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European Journal of Biochemistry May 1997Molecules containing the 33-kDa plasma protein alpha1-microglobulin were isolated from human plasma by anti-(alpha1-microglobulin) affinity chromatography. Five major...
Molecules containing the 33-kDa plasma protein alpha1-microglobulin were isolated from human plasma by anti-(alpha1-microglobulin) affinity chromatography. Five major bands could be seen after electrophoretic separation of the alpha1-microglobulin-containing proteins under native conditions. Immunoblotting demonstrated alpha1-microglobulin in all five bands. Two of these have been described previously: free alpha1-microglobulin and alpha1-microglobulin complexed with IgA (IgA x alpha1-microglobulin). The other three bands were identified as prothrombin alpha1-microglobulin, albumin x alpha1-microglobulin and dimeric alpha1-microglobulin. Prothrombin x alpha1-microglobulin were 1:2 and 1:1 complexes which carried approximately 1% of total alpha1-microglobulin, had molecular masses of about 145 kDa and 110 kDa upon SDS/PAGE and dissociated completely to free alpha1-microglobulin and prothrombin (72 kDa) when reducing agents were added, suggesting that the complexes were stabilized by disulfide bonds. The alpha1-microglobulin molecules did not inhibit cleavage of prothrombin by factor Xa and were bound to the peptides which were released upon activation of prothrombin. Albumin x alpha1-microglobulin, corresponding to 7% of total plasma alpha1-microglobulin, was a mixture between 1:1 and 1:2 complexes, with masses upon SDS/PAGE of approximately 100 kDa and 135 kDa, respectively. Both these complexes dissociated only partially to free alpha1-microglobulin and albumin when reducing agents were added. The albumin x alpha1-microglobulin complexes carried a yellow-brown chromophore similar to free alpha1-microglobulin. The complex-binding to alpha1-microglobulin did not block the fatty-acid-binding ability of albumin. The plasma concentrations of albumin x alpha1-microglobulin and prothrombin x alpha1-microglobulin were estimated to 5.2 mg/l and 1.1 mg/l, respectively.
Topics: Alpha-Globulins; Humans; Immunoglobulin A; Macromolecular Substances; Protein Binding; Prothrombin; Serum Albumin
PubMed: 9183005
DOI: 10.1111/j.1432-1033.1997.00676.x -
Cellular Physiology and Biochemistry :... 2014Des-γ-carboxy prothrombin (DCP) is a prothrombin precursor produced in hepatocellular carcinoma (HCC). Because of deficiency of vitamin K or γ-glutamyl carboxylase in... (Review)
Review
Des-γ-carboxy prothrombin (DCP) is a prothrombin precursor produced in hepatocellular carcinoma (HCC). Because of deficiency of vitamin K or γ-glutamyl carboxylase in HCC cells, the 10 glutamic acid (Glu) residues in prothrombin precursor did not completely carboxylate to γ-carboxylated glutamic acid (Gla) residues, leaving some Glu residues remained in N-terminal domain. These prothrombin precursors with Glu residues are called DCPs. DCP displays insufficient coagulation activity. Since Liebman reported an elevated plasma DCP in patients with HCC, DCP has been used in the diagnosis of HCC. Recently, its biological malignant potential has been specified to describe DCP as an autologous growth factor to stimulate HCC growth and a paracrine factor to integrate HCC with vascular endothelial cells. DCP was found to stimulate HCC growth through activation of the DCP-Met-JAK1-STAT3 signaling pathway. DCP might increase HCC invasion and metastasis through activation of matrix metalloproteinase (MMPs) and the ERK1/2 MAPK signaling pathway. DCP has also been found to play a crucial role in the formation of angiogenesis. DCP could increase the angiogenic factors released from HCC and vascular endothelial cells. These effects of DCP in angiogenesis might be related to activation of the DCP-KDR-PLC-γ-MAPK signaling pathway. In this article, we summarized recent studies on DCP in biological roles related to cancer progression and angiogenesis in HCC.
Topics: Biomarkers; Biomarkers, Tumor; Carcinoma, Hepatocellular; Growth Substances; Humans; Liver Neoplasms; Molecular Structure; Protein Precursors; Prothrombin
PubMed: 25200250
DOI: 10.1159/000366308 -
Clinical and Applied... 2021The aim of this retrospective study was to compare andexanet alfa and 4-factor prothrombin complex (4F-PCC) for reversal of factor Xa (FXa)-inhibitor bleeding. Patients...
The aim of this retrospective study was to compare andexanet alfa and 4-factor prothrombin complex (4F-PCC) for reversal of factor Xa (FXa)-inhibitor bleeding. Patients that received andexanet alfa for reversal were included. An equivalent number of patients administered 4F-PCC for FXa-inhibitor bleeding were randomly selected as historical controls. The primary outcome was effective hemostasis achievement within 12 h, defined using ANNEXA-4 criteria. Thromboembolic events and mortality within 30 days were also evaluated. A total of 32 patients were included. Baseline characteristics were not statistically different between andexanet alfa (n = 16) and 4F-PCC (n = 16). Intracranial bleeding was the primary reversal indication in 43.8% versus 62.5% of patients, respectively. Effective hemostasis was reached in 75.0% of andexanet alfa patients compared to 62.5% of 4F-PCC patients ( = .70). Thromboembolic events occurred in 4 (25.0%) patients and 3 (18.8%) patients, respectively ( = .99). Mortality incidence was 12.5% and 31.3%, respectively ( = .39). Andexanet alfa and 4F-PCC attained hemostasis in a majority of patients. A high, but a similar rate of thromboembolic events was seen with both treatments. Prospective studies are needed to elucidate comparative risks and benefits of the 2 agents.
Topics: Aged; Cohort Studies; Factor Xa; Female; Hemorrhage; Humans; Male; Prothrombin; Recombinant Proteins; Retrospective Studies
PubMed: 34541920
DOI: 10.1177/10760296211039020 -
Cellular and Molecular Life Sciences :... Dec 2006Snake envenomation is a socio-medical problem of considerable magnitude. About 2.5 million people are bitten by snakes annually, more than 100,000 fatally. However,... (Review)
Review
Snake envenomation is a socio-medical problem of considerable magnitude. About 2.5 million people are bitten by snakes annually, more than 100,000 fatally. However, although bites can be deadly, snake venom is a natural biological resource that contains several components of potential therapeutic value. Venom has been used in the treatment of a variety of pathophysiological conditions in Ayurveda, homeopathy and folk medicine. With the advent of biotechnology, the efficacy of such treatments has been substantiated by purifying components of venom and delineating their therapeutic properties. This review will focus on certain snake venom components and their applications in health and disease.
Topics: Animals; Cardiovascular System; Disintegrins; Fibrinogen; Hemostasis; Lectins, C-Type; Muscles; Neurotoxins; Phospholipases A; Platelet Aggregation Inhibitors; Protein Structure, Tertiary; Prothrombin; Signal Transduction; Snake Venoms; Thrombin
PubMed: 17103111
DOI: 10.1007/s00018-006-6315-0 -
PloS One 2016Previous studies have shown a unique method to disrupt tumor vasculature using pulsed, high-pressure amplitude therapeutic ultrasound combined with microbubbles. In this...
Previous studies have shown a unique method to disrupt tumor vasculature using pulsed, high-pressure amplitude therapeutic ultrasound combined with microbubbles. In this study, we attempted to destroy the prostate vasculature of canine prostates using microbubble-enhanced ultrasound (MEUS) and prothrombin. The prostates of 43 male mongrel canines were surgically exposed. Twenty-two prostates were treated using MEUS (n = 11) or MEUS and prothrombin (PMEUS, n = 11). The other 21 prostates, which were treated using microbubbles (n = 7), ultrasound (n = 7) or prothrombin (n = 7) only, served as the controls. Prothrombin was intravenously infused at 20 IU/kg. MEUS was induced using a therapeutic ultrasound device at a peak negative pressure of 4.47 MPa and a microbubble injection. Contrast-enhanced ultrasound was performed to assess the blood perfusion of the prostates. Then, the prostate tissue was harvested immediately after treatment and at 48 hours later for pathological examination. The contrast-enhanced ultrasound peak value of the prostate decreased significantly from 36.2 ± 5.6 to 27.1 ± 6.3 after treatment in the PMEUS group, but it remained unchanged in the other groups. Histological examination found severe microvascular rupture, hemorrhage and thrombosis in both MEUS- and PMEUS-treated prostates immediately after treatment, while disruption in the PMEUS group was more severe than in the MEUS group. Forty-eight hours after treatment, massive necrosis and infiltration of white blood cells occurred in the PMEUS group. This study demonstrated that PMEUS disrupted the normal microvasculature of canine prostates and induced massive necrosis. PMEUS could potentially become a new noninvasive method used for the treatment of benign prostatic hyperplasia.
Topics: Animals; Dogs; High-Intensity Focused Ultrasound Ablation; Male; Microbubbles; Microvessels; Prostate; Prostatic Hyperplasia; Prothrombin
PubMed: 27643992
DOI: 10.1371/journal.pone.0162398 -
Biochemia Medica Oct 2019Failure to obtain complete blood clotting in serum is a common laboratory problem. Our aim was to determine whether snake proth-rombin activators are effective in...
INTRODUCTION
Failure to obtain complete blood clotting in serum is a common laboratory problem. Our aim was to determine whether snake proth-rombin activators are effective in clotting blood and producing quality serum for analyte measurement in anticoagulated patients.
MATERIALS AND METHODS
Whole blood clotting was studied in a total of 64 blood samples (41 controls, 20 Warfarin patients, 3 anticoagulated patients using snake venom prothrombin activator (OsPA)) with plain tubes. Coagulation was analysed using a visual assay, Hyland-Clotek and thromboelastography. Healthy control blood was spiked with a range of anticoagulants to determine the effectiveness of OsPa-induced clotting. A paired analysis of a Dabigatran patient and a control investigated the effectiveness of the OsPA clotting tubes. Biochemical analytes (N = 31) were determined for 7 samples on chemistry and immunoassay analysers and compared with commercial tubes.
RESULTS
Snake venom prothrombin activators efficiently coagulated blood and plasma spiked with heparin and commonly used anticoagulants. Clotting was observed in the presence of anticoagulants whereas no clotting was observed in BDRST tubes containing 3 U/mL of heparin. Snake venom prothrombin activator enhanced heparinised blood clotting by shortening substantially the clotting time and improving significantly the strength of the clot. Comparison of 31 analytes from the blood of five healthy and two anticoagulated participants gave very good agreement between the analyte concentrations determined.
CONCLUSIONS
Our results showed that the snake venom prothrombin activators OsPA and PtPA efficiently coagulated recalcified and fresh bloods with or without added anticoagulants. These procoagulants produced high quality serum for accurate analyte measurement.
Topics: Anticoagulants; Blood Coagulation; Blood Coagulation Tests; Heparin; Humans; Prothrombin
PubMed: 31624459
DOI: 10.11613/BM.2019.030706 -
Thrombosis Research Mar 1997A dysprothrombin designated prothrombin Segovia was isolated from the plasma of an individual with normal prothrombin antigen and prothrombin activity lesser than 25% of...
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.
Topics: Blood Coagulation; Electrophoresis, Polyacrylamide Gel; Endopeptidases; Enzyme Activation; Factor V; Factor Va; Factor X; Factor Xa; Fibrinolytic Agents; Humans; Molecular Weight; Prothrombin; Thrombin; Thromboplastin; Viper Venoms
PubMed: 9101639
DOI: 10.1016/s0049-3848(97)00036-4 -
Blood Sep 1987Prothrombin synthesis and secretion were studied in a human hepatoma cell line (Hep G2) incubated with 35S-methionine for 2 to 24 hours at 37 degrees C. Extracellular...
Prothrombin synthesis and secretion were studied in a human hepatoma cell line (Hep G2) incubated with 35S-methionine for 2 to 24 hours at 37 degrees C. Extracellular and intracellular prothrombin were detected by immunoprecipitation with affinity-purified antiprothrombin antibody. Incorporation of 35S-methionine into prothrombin was monitored by counting specific bands excised from 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Prothrombin represented 0.3% to 0.7% of total newly synthesized protein secreted into the media. Warfarin had no effect on total prothrombin synthesis (extracellular plus intracellular). However, warfarin inhibited secretion of newly synthesized prothrombin by 58% to 73% over a 2 to 4 hour period. This was accompanied by the intracellular accumulation of an immunoprecipitable species of prothrombin of 78 kd, 6 kd less than extracellular prothrombin. At the end of the 4-hour incubation with warfarin, intracellular prothrombin increased from 44% to 82% (twofold) of total prothrombin, whereas extracellular prothrombin decreased from 56% to 19% (threefold) of total prothrombin. After 24-hour incubation with warfarin, intracellular and extracellular immunoprecipitable prothrombin approached control values. Deglycosylation of extracellular and intracellular prothrombin with hydrofluoric acid (HF) resulted in a decrease in mol wt for both species to 66 kd. Endoglycosidase-H treatment, which digests "early mannosyl" residues, resulted in a decrease in the mol wt of the intracellular species of 8 kd with no effect on the extracellular species. Thus, the lower mol wt intracellular species that accumulates following early warfarin treatment is due to the presence of incompletely processed carbohydrate chain. The data are compatible with the hypothesis that optimum glycosylation and secretion require Vitamin K-dependent carboxylation.
Topics: Acetylglucosaminidase; Carcinoma, Hepatocellular; Cell Line; Cycloheximide; Glycosylation; Humans; Liver Neoplasms; Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; Molecular Weight; Protein Biosynthesis; Prothrombin; Time Factors; Warfarin
PubMed: 3040156
DOI: No ID Found -
Blood May 2000Association studies suggest that the G20210A mutation (G to A substitution at nucleotide position 20210) in the prothrombin gene (PT) is associated with increased plasma...
Association studies suggest that the G20210A mutation (G to A substitution at nucleotide position 20210) in the prothrombin gene (PT) is associated with increased plasma prothrombin activity and with increased risk for venous thromboembolism. To test directly for linkage between this PT variant and plasma prothrombin activity we performed a family-based study. The G20210A genotypes and plasma prothrombin activity levels were determined in 435 individuals belonging to 22 extended Spanish families. The sample was composed of 388 homozygous (G/G) normal individuals and 43 heterozygote (G/A) and 4 homozygote (A/A) carriers for the G20210A mutation. The results of variance-component linkage analysis yielded a highly significant lod score of 3.6 (P = 2.4 x 10(-5)) between this mutation and a quantitative trait locus (QTL) that influences prothrombin activity. Importantly, a conditional linkage analysis that simultaneously accounted for association with the G20210A variant completely eliminated the linkage signal, which indicates that this mutation affects the function of the prothrombin gene. Additionally, a bivariate linkage analysis of plasma prothrombin activity and thrombosis significantly improved the linkage signal for prothrombin activity (lod score = 4.7; P = 1.5 x 10(-6)) and provided strong evidence that this QTL has a pleiotropic effect on the risk of thrombosis (lod score = 2.43; P =.0004). These results represent the first direct genetic evidence that a QTL in the PT gene influences prothrombin activity levels and susceptibility to thrombosis and strongly support the conclusion that G20210A is a functional polymorphism. (Blood. 2000;95:2780-2785)
Topics: Adult; Amino Acid Substitution; Analysis of Variance; Family; Female; Genetic Carrier Screening; Genetic Linkage; Heterozygote; Homozygote; Humans; Linkage Disequilibrium; Lod Score; Male; Point Mutation; Prothrombin; Quantitative Trait, Heritable; Risk Factors; Spain; Thrombosis
PubMed: 10779421
DOI: No ID Found -
Haemostasis 2001We have recently determined the complete amino acid sequence of trocarin, a group D prothrombin activator from the venom of Tropidechis carinatus (Australian... (Review)
Review
We have recently determined the complete amino acid sequence of trocarin, a group D prothrombin activator from the venom of Tropidechis carinatus (Australian rough-scaled snake). This proteinase is both functionally and structurally similar to mammalian blood coagulation factor Xa. It shows approximately 70% homology and possesses the characteristic Gla domain, two EGF domains and serine proteinase domain. To examine structure-function relationships, we generated a molecular model of trocarin based on a human factor Xa des-Gla crystal structure (1xka) as template. Based on known sites of interaction between mammalian factor Xa, factor Va and prothrombin, structure-function relationships of trocarin were explored. Unlike factor Xa, trocarin is glycosylated and has a large carbohydrate moiety at the entrance to its active site pocket. This might contribute to differences observed in the kinetics of hydrolysis of synthetic substrates by trocarin as compared to human factor Xa. A Ca(2+)-binding loop present in the heavy chain of factor Xa also seems to be lost in trocarin. In addition to its role in hemostasis, factor Xa shows other biological effects, including inflammation via its interaction with effector protease receptor-1 (EPR-1). Interestingly, the EPR-1 recognition site is distinctly different in trocarin, the functional consequences of which are being investigated.
Topics: Animals; Binding Sites; Coagulants; Factor Xa; Humans; Protein Processing, Post-Translational; Prothrombin; Sequence Homology, Nucleic Acid; Snake Venoms
PubMed: 11910190
DOI: 10.1159/000048068