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PubMed1999

The effects of freeze drying and freeze drying additives on the prothrombin time and the international sensitivity index.

AIM: To determine whether freezing, freeze drying protective additives, or freeze drying of plasma samples from patients on coumarin treatment and from normal individuals affects prothrombin times or the international sensitivity index (ISI) calibration. METHODS: The effect of the addition of the protective additives singly and combined on the prothrombin time of coumarin samples and normal samples before and after freeze drying was observed using high and low ISI reference thromboplastins. ISI values were also determined. RESULTS: Freezing caused a prolongation of prothrombin time in the normal plasma samples with both reagents, which was significant with the low ISI human. Prolongation (non-significant) of the prothrombin time in coumarin plasma samples occurred with the human reagent only. Significant prolongation of normal prothrombin time by some of the protective additives before and after freeze drying was observed with both thromboplastins but to a greater extent with the human. Significant prolongation of prothrombin time in coumarin plasma samples was observed, but again was more marked with human thromboplastin. An approximate ISI was determined on the 20 coumarin samples. The only marked ISI change was with the WHO human thromboplastin after freeze drying of plasma, where a decrease from 0.95 to 0.90 was observed, corresponding to a marked prothrombin ratio increase. CONCLUSIONS: Freeze drying additives and the freeze drying procedure prolong normal and coumarin prothrombin times, with low ISI thromboplastin. Less marked prolongations occurred with a high ISI rabbit reagent, coumarin samples showing more significant prolongations. Marked ISI change in freeze dried plasma was only recorded with the low ISI ECAA human reagent. Frozen normal plasma samples cannot be used with confidence for ISI calibrations.

Animals↗
PubMed1982

Studies on vitamin K-dependent factor deficiency during early childhood with special reference to prothrombin activity and antigen level.

8 young infants aged 14 days to 5 months with vitamin K-dependent factor deficiency were studied with special reference to prothrombin activity and antigen level. Among them, 3 infants had congenital bile duct atresia and 5 were breast-fed babies with severe hemorrhagic tendency of unknown cause. In the patients with both congenital bile duct atresia and breast feeding the ratio of prothrombin activity to prothrombin antigen was lower than 0.1. Furthermore, the arc of prothrombin antigen in these patients demonstrated a faster anodal shift than did normal prothrombin antigen on crossed immunoelectrophoresis. This abnormal prothrombin antigen was not consumed after recalcification of patient plasma, and absorbed poorly on BaSO4. In addition, the abnormal prothrombin antigen disappeared from the patient plasma within a few days after parenteral administration of vitamin K. These results suggest that this abnormal prothrombin is PIVKA-II.

Bile Ducts↗
PubMed1977

Comparative aspects of prothrombin activation.

The activation of the purified prothrombins from human, bovine, and chicken species have been studied. Chicken prothrombin activation, similar to bovine prothrombin, resulted in the formation of a 161 residue prothrombin fragment 1, a 113 residue prothrombin fragment 2, and chicken thrombin with a 49 residue A chain and a B chain of approximately 260 residues. When human prothrombin is converted to thrombin, the resulting thrombin is shorter from the amino-terminus of the A chain by 13 residues (human prothrombin fragment 3). The vitamin K-dependent regions of all three species are very similar in sequence (33 of 46 residues identical for all three species). The regions of internal homology observed within the human and bovine fragments are apparently also present within the chicken fragments, indicating that the partial gene duplication which resulted in the evolution of a prothrombin molecule of greater size than the other vitamin K-dependent coagulation proteins occurred prior to the divergence of birds and mammals over 300 million years ago.

Amino Acid Sequence↗
PubMed1994

Chylomicron-induced prothrombin activation and platelet aggregation.

The effects on platelet aggregation of native rat chyle chylomicrons, chylomicron remnants, and chylomicrons that had been preincubated with rat or human EDTA-plasma, serum, whole blood, or pure human prothrombin were examined. The native chyle chylomicrons did not induce platelet aggregation but decreased ADP- and thrombin-induced platelet aggregation and [14C]serotonin release. Chylomicron remnants also failed to induce platelet aggregation, but they potentiated the aggregation and the [14C]serotonin release induced by ADP and thrombin. Aggregation, after a lag phase of 15 to 20 minutes, was seen when platelets were incubated with chylomicrons that had been preincubated with plasma and then isolated as the top layer after a single centrifugation at d = 1.006. This aggregation was inhibited in a dose-dependent manner by an antiserum against prothrombin that also inhibited thrombin-induced platelet aggregation. After washing by centrifugation the plasma-preincubated chylomicrons did not induce platelet aggregation, but this effect could be restored by adding a small amount of prothrombin, which did not cause aggregation when added alone or together with native chyle chylomicrons. Addition of 2% (vol/vol) plasma, however, induced aggregation when added together with either native chyle chylomicrons or washed preincubated chylomicrons, but not when added alone. Binding of 125I-labeled prothrombin to native chyle chylomicrons was demonstrated by gradient ultracentrifugation. During incubation of washed plasma-preincubated chylomicrons with 125I-prothrombin and platelets, a significant conversion of 125I-prothrombin to 125I-prethrombin and 125I-thrombin occurred, as demonstrated by autoradiography after separation on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The interaction between chylomicrons and prothrombin, and possibly other coagulation proteins, thus enhances prothrombin activation in the presence of platelets.

Animals↗
PubMed1997

Tissue prothrombin. Universal distribution in smooth muscle.

Immunohistochemical analysis of surgically obtained porcine tissue samples reveals ubiquitous staining for prothrombin in organs rich in smooth muscle content and universal staining of smooth muscle in tissue vasculature. The native character of tissue prothrombin is verified first by chromogenic substrate hydrolysis and hirudin inhibition after incubation of tissue extracts with taipan snake venom and phospholipid. Western analysis of tissue extracts confirms the native zymogen molecular weight. In addition, prothrombin purified in good yield from porcine uterus is activated by Echis carinatus venom which, like taipan venom, is 4-carboxyglutamic acid-sensitive. After correction for blood (gross heme) and interstitial fluid (albumin), excess functional prothrombin is observed in extracts of tissues having abundant smooth muscle. In contrast with factor X, the yield of prothrombin purified from porcine uterus greatly exceeds that attributable to contamination by whole blood. Northern blot analysis from selected bovine tissues extracted for polyadenylated messenger RNA is equivocal for prothrombin mRNA with the exception of liver, which is positive. It is concluded that functionally intact prothrombin is widely distributed among tissues owing to smooth muscle content, although the mechanism of emplacement and physiologic significance of prothrombin in these tissues remains unclear.

Animals↗
PubMed1969

Human prothrombin metabolism in normal man and in hypocoagulable subjects.

The metabolism of human prothrombin labeled with radioactive iodine was studied in seven normal subjects and four hemophilic patients. Results in the normal subjects were: plasma volume, 37.6+/-3.8 ml/kg; plasma prothrombin concentration, 303+/-40 U/ml (0.153+/-0.02 mg/ml); prothrombin half-life, 2.81+/-0.51 days; total plasma prothrombin pool, 5.72+/-0.62 mg/kg, representing 64.1+/-9.1% of total body prothrombin; fractional catabolic rate, 42.5+/-12.4% of the plasma pool per day; prothrombin synthesis rate, 2.43+/-0.76 mg/kg per day. Results in the hemophilic patients did not differ significantly from normal. Circulating products of prothrombin activation could not be demonstrated in normal individuals or hemophilic subjects. The data suggest that continuous physiologic activation of the blood coagulation mechanism plays only a small part, if any, in the normal catabolism of prothrombin.

Adult↗
PubMed2001

Immunologic and hematologic properties of antibodies to prothrombin and plasminogen in a mouse model.

Antibodies to prothrombin have been associated with venous and arterial thrombosis, and they cross-react with a structurally closely related protein plasminogen. We immunised 16 mice with human prothrombin and 15 mice with human plasminogen. Mice immunised with prothrombin developed cross-reactive antibodies to plasminogen (12/16), beta2-glycoprotein I (4/16), tissue-type plasminogen activator (6/16) and cardiolipin (11/16). Mice immunised with plasminogen developed cross-reactive antibodies to prothrombin (8/15), tissue-type plasminogen activator (2/12) and cardiolipin (5/12). Functional effects of antibodies were examined. Immunisation with prothrombin induced lupus anticoagulant activity in 9/14 mice. In mice immunised with plasminogen, radial fibrinolysis was inhibited in 8/10 and plasminogen activation in the chromogenic assay was inhibited in 9/11. No cross-functionality was observed. In conclusion, antibodies to prothrombin and plasminogen cross-react in vivo. Antibodies to prothrombin and plasminogen have different functional profiles, immunisation with prothrombin leads to prolonged blood clotting time, and immunisation with plasminogen induces antibodies interfering with fibrinolysis.

Animals↗
PubMed1999

Simultaneous detection of FV Q506 and prothrombin 20210 A variation by allele-specific PCR.

BACKGROUND AND OBJECTIVE: Factor V Leiden is the most important risk factor for hereditary thromboembolism, whereas the mutation in the 3'-untranslated region of the prothrombin gene seems to be only a mild risk factor for thrombotic events. On the other hand the factor V mutation (Arg 506) is frequently coinherited with the prothrombin 3'-untranslated region G20210A variant and there is increasing evidence that the co-segregated prothrombin variant is an additional risk factor for venous thromboembolism, contributing to thrombotic manifestations. A rapid, simple and cost-effective screening method is, therefore, required for the detection of both factor V Leiden and the prothrombin variant A20210G. DESIGN AND METHODS: Eighty-eight patients were enrolled in this study. Forty-four had a previously identified factor V and/or prothrombin mutation, the remaining 44 patients served as negative controls. A multiplex allele specific oligonucleotide PCR was established for the simultaneous detection of the two genetic risk factors for thrombophilia. To test the specificity of the simultaneous ASO PCR approach, the mutated and physiological factor V and prothrombin amplification products were sequenced. RESULTS: The factor V Leiden mutation and the prothrombin variant were correctly identified in all of 44 patients with known mutations. Furthermore the test was able to detect the mutated factor V and the II variant alone, as well as in the cosegregated pattern. Five patients with a homozygous pattern of factor V Leiden or prothrombin variant were also correctly identified. The sensitivity of the test is therefore 100%. In none of the 44 control cases were false positive results seen. INTERPRETATION AND CONCLUSIONS: The ASO PCR test is a rapid, simple and cost-effective screening test for thrombophilia.

Activated Protein C Resistance↗
PubMed1999

Isolation, characterization and sequence analysis of five IgG monoclonal anti-beta 2-glycoprotein-1 and anti-prothrombin antigen-binding fragments generated by phage display.

We have isolated five monoclonal IgG anti-beta 2-glycoprotein-1 (anti-beta 2G-1) and anti-prothrombin Fab from a patient with autoantibodies to oxidized low-density lipoproteins by phage display method. Analysis of their binding specificity revealed that all three beta 2GP-1-enriched mAbs (B14, B22, B27) reacted with beta 2GP-1 while both prothrombin-isolated mAbs (P11 and P13) reacted with prothrombin. Intriguingly, mAb P11 reacted with beta 2GP-1 and prothrombin and showed comparable binding affinity to both Ags, with Kd values of 1.6 x 10-6 M for beta 2GP-1 vs 3.2 x 10-6 M for prothrombin. This clone may thus, define a hitherto unknown shared epitope between beta 2GP-1 and prothrombin. Sequence analysis of all five clones showed significant mutations of the expressed genes. One rearranged V-D-J segment was repeatedly employed by three clones (mAbs B22, B27, and P13). However, all three clones used different L chains. Of note, the pairing of VH6-D-J with the L5-Vk1 L chain in mAb P13 resulted in the loss of binding to beta 2GP-1 and specific reactivity to prothrombin. Together, these data suggest that while the VH6-D-J chain may be important in the binding to beta 2GP-1, pairing with certain L chains may influence this binding. These data are the first human IgG anti-beta 2GP-1 and anti-prothrombin sequences reported; both represent the major subsets of antiphospholipid Abs present in antiphospholipid syndrome patients.

Aged↗
PubMed2002

Effect of prothrombin and its activation fragments on calcium oxalate crystal growth and aggregation in undiluted human urine in vitro: relationship between protein structure and inhibitory activity.

In recent years there has been great interest in the putative role of prothrombin and its activation peptides, especially the urinary form of prothrombin fragment 1, in the pathogenesis of calcium oxalate (CaOx) urolithiasis. Previously, we showed that prothrombin and its activation peptides inhibit CaOx crystallization in inorganic conditions in vitro. The aim of the present study was to determine if this inhibitory activity is retained in undiluted human urine and, therefore, whether it is likely to have any influence under physiological conditions. A secondary objective was to assess the relationship between the structures of the proteins and their inhibitory activities. Prothrombin was purified from Prothrombinex-HT, cleaved with thrombin and the resulting fragment 1 (F1) and fragment 2 (F2) were purified. The purity of each protein was confirmed by SDS/PAGE, and their effects on CaOx crystallization in undiluted ultrafiltered human urine were determined at a final concentration 80.65 nmol/l using Coulter Counter and [(14)C]oxalate analysis. The precipitated crystals were visualized using scanning electron microscopy. The Coulter Counter data revealed that, whereas prothrombin and its activation peptides did not affect the urinary metastable limit and the size of the precipitated particles, F1 did significantly reduce the latter. These findings were corroborated with scanning electron microscopy which also revealed that the reduction in particle size caused by F1 resulted from a decrease in the degree of crystal aggregation, rather than in the size of the individual crystals. The [(14)C]oxalate data showed that none of the proteins added significantly inhibited the mineral deposition. It was concluded that with the exception of F1, which does inhibit CaOx crystal aggregation, prothrombin and its activation peptides do not alter the deposition and aggregation of CaOx crystals in ultrafiltered human urine in vitro. Also, the gamma-carboxyglutamic acid domain of prothrombin and F1, which is absent from thrombin and F2, is the region of the molecules that determines their potent inhibitory effects. The superior potency of F1, compared with prothrombin, probably results from the molecule's greater charge-to-mass ratio.

Calcium Oxalate↗
PubMedDate not supplied

Early diagnosis of hepatocellular carcinoma using a sensitive assay for serum des-gamma-carboxy prothrombin: a prospective study.

BACKGROUND/AIMS: Measurement of des-gamma-carboxy prothrombin by conventional methods is of limited use for the early detection of hepatocellular carcinoma for its low sensitivity. The aim of the present study was to investigate the usefulness of measuring des-gamma-carboxy prothrombin by a highly sensitive assay for the early diagnosis of hepatocellular carcinoma in patients with chronic liver disease and for the detection of recurrence after treatment of hepatocellular carcinoma. METHODOLOGY: Des-gamma-carboxy prothrombin levels by a sensitive assay and alpha-fetoprotein levels were sequentially measured in 188 patients with type B or C chronic liver disease and in 63 patients with hepatocellular carcinoma. RESULTS: The positive rate of des-gamma-carboxy prothrombin was 62% in all of hepatocellular carcinoma patients. Hepatocellular carcinoma was detected in 14 of 188 chronic liver disease patients during their follow-up period, the positive rate of des-gamma-carboxy prothrombin and of alpha-fetoprotein being 57% and 71% in these 14 patients, respectively. Des-gamma-carboxy prothrombin level normalized in 67% of 39 patients after the treatment of hepatocellular carcinoma. Of the 19 patients with tumor recurrence, 84% showed re-elevation of des-gamma-carboxy prothrombin level. CONCLUSIONS: Measurement of des-gamma-carboxy prothrombin by this highly sensitive assay combined with alpha-fetoprotein is useful for detecting hepatocellular carcinoma in chronic liver disease patients and for monitoring recurrence after treatment of hepatocellular carcinoma.

Adult↗
PubMed1991

Meizothrombin formation during factor Xa-catalyzed prothrombin activation. Formation in a purified system and in plasma.

Meizothrombin and thrombin formation were quantitated during factor Xa-catalyzed activation of human prothrombin in reaction systems containing purified proteins and in plasma. In the purified system considerable amounts of meizothrombin accumulated when prothrombin was activated by factor Xa (with or without accessory components) under initial steady state conditions. The ratio of the rates of meizothrombin and thrombin formation was not influenced by variation of the pH, temperature, or ionic strength of the reaction medium. When 2 microM prothrombin was activated by the complete prothrombinase complex (factor Xa, factor Va, Ca2+, and phospholipid) 80-90% of the initially formed reaction product was meizothrombin. Lowering the prothrombin concentration from 2 to 0.03 microM caused a gradual decrease in the ratio of meizothrombin/thrombin formation from 5 to 0.6. When the phosphatidylserine content of the phospholipid vesicles was varied between 20 and 1 mol % and prothrombin activation was analyzed at 2 microM prothrombin the relative amount of meizothrombin formed decreased from 85 to 55%. With platelets, cephalin, or thromboplastin as procoagulant lipid, thrombin was the major reaction product and only 30-40% of the activation product was meizothrombin. We also analyzed complete time courses of prothrombin activation both with purified proteins and in plasma. In reaction systems with purified proteins substantial amounts of meizothrombin accumulated under a wide variety of experimental conditions. However, little or no meizothrombin was detected in plasma in which coagulation was initiated via the extrinsic pathway with thromboplastin or via the intrinsic pathway with kaolin plus phospholipid (cephalin, platelets, or phosphatidylserine-containing vesicles). Thus, thrombin was the only active prothrombin activation product that accumulated during ex vivo coagulation experiments in plasma.

Enzyme Activation↗
PubMed1990

Studies of the role of factor Va in the factor Xa-catalyzed activation of prothrombin, fragment 1.2-prethrombin-2, and dansyl-L-glutamyl-glycyl-L-arginine-meizothrombin in the absence of phospholipid.

In order to specifically evaluate the role of Factor Va in the prothrombinase complex, studies of the activation of prothrombin, Fragment 1.2-prethrombin-2, and active-site-blocked meizothrombin were carried out, both in the absence of phospholipid and at concentrations of substrates and Factor Va sufficient to approach saturation in all components. Km values were independent of Factor Va concentrations, whereas kcat (apparent) values approached saturation with respect to Factor Va concentrations. The three respective substrates exhibited the following parameters of kinetics (Km, microM; kcat, s-1 at saturating [Factor Va]): prothrombin (9.0 +/- 0.4; 31 +/- 1); Fragment 1.2-prethrombin-2 (5.4 +/- 0.4; 13 +/- 2); and meizothrombin (3.6 +/- 0.3; 51 +/- 5). Models of kinetics were constructed to interpret the results, and two of these were formally consistent with experimental results. Both models indicated that the variation of kcat(app) with concentrations of Factor Va reflects the formation of a Factor Va-Factor Xa binary complex. Analysis of kinetics indicated Kd values for this interaction of 1.3 +/- 0.1, 3.0 +/- 0.5, and 1.0 +/- 0.1 microM for the three respective substrates. The models differed in the interpretation of Km. One indicated that Km reflects a binary interaction between Factor Xa and prothrombin, whereas the other indicated a binary interaction between Factor Va and prothrombin. Both indicated that two of the three possible binary interactions between the three components would be reflected in Km and kcat values but not the third. To distinguish these models, the binary interactions were studied by extrinsic fluorescence (Va.Xa), light-scattering (Factor Va.prothrombin), and competition kinetics (Xa.II). The first two interactions were detected and were characterized by Kd values of 2.7 +/- 0.1 microM (Va.Xa) and 8.8 +/- 0.8 microM (Factor Va.prothrombin). No active-site-dependent interaction between prothrombin and Factor Xa could be detected in the absence of Factor Va. The results of these studies suggest that Factor Va interacts with both Factor Xa and prothrombin and effectively presents one to the other in the formation of a ternary enzyme-substrate-cofactor complex. In addition, a comparison of the parameters of kinetics of conversion of prothrombin and its intermediates indicates that meizothrombin is the major intermediate of prothrombin activation in the absence, as well as in the presence of phospholipid.

Amino Acid Sequence↗
PubMed1989

Molecular defects of factor IX Chicago-2 (Arg 145----His) and prothrombin Madrid (Arg 271----cys): arginine mutations that preclude zymogen activation.

Factor IX Chicago-2 and prothrombin Madrid were purified from patients with hemophilia B and congenital dysprothrombinemia, respectively. Each protein displays defects in zymogen activation secondary to the failure to cleave one of the sessile bonds whose cleavage is necessary for full coagulant activity. These proteins were isolated by immunoaffinity chromatography using conformation-specific antibodies directed at either factor IX or prothrombin. Factor IX Chicago-2 is cleaved abnormally by factor XIa, yielding a pattern consistent with the failure to cleave the sessile bond between Arg 145 and Ala 146. Prothrombin Madrid is cleaved abnormally by factor Xa, yielding a pattern consistent with the failure to cleave the sessile bond between Arg 271 and Thr 272. Peptide mapping was performed on reduced and alkylated factor IX, factor IX Chicago-2, prothrombin, and prothrombin Madrid, and the hydrolysates were separated by high-performance liquid chromatography. The mutant peptide in factor IX Chicago-2 was identified by automated Edman degradation as residues 143 through 188 of factor IX, and had a histidine substituted for arginine at residue 145. The mutant peptide identified in prothrombin Madrid corresponds to residues 267 through 285 of prothrombin and has the substitution of cysteine for arginine at residue 271. These mutations, each occurring at arginines, are identical to those in factor IX Chapel Hill and prothrombin Barcelona. These results suggest that a limited repertoire of point mutations, many affecting arginine residues, may be responsible for hereditary defects of the vitamin K-dependent proteins in patients with normal antigen levels.

Amino Acid Sequence↗
PubMed1986

Terbium probe of calcium-binding sites on the prothrombin-membrane complex.

Terbium was used as a probe of Ca2+-binding sites on the prothrombin-phospholipid complex. Stoichiometric titrations of prothrombin binding to phospholipid vesicles with either Tb3+ or Ca2+ showed that a minimum of 8 metal ions were needed for binding prothrombin to vesicles (3 Mn2+ + 5 Ca2+ for prothrombin or 8 Tb3+ for F-1). When Ca2+ alone was used, a total of about 11 metal ions were needed for complete binding. These stoichiometries indicated 3 classes of metal ions: one class needed to induce the conformational change, a second required for protein-membrane contact, and a third class bound at other sites on the protein that are not involved in membrane binding. By adding Tb3+ to solutions containing both protein and phospholipid, undesirable Tb3+-induced events, such as irreversible aggregation of prothrombin or vesicle fusion, were avoided. Protein-vesicle binding apparently prevented protein aggregation or vesicle fusion. The protein-vesicle binding affinity was severalfold greater in the presence of Tb3+ compared to Ca2+. CoEDTA quenching of Tb3+ bound to the prothrombin-phospholipid complexes indicated that all metal ions were at least partially exposed to the quencher. Some populations of Tb3+ showed lower quenching constants when all of the prothrombin was bound. Tb3+ emission lifetimes revealed that some Tb3+ ions in the protein-membrane complex were in a different environment from those bound to the protein alone. The results indicated that the metal ions in the prothrombin-membrane complex are relatively open to the solvent yet do affect the characteristics of the protein-membrane binding equilibrium.

Binding Sites↗
PubMed1986

Isolation and characterization of staphylocoagulase chymotryptic fragment. Localization of the procoagulant- and prothrombin-binding domain of this protein.

Several strains of Staphylococcus aureus secrete a protein, staphylocoagulase, that binds stoichiometrically to human prothrombin, resulting in a coagulant complex designated staphylothrombin. In the present study, staphylocoagulase was digested with alpha-chymotrypsin and the resulting fragments were isolated by gel filtration. One fragment (Mr 43,000) exhibited a high affinity for human prothrombin (Kd = 1.7 X 10(-9) M), which is comparable to the affinity observed using intact staphylocoagulase (Kd = 4.6 X 10(-10) M). A complex of the Mr 43,000 fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. A second fragment (Mr 30,000) exhibited weaker affinity for prothrombin (Kd = 1.2 X 10(-7) M). While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. A third fragment (Mr 20,000) was found to bind to prothrombin, but the resultant complex did not exhibit clotting or amidase activity. Amino-terminal sequence analyses of these staphylocoagulase fragments revealed that the Mr 43,000 fragment constitutes the amino-terminal portion of staphylocoagulase and also contains the Mr 30,000 and 20,000 fragments. Moreover, the amino-terminal sequence of the Mr 20,000 fragment was identical to that observed for the Mr 30,000 fragment. From these results, we conclude that the functional region of staphylocoagulase for binding and activation of human prothrombin is localized in the amino-terminal region of the intact bacterial protein.

Amino Acid Sequence↗
PubMed1979

Purification and properties of prothrombin modified by asbestos filtration of human plasma.

Filtration through asbestos filter (Seitz) of human plasma modified the prothrombin molecule as previously shown. Factor II could no longer be activated by physiological activators (Ca++ + phospholipid + V and Xa) but reacted readily with staphylocoagulase. The separation and purification of the modified prothrombin allowed allowed the preparation of two fractions: A small slightly modified accessory fraction, "prothrombin-asbestos-1", lost its ability to be activated by physiological activators, and its ability to be adsorbed by barium citrate, but retained the immunological reactivity of fragment 1, as well as the mobility and molecular weight of unmodified prothrombin. A main fraction, "prothrombin-asbestos-2" appeared to be a modified prothrombin which has lost its N-terminal extremity. It was not adsorbed by barium citrate and could not be activated by physiological activators. It possessed a reduced electrophoretic mobility, as well as a reduced molecular weight (39,000), which are properties similar to those of thrombin. Both fractions 1 and 2 were devoid of thrombin activity. Asbestos was thus able to break the prothrombin molecule non enzymatically, the amputation of the N terminal extremity being responsible for the new functional and physicochemical properties of the molecule. Staphylocoagulase appeared not to need the N terminal extremity of the molecule of prothrombin to form the active thrombin-coagulase complex.

Asbestos↗
PubMed1983

Radioimmunoassay for human prothrombin.

I describe a radioimmunoassay for human prothrombin, with use of a double-antibody technique. Antiserum raised in rabbits was absorbed with Al(OH)3 and heated to 56 degrees C for 30 min. 125I-labeled prothrombin retaining more than 90% of its biological activity was prepared by the iodine monochloride method. The mean concentration of prothrombin in plasma of 12 normal individuals was 100 +/- 29.4 mg/L (2 SD). Prothrombin values were somewhat lower than those obtained by the Laurell electroimmunoassay or by two-stage biological assay of the same plasma, done the same day. The biological values were converted to protein on the basis of 1960 int. units/mg by comparison with the other two assays. The ability of activation fragments of human prothrombin to inhibit binding of labeled prothrombin to its antibody was evaluated by competitive radioimmunoassay. Although precipitin lines formed with undiluted antiserum against all the fragments tested (F-1, F-1.2, prethrombin-1, and thrombin), none of the fragments competed well with prothrombin, even in 10-fold molar excess. Evidently, the structural integrity of the prothrombin molecule is essential for its maximum binding to the antiserum, and antigenic sites are lost during its activation.

Animals↗