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Thrombosis and Haemostasis Dec 1997Plasma levels of blood coagulation zymogens are lower in the newborn than in the adult, with the lowest levels being in preterm infants. It is not known if the lower...
Plasma levels of blood coagulation zymogens are lower in the newborn than in the adult, with the lowest levels being in preterm infants. It is not known if the lower coagulation factor levels reflect differences in synthesis, secretion or catabolism. Using a rabbit model we have compared prothrombin synthesis in the fetus and adult. In previous studies we attempted to compare transcription in the adult and fetal liver by extraction of mRNA, immobilization on a membrane and hybridization with a labeled cDNA for rabbit prothrombin. Comparison was impaired by the markedly dissimilar composition of fetal and adult rabbit liver; fetal liver is approximately fifty percent hematopoietic tissue even at term (1). In the present study, to obtain a more meaningful comparison we have employed in situ hybridization to compare directly prothrombin expression in adult and fetal liver. We report here that fetal liver contains more prothrombin mRNA than does adult liver. We have further compared prothrombin levels in protein extracts of adult and fetal liver and found that per microgram of extract, fetal liver contains as much prothrombin as does the adult. We conclude that the lower plasma prothrombin levels in the fetus do not reflect a lower rate of synthesis.
Topics: Animals; Blotting, Western; Female; Humans; In Situ Hybridization; Liver; Pregnancy; Prothrombin; RNA, Messenger; Rabbits
PubMed: 9423796
DOI: No ID Found -
Thrombosis Research Oct 1986An abnormal prothrombin has been detected in a 23 yr-old healthy female and her mother. Both patients appeared to be heterozygous for the abnormality, plasma prothrombin...
An abnormal prothrombin has been detected in a 23 yr-old healthy female and her mother. Both patients appeared to be heterozygous for the abnormality, plasma prothrombin being 50% of normal using the usual one stage assay, but normal when measured either by using Echis carinatus venom or by immunoassay. No abnormality in the immunoelectrophoretic pattern was observed. Prothrombin isolation on DEAE Sephadex failed to separate the abnormal population (prothrombin Clamart) from the normal one. The rates of prothrombin activation by factor Xa, in the presence or absence of phospholipids and/or factor Va, were determined by measuring the production of both clotting and amidolytic activities. The thrombin generation rate from prothrombin isolated from the propositus plasma was 50% slower than normal whatever the method of measurement and the composition of the activation mixture. Analysis of the final activation products by SDS polyacrylamide gel electrophoresis revealed that equal amounts of prethrombin 2 and thrombin had been formed. Prethrombin 2 Clamart was shown to be resistant to proteolysis upon further incubation with factor Xa, whereas it was readily converted to thrombin by Echis carinatus venom. Prothrombin Clamart appears to be characterized by an impairment of Arg 320-IIe cleavage by factor Xa.
Topics: Adult; Arginine; Blood Coagulation Disorders; Factor X; Factor Xa; Female; Genetic Variation; Heterozygote; Humans; Immunoelectrophoresis, Two-Dimensional; Prothrombin; Prothrombin Time; Structure-Activity Relationship; Thrombin
PubMed: 3787558
DOI: 10.1016/0049-3848(86)90176-3 -
Thrombosis Research Oct 2014Prothrombin Yukuhashi (p.Arg596Leu) mutation can result in thrombophilia associated with antithrombin (AT) resistance. Mutant thrombin, an active form of prothrombin...
INTRODUCTION
Prothrombin Yukuhashi (p.Arg596Leu) mutation can result in thrombophilia associated with antithrombin (AT) resistance. Mutant thrombin, an active form of prothrombin Yukuhashi, demonstrated moderately lower clotting activity than the wild-type but substantially impaired the formation of the complex with AT. However, the effects of the mutation on the thrombomodulin (TM)-protein C (PC) anticoagulant system have not been previously elucidated.
MATERIALS AND METHODS
We prepared recombinant wild-type and mutant prothrombins, converted to thrombins using Oxyuranus scutellatus venom, and performed fibrinogen-clotting assays with or without recombinant soluble TM (rTM). We also evaluated activated PC (APC) generation activity of recombinant thrombins by measuring APC activity after incubation with human PC in the presence or absence of rTM.
RESULT AND CONCLUSIONS
rTM treatment reduced the relative fibrinogen-clotting activity of the wild-type down to 8.4% in a concentration-dependent manner, whereas the activity of the mutant was only decreased to 44%. In the absence of rTM, APC generation activity (∆A/min at 405nm) was fairly low (0.0089 for the wild-type and 0.0039 for the mutant). In the presence of rTM, however, APC generation activity was enhanced to 0.0907 (10.2-fold) for the wild-type and to 0.0492 (12.6-fold) for the mutant, and the relative activity of the mutant with rTM was 54% of that of the wild-type. These data suggested that the prothrombin Yukuhashi mutation may cause TM resistance in terms of inhibition of fibrinogen clotting; this may contribute to susceptibility to thrombosis, although the enhancing effect of APC generation can be maintained.
Topics: Antithrombin Proteins; Blood Coagulation Tests; Enzyme Activation; Fibrinogen; Humans; Mutation; Protein C; Prothrombin; Recombinant Proteins; Thrombomodulin
PubMed: 25149909
DOI: 10.1016/j.thromres.2014.07.040 -
Analytical Biochemistry Jul 1999Human prothrombin was acetylated to produce a modified prothrombin that upon activation by platelet-bound prothrombinase generates a form of thrombin that does not...
Human prothrombin was acetylated to produce a modified prothrombin that upon activation by platelet-bound prothrombinase generates a form of thrombin that does not activate platelets but retains its amidolytic activity on a chromogenic peptide substrate. If normal prothrombin is used in such an assay, the thrombin that is generated activates the platelets in a feedback manner, accelerating the rate of thrombin generation and thereby preventing accurate measurement of the initial platelet procoagulant activity. Acetylation of prothrombin was carried out over a range of concentrations of sulfo-N-succinimidyl acetate (SNSA). Acetylation by 3 mM SNSA at room temperature for 30 min at pH 8.2 in the absence of metal ions produced a modified prothrombin that has <0.1% clotting activity (by specific prothrombin clotting assay), but it is activated by factor Xa (in the presence of either activated platelets or factor Va + anionic phospholipid) to produce thrombin activity that is measurable with a chromogenic substrate. Because the feedback action on the platelets is blocked, thrombin generation is linear, allowing quantitative measurement of the initial platelet activation state.
Topics: Acetylation; Binding Sites; Blood Platelets; Feedback; Humans; In Vitro Techniques; Platelet Activation; Prothrombin; Thrombin
PubMed: 10405294
DOI: 10.1006/abio.1999.4148 -
Biochemistry Aug 1992Structural studies on a hereditary abnormal prothrombin, prothrombin Salakta, have been performed to identify the difference responsible for its reduced fibrinogen...
Structural studies on a hereditary abnormal prothrombin, prothrombin Salakta, have been performed to identify the difference responsible for its reduced fibrinogen clotting activity and its reduced esterase activity. Amino acid composition and sequence analyses of a peptide isolated from a lysylendopeptidase digest of the abnormal thrombin indicated that Glu-466 had been replaced by Ala. This amino acid substitution can result from a single nucleotide change in the codon for Glu-466 (GAG----GCG). The model building and the molecular dynamics simulation of thrombin Salakta suggest that the Glu-466----Ala substitution would change the proper conformation around the substrate binding site containing Trp-468, which is a unique surface loop on the thrombin molecule. This is the experimental and theoretical evidence supporting the role of the surface loop containing Trp-468 for the proper conformation of the substrate binding site.
Topics: Alanine; Amino Acid Sequence; Base Sequence; Codon; Esterases; Fibrinogen; Glutamates; Glutamic Acid; Humans; Models, Molecular; Molecular Sequence Data; Mutation; Peptide Fragments; Protein Conformation; Prothrombin
PubMed: 1354985
DOI: 10.1021/bi00148a005 -
The Journal of Biological Chemistry Jul 1993Prothrombin contains two kringle domains, a structural motif common to other plasma proteins involved in hemostasis and fibrinolysis. To determine the role of the...
Prothrombin contains two kringle domains, a structural motif common to other plasma proteins involved in hemostasis and fibrinolysis. To determine the role of the kringle domains of prothrombin, we prepared three recombinant human prothrombin forms lacking the first kringle domain (residues 63-144; PT/delta K1), the second kringle domain (residues 144-249; PT/delta K2), or both kringle domains (63-249; PT/delta K1,2). The isolated prothrombin proteins were greater than 95% pure by SDS-polyacrylamide gel electrophoresis and were well carboxylated. PT/delta K1 displayed 50% of the specific coagulant activity of plasma prothrombin, PT/delta K2 had 10% of the specific coagulant activity, and PT/delta K1,2 was inactive. Polyclonal antibodies directed against the Ca(II)-specific conformer of prothrombin bound PT/delta K1 and PT/delta K2 with the same affinity as prothrombin, indicating that the Ca(II)-induced conformational transition does not involve sites on the prothrombin kringle domains. Gel filtration studies demonstrated that radiolabeled plasma prothrombin and all of the prothrombin kringle deletion mutants bound to phospholipid vesicles in the presence of Ca(II) but not in the presence of Mg(II) or EDTA. Relative dissociation constants of 1.10 +/- 0.75 and 0.49 +/- 0.18 microM were obtained by quasielastic light scattering for the interaction of phospholipid vesicles with plasma prothrombin and PT/delta K1, respectively. These data indicate that neither the first nor the second kringle domain contain unique sites for the interaction of prothrombin with phospholipid vesicles and are not required for prothrombin-phospholipid binding.
Topics: 1-Carboxyglutamic Acid; Animals; Base Sequence; Binding Sites; CHO Cells; Calcium; Chromatography, Gel; Cloning, Molecular; Cricetinae; Electrophoresis, Polyacrylamide Gel; Humans; Membrane Lipids; Molecular Sequence Data; Mutation; Oligodeoxyribonucleotides; Peptide Fragments; Phospholipids; Protein Conformation; Prothrombin; Recombinant Proteins
PubMed: 8393451
DOI: No ID Found -
Nucleic Acids Research 2005The human prothrombin G20210A polymorphism located at the 3' cleavage site of the mRNA results in elevated plasma prothrombin levels and increased risk of venous...
The human prothrombin G20210A polymorphism located at the 3' cleavage site of the mRNA results in elevated plasma prothrombin levels and increased risk of venous thrombosis. This polymorphism has been shown to directly influence a variety of processes related to prothrombin mRNA metabolism. We have constructed plasmids that express the full-length prothrombin mRNA that is polyadenylated at its natural site. The A allele prothrombin variant was more efficient than the G allele at promoting cleavage at this site in the presence of a competing poly (A) sequence. In the absence of competition, both allelic variants give rise to a similar level of cleavage site heterogeneity. An upstream sequence element (USE) was also identified within the prothrombin 3'-UTR. When placed upstream of two competing poly (A) sites, the USE directed cleavage preferentially to the proximal poly (A) site. In the absence of competition, the USE had no effect on cleavage site selection. This study suggests that the basis for the increase in prothrombin expression in A allele carriers is not due to allelic changes in cleavage site selection per se. In addition, the functionality of USEs needs to be considered within the context of endogenous sequence architecture.
Topics: 3' Untranslated Regions; Base Sequence; Conserved Sequence; Humans; Molecular Sequence Data; Polyadenylation; Polymorphism, Single Nucleotide; Prothrombin; RNA 3' End Processing; RNA, Messenger; Regulatory Sequences, Ribonucleic Acid
PubMed: 15718300
DOI: 10.1093/nar/gki245 -
Thrombosis and Haemostasis Aug 1992Plasma levels of total prothrombin and fully-carboxylated (native) prothrombin were compared with results of prothrombin time (PT) assays for patients undergoing oral... (Comparative Study)
Comparative Study
Plasma levels of total prothrombin and fully-carboxylated (native) prothrombin were compared with results of prothrombin time (PT) assays for patients undergoing oral anticoagulant therapy. Mean concentrations of total and native prothrombin in non-anticoagulated patients were 119 +/- 13 micrograms/ml and 118 +/- 22 micrograms/ml, respectively. In anticoagulated patients, INR values ranged as high as 9, and levels of total prothrombin and native prothrombin decreased with increasing INR to minimum values of 40 micrograms/ml and 5 micrograms/ml, respectively. Des-carboxy-prothrombin increased with INR, to a maximum of 60 micrograms/ml. The strongest correlation was observed between native prothrombin and the reciprocal of the INR (1/INR) (r = 0.89, slope = 122 micrograms/ml, n = 200). These results indicated that native prothrombin varied over a wider range and was more closely related to INR values than either total or des-carboxy-prothrombin. Levels of native prothrombin were decreased 2-fold from normal levels at INR = 2, indicating that the native prothrombin antigen assay may be a sensitive method for monitoring low-dose oral anticoagulant therapy. The inverse relationship between concentration of native prothrombin and INR may help in identification of appropriate therapeutic ranges for oral anticoagulant therapy.
Topics: Administration, Oral; Anticoagulants; Blood Chemical Analysis; Humans; Prothrombin; Prothrombin Time; Reference Values
PubMed: 1412161
DOI: No ID Found -
Thrombosis Research Nov 2014Prothrombin deficiency is a very rare disorder caused by mutations in the F2 gene that generate hypoprothrombinemia or dysprothrombinemia and is characterized by...
INTRODUCTION
Prothrombin deficiency is a very rare disorder caused by mutations in the F2 gene that generate hypoprothrombinemia or dysprothrombinemia and is characterized by bleeding manifestations that can vary from clinically irrelevant to life-threatening.
AIM
Here we characterize a patient with a novel missense mutation in F2, c.1090T/A (p.Val322Glu), that causes severe dysprothrombinemia.
METHODS
Coagulation assays, prothrombin Western Blotting, FII activation by Ecarin, fibrinogen degradation products quantification and thrombin generation assay were carried out to assess prothrombin expression and function. PCR followed by direct sequencing was carried out to characterize the mutation. In silico analysis for missense variant and molecular modeling were applied to predict the mechanism that leads to dysprothrombinemia.
RESULTS AND CONCLUSIONS
The homozygous patient had a markedly prolonged prothrombin time, strongly reduced FII activity (0.82%) but normal antigen levels. In the thrombin generation assay the lag time and the peak height were unmeasurable, suggesting that the Val322Glu mutation results in the inability of the mutant prothrombin to be fully activated to thrombin. In fact, prothrombin activation by ecarin was defective, with a massive accumulation of the meizothrombin intermediate. Molecular modeling and dynamic simulation studies showed that the Val322Glu mutation interferes with protein flexibility at Arg271 and Arg320. This impairs the switch of the protein from zymogen to proteinase, thus preventing the formation of thrombin. Accumulated meizothrombin, however, maintains some fibrinogen-degrading activity, as shown by the formation of FDPs, and this probably explains the patient's mild bleeding phenotype.
Topics: Blood Coagulation; Blood Coagulation Disorders, Inherited; Enzyme Precursors; Female; Homozygote; Humans; Male; Middle Aged; Molecular Dynamics Simulation; Mutation, Missense; Pedigree; Prothrombin; Thrombin; Thromboplastin
PubMed: 25242243
DOI: 10.1016/j.thromres.2014.08.028 -
Journal of the American Society of... Nov 1999Prothrombin has remarkable affinity for calcium oxalate crystals. It is produced in renal tubular cells and is detected as a urinary form of prothrombin F1. The aim of...
Prothrombin has remarkable affinity for calcium oxalate crystals. It is produced in renal tubular cells and is detected as a urinary form of prothrombin F1. The aim of this basic study was (1) to isolate prothrombin mRNA from normal human and rat kidneys; (2) to confirm expression level changes in stone-forming rat kidneys; and (3) to analyze the DNA sequence of renal prothrombin. The aim of the clinical investigation was to measure the serum levels of renal prothrombin in clinical cases of various urologic diseases. The expression of prothrombin mRNA in human kidneys and male Wistar rat kidneys was investigated using reverse transcription-PCR, with prothrombin (F1, F2, and thrombin) primers. Renal prothrombin levels were measured in the sera of patients with renal cell carcinoma, renal transplant donors, patients with chronic renal failure, and renal transplant recipients, using an enzyme-linked immunosorbent assay. Expression of cyclophilin as well as prothrombin mRNA could be detected. Prothrombin mRNA expression levels seemed to be increased in stone-forming rats. The DNA sequence of renal prothrombin differed from that of liver prothrombin at three points. Repeated measurements of renal prothrombin showed that values were high during the acute tubular necrosis period and tended to decrease with the recovery of renal function. Prothrombin mRNA expression could be confirmed in human and rat kidneys, as well as in stone-forming rat kidneys. Serum concentration measurements can be considered useful for assessment of recovery from acute tubular necrosis after renal transplantation and for diagnosis of acute rejection.
Topics: Adult; Animals; Cadaver; Female; Graft Rejection; Humans; Kidney; Kidney Transplantation; Male; Middle Aged; Prothrombin; RNA, Messenger; Rats; Rats, Wistar
PubMed: 10541274
DOI: No ID Found