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PubMed1996

Soluble phospholipids enhance factor Xa-catalyzed prothrombin activation in solution.

Acidic phospholipids play an important but incompletely understood role in prothrombin activation. Here we report the effect of short-chain phosphatidylserine (dicaproylphosphatidylserine, C6PS) and the corresponding phosphatidylglycerol (C6PG) and phosphatidylcholine (C6PC) derivatives on the rate of prothrombin activation by factor Xa. The critical micellar concentrations of these short-chained phospholipids have been determined under a variety of conditions that we used for kinetic and structural studies. Under conditions for which these lipids exist in a soluble form, the results demonstrate that: (i) the rate of human prothrombin activation by human factor Xa was enhanced in a calcium-dependent fashion up to 60-fold by addition of C6PS, roughly 20% of the optimal enhancement seen with bovine phosphatidylserine/palmitoyloleoylphosphatidylcholine (25/75 PS/POPC) membranes; (ii) C6PS inhibited the rate of hydrolysis of synthetic factor Xa substrate (S-2765), an effect that was mimicked, but at much lower lipid concentrations, by PS/POPC membranes; (iii) there was no enhancement of prothrombin activation and much less inhibition of hydrolysis of S-2765 by factor Xa in the presence of C6PG or C6PC; and (iv) the thermal denaturation of prothrombin was altered in a calcium-independent but dose-dependent fashion by either C6PS or C6PG. These results have been interpreted in terms of the existence of (a) specific PS binding site(s) on factor Xa (Kd approximately 73 microM) that regulate(s) the activity of this serine protease. Our results do not rule out the possibility that the rate of prothrombin activation is also influenced by a weaker, calcium-independent, and less specific acidic lipid binding site on prothrombin, the occupancy of which results in conformational changes in this protein. The results clearly suggest that PS binding regulates the rate of prothrombin activation.

Animals↗
PubMed1996

A binding site expressed on the surface of activated human platelets is shared by factor X and prothrombin.

We have demonstrated the presence of a saturable, reversible, and Ca(2+)-dependent binding site for 125I-labeled factor X ([125I]factor X) on human platelets (16000 +/- 2000 sites per platelet, Kd = 320 +/- 40 nM, n = 12) activated with either thrombin or the thrombin receptor agonist peptide, SFLLRN-amide, but not with ADP. Bound [125I]factor X could be completely removed by the addition of a Ca2+ chelator or an excess of unlabeled factor X. Antibodies that inhibit binding of factor X to the MAC-1 integrin receptor of monocytes and those directed against human factor V, failed to disrupt [125I]factor X binding to platelets. Prothrombin, but neither factor VII, factor IX, protein C, nor protein S, was an effective competitor of [125I]factor X binding with a K1 approximately Kd. [125I]Prothrombin also binds to activated (but not unactivated) platelets in a saturable, reversible, and Ca(2+)-dependent manner (20500 +/- 1500 sites, Kd = 470 +/- 110 nM, n = 3). Annexin V potently inhibited the binding of both [125I]factor X and [125I]prothrombin (IC50 approximately 3 nM). Factor X, prothrombin, and prothrombin fragment 1 (residues 1-155) were equipotent inhibitors of [125I]prothrombin and [125I]factor X binding, whereas Gla-domain-less factor X was unable to compete with [125I]factor X for platelet binding sites. Thus, it is the Gla-domains of factor X and prothrombin that appear to contain the regions necessary for platelet binding. The results of studies utilizing artificial phospholipid surfaces have led to the hypothesis that the substrates (FX and prothrombin) for the intrinsic pathway FXase and prothrombinase complexes are bound to the phospholipid surface. The factor X/prothrombin binding site we have described on the surface of activated platelets permits the utilization of surface-bound substrates by these complexes when they are assembled on a physiologic surface.

1-Carboxyglutamic Acid↗
PubMed1997

Fluorescence resonance energy transfer study of shape changes in membrane-bound bovine prothrombin and meizothrombin.

Prothrombin activation to thrombin is a key control reaction in blood coagulation. During the process, prothrombin is sequentially cleaved at two peptide bonds (Arg323-Ile and Arg274-Thr) by factor X(a) to generate meizothrombin and then thrombin. Phosphatidylserine (PS)-containing membranes from platelets are believed to facilitate this two-step process. Using fluorescence energy transfer (FRET), we determined the distances of closest approach between a specifically located C-terminal fluorescein of a double mutant bovine prothrombin (P(S528A, G581C)-FM) or meizothrombin (M(S528A, G581C)-FM) and phosphatidylethanolamine-N-rhodamine B (PE-Rh; 0-8.7 mol %) contained in membranes composed of PS (25 mol %) and phosphatidylcholine (66.3-75 mol %). Plots of the energy transfer efficiency as a function of membrane concentration, at six PE-Rh surface densities, were analyzed globally to obtain dissociation constants and binding stoichiometries as global parameters and saturating energy transfer efficiencies characteristic of each surface density. From the global analysis, the dissociation constants were estimated to be 0.32 +/- 0.10 and 0.28 +/- 0.12 microM with stoichiometries of 42 +/- 12 and 44 +/- 9 lipid/protein for prothrombin and meizothrombin, respectively. The distance of closest approach was obtained from the dependence of the saturating energy transfer efficiency on the acceptor (PE-Rh) surface density. With the assumptions of kappa2 = 2/3 and n = 1.4, the distances were 94 +/- 3 A for prothrombin and 114 +/- 2 A for meizothrombin. Since both prothrombin and meizothrombin behave in solution as oblate ellipsoids of revolution with a long axis of 120 A, our FRET measurements suggest that binding to PS-containing membranes induced tighter folding of the prothrombin molecule but not of the meizothrombin intermediate. This observation is consistent with our hypothesis that membrane binding plays an essential role in the sequential alignment of the bond Arg323-Ile in prothrombin and Arg274-Thr in meizothrombin with the active site of the membrane-bound prothrombinase in the two-step thrombin-generating process.

Amino Acid Sequence↗
PubMed1998

Prothrombin is a cofactor for the binding of factor XI to the platelet surface and for platelet-mediated factor XI activation by thrombin.

To study the physiological significance of thrombin as an initiator of intrinsic blood coagulation, activated human platelets were compared with dextran sulfate as a surface for thrombin-catalyzed factor XI activation. Activated gel-filtered platelets promoted factor XI activation by thrombin at initial rates 2-5-fold greater than dextran sulfate in the presence of high molecular weight kininogen (HK, 45 nM), ZnCl2 (25 microM), and CaCl2 (2 mM), conditions optimal for factor XI binding to platelets. Physiological concentrations of HK (636 nM) inhibited factor XI activation by thrombin in a concentration-dependent manner, and this inhibition was reversed by prothrombin (1-3 microM) and by prothrombin fragment 1.2 (PF1.2), but not by prothrombin fragment 1 (PF1). Since prothrombin and PF1.2 (but not PF1) also displaced HK from its binding site on the Apple 1 domain of factor XI, we conclude that the Kringle II domain of prothrombin competes with HK for binding to the Apple 1 domain of factor XI. Prothrombin (1-3 microM) and PF1.2 (but not PF1) in the presence of CaCl2 (2 mM) were able to replace HK (45 nM) in the presence of ZnCl2 (25 microM) as a cofactor for the specific, reversible, high-affinity (Kd approximately 25 nM) binding of factor XI to 947 +/- 150 sites per platelet. This binding is mediated by residues Asn 235-Arg 266 in the Apple 3 domain since a conformationally constrained, synthetic peptide analogue of this sequence inhibits both factor XI binding to activated platelets and platelet-mediated, thrombin-catalyzed factor XI activation in the presence of prothrombin and CaCl2. Finally, prothrombin (1.2 microM) and CaCl2 (2 mM) could substitute for HK (45 nM) and ZnCl2 (25 microM) in promoting optimal rates of thrombin-catalyzed factor XI activation on the platelet surface, thereby initiating the intrinsic coagulation pathway by mechanisms completely independent of the contact phase proteins, factor XII, HK, and prekallikrein.

Binding Sites↗
PubMed1991

Processing of prothrombin in the secretory pathway.

Antibodies raised against plasma prothrombin and the prothrombin propeptide were used to identify prothrombin precursors in rough and smooth microsomes and in the Golgi apparatus. The data demonstrate that the propeptide is part of the prothrombin molecule when undergoing a variety of modifications in the Golgi apparatus. It is shown that these modifications result in an increase in the apparent molecular mass of the prothrombin precursor from 78 kDa in early processing to 83 kDa in late processing. The 83 kDa prothrombin precursor was not recognized by the anti-propeptide antiserum and most likely represents the final product of the precursor in the secretory pathway. Evidence is presented that the propeptide is released from the parent molecule in the Golgi apparatus by a membrane-bound Ca(2+)-dependent serine proteinase(s) with characteristics similar to those of the proalbumin-to-albumin-converting enzyme. Vitamin K-dependent carboxylase activity was measured in membrane fragments obtained from the Golgi apparatus preparation. Sucrose-density-gradient centrifugation and the use of marker enzymes showed that carboxylase activity was highest in fractions enriched in cis-Golgi cisternae. Two different synthetic peptides were used as substrates for the carboxylase. These peptides were from the N-terminal and the C-terminal part of the gamma-carboxyglutamic acid (Gla) region of prothrombin. It is shown that the N-terminal and the C-terminal peptides were preferred as substrates for the carboxylase in the microsomal and the Golgi apparatus preparations respectively. It is also shown that the prothrombin precursor acquires negative charges in the Golgi apparatus that do not result from addition of sugars in late processing. These negative charges could be eliminated by thermal decarboxylation, suggesting that Gla residues may also be synthesized in late processing.

Amino Acid Sequence↗
PubMed1995

The urinary F1 activation peptide of human prothrombin is a potent inhibitor of calcium oxalate crystallization in undiluted human urine in vitro.

1. The urinary F1 activation peptide of prothrombin is the predominant protein incorporated into calcium oxalate crystals precipitated from human urine. The aim of this study was to examine the effect of pure urinary prothrombin F1 on calcium oxalate crystallization in human urine. 2. Urinary prothrombin F1 was purified from demineralized calcium oxalate crystals precipitated from human urine, and its effects on calcium oxalate crystallization induced by addition of an oxalate load were tested in undiluted, ultrafiltered urine from healthy men, at final concentrations of 0 to 10 mg/l. 3. Urinary prothrombin F1 did not affect the amount of oxalate required to induce crystallization, but the volume of material deposited increased in proportion to increasing concentrations of urinary prothrombin F1. However, the mean particle size decreased in reverse order: this was confirmed by scanning electron microscopy, which showed it to be the result of a reduction in crystal aggregation rather than in the size of individual crystals. Analysis of 14C-oxalate data revealed a dose-dependent decrease in calcium oxalate deposition with an increase in urinary prothrombin F1 concentration, indicating that the increase in particle volume recorded by the Coulter Counter resulted from inclusion of urinary prothrombin F1 into the crystalline architecture, rather than increased deposition of calcium oxalate. 4. It was concluded that urinary prothrombin F1 may be an important macromolecular determinant of stone formation.

Calcium Oxalate↗
PubMed1999

The 20210 G-->A mutation in the 3'-untranslated region of the prothrombin gene and the risk for arterial thrombotic disease.

A sequence variation in the 3'-untranslated region of the prothrombin (PT) gene (20210 G-->A) was recently claimed to be associated with elevated plasma prothrombin levels and an increased risk for venous and arterial thrombosis. We examined the prevalence of the 20210 A allele in the prothrombin gene in 400 healthy controls and in 263 patients with proven premature atherosclerotic disease. In addition, we measured prothrombin, prothrombin fragment 1 + 2, thrombin-antithrombin (TAT) complex and D-dimer levels in plasma from carrier and non-carrier patients. The frequency of the variant allele was 1% in the control subjects and 2.7% in the patient group, yielding a relative risk (RR) for the 20210 A allele of 2.7 (95% CI 0.8-9.4). All heterozygotes in the patient group were found to have had a myocardial infarction (MI), yielding a RR for MI of 4.2 (95% CI 1.2-14.6). Plasma prothrombin levels in carriers (126+/-10) were higher than in non-carriers (103+/-1, P=0.02). The levels of TAT complexes (16+/-9 v 6+/-1 microg/ml, P=0.02) as well as of prothrombin fragment 1 + 2 (1.5+/-0.3 v 1.0+/-0.1 nmol/l, P=0.02) were also elevated in carriers of the mutation. Our findings suggest that the 20210 G-->A mutation in the prothrombin gene is a genetic risk factor for MI. In addition, our data provide evidence for an association of the mutation with excessive thrombin generation, which may contribute to the understanding of its role in venous and arterial disease.

Adult↗
PubMed2001

Anti-prothrombin antibodies combined with lupus anti-coagulant activity is an essential risk factor for venous thromboembolism in patients with systemic lupus erythematosus.

Anti-prothrombin antibodies (anti-prothrombin) and anti-beta2-glycoprotein I antibodies (anti-beta2-GP I) are the most common and characterized anti-phospholipid antibodies (aPL) detected using specific enzyme-linked immunosorbent assay (ELISA) systems. Recently, lupus anti-coagulant (LA) activity detected by a phospholipid-dependent coagulation assay was reported to be associated with anti-prothrombin and/or anti-beta2-GP I. Here we show that the co-existence of IgG anti-prothrombin and LA activity might be an essential risk factor for venous thromboembolism (VTE) in patients with systemic lupus erythematosus (SLE). We examined not only the levels of antibodies to prothrombin and anti-beta2-GP I (both IgG and IgM isotypes) using an ELISA system, but also LA activity detected using both diluted Russell's viper venom time (dRVVT) and STACLOT LA test in 124 patients with SLE. The SLE patients were divided into four groups according to the results of ELISA and LA assay results for each aPL: group A, ELISA+ and LA+ group B, ELISA+ and LA-; group C, ELISA- and LA+ group D, ELISA- and LA-. Regarding IgG anti-prothrombin, the prevalence of VTE was significantly higher in group A (16/35 cases, 45.7%, P < 0.001, Fisher's exact probability test) than in the other groups (B, 2/30, 6.7%; C, 1/22, 4.5%; D, 1/37, 2.7%). With respect to IgM anti-prothrombin and IgG or IgM anti-beta2-GP I, the prevalence of VTE was higher in both groups A and C than in group D, but no statistical difference in prevalence was found between groups A and C. Multivariate logistic regression analysis of risk factors for VTE confirmed that the co-existence of IgG anti-prothrombin and LA activity was the only significant risk factor for VTE (odds ratio, 19.13; 95% confidence intervals, 4.74-77.18).

Adolescent↗
PubMed2003

A common mutation, Arg457-->Gln, links prothrombin deficiencies in the Puerto Rican population.

Five unrelated families with Puerto Rican ancestry were identified as having at least one member with bleeding due to a prothrombin deficiency. Genetic prothrombin deficiencies are extremely rare, but at the University of Puerto Rico Hemophilia Center, prothrombin deficiency is the third most common congenital coagulation factor deficiency. Because Puerto Rico is relatively isolated, there was a reasonable expectation of a founder effect. Prothrombin genes from probands and their parents were directly sequenced from PCR amplified exons using forward and reverse primers. Four novel prothrombin mutations were identified. The first, a G-->A substitution at DNA position 10150 predicting an Arg457-->Gln (R457Q) replacement, is common to all five families. In two of the families, the proband children are homozygous for R457Q. In the other three families, the probands are compound heterozygotes for R457Q and one of the other three mutations, which include another point mutation (gamma16Q), a deletion and a splice junction mutation. The two point mutations have been designated Puerto Rico I and Puerto Rico II. The crystal structure of alpha-thrombin predicts that the R457Q mutation removes a salt bridge that links the A- and B-chains of thrombin. The primary effect of this defect appears to be destabilization of the circulating prothrombin, creating a moderate hypoprothrombinemia. However, prothrombin antigen/activity ratios indicate a dysprothrombinemia as well, most likely due to the inability of R457Q prothrombin to activate fully to thrombin.

Adolescent↗
PubMed1971

Mechanism of action of vitamin K: evidence for the conversion of a precursor protein to prothrombin in the rat.

Previous studies have shown that the vitamin K-induced synthesis of prothrombin in a vitamin K-deficient rat is only slightly inhibited by cycloheximide treatment. Rat prothrombin has now been purified by disc electrophoresis after BaSO(4) adsorption and citrate elution. When cycloheximide (5 mg/kg) was given to vitamin K-deficient rats 30 min before vitamin K, about 70% of the amount of prothrombin seen in rats not treated with cycloheximide was produced (two-stage assay), and the prothrombin band could be seen on the electrophoretic gels. However, if radioactive amino acids are administered to the rats after cycloheximide treatment, the newly formed prothrombin contains no radioactivity. The isolated prothrombin does contain radioactivity if the vitamin K-deficient rats are treated with vitamin K but no cycloheximide. When radioactive amino acids were given to deficient rats 1 hr before cycloheximide and vitamin K, radioactivity was found in prothrombin. These data suggest that, in the intact rat, the action of vitamin K is to convert a protein precursor with a short biological half life to prothrombin.

Amino Acids↗
PubMed1995

Prothrombin contributes to the assembly of the factor Va-factor Xa complex at phosphatidylserine-containing phospholipid membranes.

The activation of prothrombin is catalyzed by prothrombinase, a complex of factor Xa and factor Va assembled on a negatively charged phospholipid membrane. We used a tubular flow reactor to identify the relative contributions of factor Va, prothrombin, and the negatively charged phosphatidylserine to the assembly of prothrombinase. Perfusion of phospholipid-coated capillaries with a mixture of factor Xa, factor Va, and prothrombin resulted in a steady-state rate of thrombin production that increased with (i) the phosphatidylserine content of the phospholipid bilayer, (ii) the factor Va concentration, and, most interestingly, (iii) the prothrombin concentration of the perfusion solution. Incorporation of 20 mol % phosphoatidylethanolamine, a phospholipid with poor ability to promote prothrombinase activity, into a 5 mol % phosphatidylserine membrane also increased the steady-state rate of thrombin production. Direct measurements of the amount of prothrombinase in the flow reactor demonstrated that increased catalytic activities were the result of an increased steady-state amount of membrane-associated prothrombinase. Thus, similar turnover numbers of prothrombin activation (3100 min-1) were calculated, irrespective of the phosphatidylserine content of the membrane. We established for membranes with low phosphatidylserine content (< 10 mol%) a linear relationship between the prothrombinase activity and the arithematical product of the factor Va concentration in the perfusion solution and the prothrombin concentration near the catalytic surface. Our results indicate that, in addition to factor Va, prothrombin also is essential to the assembly of prothrombinase at macroscopic surfaces with low phosphatidylserine content. The data further suggest that the prothrombin concentration near the surface, controlled by the prothrombinase activity and mass transfer, is an important regulator of the prothrombinase surface density.

Factor Va↗
PubMed1998

Activation of alphaVbeta3 on vascular cells controls recognition of prothrombin.

Regulation of vascular homeostasis depends upon collaboration between cells of the vessel wall and blood coagulation system. A direct interaction between integrin alphaVbeta3 on endothelial cells and smooth muscle cells and prothrombin, the pivotal proenzyme of the blood coagulation system, is demonstrated and activation of the integrin is required for receptor engagement. Evidence that prothrombin is a ligand for alphaVbeta3 on these cells include: (a) prothrombin binds to purified alphaVbeta3 via a RGD recognition specificity; (b) prothrombin supports alphaVbeta3-mediated adhesion of stimulated endothelial cells and smooth muscle cells; and (c) endothelial cells, either in suspension and in a monolayer, recognize soluble prothrombin via alphaVbeta3. alphaVbeta3-mediated cell adhesion to prothrombin, but not to fibrinogen, required activation of the receptor. Thus, the functionality of the alphaVbeta3 receptor is ligand defined, and prothrombin and fibrinogen represent activation- dependent and activation-independent ligands. Activation of alphaVbeta3 could be induced not only by model agonists, PMA and Mn2+, but also by a physiologically relevant agonist, ADP. Inhibition of protein kinase C and calpain prevented activation of alphaVbeta3 on vascular cells, suggesting that these molecules are involved in the inside-out signaling events that activate the integrin. The capacity of alphaVbeta3 to interact with prothrombin may play a significant role in the maintenance of hemostasis; and, at a general level, ligand selection by alphaVbeta3 may be controlled by the activation state of this integrin.

Adenosine Diphosphate↗
PubMed1990

Characterization of the cDNA coding for mouse prothrombin and localization of the gene on mouse chromosome 2.

A series of overlapping cDNAs coding for mouse prothrombin (coagulation factor II) have been isolated and the composite DNA sequence has been determined. The complete prothrombin cDNA is 1,987 bp in length [excluding the poly(A) tail] and codes for 18 bp of 5' untranslated sequence, an open reading frame coding for 618 amino acids, a stop codon, and a 3' untranslated region of 112 bp followed by a poly(A) tail. The translated amino acid sequence predicts a molecular weight of 66,087, which includes 10 residues of gamma-carboxyglutamic acid. There are five potential N-linked glycosylation sites. Mouse prothrombin is 81.4% and 77.3% identical to the human and bovine proteins, respectively. Comparison of the cDNA coding for mouse prothrombin to the human and bovine cDNAs indicates 79.9% and 76.5% identity, respectively. Amino acid residues important for the structure and function of human prothrombin are conserved in the mouse and bovine proteins. In the adult mouse and rat, prothrombin is primarily synthesized in the liver, where is constitutes 0.07% of total mRNA as determined by solution hybridization analysis. The genetic locus for mouse prothrombin, Cf-2, has been mapped using an interspecies backcross and DNA fragment differences between the two species. The prothrombin locus lies on mouse chromosome 2, 1.8 +/- 1.3 map units proximal to the catalase locus. The gene order in this region is Cen-Acra-Cf-2-Cas-1-A-Tel. This localization extends the proximal boundary of the known region of homology between mouse chromosome 2 and human chromosome 11p from Cas-1 about 2 map units toward the centromere.

Amino Acid Sequence↗
PubMed2000

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.

Adolescent↗
PubMed2006

Manipulation of prothrombin concentration improves response to high-dose factor VIIa in a cell-based model of haemophilia.

Clinical reports suggest that treatment regimens employing both activated prothrombin complex concentrates (aPCCs) and recombinant activated factor VII (rFVIIa) may control the bleeding in patients with haemophilia who fail to respond to either agent alone. We hypothesised that increased concentrations of prothrombin, as may be observed after the infusion of aPCCs, favourably influence parameters of thrombin generation in haemophilia treated with high-dose rFVIIa. We examined the effect of varied prothrombin and rFVIIa concentrations on thrombin generation in a model of haemophilia. At all concentrations of rFVIIa, increased prothrombin concentrations led to increases in the peak and rate of thrombin generation. In assays with the highest concentrations of prothrombin and rFVIIa, peak thrombin actually equalled that measured in the model of normal haemostasis. The significant impact of prothrombin concentration on the effect of rFVIIa in vitro may explain the improved haemostasis reported with concurrent use of aPCCs and rFVIIa. These results imply that persons with plasma prothrombin levels at either end of the 'normal' range could have significantly different responses to similar rFVIIa doses. Furthermore, these results suggest that increasing plasma prothrombin concentration prior to rFVIIa administration may offer advantages over the use of rFVIIa alone in the treatment of haemophilic bleeding.

Blood Platelets↗
PubMed2004

G20210A is a functional mutation in the prothrombin gene; effect on protein levels and 3'-end formation.

BACKGROUND: The prothrombin G20210A mutation is associated with increased plasma prothrombin levels and risk of thrombosis. The mechanism by which this mutation leads to increased prothrombin expression is as yet unclear and still the subject of debate. OBJECTIVES: The aim of this study was to investigate the effect of the G20210A mutation on mRNA and protein expression. METHODS: We made a set of constructs containing the prothrombin 5'-regulatory region, the firefly luciferase reporter gene and the prothrombin 3'-UTR+ downstream region. The latter element contained either the 20210G or A allele and was inserted either as a single unit (constructs G1 and A1) or in tandem (A1A2, G1G2, A1G2, G1A2). Constructs were transiently expressed in HepG2 cells. Expression was evaluated by luciferase assays and reverse transcriptase-polymerase chain reaction (RT-PCR), followed by quantification of the products and determination of the ratio of poly(A)site usage. RT-PCR sequencing was used for determination of the actual site of polyadenylation in mRNAs from constructs G1 and A1 and from endogenous prothrombin mRNAs from HepG2 cells and human liver tissue. RESULTS: The A1 constructs expressed 1.2-fold more protein than the G1 constructs. The double constructs expressed 1.4-fold more protein (A1A2 vs. G1G2). Similar results were found in a set of constructs in which an SV40 promoter replaced the prothrombin 5'-regulatory region. Ratios of poly(A) site usage (expressed as ratio poly(A) site 1 and 2) for the tandem constructs were similar for constructs with two Gs or As at both poly(A)sites; 2.92 (95% confidence interval 2.39-3.45) and 2.75 (2.55-2.95). pA1/pA2 ratios were 1.46 (1.11-1.81) for G1A2 and 6.29 (5.48-7.10) for A1G2 constructs with different poly(A) sites, indicating that the poly(A)site with the 20210A variant is favored over the normal site. In 20210G mRNAs, the G at 20210 was the last non-A nucleotide in the majority of mRNAs, whereas in most 20210A mRNAs, the last non-A nucleotide was the C at 20209. Over 70% of the prothrombin 20210G mRNAs from HepG2 cells and human liver tissue is polyadenylated at position 20210. CONCLUSIONS: The 20210A variant has a more effective poly(A) site, leading to increased mRNA and protein expression, irrespective of the promoter and gene. It does not affect the position of poly(A) attachment.

3' Untranslated Regions↗
PubMed2006

Type and location of venous thromboembolism in patients with factor V Leiden or prothrombin G20210A and in those with no thrombophilia.

BACKGROUND: Patients with factor (F) V Leiden or the prothrombin G20210A polymorphism are at increased risk of developing deep vein thrombosis (DVT). On the other hand, the risk of developing pulmonary embolism (PE) appears to be low in carriers of FV Leiden, perhaps because of a lower tendency to develop iliofemoral DVT than non-carriers. For prothrombin G20210A, data are scanty and controversial. METHODS: The clinical manifestations (isolated DVT, DVT and PE, and isolated PE), the extension of DVT, and the presence of transient risk factors were retrospectively investigated in 115 patients with heterozygous FV Leiden, 87 with prothrombin G20210A and 200 with no thrombophilia marker. RESULTS: Isolated symptomatic PE was less prevalent in patients with FV Leiden (6%) than in those with prothrombin G20210A (21%) and no thrombophilia (23%) (P > 0.0001). The rate of distal DVT was higher in patients with no thrombophilia (16% vs. 7% for FV Leiden and 6% for prothrombin G20210A) (P = 0.02). No difference in the incidence of PE from distal and proximal DVT, the extension of proximal DVT and the type of transient risk factors for venous thromboembolism (VTE) was found in the three groups. Patients with prothrombin G20210A had a younger age at their first VTE (24 years, P < 0.0001) and a higher rate of DVT accompanying PE (P = 0.04) than those with FV Leiden or no thrombophilia. CONCLUSIONS: Carriers of prothrombin G20210A, unlike those of FV Leiden, have an increased risk of developing isolated PE. This difference was not explained by a different rate of distal DVT, extension of proximal DVT, or distribution of transient risk factors in the two groups. Patients with prothrombin G20210A have more severe clinical manifestations than those with FV Leiden or no thrombophilia.

Adenine↗
PubMed2004

The role of genetics in the risk of thromboembolism: prothrombin 20210A and oral contraceptive therapy.

PURPOSE: To provide an overview of the prothrombin 20210A mutation, its effects on the incidence of venous thromboembolism (VTE) in users of oral contraceptive therapy (OCT), and screening recommendations for the primary care practice setting. DATA SOURCES: Several databases, including MEDLINE, EMBASE, BIOSIS, Cochrane Library, and SciSearch, were searched for articles published between 1996 and 2003. An integrative review of studies addressing prothrombin 20210A was done using available case-series and case-control studies. No randomized controlled trials on prothrombin 20210A were available in the literature from the years searched. CONCLUSIONS: Prothrombin 20210A increases the risk of developing a VTE for those who carry the genetic mutation. The presence of either concomitant circumstantial factors (e.g., surgery or immobilization) or a combination of genetic factors (e.g., factor V Leiden and prothrombin 20210A) raises the frequency of VTEs to an even greater extent. The risk increases exponentially in users of OCT. Screening guidelines for the use of OCT in prothrombin 20210A carriers remain unclear due to the paucity of empirical evidence related to the topic. IMPLICATIONS FOR PRACTICE: Practitioners caring for a prothrombin 20210A carrier should maintain a high degree of suspicion with even vague signs or symptoms of a possible VTE. Practitioners should consider completing a full diagnostic workup for VTE on that patient, particularly if that patient is taking OCT. Until evidence becomes available as to the best anticoagulation practice after an initial or recurrent VTE in a prothrombin 20210A carrier, standard treatment guidelines for anticoagulation should be followed.

Contraceptives, Oral, Hormonal↗