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PubMed1987

Homo- and heterodimer formation with prothrombin and prothrombin fragment 1 in the presence of calcium ions.

The purpose of the current study is to present further evidence for prothrombin self-association as assessed by chemical crosslinking. When the self-association (evaluated by covalent crosslinking with dithiobis(succinimidylpropionate) of prothrombin or fragment 1 was evaluated at the same molar concentration of protein, similar rates of dimer formation were observed for either protein. When prothrombin and fragment 1 were incubated together with the crosslinking reagent and calcium ions, a heterodimer consisting of prothrombin and fragment 1 was observed in addition to prothrombin dimer and fragment 1 dimer. Similar experiments with prethrombin 1 showed neither significant self-association nor effect on prothrombin self-association. Comparison of the formation of prothrombin fragment 1 heterodimer formation with the effect of fragment 1 on prothrombin activation by factor Xa suggests that the anticoagulant activity of fragment 1 is not solely a result of the formation of a heterodimer between prothrombin and fragment 1.

Animals↗
PubMed1991

Fourier transform infrared spectroscopic study of Ca2+ and membrane-induced secondary structural changes in bovine prothrombin and prothrombin fragment 1.

Fourier transform infrared (FTIR) spectroscopy was used to monitor secondary structural changes associated with binding of bovine prothrombin and prothrombin fragment 1 to acidic lipid membranes. Prothrombin and prothrombin fragment 1 were examined under four different conditions: in the presence of (a) Na2EDTA, (b) 5 mM CaCl2, and in the presence of CaCl2 plus membranes containing 1-palmitoyl-2-oleoyl-3-sn-phosphatidylcholine (POPC) in combination with either (c) bovine brain phosphatidyl-serine (bovPS) or (d) 1,2-dioleoyl-phosphatidylglycerol (DOPG). The widely reported Ca(2+)-induced conformational change in bovine prothrombin fragment 1 was properly detected by our procedures, although Ca(2+)-induced changes in whole prothrombin spectra were too small to be reliably interpreted. Binding of prothrombin in the presence of Ca2+ to procoagulant POPC/bovPS small unilamellar vesicles produced an increase in ordered secondary structures (2% and 3% increases in alpha-helix and beta-sheet, respectively) and a decrease of random structure (5%) as revealed by spectral analysis on both the original and Fourier-self-deconvolved data and by difference spectroscopy with the undeconvolved spectra. Binding to POPC/DOPG membranes, which are less active as procoagulant membranes, produced no detectable changes in secondary structure. In addition, no change in prothrombin fragment 1 secondary structure was detectable upon binding to either POPC/bovPS or POPC/DOPG membranes. This indicates that a membrane-induced conformational change occurs in prothrombin in the nonmembrane-binding portion of the molecule, part of which is activated to form thrombin, rather than in the membrane-binding fragment 1 region. The possible significance of this conformational change is discussed in terms of differences between the procoagulant activities of different acidic lipid membranes.

Animals↗
PubMed1995

The second kringle domain of prothrombin promotes factor Va-mediated prothrombin activation by prothrombinase.

The incorporation of factor Xa into the prothrombinase complex, factor Xa-factor Va-phospholipid-Ca(II), results in an approximately 10(5)-fold higher rate of substrate activation than that of the enzyme alone. To examine the role of the prothrombin kringle domains in the interaction with prothrombinase we have employed site-directed mutagenesis to produce prothrombin species that lack either the first kringle domain, PT/delta K1, or the second kringle domain, PT/delta K2. Previously, we have shown that these proteins are fully carboxylated and that they bind to phospholipid vesicles. In this investigation we demonstrate that cleavage at Arg271-Thr272 and Arg320-Ile321 peptide bonds occurs upon activation with prothrombinase to yield normal thrombin from both PT/delta K1 and PT/delta K2. In the absence of factor Va, the Km(app) for the activation of PT/delta K1, PT/delta K2, or plasma-derived prothrombin by factor Xa-phospholipid-Ca(II) are equivalent. The Km(app) for the activation of PT/delta K2 by prothrombinase is approximately 4-5-fold higher than that obtained for plasma-derived prothrombin or PT/delta K1. These data demonstrate that the prothrombin kringle domains do not contribute significantly to the binding affinity of the substrate-enzyme interaction. In the absence of factor Va, equivalent kcat values were obtained for all of the prothrombin species when they were activated by factor Xa-Ca(II)-phospholipid. In contrast, a 7-fold lower kcat value was obtained for the activation of PT/delta K2 by prothrombinase as compared with that obtained for plasma prothrombin or PT/delta K1. Collectively, these data suggest that determinants within the second prothrombin kringle domain interact with factor Va to elicit a significant acceleration in the catalytic rate of substrate turnover. Indeed, in contrast to plasma-derived prothrombin, no direct binding of PT/delta K2 to factor Va to form the PT/delta K2-factor Va complex could be demonstrated by 90 degrees light scattering.

Animals↗
PubMed1990

Chemical modification of gamma-carboxyglutamic acid residues in prothrombin elicits a conformation similar to that of abnormal (des-gamma-carboxy)prothrombin.

Chemical modification of gamma-carboxyglutamic acid (Gla) residues in human prothrombin to gamma-methyleneglutamic acid (gamma-MGlu) residues elicited a conformation similar, if not identical, to that of des-gamma-carboxy prothrombin or PIVKA-II, i.e., prothrombin molecules induced by vitamin K antagonists or vitamin K deficiency states. The reaction seems to proceed sequentially by preferentially modifying a Gla at residue 32 that is located innermost among 10 Gla residues of human prothrombin. The initial modification resulted in nearly 50% losses of barium salt adsorption, the procoagulant activity and thrombin generation by the prothrombinase complex. The subsequent modification of two Gla residues at positions 6 and 16 gave rise to the immunoreactivity to an established monoclonal antibody that specifically recognizes the des-gamma-carboxy prothrombin. Further modification of Gla residues increased the reactivity to the antibody, indicating that the conformation recognized by the antibody was stabilized so as to more readily fit the recognition site of the antibody. The appearance of the immunoreactivity was obviously related to the modification of Gla residues in prothrombin, since all other similarly treated derivatives of prothrombin lacking the Gla-domain failed to react with the antibody. Such chemically modified prothrombins may serve as models for studying abnormal des-gamma-carboxy prothrombin produced in vitamin K deficiency states.

1-Carboxyglutamic Acid↗
PubMed1990

Measurement of immunoreactive prothrombin precursor and vitamin-K-dependent gamma-carboxylation in human hepatocellular carcinoma tissues: decreased carboxylation of prothrombin precursor as a cause of des-gamma-carboxyprothrombin synthesis.

Des-gamma-carboxyprothrombin is an abnormal prothrombin which is drastically increased in the plasma of patients with hepatocellular carcinoma. To investigate the process of the abnormal prothrombin synthesis, the amount of prothrombin precursor was measured with an enzyme-linked immunosorbent assay using a specific antibody directed to human prothrombin; the vitamin-K-dependent gamma-carboxylation of prothrombin precursor was determined in human liver tissues. The tissue content of prothrombin precursor was increased in hepatoma tissues compared with noncancerous liver tissues, while the vitamin-K-dependent carboxylation of prothrombin precursor was markedly decreased in hepatoma tissues of the patients with increased plasma des-gamma-carboxyprothrombin. The present study indicates that in hepatocellular carcinoma an increase in prothrombin precursor concentration does not induce vitamin-K-dependent carboxylase activity, which is ordinarily observed in normal liver; probably an overproduction of prothrombin precursor with reduced gamma-carboxylation causes an increase in plasma des-gamma-carboxyprothrombin in patients with hepatocellular carcinoma.

Adult↗
PubMed1985

Modification of histidines in human prothrombin. Effect on the interaction of fibrinogen with thrombin from diethyl pyrocarbonate-modified prothrombin.

Diethyl pyrocarbonate (ethoxyformic anhydride) was used to modify histidyl residues in prothrombin. Diethyl pyrocarbonate inactivated the potential fibrinogen-clotting activity of prothrombin with a second-order rate constant of 70 M-1 min-1 at pH 6.0 and 25 degrees C. The difference spectrum of the modified protein had a maximum absorption at 240 nm which is characteristic of N-carbethoxyhistidine. The pH dependence for inactivation suggested the participation of a residue with a pKa of 6.2. Addition of hydroxylamine to ethoxyformylated prothrombin reversed the loss of fibrinogen-clotting activity. No structural differences were detected between the native and modified proteins using fluorescence emission and high-performance size-exclusion chromatography. The tyrosine and tryptophan content was not altered, but approximately 1-2 amino groups were modified. Statistical analysis of residual enzyme activity and extent of modification indicates that among 7 histidyl residues modified per molecule, there is 1 essential histidine (not in the active site) involved in the potential fibrinogen-clotting activity of prothrombin. To further examine its properties, the modified prothrombin was activated to thrombin using Echis carinatus venom protease. There was no difference in the catalytic activity of thrombin obtained from either native or ethoxyformylated prothrombin, as measured by H-D-Phe-pipecolyl-Arg-p-nitroanilide (D-Phe-Pip-Arg-NA) hydrolysis. However, thrombin produced from the modified protein showed a loss of fibrinogen-clotting activity but had a comparable apparent Ki value (about 20 microM) to thrombin from native prothrombin when fibrinogen was used as a competitive inhibitor during D-Phe-Pip-Arg-NA hydrolysis. The similarity in Ki values indicated that thrombin derived from diethyl pyrocarbonate-modified prothrombin does not have an altered fibrinogen-binding site. Although the histidyl residue involved during inactivation has not been identified, the results suggest that a histidyl residue in the thrombin portion of prothrombin is essential for interaction with fibrinogen.

Diethyl Pyrocarbonate↗
PubMed1995

Catabolism of rabbit prothrombin in rabbits: uptake of prothrombin by the aorta wall before and after a de-endothelializing injury in vivo.

After an injury to the vascular endothelium, certain blood proteins collect rapidly at the site of damage to prevent blood loss and maintain blood flow. The uptake of fibrinogen, plasminogen, and antithrombin--but not prothrombin--have been measured previously at the rabbit aorta wall after injury in vivo. This report describes the clearance of rabbit iodine 131-labeled prothrombin from the rabbit circulation to measure the distribution and fractional catabolic rate and compares the behavior of 131I-labeled prothrombin with either iodine 125-labeled fibrinogen or 125I-labeled antithrombin at the balloon catheter-injured aorta wall. When injected into young rabbits, 131I-labeled prothrombin was cleared from the intravascular space to yield a plasma curve that was best described by three exponentials. Fractional plasma and whole body catabolic rates were 2.0 day-1 and 0.41 day-1, respectively, equivalent to a catabolic half-life of 1.7 days. Fractional distribution of prothrombin amounted to 0.21, 0.24, and 0.55 within the intravascular, vascular endothelial, and extravascular compartments, respectively. Samples of 131I-labeled prothrombin and either 125I-labeled fibrinogen or 125I-labeled antithrombin were injected into anesthetized rabbits before balloon de-endothelialization of the thoracic aorta. The uptake of each radiolabeled protein by the aorta intima-media was measured at various times (5 to 60 minutes) after injury. Whereas uptake of plasma fibrinogen by the balloon-injured intima-media was maximal (20 pmol/cm2) in less than 5 minutes after injury, maximum uptake of prothrombin (5 to 6 pmol/cm2) took approximately 15 minutes. Uptake of prothrombin was initially faster than that of antithrombin, although approximately equimolar amounts of prothrombin and antithrombin were bound by the intimamedia by 60 minutes. The results are discussed in relation to thrombin production and the demand for antithrombin by the damaged aorta wall in vivo.

Albumins↗
PubMed1995

Prothrombin Frankfurt: a dysfunctional prothrombin characterized by substitution of Glu-466 by Ala.

We have identified a patient with a dysfunctional prothrombin that we have designated Prothrombin Frankfurt. The proband was characterized by a prothrombin activity level of 13% and 20% compared to normal controls using two different assays with a normal prothrombin antigen level of 91% of normal controls. The genetic defect responsible for the abnormal prothrombin activity was determined by the polymerase chain reaction followed by single-strand conformation polymorphism (PCR-SSCP) analysis and by DNA sequence analysis of the human prothrombin gene. Substitution of a C for an A at nucleotide 10177 in the human prothrombin gene of the proband was identified, which results in the replacement of Glu-466 by Ala. The proband and one sister were homozygous for this mutation. Both parents, as well as one brother, were found to be heterozygous for this mutation. The same amino acid substitution was previously identified to be responsible for the dysfunctional protein Prothrombin Salakta and was hypothesized to result in altered substrate specificity. Four polymorphisms were also identified in the prothrombin gene from the proband when compared to the published sequence at nucleotides 554, 4048, 4272 and 10253.

Adult↗
PubMed1995

A point mutation (Arg271-->Cys) of a homozygote for dysfunctional prothrombin, prothrombin Obihiro, which has a region of high sequence variability.

The molecular defect of a congenitally dysfunctional form of prothrombin, prothrombin Obihiro, was identified in a patient with a severe bleeding tendency. He showed reduced fibrinogen clotting activity, despite a normal prothrombin antigen level. Nucleotide sequencing of amplified DNA revealed a C-->T change at nucleotide 7311 of exon VIII of the prothrombin gene. This resulted in the substitution of Arg271 by Cys at the factor Xa cleavage site which precludes normal activation of prothrombin Obihiro by factor Xa and the generation of thrombin. The proband was homozygous for this mutation. In addition, seven new nucleotide changes were identified in the prothrombin gene of this patient by comparison with the published gene sequence. Three polymorphisms at nucleotides 4291, 4298 and 7223 were exclusively identified in the prothrombin gene from Japanese. Particularly in the region containing exon VI and introns E and F (nucleotides 4048-4303), the prothrombin gene proved to be of high-sequence variability.

Base Sequence↗
PubMedDate not supplied

Levels of vitamin K, immunoreactive prothrombin, des-gamma-carboxy prothrombin and gamma-glutamyl carboxylase activity in hepatocellular carcinoma tissue.

To clarify the mechanism of production of des-gamma-carboxy (abnormal) prothrombin (DCP) by hepatocellular carcinoma (HCC), we measured the levels of vitamin K, DCP, immunoreactive prothrombin and the activity of gamma-glutamyl carboxylase in liver tissues from HCC patients and in the medium of cultured human hepatoma cells. There was no significant difference in vitamin K (K1, MK-4) contents between HCC and non-HCC cirrhotic liver tissues. The activity of gamma-glutamyl carboxylase per unit amount of endogenous microsomal prothrombin precursor was decreased in HCC tissue compared with non-HCC liver tissue (positive plasma DCP: 335 +/- 72 vs 372 +/- 67, negative plasma DCP: 370 +/- 84 vs 393 +/- 56 nmol/min per mg prothrombin precursor, P > 0.05), although the total incorporation of 14COOH into microsomal precursor protein was higher in the former. By contrast, levels of DCP and immunoreactive prothrombin in HCC tissue were greater (P < 0.05) than those in non-HCC cirrhotic liver tissue. Furthermore, production of large amounts of immunoreactive prothrombin was observed in human hepatoma cells huH-1 and huH-2, which produced large amounts of DCP. These results suggest that there was excessive synthesis of prothrombin precursors by human HCC tissue and hepatoma cell lines huH-1 and huH-2. Thus, excessive synthesis of prothrombin precursors seems to be the main mechanism of DCP production by HCC.

Aged↗
PubMed1987

Prothrombin Tokushima: characterization of dysfunctional thrombin derived from a variant of human prothrombin.

A mutant prothrombin, designated prothrombin Tokushima, was purified from plasma of a proband with 12% of normal plasma clotting activity and 42% of normal prothrombin antigen. The purified preparation gave a single band with the same mobility as that of "prothrombin" by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The factor Xa-catalyzed proteolysis of prothrombin Tokushima examined by SDS-PAGE was found to be identical to that of "prothrombin." Subsequently thrombin Tokushima was prepared by CM-Sepharose CL-6B column chromatography after prothrombin activation by factor Xa. The molecular weight of thrombin Tokushima estimated by SDS-PAGE was identical to that of "thrombin." Thrombin Tokushima exhibited less than 22% of normal clotting activity, and the value of kcat/Km (mumol/L-1 second-1) was less than one tenth of that of "thrombin" when Boc-Val-Pro-Arg-4-methylcoumaryl-7-amide was used as a substrate. However, active site titration using p-nitrophenyl-p'-guanidinobenzoate failed to detect any difference between the two. Thrombin Tokushima was 2.5% as effective as "thrombin" in inducing platelet aggregation. Interaction of thrombin Tokushima with antithrombin III was much slower than "thrombin" when followed by SDS-PAGE. Based on the residual thrombin activity, it was 33% as effective as "thrombin" in forming a complex with antithrombin III. These results indicate that the molecular defect resides in the thrombin portion of prothrombin Tokushima and that the binding sites for various substrates appear to be greatly impaired.

Blood Coagulation Disorders↗
PubMed1986

Prothrombin Clamart: prothrombin variant with defective Arg 320-IIe cleavage by factor Xa.

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.

Adult↗
PubMed1994

Evolution of prothrombin: isolation and characterization of the cDNAs encoding chicken and hagfish prothrombin.

The cDNA sequences of chicken and hagfish prothrombin have been determined. The sequences predict that prothrombin from both species is synthesized as a prepro-protein consisting of a putative Gla domain, two kringle domains, and a two-chain protease domain. Chicken and hagfish prothrombin share 51.6% amino acid sequence identity (313/627 residues). Both chicken and hagfish prothrombin are structurally very similar to human, bovine, rat, and mouse prothrombin and all six species share 41% amino acid sequence identity. Amino acid sequence alignments of human, bovine, rat, mouse, chicken, and hagfish prothrombin suggest that the thrombin B-chain and the propeptide-Gla domain are the regions most constrained for the common function(s) of vertebrate prothrombins.

Amino Acid Sequence↗
PubMed1993

Vitamin K-dependent carboxylase activity, prothrombin mRNA, and prothrombin production in two cultured rat hepatoma cell lines.

The presence of under-gamma-carboxylated forms of plasma prothrombin is a marker for human primary hepatocellular carcinoma. A rat hepatoma cell line (7777) which was previously shown to secrete undercarboxylated prothrombin when grown as a solid tumor has now been grown in monolayer culture. This cell line has a decreased activity of the microsomal vitamin K-dependent carboxylase when compared to a control (H4IIEC3) hepatoma line, does not increase intracellular prothrombin concentrations in response to vitamin K depletion, and secretes undercarboxylated prothrombin even when grown in vitamin K supplemented media. Prothrombin gene expression in the 7777 cell line, as measured by prothrombin mRNA levels, was not altered in the 7777 cell line. This cell line appears to be a model for assessing the cellular alterations responsible for undercarboxylated prothrombin excretion by human hepatocellular tumors.

Alkylation↗
PubMed1997

Purification and characterization of a variant of human prothrombin: prothrombin Segovia.

A dysprothrombin designated prothrombin Segovia was isolated from the plasma of an individual with normal prothrombin antigen and prothrombin activity lesser than 25% of the control prothrombin activity. Activation by prothrombinase complex showed a lower amidolytic than clotting activity, which suggests a lesser generation of active intermediates than normal prothrombin. When prothrombin Segovia was activated by prothrombinase complex in the absence of factor Va, no thrombin formation was found by functional activities. SDS-PAGE analysis of the molecules derived by activation with prothrombinase complex, Taipan snake venom and Echis carinatus venom showed an accumulation of molecules not cleaved at bond Arg320-Ile321. This was more evident with Echis carinatus venom, which only acts on this bond. Our data suggest that the alteration of prothrombin Segovia impairs the scission of bond Arg320-Ile321.

Blood Coagulation↗