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PubMed1998

Induction of prothrombin synthesis by K-vitamins compared in vitamin K-deficient and in brodifacoum-treated rats.

Vitamin K is a group name for a number of prenylated 2-methyl-1,4-naphtoquinones, which may differ in their ability to function as a cofactor for prothrombin biosynthesis. To quantify the bioactivity of different forms of vitamin K, two experimental animal systems are frequently used: vitamin K-deficient rats and anticoagulated rats. In this paper both models are compared, and it is shown that the results obtained depend on the model used. The main reason for this discrepancy is the difference in recycling of vitamin K-epoxide, which results in a 500 times higher vitamin K requirement in anticoagulated rats. Absorption and hepatic accumulation of long chain menaquinones seem to be restricted to a maximum, whereas also the lipophilic nature of long chain menaquinones may hamper the quinone-quinol reduction in anticoagulated animals. If these data may be extrapolated to patients, food items rich in K1 and MK-4 would be expected to influence the stability of oral anticoagulation to a much larger extent than food items primarily containing higher menaquinones.

4-Hydroxycoumarins↗
PubMed2000

Purification of human prothrombin from Nitschmann fraction III using DEAE membrane radial flow chromatography.

A DEAE membrane radial column (4x5 cm) was used to purify human prothrombin from Nitschmann fraction III and performances of the column were investigated. Sample flow-rate of 20-30 ml/min (i.e., 24-36 bed volumes/h) and elution velocity up to 40 ml/min (i.e., 48 bed volumes/h) were obtained without compromising the separation efficiency. Its breakthrough capacity was one-third to half that of the commercial DEAE Sepharose Fast Flow media. It was concluded that the novel column is an attractive alternate to traditional axial column.

Chromatography, Liquid↗
PubMed2001

Effects of Mucuna pruriens extract on activation of prothrombin by Echis carinatus venom.

Mucuna pruriens (L.) DC has long been used as a medicinal plant by traditional healers. The validity of the claims made for this plant has also been tested scientifically. Some of its properties are probably linked to high concentrations of dopa since it is useful in the treatment of Parkinson's disease. The antisnake properties of an extract of Mucuna pruriens' seeds (MP101UJ) in vivo were recently demonstrated and one is now investigating its biochemical mechanism. Echis carinatus venom (EV) contains a mixture of proteins that affect the coagulative cascade, causing severe bleeding and haemorrhage. Here the effect of an extract of MP101UJ in prothrombin activation by EV in vitro by clotting and chromogenic assay is studied. An increase in procoagulant activity was found. This could explain the protective effect in vivo.

Animals↗
PubMed2000

[Mesenteric vein and inferior vena cava thrombosis: disseminated intravascular coagulation and/or G 20210 A mutation of the prothrombin gene?].

The authors report the case of a 30-year-old man who died from pulmonary embolism and multiorgan failure caused by mesenteric and inferior vena cava thrombosis. The patient was found heterozygous for the prothrombin gene variant (G 20210 A). The family study showed the same asymptomatic anomaly in his brother. This recently described mutation is associated with an increased risk for venous thrombosis. The investigations and treatment of mesenteric venous thrombosis are discussed.

Adult↗
PubMed2002

Expression of recombinant rabbit tissue factor in Pichia pastoris, and its application in a prothrombin time reagent.

Tissue factor (TF), or thromboplastin, is a cell membrane-associated glycoprotein composed, in full length, of cytoplasmic, transmembrane, and extracellular domains. It functions as a cofactor in a complex with factor VII (FVII), generating activated factor VII (FVIIa) and initiating blood coagulation. The prothrombin time (PT) assay uses TF as the in vitro activator of coagulation under defined conditions, and it is primarily used to diagnose and manage the extrinsic-pathway factor defficiencies. To overcome the limitations of natural-source TF, we have expressed the mature full-length recombinant rabbit TF (rRTF) protein in Pichia pastoris. Isolation, by purification by immobilized metal-affinity chromatography, of full-length rRTF was facilitated by engineering a (His)(6) tail on its C-terminus, which maximizes the selection of rRTF with intact transmembrane and cytoplasmic domains, critical for proper activity. A PT reagent that incorporates this purified rRTF has performance characteristics similar to those of PT reagents made with natural TF as indicated in method comparison studies, and shows lot-to-lot consistency and reproducibility.

Animals↗
PubMed1995

Determination of normal versus abnormal activated partial thromboplastin time and prothrombin time after cardiopulmonary bypass.

OBJECTIVE: The study's objective was to determine the prothrombin time (PT) and activated partial thromboplastin time (aPTT) values that differentiated normal from excessively bleeding patients immediately after cardiopulmonary bypass (CPB). DESIGN: A prospective blinded study. SETTING: A large academic medical center. PARTICIPANTS: 148 patients were studied. INTERVENTIONS: aPTT and PT were determined by the hospital laboratory and the Biotrack 512 Coagulation Monitor (Ciba Corning Diagnostics, Medfield, MA) from an arterial whole blood sample obtained 10 minutes after protamine administration. Patients were subjectively and objectively defined as "bleeders" or "nonbleeders" with blinded observers. MEASUREMENTS AND MAIN RESULTS: The specificity and sensitivity were determined by a receiver operating characteristic (ROC) analysis. Twenty-three of 148 patients (14.9%) were characterized as bleeders. The laboratory PT had a maximal specificity and sensitivity of 78% and 75%, respectively, at a value of 15.4 s, with a negative and positive predictive value of 93% and 33%, respectively. The maximal specificity and sensitivity of the laboratory aPTT was 64% and 76%, respectively, at a value of 46 s, with a negative and positive predictive value of 89% and 33%, respectively. aPTT and PT approached normal values after 12 hours in the intensive care unit. CONCLUSION: The aPTT and PT values that produce the maximal sensitivity and specificity in the ROC analysis may be helpful to differentiate patients who are bleeding excessively from those patients who are not after CPB and to guide transfusion of blood products. New whole blood coagulation devices with rapid turn-around times had similar predictive value for bleeding tendency compared with standardized laboratory tests.

Adult↗
PubMed1991

Structure of the propeptide of prothrombin containing the gamma-carboxylation recognition site determined by two-dimensional NMR spectroscopy.

The propeptides of the vitamin K dependent blood clotting and regulatory proteins contain a gamma-carboxylation recognition site that directs precursor forms of these proteins for posttranslational gamma-carboxylation. Peptides corresponding to the propeptide of prothrombin were synthesized and examined by circular dichroism (CD) and nuclear magnetic resonance spectroscopy (NMR). CD spectra indicate that these peptides have little or no secondary structure in aqueous solutions but that the addition of trifluoroethanol induces or stabilizes a structure containing alpha-helical character. The maximum helical content occurs at 35-40% trifluoroethanol. This trifluoroethanol-stabilized structure was solved by two-dimensional NMR spectroscopy. The NMR results demonstrate that residues -13 to -3 form an amphipathic alpha-helix. NMR spectra indicate that a similar structure is present at 5 degrees C, in the absence of trifluoroethanol. Of the residues previously implicated in defining the gamma-carboxylation recognition site, four residues (-18, -17, -16, and -15) are adjacent to the helical region and one residue (-10) is located within the helix. The potential role of the amphipathic alpha-helix in the gamma-carboxylation recognition site is discussed.

Amino Acid Sequence↗
PubMed1992

Surface binding kinetics of prothrombin fragment 1 on planar membranes measured by total internal reflection fluorescence microscopy.

Total internal reflection fluorescence microscopy (TIRFM) has been employed to investigate the Ca(2+)-dependent membrane-binding characteristics of fluorescein-labeled bovine prothrombin-fragment 1 (F-BF1). Light scattering measurements demonstrated that F-BF1 bound to small unilamellar phosphatidylserine/phosphatidylcholine (25/75, mol/mol) vesicles with an apparent dissociation constant (1.5 +/- 0.2 microM) similar to that of unlabeled protein (1.1 +/- 0.1 microM). Negatively charged supported planar membranes were constructed by fusing small unilamellar vesicles at quartz surfaces. TIRFM measurements under equilibrium conditions showed that F-BF1 bound to planar membranes with an apparent dissociation constant (0.9 +/- 0.2 microM) approximately equal to that on vesicles. Total internal reflection/fluorescence photobleaching recovery (TIR/FPR) curves for F-BF1 on 25 mol% PS planar surfaces were diffusion-influenced at F-BF1 solution concentrations less than or equal to 5 microM. Fluorescence recovery rates from samples of high F-BF1 concentrations were slowed by increasing the solution viscosity with glycerol, thus providing further support for a diffusion-limited effect at low F-BF1 concentrations. Analysis of the reaction-limited fluorescence recovery curves at F-BF1 solution concentrations greater than or equal to 10 microM gave average association and dissociation kinetic rates of approximately 10(5) M-1 s-1 and approximately 0.1 s-1, respectively. Kinetic association rates increased significantly with increasing PS, whereas kinetic dissociation rates increased only slightly. Fluorescence recovery curves were nonmonoexponential; possible mechanisms for this behavior are described.

Animals↗
PubMed1992

Molecular dynamics simulation of bovine prothrombin fragment 1 in the presence of calcium ions.

Early solvation-induced structural reorganization of calcium prothrombin fragment 1 is simulated with molecular dynamics. Initial coordinates are those of the 2.2-A resolution crystal structure [Soriano-Garcia, M., Padmanabhan, K., de Vos, A. M., & Tulinsky, A. (1992) Biochemistry 31, 2554-2556]. The molecular dynamics code AMBER, appropriately modified to include long-range (less than or equal to 22.0 A) ionic forces, was employed. The solution structure appears to equilibrate within 100 ps. Although minor changes are seen in various structural domains, the early solution structure basically maintains an intricate network of nine gamma-carboxyglutamic acid (Gla) residues encapsulating seven calcium ions. However, the Gla domain moves with respect to the kringle domain. This motion is mainly due to the movement of Ser34-Leu35 that appears to be a flexible hinge between the domains. The N-terminus of Ala 1 is in a tightly bound complex with three Gla residues that remains stable in the solution structure when the long-range electrostatic cutoff is employed and the near planar alignment of the seven calcium ions is only slightly distorted. The simulation structure is discussed in terms of experiments that studied calcium ion-induced quenching of the intrinsic fluorescence, protection of the N-terminal amino group from acetylation by calcium ions, chemical modification of the N-terminus to a trinitrophenyl derivative, and the possibility of a calcium-binding site(s) in the kringle domain.

Amino Acid Sequence↗
PubMed1994

Differences in the metal ion structure between Sr- and Ca-prothrombin fragment 1.

The structure of Sr-prothrombin fragment 1 has been solved and refined by restrained least-squares methods at 2.5-A resolution to a crystallographic R value of 0.167. The protein structure is very similar to that of Ca-fragment 1. A polymeric array of five Sr2+ ions separated by about 4.0 A is buried among six gamma-carboxyglutamic acid (Gla) residues; three other Sr2+ ions interact with other Gla residues and are located further apart. One of these was not found in the Ca-fragment 1 structure. The coordination of the Sr2+ ions resembles that of Ca2+, but there are some significant differences between them. The most notable is the lack of water coordination with Sr2+ ions and two conformations for Gla 8, which change the coordination of Sr-2 and Sr-3. A hexose moiety of an oligosaccharide was located in the vicinity of Asn101 that was flexibly disordered in Ca-fragment 1. The new Sr2+ ion found may be involved in metal ion phospholipid binding interactions along with Sr-1, and Sr-7, Sr-8.

Amino Acid Sequence↗
PubMed1986

Phospholipid lateral organization in synthetic membranes as monitored by pyrene-labeled phospholipids: effects of temperature and prothrombin fragment 1 binding.

Pyrene-labeled phospholipids have been used to test for the existence of lateral domains due to temperature-induced phase separations and binding of prothrombin fragment 1 to charged lipid vesicles. When in close proximity, pyrene-containing probes can exchange excited-state energy to form excimers; the ratio of the excimer to monomer fluorescence intensity (E/M) is proportional to the local concentration of probe in the membranes, as well as to the excimer lifetime and the probe's lateral diffusion coefficient. The ability of the pyrene-labeled phospholipids to quantitatively report the coexistence of multiple environments was demonstrated in dipalmitoylphosphatidylcholine/palmitoyloleoylphosphatidylcholine multilamellar vesicle preparations of varying compositions, each of which contained coexisting fluid and gel phases. In this system, pyrene-labeled phosphatidylcholine was found to favor the fluid relative to the gel phase with a partition coefficient of 7. At 37 degrees C, in dioleoylphosphatidylglycerol (DOPG)/palmitoyloleoylphosphatidylcholine (POPC) large, unilamellar vesicles containing either pyrene-labeled phosphatidylglycerol (py-PG) or pyrene-labeled phosphatidylcholine (py-PC), the excimer lifetime (37 ns) and the lateral diffusion constant of the probe (5.8 X 10(-8) cm2/s) were independent of the membrane composition and the presence of fragment 1 and Ca2+. Consequently, E/M was directly proportional to only the local concentration of the py-PG or py-PC probes. When saturating amounts of fragment 1 and 5 mM Ca2+ were added to DOPG/POPC vesicles that contained either probe, no change in E/M and hence the local probe concentration was observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1986

Three-dimensional structure of the kringle sequence: structure of prothrombin fragment 1.

The three-dimensional structure of bovine prothrombin fragment 1 has been solved at 2.8-A resolution. The electron density clearly reveals four disulfide bridges along with more than 80% of the side chains completely in density, which correspond faithfully to the kringle sequence, its preceding 30 residues, and the dodecapeptide carboxy terminal; the polysaccharide and the first 35 residues of the amino terminal of fragment 1 are disordered or about 40% of the structure. The folding of the kringle sequence is based upon close disulfide van der Waals contacts between Cys-87-Cys-127 and Cys-115-Cys-139 (4.1 A between midpoints of the bridges), two antiparallel strands of highly conserved (113-118, 124-129) beta-structure, and the stacking of some conserved aromatic residues, all near the center of the folded structure. Moreover, the overall folding appears to be duplicated as a pair of stacked duplex loops with an antiparallel open loop. The overall shape of the kringle structure approximates an eccentric oblate ellipsoid of dimensions 11 X 28 X 30 A. The residues immediately preceding the kringle are dominated by alpha-helical structure (Phe-41-Cys-48; Leu-56-Glu-63). Residues Phe-41-Trp-42 and Tyr-45, which are conserved in factor IX, factor X, protein C, and protein Z, form another aromatic stacked cluster while the Cys-48-Cys-61 disulfide loop corresponds to the well-known alpha/beta structural unit. The dodecapeptide carboxy-terminal interkringle chain extends along the periphery of the kringle in its plane and forms a beta-structure with the kringle-closing Ser-140-Val-143 tetrapeptide.

Amino Acid Sequence↗
PubMed1988

Irreversible degradation of histidine-96 of prothrombin fragment 1 during protein acetylation: another unusually reactive site in the kringle.

Acetylation of prothrombin fragment 1 in acetate-borate buffer at pH 8.5 resulted in the appearance of increased light absorbance at about 250 nm. Protease digestions resulted in isolation of a single peptide (residues 94-99) with intense absorbance at about 250 nm (estimated extinction coefficient of 5000 M-1 cm-1). Amino acid analysis showed the expected composition except for the absence of His-96. Instead, an unidentified amino acid which had a ninhydrin product with absorption properties similar to those of proline eluted near aspartate. When sequenced, this peptide (YP?KPE containing epsilon-amino-acetyllysine) lacked histidine at the third position but gave a high yield of a PTH derivative that eluted near PTH-Gly from the HPLC column. Fast atom bombardment mass spectrometry of the derivatized 94-99 peptide showed a mass that was 74 units higher than expected. The histidine degradation product was identified as a di-N-acetylated side chain with an opened imidazole ring and loss of C2 of the ring. While a similar degradation pattern has previously been reported during acylation of histidine, the high chemical reactivity exhibited by His-96 was unusual. For example, under conditions sufficient for quantitative derivatization of His-96, His-105 of fragment 1 was not derivatized to a detectable level. Furthermore, His-96 in fragment 1 was at least an order of magnitude more susceptible to degradation than His-96 in the isolated 94-99 peptide. His-96 is therefore one of several neighboring amino acids of the kringle portion of fragment 1 that displays highly unusual chemistry (see also Asn-101 [Welsch, D.J., & Nelsestuen, G. L. (1988) Biochemistry 27 4946-4952] and Lys-97 [Pollock, J.S., Zapata, G.A., Weber, D.J., Berkowitz, P., Deerfield, D.W., II, Olson, D.L., Koehler, K.A., Pedersen, L.G., & Hiskey, R.G. (1988) in Current Advances in Vitamin K Research (Suttie, J.W., Ed.) pp 325-334, Elsevier Science, New York]).(ABSTRACT TRUNCATED AT 250 WORDS)

Acetic Anhydrides↗
PubMed1989

Structure of Ca2+ prothrombin fragment 1 including the conformation of the Gla domain.

The structure of Ca2+ prothrombin fragment 1 has been solved at 2.8-A resolution by X-ray crystallographic methods. Most of the Gla domain of fragment 1 (residues 1-48), which is high homologous with the N-terminal regions of six other blood proteins, cannot be identified in the electron density map of the apo structure. This is not the case when crystals are grown in the presence of Ca2+ ions where the Gla domain exhibits a well-defined folded structure. The folding of the Gla domain is dominated by secondary structure: (a) 3.0 turns of alpha-helix (25%) and (b) five short beta-strands arranged into two beta-structural units (40%). The Cys18-Cys23 disulfide of the small conserved loop of Gla domains is close to a cluster of conserved aromatic residues. The resulting interaction is probably responsible for the fluorescence quenching event accompanying Ca2+ ion binding. Since the Gla domain approximates a discoid, all the Gla residues are easily accessible to solvent. The arrangement of the paired Gla residues (7-8, 20-21, 26-27) is highly suggestive in that they essentially line one edge of the Gla domain creating a potentially intense electronegative environment. This region might well be that associated with phospholipid binding. The kringle structure of Ca2+ fragment 1 is essentially indistinguishable from that of the apoprotein at this stage.

Amino Acid Sequence↗
PubMed1981

Effects of temperature and pH on prothrombin fragment 1 conformation as determined by nuclear magnetic resonance.

The effects of temperature and pH on the solution conformation of native prothrombin fragment 1 were examined with 1H NMR spectroscopy. A calcium-dependent quenching of the intrinsic protein fluorescence was used to monitor calcium binding to fragment 1 as an indicator of functional protein. The native fragment 1 NMR spectrum contained several features indicative of a folded protein: (a) nonequivalent histidyl C-2 resonances at 7.9 and 8.1 ppm, (b) two resonances of nearly equal intensity at 7.26 and 7.32 ppm, and (c) a resonance at -1.04 ppm. Temperature studies showed that thermal unfolding of fragment 1 (even at 80 degrees C) was reversible; however, there was an irreversible inactivation step which occurred subsequent to the unfolding. The basis for this inactivation appeared to include disulfide exchange reactions. On the basis of NMR spectra, fragment 1 retained its conformation from pH 7.0 to pH 11.5. From pH 7.0 to pH 5.0, the protein showed a reversible conformational change, and below pH 5, the protein self-associated. The pH dependence of the chemical shift of the tyrosyl resonances indicated a pKa of approximately 10 for the tyrosyl residues. These data suggest that the tyrosyl residues are accessible to solvent in the native protein.

Humans↗
PubMed1998

Trans-cis isomerization of proline 22 in bovine prothrombin fragment 1: a surprising result of structural characterization.

The calcium ion-mediated interaction of bovine prothrombin (BF1) with negatively charged phospholipid membranes is assumed to be largely via the Gla domain of BF1 with the fold of the Gla domain essential for binding. It has been reported that Pro22 undergoes classical trans to cis isomerization in the presence of calcium ions with the cis conformation of Pro22 of BF1 responsible for membrane binding [Evans, T. C., Jr., and Nelsestuen, G. L. (1996) Biochemistry 35, 8210-8215]. However, Pro22 was found to be in the trans conformation in the crystal structure of BF1. In the present work, we have used molecular dynamics simulations to investigate the relative importance of the two conformations of Pro22 to the structural and dynamical properties of BF1. The initial trans conformation of Pro22 in BF1 was slowly converted to cis-Pro22 using constrained dynamics. The second-generation AMBER force field in conjunction with the particle mesh Ewald method to accommodate long-range interaction was employed in the trajectory calculations. Comparison of the BF1(trans-Pro22) and BF1(cis-Pro22) equilibrated structures reveals surprisingly that the overall structural changes associated with the trans-cis isomerization is minimal and only minor modifications to the hydrogen bond network and the network of N-terminus Ala1 take place. The calculated electrostatic potential energy surfaces of the two protein structures also appear to be very similar, indicating the near equality of the local interaction site environments in the protein prior to lipid binding.

1-Carboxyglutamic Acid↗
PubMed1981

Synthesis of peptide analogues of prothrombin precursor sequence 5-9. Substrate specificity of vitamin K dependent carboxylase.

Thirty-five analogues of Phe-Leu-Glu-Glu-Leu, the pentapeptide sequence 5-9 of bovine prothrombin precursor, were synthesized and assayed as potential substrates or inhibitors of rat liver vitamin K dependent carboxylase. Carboxylation of substrate was determined by measuring the incorporation of carbon-24 labeled bicarbonate into product. Changes in substrate carboxylation produced by changing peptide chain length, amino acid chirality, or the distance separating the peptide chain backbone from the carboxyl group were measured. The data suggest that the carboxylase carboxylates L-glutamic acid residues and does not carboxylate L-aspartic acid, L-homoglutamic acid, glutamine, or D-glutamic acid residues; tri-through pentapeptides are better substrates than mono- or bis(amino acid) derivatives, and hydrophobic groups added to the N-terminus can produce better substrates for the enzyme. None of the synthetic substrates is carboxylated as effectively as the endogenous protein substrates for the enzyme. The effect of structure on additional parameters affecting carboxylation is discussed.

Amino Acid Sequence↗
PubMed1983

Synthesis and biological properties of new hexapeptide substrates for vitamin K dependent carboxylase. Evidence for X-Pro cis/trans amide bond interconversions in prothrombin precursor fragment 18-23.

Three hexapeptide analogues, corresponding to sequence 18-23 of bovine prothrombin precursor [-Cys-Leu-Glu-Glu-Pro-Cys-] have been synthesized and evaluated as substrates for vitamin K dependent carboxylase. These new hexapeptides are moderately good substrates for the carboxylase but do not significantly inhibit carboxylation of Phe-Leu-Glu-Glu-Leu, a good substrate for the enzyme. Based on proton and carbon-13 NMR experiments, it is established that the conformation of sequence 18-23, which contains proline at position 22, has a trans amide bond for the Glu-Pro22 sequence in chloroform-d. This amide bond is converted to the cis amide geometry in Me2-SO-d6. It is proposed that good substrates for the carboxylase require a trans amide bond between residues 21 and 22.

Amino Acid Sequence↗