Prothrombin Antigen Levels Research Articles

Antithrombin Resistance Rescues Clotting Defect of Homozygous Prothrombin-Y510N Dysprothrombinemia.

A patient with hematuria in our clinic was diagnosed with urolithiasis. Analysis of the patient's plasma clotting time indicated that both activated partial thromboplastin time (52.6 seconds) and prothrombin time (19.4 seconds) are prolonged and prothrombin activity is reduced to 12.4% of normal, though the patient exhibited no abnormal bleeding phenotype and a prothrombin antigen level of 87.9%. Genetic analysis revealed the patient is homozygous for prothrombin Y510N mutation. We expressed and characterized the prothrombin-Y510N variant in appropriate coagulation assays and found that the specificity constant for activation of the mutant zymogen by factor Xa is impaired approximately fivefold. Thrombin generation assay using patient's plasma and prothrombin-deficient plasma supplemented with either wild-type or prothrombin-Y510N revealed that both peak height and time to peak for the prothrombin mutant are decreased; however, the endogenous thrombin generation potential is increased. Further analysis indicated that the thrombin mutant exhibits resistance to antithrombin and is inhibited by the serpin with approximately 12-fold slower rate constant. Protein C activation by thrombin-Y510N was also decreased by approximately 10-fold; however, thrombomodulin overcame the catalytic defect. The Na+-concentration-dependence of the amidolytic activities revealed that the dissociation constant for the interaction of Na+ with the mutant has been elevated approximately 20-fold. These results suggest that Y510 (Y184a in chymotrypsin numbering) belongs to network of residues involved in binding Na+. A normal protein C activation by thrombin-Y510N suggests that thrombomodulin modulates the conformation of the Na+-binding loop of thrombin. The clotting defect of thrombin-Y510N appears to be compensated by its markedly lower reactivity with antithrombin, explaining patient's normal hemostatic phenotype.

Prothrombin “Carmel”: Identification of a Novel Homozygous Mutation in the Prothrombin Gene Causing Coagulation Factor II Deficiency and Inherited Bleeding Disorder in an Israeli Arab Family

Congenital prothrombin deficiency, also known as hypoprothrombinemia is a rare autosomal recessive disorder characterized by severe bleeding manifestations and decreased prothrombin antigen levels below 10% activity of normal. Dysprothrombinemia, which is characterized by normal antigen levels but a dysfunctional prothrombin molecule, shows a more variable level in bleeding tendency, and there is often a good correlation between the levels of prothrombin activity and clinical severity. We have studied a large consanguineous family of thirteen members contains two patients with a prolonged protrombine time (PT) and activated partial thromboplastin time (aPTT) with prothrombin activity of 11%. DNA was isolated from leukocytes and the thrombin exons were amplified by polymerase chain reaction and sequenced. Exon 14 revealed a point mutation in the codon of Tyrosine 560 which is substituted by Histidine (Tyr560His). Mutation at this residue most probably distorts the structure of the peptidase catalytic domain. The variation segregated as expected in the family, it did not appear in dbSNP, EVS or the 1000 genome project. Further investigation is needed to determine the antigen level and to elucidate the potential effect of the mutation on the structure and the activity of the enzyme. This prothrombin is designated as Prothrombin “Carmel”. DisclosuresNo relevant conflicts of interest to declare.

Clinical and biochemical characterization of the prothrombin Belgrade mutation in a large Serbian pedigree: new insights into the antithrombin resistance mechanism

Background The recently reported c.1787G>A mutation in the prothrombin gene leads to Arg596Gln replacement in the protein molecule (prothrombin Belgrade). This substitution impairs binding of antithrombin to thrombin and results in inherited thrombophilia, known as antithrombin resistance. Objectives We aimed to elucidate the clinical and biochemical characteristics of thrombophilia associated with antithrombin resistance in a large Serbian family with the prothrombin Belgrade mutation. Patients and methods Nineteen family members were investigated, among whom 10 were carriers of the c.1787G>A mutation. In all subjects the clinical phenotype was determined and laboratory investigations of hemostatic parameters were performed. Results Six out of the 10 mutation carriers developed thromboembolic events, mainly deep venous and mesenteric vein thrombosis. The median age of the first thrombotic event was 26.5 (12-41) years, whereas the incidence rate of first thrombosis was 2.2% per year. In all mutation carriers prothrombin activity was significantly decreased in comparison with non-carriers, clearly distinguishing each group. However, the presence of the mutation did not affect the prothrombin antigen level in plasma. The endogenous thrombin potential was significantly increased in all carriers in comparison with non-carriers, indicating the presence of blood hypercoagulability. Interestingly, levels of D-dimer and the F1+2 fragment were similar in both groups. Conclusions Although rare, the prothrombin Belgrade mutation represents strong thrombophilia with early onset of thrombosis in the investigated family. According to our results, decreased prothrombin activity may be a simple screening test for detection of this mutation in thrombotic patients.

Asymptomatic Dysprothrombinemia (Prothtombin Himi) with p.M380T and p.R431H Shows Severely Reduced Clotting Activity, Moderate Antithrombin Resistance and Severe Thrombomodulin Binding Defect

Background and purpose:Congenital prothrombin (PT) deficiency is categorized into two phenotypes, true hypoprothrombinemia and dysprothrombinemia. A strong discrepancy between observed values from coagulation tests and bleeding severity can be found in some forms of dysprothrombinemia. Some dysprothrombinemias, such as the PT Himi that we reported previously (Morishita E, et al: Blood 1992), are asymptomatic despite low PT activity. PT Yukuhashi disturbs the interaction between thrombin and antithrombin (AT), causing a paradoxical thrombotic phenotype, called "AT resistance (ATR)". To investigate the mechanisms by which patient with PT Himi is asymptomatic, mutant PTs reflecting each of the two different gene sites identified in gene analysis were prepared, and functional analyses of fibrinogen (Fbg) degradation, ATR, and thrombomodulin (TM)-binding capacity were conducted. This study was approved by the ethics committee at Kanazawa University School of Medicine.Case report: The proband was a 26-year-old Japanese woman who had been diagnosed with congenital dysprothrombinemia at the time of first pregnancy, based on PT activity and PT antigen levels of 10% and 88%, respectively. Although cesarean section was performed for the second delivery, abnormal bleeding did not develop. Genomic DNA analysis revealed compound heterozygosity for two missense mutations in the F2gene (c.1139T>C, p.M380T and c.1292G>A, p.R431H).Methods: We established stable transformants of Chinese hamster ovary cells that expressed wild-type and two mutant recombinant PTs (rPTs), M380T PT and R431H PT. Conditioned media of stable transformants expressing rPTs in serum-free medium were collected, concentrated, and stored at -80°C until use. 1) Fbg clotting assays: We performed a one-stage clotting assay and a chromogenic assay. 2) Kinetic analysis of thrombin inactivation using AT and formation of thrombin-AT complex (TAT): After incubation with PT activator components, samples were treated with excess AT in the absence of heparin for various durations and changes in absorbance per minute were measured. Formed TAT was measured using an enzyme-linked immunosorbent assay (ELISA) kit. 3) TM-binding assay: To evaluate the ability of wild-type and mutant rPTs to bind to TM, after activating PT, we detected TM-binding thrombin using a TM ELISA method.Results:1) M380T PT activity in the one-stage assay was below the threshold of sensitivity (clot not detected), whereas R431H PT activity was decreased to 10% of that of wild-type rPT (100%). On the other hand, both mutant PT activities in the chromogenic assay were slightly decreased compared to wild-type rPT. 2) After 30 min without heparin, relative residual thrombin activity of M380T PT (74%) was higher than that of wild-type (10%), indicating severe ATR, whereas that of R431H PT was slightly increased (21%), indicating mild ATR. TAT formation by wild-type PT increased over time. TAT formation by R431H PT was about 40% of that of wild-type PT, but that by M380T PT was almost negligible after 120 min. 3) TM-binding thrombins of both mutant rPTs were barely detected.Discussion: M380T PT has high ATR and fails to bind with TM, so a large amount of thrombin with greater clotting activity than normal is thought to be present. With this abnormal thrombin, however, fibrin-forming ability is greatly decreased and almost no clotting ability is thought to exist in the body. Hence, R431H PT, which made up about half of the PT protein in the proband, was considered responsible for hemostasis in the body. The mutant thrombin activated from the R431H PT shows decreased clotting activity, but is resistant to inactivity from AT and thus maintains activity longer than ordinary thrombin. At the same time, the activation of protein C is also decreased due to failure to bind with TM, so ultimately the clotting ability necessary for hemostasis was conjectured to have been maintained, resulting in the proband being asymptomatic.Conclusion:Although neither PT position M380 nor R431 are AT binding sites, M380T PT and R431H PT showed ATR. These mutants are the first AT-resistant PTs seen from mutations other than at the Arg596 site. In recent years, TM resistance has been newly proposed as a mechanism producing thrombotic tendencies, and may also apply to the present patient. The reason the proband did not show a bleeding tendency may have been the combined contributions of AT and TM resistance. DisclosuresNo relevant conflicts of interest to declare.

Pathogenic antibodies to coagulation factors. Part II. Fibrinogen, prothrombin, thrombin, factor V, factor XI, factor XII, factor XIII, the protein C system and von Willebrand factor.

Pathogenic antibodies to coagulation factors. Part II. Fibrinogen, prothrombin, thrombin, factor V, factor XI, factor XII, factor XIII, the protein C system and von Willebrand factor.

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.

Clinical and laboratory expression of associated thrombophilic conditions (homozygous/heterozygous factor V Leiden mutation and heterozygous prothrombin variant 20210A) in an Italian family.

Prothrombin variant 20210A is maintained to be a mild risk factor for venous thromboembolism (VTE). The association of this defect with other inherited thrombophilic conditions may result in an increased risk of developing VTE. In this article, a family is described in which prothrombin variant was associated with either homozygous or heterozygous factor V Leiden (FV Leiden) mutation. All family members except the proband were asymptomatic despite the presence and the severity of the underlying genetic defect(s). The proband, who carried homozygous FV Leiden mutation and heterozygous prothrombin variant, experienced recurrent VTE during pregnancies, whereas one brother, with the same defect, was asymptomatic. Mean prothrombin antigen and activity levels were higher in carriers of the prothrombin variant as compared with noncarriers. Thrombin generation was assessed in family members, in carriers of prothrombin variant or homozygous FV Leiden mutation and in a control group. Most of the family members presented with increased prothrombin fragment 1+2 levels possibly because of the presence of the FV Leiden mutation. Although it is conceivable that the co-inheritance of prothrombin variant and FV Leiden mutation may increase the risk of VTE, patients with these combined defects may remain asymptomatic. It is likely that acquired triggering conditions play a major role in determining VTE in carriers of a mild genetic predisposition. This has to be taken into account when recommendation for thromboprophylaxis is given.

Prothrombin antigen levels in symptomatic and asymptomatic carriers of the 20210A prothrombin variant.

A recently discovered variant in the prothrombin gene (20210A) has been found in approximately 5-10% of patients with venous thromboembolism. It has been shown that patients with this variant present with high levels of prothrombin in plasma and this is maintained to be the most likely mechanism by which the risk of thrombosis is increased. We have evaluated prothrombin antigen levels in 50 carriers of the 20210A allele and compared with non-carriers. 327 subjects were subdivided according to deficiency status and previous thrombosis. 30 symptomatic and 20 asymptomatic carriers had increased mean prothrombin levels as compared to symptomatic (n=178) or asymptomatic (n=99) non-carriers. The percentage of subjects with prothrombin levels above cut-off values of 1.15u/ml or 1.30u/ml was significantly higher in carriers of the prothrombin variant as compared to non-carriers, regardless of a previous thrombosis. However, among non-carriers the percentage of those with prothrombin levels above cut-off values was significantly higher in the group of symptomatic as compared to asymptomatic individuals. In conclusion, increased prothrombin antigen levels, as detected by a specific ELISA, were found among 50 symptomatic and asymptomatic carriers of the 20210A prothrombin variant as well as among a large group of symptomatic non-carriers. The data are in agreement with those found by using functional tests for the determination of prothrombin levels in these patients.

Increased Tissue Factor Pathway Inhibitor (TFPI) and Coagulation in Patients with Insulin-dependent Diabetes mellitus

Recently, we found an increase in tissue factor pathway inhibitor (TFPI) activity in patients with insulin-dependent diabetes mellitus (IDDM). This increase in TFPI activity could be the result of increased thrombin formation and/or altered binding of TFPI to glycosaminoglycans. We studied TFPI activity (chromogenic assay) in relation to prothrombin F1 + 2 fragments and endogenous thrombin potential (ETP), in 46 IDDM patients, and 18 age and sex-matched healthy controls. Prothrombin, antithrombin and thrombomodulin were also determined. In IDDM patients, TFPI activity and F1 + 2 levels were significantly higher, while ETP, prothrombin antigen levels, and antithrombin activity were lower as compared to the controls. In IDDM patients with microalbuminuria, a manifestation of generalized angiopathy, TFPI activity, F1 + 2 and thrombomodulin levels were higher than in patients with only retinopathy or patients without complications. No correlation between TFPI activity, F1 + 2 levels and thrombomodulin was found, while TFPI activity was negatively correlated with ETP (r = -0.27). Microalbuminuria was significantly correlated with TFPI activity (r = 0.46), F1 + 2 (r = 0.56), and thrombomodulin (r = 0.52). In TFPI-depleted plasma, ETP increased, indicating that ETP is affected by TFPI. In conclusion, the increase in TFPI activity in IDDM patients may not be considered to be a reaction on a procoagulant state. It is hypothesized that vascular damage, leading to alterations in glycosaminoglycans, is in part responsible for the changes in TFPI activity, F1 + 2 levels and ETP.

Prothrombin Frankfurt: A Dysfunctional Prothrombin Characterized by Substitution of Glu-466 by Ala

We have identified a patient with a dysfunctional prothrombin that we have designated Prothrombin Frankfurt. The proband was characterized by a prothrombin activity level of 13% and 20% compared to normal controls using two different assays with a normal prothrombin antigen level of 91% of normal controls. The genetic defect responsible for the abnormal prothrombin activity was determined by the polymerase chain reaction followed by single-strand conformation polymorphism (PCR-SSCP) analysis and by DNA sequence analysis of the human prothrombin gene. Substitution of a C for an A at nucleotide 10177 in the human prothrombin gene of the proband was identified, which results in the replacement of Glu-466 by Ala. The proband and one sister were homozygous for this mutation. Both parents, as well as one brother, were found to be heterozygous for this mutation. The same amino acid substitution was previously identified to be responsible for the dysfunctional protein Prothrombin Salakta and was hypothesized to result in altered substrate specificity. Four polymorphisms were also identified in the prothrombin gene from the proband when compared to the published sequence at nucleotides 554, 4048, 4272 and 10253.

Non-specific effects of aquaMEPHYTON (vitamin K 1) on prothrombin expression in human hepatoblastoma (HEPG2) cells

In order to determine the effects of vitamin K 1 on prothrombin production, we have treated cultures of human hepatoblastoma cells with an aqueous colloidal suspension of vitamin K 1. Dose-response analysis demonstrated increases in secreted prothrombin antigen levels ranging from 3 to 3.7-fold over controls. Time-course analysis demonstrated increases in secreted prothrombin antigen levels over controls up to 6 hours of treatment. Between 6 and 24 hours, secreted prothrombin antigen levels increased at a rate parallel to controls. Vitamin K 1 treatment also resulted in a parallel increase in total secreted protein levels. Prothrombin mRNA size (approximately 2.1 kb) and levels (ranging from 390–480 prothrombin mRNA molecules per cell) were determined by Northern and quantitative solution hybridization analysis, respectively, and were unaffected by vitamin K 1 treatment. The increases in secreted prothrombin antigen levels most likely result from non-specific effects of vitamin K 1 or agents used to emulsify vitamin K 1 on protein release from HepG2 cells.

The Effects of Vitamin K1 and Warfarin on Prothrombin Expression in Human Hepatoblastoma (HepG2) Cells

Cultures of human hepatoblastoma (HepG2) cells were treated with vitamin K1 or warfarin and prothrombin antigen and mRNA levels were determined. With 3 and 6 h of 10 micrograms vitamin K1 treatment secreted prothrombin antigen levels, relative to total secreted protein levels, were increased 1.5-fold and 2.1-fold, respectively, over ethanol-treated control levels as determined by an enzyme-linked immunosorbent assay. Dose-response analysis with 3 h of 25 micrograms/ml vitamin K1 treatment demonstrated a maximal increase of 2.0-fold in secreted prothrombin antigen levels, relative to total secreted protein levels, over ethanol-treated control levels. Pulse-chase analysis with 35S-methionine and immunoprecipitation of 35S-labelled prothrombin demonstrated that, with vitamin K1 treatment (25 micrograms/ml, 3 h), the rate of prothrombin secretion increased approximately 2-fold and the total amount (intra- and extracellular) of prothrombin synthesized increased approximately 50% over ethanol-treated control levels. Warfarin treatment (1, 5, or 10 micrograms/ml, 24 h) resulted in decreases in secreted prothrombin antigen levels, relative to total protein levels to approximately 85%, 87% or 81% of ethanol-treated control levels. Analysis of total RNA isolated from these cultures by Northern and solution hybridization techniques demonstrated that prothrombin mRNA was approximately 2.1 kb and that neither vitamin K1 nor warfarin treatment affected the quantity of prothrombin mRNA (ranging from 240-350 prothrombin mRNA molecules per cell). These results demonstrate that vitamin K1 and warfarin, in addition to effects on gamma-carboxylation, affect prothrombin synthesis post-transcriptionally, perhaps influencing translation, post-translational processing and/or secretion mechanisms.

Prothrombin Himi; An abnormal prothrombin characterized by a defective thrombin activity

An abnormal prothrombin has been detected in a 26-year-old female, who had no history of excessive bleeding. Prothrombin activity was approximately 10% when measured using either the classical one-stage assay or the assay with Echis carinatus venom, whereas prothrombin antigen level was normal. In keeping with current nomenclature practices, the abnormal prothrombin was designated “Prothrombin Himi”. The electrophoretic behavior and calcium binding properties of Prothrombin Himi did not differ significantly from normal. Prothrombin Himi was isolated by chromatography on Q-Sepharose. Electrophoretic migration of the purified abnormal prothrombin on SDS -PAGE was normal. Upon prothrombin activation by Echis carinatus venom, the clotting activity produced from Prothrombin Himi was only 37% of the normal level after 90 minutes of the activation time, where as the amidolytic activity was almost the same as normal. The cleavage patterns of Prothrombin Himi by factor Xa or Echis carinatus venom investigated by SDS-PAGE, were found to be normal. These results indicate that Prothrombin Himi was characterized by a defective thrombin enzymatic activity.

Molecular defect of prothrombin Barcelona. Substitution of cysteine for arginine at residue 273.

Prothrombin Barcelona has been isolated from a patient with a normal prothrombin antigen level but low prothrombin coagulant activity. The activation of this protein is impaired by the absence of one of the two factor Xa-catalyzed cleavages that normally lead to the formation of thrombin. Prothrombin Barcelona and prothrombin were isolated from patient plasma and normal plasma, respectively, in a single-step, high-yield immunoaffinity purification using conformation-specific antibodies immobilized on Sepharose. After reduction and alkylation, the purified proteins were subjected to trypsin hydrolysis. The resulting peptides were separated by reverse-phase high performance liquid chromatography. Comparison of the peptide maps of prothrombin Barcelona and prothrombin demonstrated that a peptide, identified as fragment 274-287 in prothrombin by automated Edman degradation, was missing in the prothrombin Barcelona digest. In the chromatogram derived from prothrombin Barcelona, an additional peptide was observed. The amino acid sequence of this peptide was Ala-Ile-Glu-Gly-Cys-Thr-Ala-Thr-Ser-Glu-Tyr-Gln-Thr-Phe-Phe-Asn-Pro-Arg, corresponding to residues 269-287 in prothrombin except for the substitution of cysteine for arginine at residue 273. The substitution of cysteine for arginine was confirmed by tryptic digestion of 14C-carboxymethylated prothrombin Barcelona. Edman degradation of fragment 269-287 indicated the association of 14C with the cysteine at residue 273. The replacement of arginine by cysteine at residue 273, adjacent to the known factor Xa cleavage site, precludes normal activation of prothrombin Barcelona by factor Xa and the generation of thrombin.

Prothrombin Houston: a dysprothrombin identifiable by crossed immunoelectrofocusing and abnormal Echis carinatus venom activation

A 72-yr-old male with a lifelong history of easy bruisability and posttraumatic bleeding had a prolonged prothrombin time and activated partial thromboplastin time. His plasma Stypven, Taipan, and Echis carinatus venom clotting times were prolonged. The presence of a dysprothrombin was confirmed by the discrepancy between plasma prothrombin coagulant activity and prothrombin antigen levels. His plasma prothrombin was capable of being completely absorbed onto and then eluted from barium sulfate. Crossed immunoelectrophoresis of his plasma prothrombin, and normal plasma prothrombin, into agarose containing rabbit anti-human factor II antibody were similar. Crossed immunoelectrofocusing, a procedure combining isoelectric focusing in disc gels with electroimmunoassay in the second dimension, demonstrated that the patient's prothrombin antigen was more basic than normal. The eluate from barium sulfate absorbtion of patient plasma, when reacted with Echis carinatus venom (which directly cleaves prothrombin to thrombin) clotted purified fibrinogen at a rate slower than normal plasma eluate. SDS-slab gel electrophoresis revealed that the prothrombin present in the patient's eluate was cleaved by Echis carinatus venom. These studies suggest that the coagulopathy of prothrombin Houston results from the generation of a dysfunctional thrombin.

Prothrombin Houston: a dysprothrombin identifiable by crossed immunoelectrofocusing and abnormal Echis carinatus venom activation

A 70-yr-old male with a lifelong history of easy bruisability and posttraumatic bleeding had a prolonged prothrombin time and activated partial thromboplastin time. His plasma Stypven, Taipan, and Echis carinatus venom clotting times were prolonged. The presence of a dysprothrombin was confirmed by the discrepancy between plasma prothrombin coagulant activity and prothrombin antigen levels. His plasma prothrombin was capable of being completely absorbed onto and then eluted from barium sulfate. Crossed Immunoelectrophoresis of his plasma prothrombin, and normal plasma prothrombin, into agarose containing rabbit anti-human factor II antibody were similar. Crossed immunoelectrofocusing, a procedure combining isoelectric focusing in disc gels with electroimmunoassay in the second dimension, demonstrated that the patient's prothrombin antigen was more basic than normal. The eluate from barium sulfate absorbtion of patient plasma, when reacted with Echis carinatus venom (which directly cleaves prothrombin to thrombin) clotted purified fibrinogen at a rate slower than normal plasma eluate. SDS-slab gel electrophoresis revealed that the prothrombin present in the patient's eluate was cleaved by Echis carinatus venom. These studies suggest that the coagulopathy of prothrombin Houston results from the generation of a dysfunctional thrombin.