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PubMedDate not supplied

Purification and characterization of ostrich prothrombin.

The work focused on the penultimate enzyme, prothrombin, in the coagulation cascade. Prothrombin was purified and characterized from ostrich plasma. The results obtained contribute to a better understanding of blood coagulation in the ostrich and the evolution of prothrombin and the coagulation cascade. Prothrombin was purified from ostrich plasma by barium chloride precipitation, ammonium sulfate fractionation, and DEAE-cellulose and Cu(2+)-chelate Sepharose chromatography. Ostrich prothrombin exhibited a M(r) of 72,800 and a pI of 6.9 using SDS-PAGE and PAG-isoelectrofocusing, respectively. The N-terminal sequence of ostrich prothrombin showed 78 and 87% identity with human and bovine, respectively. The cDNA was isolated from ostrich liver and the predicted amino acid sequence compared with those from other species. Ostrich prothrombin shares sequence identity with chicken (84%), human (60%), bovine (59%), rat (60%), mouse (59%) and hagfish (50%) prothrombin, suggesting a common function of prothrombin in these vertebrates. Amino acid sequence identities indicate that the thrombin beta-chain (62%) and the propeptide-Gla (75%) domains are the regions most constrained for the common functions of vertebrate prothrombins. Ostrich prothrombin, therefore, shows similarity in structure to other vertebrate prothrombins.

Amino Acid Sequence↗
PubMed2002

Kinetics of prothrombin-mediated binding of lupus anticoagulant antibodies to phosphatidylserine-containing phospholipid membranes: an ellipsometric study.

Antiphospholipid antibodies interact with phospholipid membranes via lipid binding plasma proteins, mostly, prothrombin and beta(2)-glycoprotein I. Using ellipsometry, we characterized prothrombin-mediated binding of lupus anticoagulant (LA) positive IgG, isolated from patients with antiphospholipid syndrome, to phosphatidylserine (PS)-containing membranes. LA IgG did not bind to membranes in the absence of prothrombin, but addition of prothrombin resulted in high-affinity binding of prothrombin-LA IgG complexes; half-maximal binding was attained at IgG and prothrombin concentrations of 10 microg/mL and 4 nM, respectively. Adsorption to membranes containing 10-40 mol % PS revealed that membrane-bound rather than solution-phase prothrombin determines the adsorption kinetics. Depletion of prothrombin and LA IgG from the solution results in rapid desorption which is strongly inhibited by addition of prothrombin but not of LA IgG. Prothrombin-mediated adsorption of monovalent Fab1 fragments prepared from patient LA IgG was negligible, indicating that monovalent interaction between prothrombin and LA IgG is weak. The kinetics of adsorption and desorption indicate that divalent binding of LA IgG to prothrombin at the lipid membrane occurs.

Antibodies, Antiphospholipid↗
PubMed1997

Prothrombin kringle 1 domain interacts with factor Va during the assembly of prothrombinase complex.

The kringle 2 domain of prothrombin has been shown to interact with factor Va during the activation of prothrombin by the prothrombinase complex composed of factor Xa, factor Va, negatively charged phospholipids and Ca2+ ions. However, contradictory results have been reported about the role of the kringle 1 domain of prothrombin during the assembly of the prothrombinase complex. In an attempt to clarify the role of the kringle 1 domain of prothrombin, its effect on the activation of prothrombin by the prothrombinase complex and its direct binding to human factor Va were assessed. Comparative evaluation with the effects caused by other prothrombin structural components [a fragment 1 (gamma-carboxyglutamic acid and kringle 1 domains), a kringle 2 domain and a catalytic protease domain] was also performed. In the presence of factor Va, each kringle 1 and kringle 2 fragment significantly inhibited the factor Xa-catalysed prothrombin activation in the absence of phospholipids. However, in the absence of both factor Va and phospholipids, kringle 2 fragment, but not kringle 1 fragment, inhibited prothrombin activation. Evaluation of the molecular interaction of the kringle domains with factor Va in assays with solid-phase phospholipid vesicles showed that each kringle 1 and kringle 2 fragment inhibited the prothrombinase complex activity. Assessment of the direct binding of prothrombin and each kringle domain of prothrombin with factor Va by fluorescence polarization showed that prothrombin, kringle 1 and kringle 2 fragments bind directly to factor Va with dissociation constants of 1.9+/-0.1, 2.3+/-0.1 and 2.0+/-0.4 microM (means+/-S.D.) respectively. These findings suggest that both kringle 1 and 2 domains of prothrombin interact with factor Va during the assembly of the prothrombinase complex.

Blood Coagulation↗
PubMed1997

Antiphospholipid antibody: functional specificity for inhibition of prothrombin activation by the prothrombinase complex.

The antiphospholipid syndrome (APS) is associated with production of autoantibodies with lupus anticoagulant (LA) activity. These antibodies cause prolongation of in vitro clotting tests by inhibition of the conversion of prothrombin to thrombin in the presence of anionic phospholipid (PL). The extent to which this inhibition reflects antibody binding to, or functional inhibition of, phospholipids alone, prothrombin alone, or a prothrombin-phospholipid complex is pertinent to our understanding of the pathophysiology of this syndrome. Immunoglobulin fractions (IgG) from 18 patients with LA activity were tested for inhibitory activity against prothrombin activation by either factor Xa, in a purified prothrombinase system, or by purified fractions of snake venoms (E. carinatus, E. multisquamatus) which cleave prothrombin at the same initial site as the prothrombinase complex but do not require anionic phospholipid as a cofactor. Parallel testing of the same IgG samples for prothrombin binding by immunoassay was performed. Although all IgG samples inhibited the prothrombinase reaction, only three exhibited any inhibition of venom protease prothrombin activation in either the presence or absence of PL. Only one sample exhibited prothrombin binding by Western blot. These results suggest that lupus anticoagulant antibodies are heterogenous and that many, if not most, of the autoantibody populations responsible for LA activity impair prothrombin activation by interaction either with phospholipid alone or with a restricted range of prothrombin-phospholipid epitopes expressed by prothrombin only as part of the intact prothrombin-prothrombinase complex.

Antibodies, Anticardiolipin↗
PubMed2001

Interaction of estrogen replacement therapy with the thrombophilic 20210 G/A prothrombin gene mutation for atherothrombotic vascular disease: a cross-sectional study of 275 hyperlipidemic women.

In a consecutive case series, cross-sectional study of 275 women referred for therapy of hyperlipidemia, (75 [27%] on estrogen replacement therapy [ERT]), our specific aim was to determine whether ERT-mediated thrombophilia and heterozygosity for the thrombophilic 20210 G/A prothrombin gene mutation interacted as risk factors for atherothrombotic cardiovascular disease (ATCVD). Of the 275 women, 100 (36%) had ATCVD; 10 (3.6%) were heterozygous for the 20210 G/A prothrombin gene mutation. In women without the 20210 G/A prothrombin gene mutation, 15 of 71 (21%) on ERT had ATCVD versus 78 of 194 (40%) not on ERT (X(2) = 8.31, P =.004). By stepwise logistic regression, in 261 women with ATCVD risk factor data, positive explanatory variables for ATCVD included the 20210 G/A prothrombin mutation (risk odds ratio, 5.8; 95% confidence intervals [CI], 1.4 to 30.2; P =.021) and a 20210 G/A prothrombin gene mutation*ERT interaction term (risk odds ratio, 2.70; 95% CI, 1.4 to 5.4; P =.004). ATCVD events were more likely in 2 subgroups of women (ERT minus [-] and 20210 G/A prothrombin gene mutation -) or (ERT plus [+] and 20210 G/A prothrombin gene mutation +), P =.004. Other positive explanatory variables for ATCVD events included age (P =.004), triglycerides (P =.012), lipoprotein (a) (P =.03), and homocysteine (P =.032). ERT may be protective against ATCVD when the thrombophilic 20210 G/A prothrombin gene mutation is absent, whereas the 20210 G/A prothrombin gene mutation may increase risk for ATCVD, particularly in the presence of ERT. We suggest that the 20210 G/A prothrombin gene mutation be measured in all women on ERT or before beginning ERT to identify those heterozygous for the thrombophilic prothrombin gene mutation (4%) in whom ERT is contraindicated because of increased risk for ATCVD and thromboembolism, and a second, much larger group of women without the 20210 G/A prothrombin gene mutation (96%) in whom ERT may possibly reduce risk for ATCVD.

Aged↗
PubMed2000

Role of proexosite I in factor Va-dependent substrate interactions of prothrombin activation.

Regulatory exosite I of thrombin is present on prothrombin in a precursor state (proexosite I) that specifically binds the Tyr(63)-sulfated peptide, hirudin(54-65) (Hir(54-65)(SO(3)(-))) and the nonsulfated analog. The role of proexosite I in the mechanism of factor Va acceleration of prothrombin activation was investigated in kinetic studies of the effects of peptide binding. The initial rate of human prothrombin activation by factor Xa was inhibited by the peptides in the presence of factor Va but not in the absence of the cofactor. Factor Xa and factor Va did not bind the peptide with significant affinity compared with prothrombin. Maximum inhibition reduced the factor Va-accelerated rate to a level indistinguishable from the rate in the absence of the cofactor. The effect of Hir(54-65)(SO(3)(-)) on the kinetics of prothrombin activation obeyed a model in which binding of the peptide to proexosite I prevented productive prothrombin interactions with the factor Xa-factor Va complex. Comparison of human and bovine prothrombin as substrates demonstrated a similar correlation between peptide binding and inhibition of factor Va acceleration. Inhibition of prothrombin activation by hirudin peptides was opposed by assembly on phospholipid vesicles of the membrane-bound factor Xa-factor-Va-prothrombin complex. Factor Va interactions of human and bovine prothrombin activation are concluded to share a common mechanism in which proexosite I participates in productive interactions of prothrombin as the substrate of the factor Xa-factor Va complex, possibly by directly mediating productive prothrombin-factor Va binding.

Animals↗
PubMed1975

Prothrombin fragments. Ca2+ binding and activation kinetics.

The binding of Ca2+ to prothrombin and the intermediates of prothrombin activation was investigated by equilibrium dialysis using 45Ca2+ as the ligand. Scatchard plots of these data indicate that prothrombin (Mr = 70,000) has 10 to 11 Ca2+ binding sites which can be differentiated in terms of their binding affinity. Six of these Ca2+ binding sites have log Kassoc = 3.5 and all are found intact in the NH2-terminal segment (activation intermediate 3, Mr = 23,000) of the prothrombin molecule. Four or five additional weaker binding sites for Cz2+ with log Kassoc = 2.7 present in prothrombin are found intact in the remaining COOH-segment (activation intermediate 1, Mr = 51,000) of the prothrombin molecule. Upon further activation the Ca2+ binding sites residing in intermediate 1 are found intact in activation intermediate 4 (which constitutes the NH2-terminal segment of the intermediate 1 molecule). The remaining COOH-terminal portion (activation intermediate 2, Mr = 41,000) of the intermediate 1 molecule has no affinity for Ca2+. The activation of prothrombin and activation intermediates 1 and 2 was studied using these activators: Factor Xa alone, Factor Xa-Ca+, AND Factor Xa-Ca2+-phospholipid. The rate of thrombin production from prothrombin was progressively increased as Ca2+ and phospholipid were added to the system, whereas no significant increase in the rates of activation of intermediate 1 and 2 was observed. When Factor V was added to the Factor Xa-Ca2+-phospholipid system, the rate of activation of intermediate 1 was greatly enhanced. In the absence of Ca2+, Factor V had no effect on the rate of thrombin formation from intermediate 1. Factor V had no stimulatory effects on the rate of intermediate 2 activation. However, in the presence of an equimolar amount of intermediate 4, Factor V accelerated the conversion of intermediate 2 to thrombin. These studies indicate that the Ca2+ binding sites of the prothrombin molecule are contained in the "pro" fragment (intermediates 3 and 4) of the prothrombin molecule. Intermediate 1 and intermediate 2, both of which lack the strong Ca2+ binding sites of prothrombin, are poor substrates for the Factor Xa-Ca2+-phospholipid complex activation when compared to prothrombin. The addition of Factor V to the catalyst results in acceleration of the activation rate of intermediate 1 and an equimolar mixture if intermediates 2 and 4. These results lead us to conclude that the strong Ca2+ binding sites are the sites of phospholipid binding (intermediate 3), whereas the seak binding sites are the sites of Factor V binding (intermediate 4).

Binding Sites↗
PubMed1991

Expression, isolation, and characterization of an active site (serine 528----alanine) mutant of recombinant bovine prothrombin.

An active site mutant bovine prothrombin cDNA (Ser528----Ala) has been constructed, subcloned, and expressed in Chinese hamster ovary cells. The recombinant mutant prothrombin, expressed at the level of 1.5-2.0 micrograms/ml of cell medium, was fully carboxylated (9.9 +/- 0.4 mol of gamma-carboxyglutamic acid/mol of prothrombin). The mutant prothrombin could be activated to thrombin by Taipan snake venom and activated to meizothrombin by ecarin in a manner comparable to native bovine prothrombin or recombinant wild-type bovine prothrombin. The mutant meizothrombin thus formed was stable and did not autolyze. The initial rate of cleavage of mutant prothrombin catalyzed by the full prothrombinase was only 28% of the rate of cleavage of native prothrombin, while recombinant wild-type prothrombin was cleaved at the same rate as the native molecule. The mutant thrombin, obtained from the mutant prothrombin in situ by prothrombinase or Taipan snake venom activation, showed no enzymatic activity toward either fibrinogen or a synthetic chromogenic substrate, D-phenylalanyl-L-pipecolyl-L-arginine-p-nitroanilide dihydrochloride (S2238). The mutant thrombin also bound dansylarginine-N-(3-ethyl-1,5-pentanediyl)amide, a specific fluorescent inhibitor of the thrombin active site, with a weaker binding affinity (kd = 5.4 x 10(-8) M) than did native thrombin (kd = 1.7 x 10(-8) M). These results indicate that the mutant recombinant prothrombin described here is a useful tool for the study of meizothrombin or thrombin without the complications arising from the proteolytic activities of these molecules. Study of the activation of this mutant has already revealed a functional link between the site of initial cleavage by the prothrombinase and the conformation at the nascent active site of prothrombin.

1-Carboxyglutamic Acid↗
PubMed1984

Prothrombin evaluation as obtained by kinetics studies of antigen-antibody reaction in a laser nephelometer.

Laser nephelometry is a technique which allows the evaluation of the concentration of several serum proteins and clotting factors. By means of this technique it is also possible to study the kinetics of the reaction between antigen and antibody. We studied the kinetics of the reaction between prothrombin and an antiprothrombin antiserum using several prothrombins namely: Prothrombin Padua, prothrombin Molise, which are two congenital dysprothrombinemias, cirrhotic, coumarin or normal prothrombins. Different behaviors in the kinetics of the reactions were shown even when the concentration of prothrombins was about the same in all plasma tested. These differences were analyzed by means of a computer (Apple II 48 RAM) programmed to solve four unknown equations (Rodbard's equation). From the data so obtained one can see that when voltages at the beginning and at the end of the reaction are in all cases about the same, a clear difference in the time required to reach half the maximum value of the voltage can still be demonstrated. This parameter, which is expressed in minutes, is longer in coumarin and prothrombin Molise than in controls. On the contrary it is shorter in prothrombin Padua and has about the same value of controls in the cirrhotic patient. Moreover the time at which the maximum rate is obtained is longer in coumarin and prothrombin Molise than in controls and shorter in liver cirrhosis and prothrombin Padua. In conclusion data obtained show that coumarin prothrombin behaves in a different way from cirrhotic prothrombin and also that there is a different behaviour between the two congenital dysprothrombinemias.

Anticoagulants↗
PubMed1977

Differentiation of metal ion-induced transitions of prothrombin fragment 1.

The intrinsic fluorescence of human and bovine prothrombin fragment 1 is quenched (approximately -35%) when calcium ions are bound. The intrinsic fluorescence of prothrombin is also quenched by the binding of Ca2+ but to a lesser extent (total change is approximately -6%). The prothrombin fragment 1 fluorescence transition is also effected by the binding of Mg2+, Mn2+, Gd3+, and Tb3+. With all ions the fluorescence quenching is readily reversed by titration with EDTA. Titration with any of the above ions results in a sigmoidal titration curve. The fluorescence transition midpoints (expressed as Tm) occur at the following concentrations of ions: Ca2+, 0.22 (0.35) mM; Mg2+, 0.22 (0.45) mM; Mn2+, 12.6 (12.6) muM; and Gd3+, 3.6 (5.3) muM (the values in parentheses indicate the concentration of the respective ions bound to bovine prothrombin fragment 1). The presence of phospholipid steepens the Ca2+ titration curve, significantly more so for the bovine system than for the human. Tm values for Ca2+ binding in the presence of phospholipid are 0.21 mM and 0.22 mM for binding to human and bovine prothrombin fragment 1, respectively. Sedimentation equilibrium and velocity studies indicate that prothrombin fragment 1 which is monomeric in the presence of EDTA undergoes concentration-dependent association in the presence of Ca2+. A plot of fraction monomer versus Ca2+ concentration is sigmoidal, with a Tm of approximately 1 mM Ca2+. Mg2+ is only marginally effective in promoting the dimerization, and Mn2+ and Gd3+ are even less effective. However, sedimentation studies performed in the presence of 1 mM Mg2+ at varying Ca2+ concentrations shifted the apparent dimerization transition markedly to the left, the midpoint of the transition occurring at 0.25 mM. Kinetic studies reveal that while Mg2+ by itself does not promote prothrombin activation to thrombin, Mg2+ will partially substitute for Ca2+ in prothrombin activation. In the presence of 1 mM Mg2+ maximal activation rates are attained with 0.3 mM Ca2+; higher Mg2+ concentrations (at this Ca2+ concentration) are inhibitory. Only Ca2+ and Gd3+ permit significant prothrombin-phospholipid binding. Scatchard plots derived from equilibrium dialysis experiments performed with bovine prothrombin fragment 1 suggest marked cooperativity in calcium binding and the existence of approximately six Ca2+ binding sites. Similar studies, but with 1 mM Mg2+ also present in the dialysate, suggested that Mg2+ promotes a greater degree of cooperativity in Ca2+ binding to prothrombin fragment 1 with an apparent decrease in the number of Ca2+ sites occupied. It therefore appears that prothrombin fragment 1 has two classes of metal ion binding sites. One class, probably comprising two of six sites, is apparently fairly nonselective with respect to which metal ion is bound; binding of metal ion to these sites is responsible for the fluorescence change and apparently triggers a conformational transition...

Animals↗
PubMed1996

Catabolism of prothrombin-chylomicron complexes in vivo in the rat.

The metabolism in vivo in the rat of 125I-prothrombin-chylomicron complexes was compared to that of free 125I-prothrombin injected in saline or together with chyle chylomicrons. The group that obtained 125I-prothrombin-chylomicron complexes exhibited a faster initial decrease of plasma 125I-prothrombin, a higher radioactivity in the liver and more trichloroacetic acid soluble 125I in plasma than in the other groups that did not differ. In the group injected with 125I-prothrombin-chylomicron complexes, 4% was in the HDL, 0.7% LDL, and 0.8% in VLDL 2 hours after injection. The data thus indicate that although most of the 125I-prothrombin is released during the metabolism of the prothrombin-chylomicron complexes, some is rapidly degraded and some is transferred to plasma lipoproteins and remains circulating for a considerable time period. The observations demonstrate that binding to chylomicrons influences the catabolism of prothrombin in vivo. Since chylomicrons did not influence the clearance of simultaneously injected free 125I-prothrombin, the prothrombin binding to chylomicrons or chylomicron remnants in vivo may, however, be too slow to influence the prothrombin catabolism under normal conditions.

Animals↗
PubMed2006

Decreased inhibitory activity of prothrombin to calcium oxalate crystallization by specific chemical modification of its gamma-carboxyglutamic acid residues.

OBJECTIVES: To investigate the inhibitory role of gamma (gamma)-carboxyglutamic acid (Gla) in the Gla domain of urinary prothrombin in the formation of calcium oxalate crystals. METHODS: Morpholine and formaldehyde at different concentrations were added to the solution of prothrombin to cause the conversion of Gla to gamma-methyleneglutamic acid (MGlu). The extent of the modification was controlled by the relative amount of modification reagents to prothrombin. The 100-fold molar excess of modification reagents to prothrombin produced the prothrombin molecule containing 2gamma-MGlu residues, and the 10,000-fold molar excess produced the molecule containing 8gamma-MGlu residues. The inhibitory activities of prothrombin and modified prothrombin to calcium oxalate crystallization were evaluated by the seeded crystallization technique. RESULTS: The inhibitory index of Gla prothrombin to calcium oxalate crystallization was 14.2%, that of 2gamma-MGlu prothrombin was 12.8%, decreased by about 10%, and that of 8gamma-MGlu prothrombin was 5.0%, decreased by about 65%. CONCLUSIONS: The Gla in the Gla domain of urinary prothrombin may play an important role in inhibiting the formation of renal calcium oxalate calculus.

1-Carboxyglutamic Acid↗
PubMed1997

Purification and characterization of multisquamase, the prothrombin activator present in Echis multisquamatus venom.

The venom of Echis multisquamatus (Central Asian sand viper) contains a single prothrombin activator, designated multisquamase, which is structurally and functionally different from ecarin, the prothrombin activator from the venom of Echis carinatus (saw-scaled viper). Multisquamase is comprised of a 58000 Mr and a 23000 Mr subunit that consists of two disulfide-linked chains of 12000 Mr and 10000 Mr, respectively. In contrast to ecarin, which activates prothrombin and prethrombin 1 at comparable rates, and whose activity is hardly affected by Ca2+ or by changes in ionic strength, multisquamase hardly activates prethrombin 1; prothrombin activation requires Ca2+ and is strongly inhibited at high ionic strength. The most favourable kinetic parameters are observed at 1 mM Ca2+ and at low ionic strength (Km=0.085 microM and kcat=0.68 s(-1) at I approximately 0.04). An increase in ionic strength considerably reduces the rate of prothrombin activation, due to an increase of the Km (Km=0.8 microM and kcat=1.03 s(-1) at I approximately 0.2). Studies in plasmas from patients on oral anticoagulant therapy show that E. Multisquamatus venom only activates carboxylated prothrombin, whereas E. carinatus activates both prothrombin and descarboxyprothrombin. Thus, multisquamase-dependent prothrombin activation appears to require post-translational modification of the gla-domain. This venom prothrombin activator may, therefore, become a useful tool to quantitate prothrombin and descarboxyprothrombin in cases where vitamin K-dependent carboxylation of prothrombin is impaired.

Enzymes↗
PubMed2004

Lupus-derived antiprothrombin autoantibodies from a V gene phage display library are specific for the kringle 2 domain of prothrombin.

Autoantibodies to prothrombin are common in patients with systemic lupus erythematosus. Although their presence is a risk factor for thrombosis, neither their origin nor their precise role in inducing the procoagulant state is known. We have developed a phage-display antibody library from patients with systemic lupus erythematosus with antiprothrombin antibodies, and we have selected two single-chain Fv antibody fragments (ScFvs) by panning on a prothrombin-coated surface. In prothrombin activation assays using purified components, these antibodies promoted prothrombin activation. These ScFvs, termed AN78 and AN129, bound to immobilized prothrombin in a concentration-dependent specific manner but not to other anionic phospholipid binding proteins such as beta2-glycoprotein I or annexin V. Phosphatidylserine-bound prothrombin, but not soluble prothrombin, inhibited the binding suggesting that the epitope is available only on immobilized prothrombin. To localize the epitope, prothrombin was treated with thrombin or factor Xa and various prothrombin activation fragments were subsequently isolated and tested in ELISA with the ScFvs. Both AN78 and AN129 bound to prethrombin I (the fragment lacking the Gla domain and the first kringle domain), to fragment 1.2 (containing Gla and the two kringle domains only) and to fragment 2 but not to thrombin, thus localizing the cognate epitope to the kringle 2 domain in prothrombin. Analysis of the cDNA sequences of these antibodies show clustered mutational patterns in the complementarity determining region, suggesting that variable domains are the products of antigen-driven B cell clonal maturation.

Amino Acid Sequence↗
PubMed1996

Isolation and characterization of carinactivase, a novel prothrombin activator in Echis carinatus venom with a unique catalytic mechanism.

The venom of the viper Echis carinatus contains a metalloprotease, ecarin, that is a potent prothrombin activator. We here show that the venom is also rich in another prothrombin activator, which does not belong to any known category of prothrombin activators. The novel enzyme, designated carinactivase-1 (CA-1), consists of two subunits held together non-covalently but very tightly. One subunit is a 62-kDa polypeptide that has metalloprotease activity and is homologous to the single-chain enzyme ecarin; the other subunit of 25 kDa consists of two disulfide-linked polypeptides of 17 and 14 kDa, and this subunit resembles the anticoagulant in the habu snake venom, IX/X-bp, that specifically binds the Gla domains of coagulation factors IX and X in a Ca2+-dependent fashion. The activation of prothrombin by CA-1 requires Ca2+ ions at millimolar concentrations and in the absence of Ca2+ ions this enzyme is virtually inactive. By contrast, activation by ecarin is completely independent of Ca2+ ions. CA-1, unlike ecarin, does not activate prothrombin derivatives, in which binding of Ca2+ ions has been perturbed, namely prethrombin-1 and acarboxyprothrombin. Furthermore, the isolated catalytic subunit, although its activity is greatly reduced as compared to that of the holoenzyme, no longer requires Ca2+ ions for the activation of prothrombin. Reconstitution with the non-catalytic 25-kDa subunit restores high level activity and the dependence on Ca2+ ions. Finally, prothrombin activation by CA-1 is inhibited by prothrombin fragment 1, and the isolated non-catalytic subunit is capable of binding fragment 1 in the presence of Ca2+ ions. From these observations, we postulate the following unique mechanism for the activation of prothrombin by CA-1. The enzyme primarily recognizes the Ca2+-bound conformation of the Gla domain in prothrombin via the 25-kDa regulatory subunit, and the subsequent conversion of prothrombin to active thrombin is catalyzed by the 62-kDa catalytic subunit.

Amino Acid Sequence↗
PubMed2005

Effects of urinary prothrombin fragment 1 in the formation of calcium oxalate calculus.

PURPOSE: We investigated the effects of urinary prothrombin fragment 1 in the formation of calcium oxalate urolithiasis. MATERIALS AND METHODS: Fresh urine and renal parenchyma from patients with calcium oxalate calculus and normal controls were collected. Urinary prothrombin fragment 1 was isolated and purified from urine. It was identified by sodium dodecyl sulfide-polyacrylamide gel electrophoresis and analysis of its first 13 N-amino acids. The inhibitory activity of urinary prothrombin fragment 1 on calcium oxalate crystal growth was tested by the seeded crystallization technique. Meanwhile, the gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 was analyzed by a previously described method and genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1 from renal parenchyma was detected by polymerase chain reaction-single strand conformational polymorphism sequencing. RESULTS: The gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 was significantly decreased from normal (24.4 to 1.7 mol/1,000 amino acids) in patients with calcium oxalate calculus. The mean growth index +/- SD of urinary prothrombin fragment 1 to calcium oxalate crystals was 42.3 +/- 4.2 compared with the normal index of 19.2 +/- 2.8 (p <0.01). The polymerase chain reaction-single strand conformational polymorphism sequencing technique revealed no genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1 in patients with calcium oxalate calculus. CONCLUSIONS: The gamma-carboxyglutamic acid composition of urinary prothrombin fragment 1 as well as its ability to inhibit calcium oxalate crystal growth was significantly decreased in patients with calcium oxalate calculus. This was not caused by genetic mutation of the gamma-carboxyglutamic acid domain of urinary prothrombin fragment 1. It is important to elucidate the mechanisms of calcium oxalate stones in view of urinary prothrombin fragment 1.

1-Carboxyglutamic Acid↗
PubMed2001

Characterization of bothrojaracin interaction with human prothrombin.

Bothrojaracin (BJC) is a 27-kD snake venom protein from Bothrops jararaca that has been characterized as a potent thrombin inhibitor. BJC binds to exosites I and II, with a dissociation constant of 0.7 nM, and influences but does not block the proteinase catalytic site. BJC also binds prothrombin through an interaction that has not been characterized. In the present work we characterize the interaction of BJC with prothrombin quantitatively for the first time, and identify the BJC binding site on human prothrombin. Gel filtration chromatography demonstrated calcium-independent, 1:1 complex formation between fluorescein-labeled BJC ([5F]BJC) and prothrombin, whereas no interactions were observed with activation fragments 1 or 2 of prothrombin. Isothermal titration calorimetry showed that binding of BJC to prothrombin is endothermic, with a dissociation constant of 76 +/- 32 nM. The exosite I-specific ligand, hirudin(54-65) (Hir(54-65) (SO(3)(-)), displaced competitively [5F]BJC from prothrombin. Titration of the fluorescent hirudin(54-65) derivative, [5F]Hir(54-65)(SO(3)(-)), with human prothrombin showed a dissociation constant of 7.0 +/- 0.2 microM, indicating a approximately 100-fold lower binding affinity than that exhibited by BJC. Both ligands, however, displayed a similar, approximately 100-fold increase in affinity for exosite I when prothrombin was activated to thrombin. BJC efficiently displaced [5F]Hir(54-65)(SO(3)(-)) from complexes formed with thrombin or prothrombin with dissociation constants of 0.7 +/- 0.9 nM and 11 +/- 80 nM, respectively, indicating that BJC and Hir(54-65)(SO(3)(-)) compete for the same exosite on these molecules. The results indicate that BJC is a potent and specific probe of the partially exposed anion-binding exosite (proexosite I) of human prothrombin.

Animals↗
PubMed1994

Prothrombin activation on dioleoylphosphatidylcholine membranes.

Factor-Xa-catalyzed prothrombin activation is greatly accelerated by negatively charged phospholipids plus calcium ions. In 1990, we reported that neutral phosphatidylcholine membranes also stimulated prothrombin activation [Gerads, I., Govers-Riemslag, J.W.P., Tans, G., Zwaal, R. F. A. & Rosing, J. (1990) Biochemistry 29, 7967-7974]. In the present study, we have performed a detailed analysis of the prothrombin-converting activity of phosphatidylcholine membranes. Stimulation of prothrombin activation by phosphatidylcholine vesicles was particularly observed (a) with phosphatidylcholine molecules that contained unsaturated hydrocarbon side chains, (b) in the presence of factor Va, (c) at low ionic strength and (d) when Ca2+ were present in the reaction medium. It is unlikely that the prothrombinase activity of phosphatidylcholine preparations was due to contaminating anionic phospholipids. This is concluded from the fact that thin-layer chromatographic analysis showed that dioleoylphosphatidylcholine [(Ole)2GroPCho] contained less than 0.1 mol/100 mol anionic phospholipid, and that incorporation of such amounts of anionic lipids in (Ole)2-GroPCho membranes hardly increased their prothrombin-converting activity. At low ionic strength and in the presence of factor Va and Ca2+ (Ole)2GroPCho membranes accelerated prothrombin activation about 100-fold. At ionic strength (I) 0.06, prothrombin activation on 100 microM (Ole)2-GroPCho was characterized by a Km for prothrombin of 2 microM, a Vmax of 3020 IIa min-1.Xa-1 and a Kd for factor XaVa complex formation at the membrane surface of 7.5 nM. Prothrombin activation on (Ole)2GroPCho membranes was drastically reduced when the ionic strength was increased. The inhibition at high ionic strength could be explained by an effect on the Kd for XaVa complex formation which increased from 7.5 nM at I = 0.06 to 100 nM at I = 0.22. Prothrombin activation on (Ole)2GroPCho required Ca2+ and was dependent on the presence of gamma-carboxyglutamic acid domains in prothrombin and factor Xa. This indicates that similar interactions may account for the assembly of prothrombinase complexes on phosphatidylcholine and an anionic lipid-containing membranes.

1-Carboxyglutamic Acid↗