity become detectable in both lysates and culture medium of treated cells. These results are consistent with the notion that certain cells of myelomonocytic lineage are capable of synthesizing proteins relevant to coagulation.

Carcinogens↗
PubMed1988

Circular dichroism analysis of the secondary structures of bovine blood coagulation factor IX, factor X, and prothrombin.

Analysis of the far-ultraviolet circular dichroism spectrum of bovine blood coagulation factor IX reveals the presence of approximately 14% helical structures 26% beta-sheets, 20% beta-turns, and 40% coils. These values are essentially the same for the activation products of this zymogen, factor IX alpha alpha and factor IX alpha beta. Similar analysis for bovine factor X permits calculation of these secondary structural as approximately 11% helices, 31% beta-structures, 22% beta-turns, and 36% random structures. Bovine prothrombin contains approximately 12% helical structures, 35% beta-structures, 24% beta-turns, and 29% coils. None of these values is substantially altered as a result of increase of the pH from 7.4 to 10.5, or upon addition of Ca2+ to a concentration of at least 20 mM. Analysis of the near-ultraviolet spectra of factor IX and prothrombin suggests that several aromatic amino acid residues and the disulfide bond present in their gamma-carboxyglutamic acid-containing regions are exposed to solvent and are perturbed by the above pH adjustment and Ca2+ addition. Similar effects are observed in the case of factor X; in addition, the Trp residue at the amino terminus of the heavy chain appears to be influenced by the above pH alteration. The results reported in this paper show that these vitamin K-dependent blood coagulation proteins are similar in their ordered secondary structures, which are dominated by beta-sheets and beta-turns. Their overall secondary structures are not influenced by Ca2+ binding and are stable to alkaline pH changes. However, these same environmental alterations appear to be effective probes of aromatic residues in the gamma-carboxyglutamic acid regions.

Animals↗
PubMed1997

Are prothrombin fragment 1 + 2 and thrombin-antithrombin complexes useful in the management of oral anticoagulant therapy?

We investigated the behavior of prothrombin fragment F1 + 2 and thrombin-antithrombin complexes in 70 patients treated with chronic anticoagulant therapy. Moreover, in a longitudinal study 37 patients were evaluated twice and 16 patients three times. Twenty-eight age- and sex-matched healthy subjects were also studied as a control group. Prothrombin fragment F1 + 2 or thrombin-antithrombin values among patients with different International Normalized Ratios, nor in the same patients studied two or three times. Our results confirm that oral anticoagulant treatment can effectively reduce thrombin activity. However, strong anticoagulation does not induce a further significant decrease in fragment F1 + 2 values. Therefore, we feel measurement of fragment F1 + 2 might be less useful than thought in optimizing oral anticoagulant therapy.

Administration, Oral↗
PubMedDate not supplied

Quality control of the prothrombin time and international normalized ratios. National and international schemes.

The international normalized ratios (INR) system allows valid comparisons in results and quality of performance to be made between users of different thromboplastin reagents. In the international quality control surveys currently over 80% of the 53 countries participating report INR. Stated local international sensitivity index (ISI) values show fair agreement with values calculated from quality control returns obtained with local and reference reagents. The coefficient of variations (CV) of the INR in these surveys are between 11-22% depending upon INR values. In comparison, CV of the UK national scheme are currently 7-13%. However, analysis of UK results has shown high CV with high ISI reagents. This is due to the ISI effect as CV of INR is CV of prothrombin ratio (PR) multiplied by the ISI. Ideal thromboplastins should show good precision of PR and a low ISI to prevent this apparent deterioration when PR results are transformed into the INR scale. Instrumentation has a further effect on the INR result. Unfortunately, the effect is not uniform even within instrument type and model or even between normal and therapeutic results. Local instrument adjustment or local calibration is therefore necessary. Thus, quality control surveys continue to highlight problems in prothrombin time standardization.

Automation↗
PubMed2001

Simultaneous allele-specific amplification: a strategy using modified primer-template mismatches for SNP detection--application to prothrombin 20210A (factor II) and factor V Leiden (1691A) gene mutations.

BACKGROUND: Inherited thrombophilia is caused by mutations in genes central to the clotting cascade. Analysis of the factor V Leiden (FVL) and prothrombin G20210A mutations are the most prevalent in thrombophilia. METHODS AND RESULTS: We have optimized an allele-specific PCR assay for the simultaneous detection of both wild-type and mutant alleles. This method is adapted for clinical use with the FVL and prothrombin G20210A assays and is significant in its intentional use of nucleotide mismatches at the 3' end of allele-specific primers. Two internal allele-specific primers are designed to amplify in opposite directions on opposite strands that reduce differential amplification. Our results show concordance with methods involving PCR with restriction endonuclease digestion, yet are simpler to perform. CONCLUSION: The simultaneous allele-specific amplification method allows simultaneous detection of wild-type and mutant alleles by PCR using four distinct primers. Nucleotide mismatches in the primers reduce competitive amplification.

Alleles↗
PubMed2000

Prothrombin gene G20210A mutation and elevated anticardiolipin antibodies in a patient with combined portal-mesenteric vein thrombosis.

A 29-year-old man was admitted to the ICU after emergency laparotomy for portal-mesenteric vein thrombosis. Under continuous intravenous heparin therapy the portal-mesenteric shunt occluded on the first postoperative day. After thrombectomy the heparin dose was increased, and the patient remained free of symptoms (partial thromboplastin time 53 s). Two days later abdominal distension developed concomitantly with ventilatory distress due to a large retroperitoneal hematoma. The patient was mechanically ventilated and underwent the third consecutive laparotomy for the hematoma removal on the fifth day. During the surgical procedure the abdomen was packed with towels to stop multiple bleeding sites. The heparin dose was reduced, aiming for a partial thromboplastin time of 30-35 s. Initial coagulation tests revealed increased levels of anticardiolipin immunoglobulin G. After removal of the surgical towels the patient was successfully weaned from mechanical ventilation and discharged from the ICU. Two weeks later genomic testing revealed that he also had a G20210A mutation of the prothrombin gene. Both, increased levels of anticardiolipin immunoglobulin G and the G20210A mutation of the prothrombin gene predispose to thrombosis. Increased levels of anticardiolipin immunoglobulin G may also cause bleeding. Long-term anticoagulation therapy was started with a vitamin K antagonist, and 2 months later a follow-up showed that the patient had no further symptoms of portal-mesenteric vein thrombosis or bleeding. This case illustrates that the convergence of multiple risk factors, including genetic defects, must be considered in patients suffering from thrombosis in unusual sites

Adult↗
PubMed1998

Donor des-gamma-carboxy prothrombin positivity is a risk factor for poor early graft function in liver transplantation.

Des-gamma-carboxy prothrombin (DCP) is an abnormal prothrombin that lacks coagulating activity. The aim of this study was to determine if the presence of DCP in the donor could be used as a marker of post-transplant graft function. We collected data and serum samples on 90 organ donors. DCP level was correlated with donor-specific factors and with graft function intraoperatively and in the early post-transplant period. Twenty-seven donors (30.0%) had positive DCP levels before harvesting. Although recipients were similar in demographics, preoperative liver function, and primary disease distribution, patients transplanted with livers from DCP-positive donors needed significantly more intraoperative transfusion. Furthermore, donor DCP positivity was identified as a preoperative risk factor for poor early graft function based on multivariate analysis (odds ratio = 6.58, P = 0.0032). Our findings suggest that DCP is another valuable marker for evaluating the quality of donor livers.

Adolescent↗
PubMed2003

Purpura fulminans in a child with combined heterozygous prothrombin G20210A and factor V Leiden mutations.

Although thrombosis is relatively rare in children, reports of young patients with thrombosis are becoming more frequent with time. Activated protein C resistance and prothrombin 20210 A mutation are results of point mutations described in the last decade. This article highlights a case of a child with severe arterial thrombosis who was heterozygous for the factor V Leiden (FVL) and prothrombin G20210A mutations. The patient diagnosed with purpura fulminans was an 8-year-old boy who was referred to our hospital with purpuric lesions on the extremities and necrosis of the penis. We believe that the coexistence of more than one thrombophilic mutation contributed to the occurrence of severe thrombosis at a young age in this patient.

Age of Onset↗
PubMed2003

Fructose-1,6-diphosphatase deficiency: a rare cause of prolonged prothrombin time.

A 20-year-old woman presented with severe life-threatening metabolic acidosis and hypoglycemia. In addition, her blood tests revealed elevated hepatic enzymes and a prolonged prothrombin time, with a reduction in factor VII activity. After treatment with a glucose and bicarbonate-containing intravenous infusion, there was a dramatic clinical improvement and normalization of the prothrombin time within 2 days. The patient was found to have fructose-1,6-diphosphatase deficiency, a rare metabolic disorder which has not been described previously as causing coagulation defects.

Acidosis↗
PubMed2000

Prevalence of prothrombin 20210A allele and methylenetetrahydrofolate reductase C677T genetic mutations in the Chinese population.

From July 1997 to June 1998, a total of 1323 subjects, including 1180 controls, 94 patients with diabetes mellitus, and 49 patients with deep-vein thrombosis, varying in age and gender, were consecutively entered into our study. Their mean (+/- SD) age was 50.0 +/- 18.0 years, range 1-99 years; 930 were male and 393 were female. None of the subjects was found to have abnormal prothrombin 20210A allele mutation. In total, 150 subjects (11.3%) were found to have a homozygous 677 C-->T mutation of the methylenetetrahydrofolate reductase gene, in which 125 were controls (10.6%), 17 were diabetics (18.1%) and 8 were patients with deep-vein thrombosis (16.3%). However, 524 subjects (39.6%) were found to have a heterozygous methylenetetrahydrofolate reductase 677 C-->T mutation. We suggested that the Chinese race dose not have the prothrombin 20210A allele, but can carry the 677 C-->T mutation of the methylenetetrahydrofolate reductase gene.

Adolescent↗
PubMed2002

Three-dimensional solution structure of Tropidechis carinatus venom extract trocarin: a structural homologue of Xa and prothrombin activator.

Trocarin belongs to group D of prothrombin activators derived from snake venom of Tropidechis carinatus and is a rich non-hepatic source of Xa, the only known hepatic prothrombin activator. The structural and functional similarity with Xa makes trocarin an interesting target for exploring the structure-functional relationship with Xa. Herein we report a predicted complete three-dimensional all-atom structural model of trocarin equilibrated in explicit water using 4 ns of molecular dynamics simulation. The tertiary structure was modeled using the structure of human blood coagulation factor Xa. The conformational and structural features of trocarin are then compared with the X-ray crystal and solution simulation structures of human factor Xa. The modeled structure of trocarin has four individual domains (Gla, EGF1, EGF2 and SP) connected along the long axis with similar secondary structural elements to Xa. The simulations suggest that sodium ion binding in the serine protease domain is impaired in trocarin as compared to Xa. In contrast to Xa, for which the sodium ion forms an octahedral coordination network that brings two loop regions connecting four anti-parallel beta-sheets together, we do not find a similar pattern of network in trocarin. We observe that the difference in the binding pattern of sodium ion leads to a approximately 2-A "shrinkage" of the beta2 strand (B2), in comparison to human Xa, as marked by a shorter distance between 189Asp373 (S1-site residue) and 195Ser379 (active-site residue) in the B2 strand. We propose that these differences may be linked to the experimentally observed lower amidolytic activity of trocarin as compared to Xa.

Amino Acid Sequence↗
PubMed1984

The separation of bovine prothrombin and descarboxyprothrombin by high-performance liquid chromatography.

Prothrombin contains 10 gamma-carboxyglutamic acid (Gla) residues which are absent in the warfarin-induced descarboxyprothrombin; hence prothrombin has 10 more negative groups than has descarboxyprothrombin. The two proteins can be separated by HPLC with the aid of an anion-exchange column. Plasma from warfarin-treated animals could be analyzed without pretreatment of the samples and a full analysis was obtained in 30 min.

Animals↗
PubMed1986

Secondary structure predictions of human plasminogen and the bovine prothrombin kringle loops.

Secondary structural predictions, based upon the statistical methodology of Chou and Fasman, for the kringle loops of human plasminogen and bovine prothrombin suggest a "winding staircase" pattern of beta-turns, spaced by short regions of ordered and coil structures. Analysis of the predicted structures of the regions containing the two His (113 and 387) and Asp (136 and 410) residues in plasminogen kringles 1 and 4, which have been found to be important in binding the ligand, epsilon-aminocaproic acid, shows that all are localized at the same positions on beta-turns. In addition, both of the two Asp residues occur at the end of homologous nonapeptide regions common to all of the five human plasminogen and two bovine prothrombin kringles, indicating evolutionary conservation to preserve biologically critical conformations. Examination of the protein conformation in the region of Asn288, the residue which is glycosylated in one of the two circulating variants of human plasminogen, shows that it most likely exists in a position which may present topographical hindrance to post-translational attachment of carbohydrate, thus, possibly, explaining the incomplete glycosylation of human plasminogen with complex-type carbohydrate.

Amino Acid Sequence↗
PubMed1976

Sheep prothrombin: purification and partial characterization.

A procedure for the preparation of highly purified sheep prothrombin is described. The purified zymogen, when subjected to disc gel electrophoresis in polyacrylamide, gave rise to one single band. Only alanine was found as N-terminal residue. Carboxypeptidases A and B failed to release any C-terminal residue. The isoelectric point, as determined by isoelectric focusing in polyacrylamide gel slab, was shown to be 4.9-5.0. Non-chromatographed, but not the purified zymogen, could be converted into active thrombin in half-saturated trisodium citrate seeded with thrombin. Pure sheep prothrombin showed 5.6% of neutral sugars and the following amino acid composition: Ala35, Arg44, Asx54-55, -Cys24, Glx72, Gly53-54, His8, Ile19, Leu45, Lys31, Met7, Phe23, Pro36, Ser34, Thr29-29, Trp16, Tyr19 and Val33, which accounts for a molecular weight of about 66 000 (amino acids only). The molecular weight as determined by sodium dodecyl sulphate-polyacrylamide gel electrophoresis after reduction by 2-mercaptoethanol, was shown to be 77 000 +/- 3000 (carbohydrates included).

Amino Acids↗
PubMed1977

The gamma-carboxy glutamic acid content of human and bovine prothrombin following warfarin treatment.

A form of prothrombin induced by Warfarin therapy, has been isolated which is adsorbed onto insoluble barium salts, but has a reduced biological activity. This protein contains, on average, seven out of a possible ten gamma-carboxy glutamic acid residues. A second form of prothrombin is also described, which is not adsorbed into barium slats, and has less than 1% the activity of the normal protein, contains only four gamma-carboxy glutamic acid residues. The significance of these results is discussed.

Amino Acid Sequence↗
PubMed1988

Structure and evolution of the bovine prothrombin gene.

The cloned bovine prothrombin gene has been characterized by partial DNA sequence analysis, including the 5' and 3' flanking sequences and all the intron-exon junctions. The gene is approximately 15.4 x 10(3) base-pairs in length and comprises 14 exons interrupted by 13 introns. The exons coding for the prepro-leader peptide and the gamma-carboxyglutamic acid-containing region are similar in organization to the corresponding exons in the factor IX and protein C genes. This region has probably evolved as a result of recent gene duplication and exon shuffling events. The exons coding for the kringles and the serine protease region of the prothrombin gene are different in organization from the homologous regions in other genes, suggesting that introns have been inserted into these regions after the initial gene duplication events.

Animals↗