Study of Optimum Condition for Rapid Preparation of Thrombin using Russell’s Viper Venom Factor X Activator

Background: The purpose of this study is to investigate a simple method with the optimum condition for rapid thrombin preparation from Cryoprecipitate-depleted Plasma (CDP) using RVV-X in the process. Methods: Thrombin preparation from human CDP was studied with the presence of different factors in batch condition including: 1) RVV-X; 2) volume of calcium chloride solution; 3) volume of sodium chloride solution for final extraction; and 4) incubation time. The properties of the prepared sample were analyzed for fibrin clot formation, total protein by Kjeldahl method, thrombin time, molecular weight and protein patterns by SDS-PAGE, and thrombin concentration by coagulation analyzer. The method and process of preparing thrombin and the study of optimum condition for rapidly preparing the highest yield of thrombin from starting CDP 100 ml were introduced. Results: The best four conditions were concluded: 1) RVV-X 50 mcg should be present in the process; 2) volume of 0.25 M calcium chloride should be 3 ml; 3) volume of 0.85% sodium chloride for the final protein precipitate extraction should be 10 ml and; 4) no incubation time needed for prothrombin activation process. A solution prepared from the optimum condition showed an obvious band on SDS-PAGE at a molecular weight about 36,000 Da which is our target protein thrombin. The prepared solution had a total protein content of 0.065 g/dl and gave satisfactory results of thrombin time (9 seconds) and fibrin clot formation. The test results of thrombin concentration between the method with and without incubation time were 269.4 and 295.2 IU/ml, respectively. Conclusion: This result showed that the method with RVV-X but without incubation time for prothrombin activation (optimum condition) gave the highest yield of thrombin.

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Plasmin effect on platelet glycoprotein Ib-von Willebrand factor interactions

Blood ◽

10.1182/blood.v65.1.32.bloodjournal65132 ◽

1985 ◽

Vol 65 (1) ◽

pp. 32-40 ◽

Cited By ~ 20

Author(s):

B Adelman ◽

AD Michelson ◽

J Loscalzo ◽

J Greenberg ◽

RI Handin

Keyword(s):

Molecular Weight ◽

Von Willebrand Factor ◽

Maximum Rate ◽

Enzyme Linked Immunosorbent Assay ◽

Thrombin Time ◽

Proteolytic Fragment ◽

Platelet Surface ◽

Sds Page ◽

Von Willebrand ◽

Willebrand Factor

We have studied the effect of streptokinase on platelets in platelet- rich plasma (PRP) and of plasmin on washed platelets. By three and one- half minutes after the addition of 50,000 IU/mL streptokinase to PRP, the maximum rate of ristocetin-induced platelet agglutination declined 40%, and by 60 minutes, it declined 70%. During the same time interval, the thrombin time increased from 20 seconds to over 120 seconds. At a concentration as low as 50 IU/mL, streptokinase reduced the maximum rate of ristocetin-induced platelet agglutination by 50% and prolonged the thrombin time to 1.5 times control value. Streptokinase added to PRP also caused inhibition of platelet aggregation following stimulation by 2.9 mumol/L adenosine diphosphate, 0.25 U/mL thrombin, and 0.025 mg/mL collagen. Plasmin, 0.05 to 1.0 CU/mL, reduced ristocetin-mediated agglutination of washed platelets in the presence of von Willebrand factor (vWF) from 66% of control to 2% of control, following a one-hour incubation. Autoradiograms produced following sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) of plasmin- treated 125I-surface-labeled platelets demonstrated progressive loss of a protein with a molecular weight (mol wt) of 180,000; simultaneously, a protein with mol wt 135,000 appeared on autoradiograms produced following SDS-PAGE of the surrounding platelet medium. These proteins are similar in molecular weight to glycoprotein (gp) Ib, a platelet surface receptor for vWF, and glycocalicin, a proteolytic fragment of gpIb. By use of an enzyme-linked immunosorbent assay (ELISA) based immunoinhibition assay for glycocalicin, we were able to demonstrate that plasmin treatment of washed platelets released a glycocalicin- related antigen into the surrounding medium and that appearance of this material corresponding to loss of vWF-dependent, ristocetin-induced agglutination.

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A novel fibrinogen γ chain frameshift deletion (c.637delT) in a patient with hypodysfibrinogenemia associated with thrombosis

Hämostaseologie ◽

10.1055/s-0037-1619826 ◽

2015 ◽

Vol 35 (S 01) ◽

pp. S27-S31

Author(s):

A. Biswas ◽

H. Ensikat ◽

U. Schmitt ◽

S. Horneff ◽

A. Pavlova ◽

...

Keyword(s):

Direct Sequencing ◽

Amino Acid Position ◽

Fibrin Clot ◽

Time Shift ◽

Thrombin Time ◽

Vein Thrombosis ◽

Increased Risk ◽

Sds Page ◽

Chain Polymerization ◽

Clot Structure

SummaryInherited fibrinogen (FG) disorders are rare and result in quantitative or/and qualitative FG deficiency. While the majority of patients with clinically relevant FG deficiencies demonstrate a bleeding phenotype, a subset of patients are at increased risk of thrombosis.We report a 54-years old man presenting with a thrombophilic phenotype characterized by two episodes of unprovoked venous thrombosis and a deep vein thrombosis several weeks after myocardial infarction. Recently, he developed A. carotis communis thrombosis and died. Coagulation tests were done using standard procedures. FG genes were screened using direct sequencing. Effect on fibrin clot structure was analyzed by scanning electron microscopy (SEM) and FG chain polymerization was analysed using SDS-PAGE.While thrombophilia testing was negative, we found a decreased concentration of clottable FG (126-148 mg/dl) compared to FG antigen (182-194 mg/dl of normal). The thrombin time was slightly prolonged, while aPTT and reptilase time were within the normal range. A novel deletion in FGG gene (c.637delT) resulting in a frameshift and the premature termination of the γ chain at amino acid position p.228 was identified. SDS-PAGE showed a time-shift in γ-γ and α-α cross linking. SEM showed no statistically significant differences between the patient´s and a healthy control´s fibrin clot structure.In addition to the reduction of FG concentration expected by the nature of the mutation also a functional defect (hypodysfibrinogenemia) was found. Moreover this mutation seems to increase the risk of thrombosis warranting long term anticoagulation possibly in a combination with antiplatelet drugs.

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Plasmin effect on platelet glycoprotein Ib-von Willebrand factor interactions

Blood ◽

10.1182/blood.v65.1.32.32 ◽

1985 ◽

Vol 65 (1) ◽

pp. 32-40 ◽

Cited By ~ 134

Author(s):

B Adelman ◽

AD Michelson ◽

J Loscalzo ◽

J Greenberg ◽

RI Handin

Keyword(s):

Molecular Weight ◽

Von Willebrand Factor ◽

Maximum Rate ◽

Enzyme Linked Immunosorbent Assay ◽

Thrombin Time ◽

Proteolytic Fragment ◽

Platelet Surface ◽

Sds Page ◽

Von Willebrand ◽

Willebrand Factor

Abstract We have studied the effect of streptokinase on platelets in platelet- rich plasma (PRP) and of plasmin on washed platelets. By three and one- half minutes after the addition of 50,000 IU/mL streptokinase to PRP, the maximum rate of ristocetin-induced platelet agglutination declined 40%, and by 60 minutes, it declined 70%. During the same time interval, the thrombin time increased from 20 seconds to over 120 seconds. At a concentration as low as 50 IU/mL, streptokinase reduced the maximum rate of ristocetin-induced platelet agglutination by 50% and prolonged the thrombin time to 1.5 times control value. Streptokinase added to PRP also caused inhibition of platelet aggregation following stimulation by 2.9 mumol/L adenosine diphosphate, 0.25 U/mL thrombin, and 0.025 mg/mL collagen. Plasmin, 0.05 to 1.0 CU/mL, reduced ristocetin-mediated agglutination of washed platelets in the presence of von Willebrand factor (vWF) from 66% of control to 2% of control, following a one-hour incubation. Autoradiograms produced following sodium dodecyl-polyacrylamide gel electrophoresis (SDS-PAGE) of plasmin- treated 125I-surface-labeled platelets demonstrated progressive loss of a protein with a molecular weight (mol wt) of 180,000; simultaneously, a protein with mol wt 135,000 appeared on autoradiograms produced following SDS-PAGE of the surrounding platelet medium. These proteins are similar in molecular weight to glycoprotein (gp) Ib, a platelet surface receptor for vWF, and glycocalicin, a proteolytic fragment of gpIb. By use of an enzyme-linked immunosorbent assay (ELISA) based immunoinhibition assay for glycocalicin, we were able to demonstrate that plasmin treatment of washed platelets released a glycocalicin- related antigen into the surrounding medium and that appearance of this material corresponding to loss of vWF-dependent, ristocetin-induced agglutination.

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Isolasi dan Karakterisasi Inulinase dari Aspergillus niger Gmn11.1 Galur Lokal

Jurnal Natur Indonesia ◽

10.31258/jnat.11.1.19-23 ◽

2012 ◽

Vol 11 (1) ◽

pp. 19 ◽

Cited By ~ 1

Author(s):

Saryono Saryono

Keyword(s):

Molecular Weight ◽

Aspergillus Niger ◽

Incubation Time ◽

Gel Filtration ◽

Deae Cellulose ◽

Salt Precipitation ◽

Sds Page ◽

Optimum Ph ◽

Relative Molecular Weight ◽

Optimum Ph And Temperature

Inulin is a naturally potential polysaccharide used to produced fructose and fructooligosaccharide. Inulinaseknown also as ß-fructosidase can hydrolise inulin to fructose or fructooligosaccharide. Inulinase production fromAspergillus niger Gmn11.1 isolated from dahlia tubers is conducted using medium containing 1% inulin and 0,2%yeast extract. The crude enzyme (filtrate culture) is purified by means of ammonium sulphate salt precipitation,followed by Sephadex G25 gel filtration column chromatography and DEAE cellulose anion exchanger columnchromatography. The result indicated that the enzyme had optimum pH and temperature of 4,6 and 450C, respectivelywith incubation time of 15 hours. The Km and Vmaxs values obtained from this experiment are 20 mg/ml and 0,769mg/ml/hours, respectively. Whereas the relative molecular weight of inulinase was monitored by SDS PAGE is 63KDa.

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Parallel Reduction of Plasma Levels of High and Low Molecular Weight Kininogen in Patients with Cirrhosis

Thrombosis and Haemostasis ◽

10.1055/s-0037-1614849 ◽

1999 ◽

Vol 82 (11) ◽

pp. 1428-1432 ◽

Cited By ~ 9

Author(s):

Cheryl Scott ◽

Francesco Salerno ◽

Elettra Lorenzano ◽

Werner Müller-Esterl ◽

Angelo Agostoni ◽

...

Keyword(s):

Molecular Weight ◽

Liver Disease ◽

Liver Function ◽

Plasma Levels ◽

Plasma Concentrations ◽

Normal Subjects ◽

Low Molecular Weight ◽

Major Band ◽

Sds Page ◽

Normal Controls

SummaryLittle is known about the regulation of high-molecular-weight-kininogen (HK) and low-molecular-weight-kininogen (LK) or the relationship of each to the degree of liver function impairment in patients with cirrhosis. In this study, we evaluated HK and LK quantitatively by a recently described particle concentration fluorescence immunoassay (PCFIA) and qualitatively by SDS PAGE and immunoblotting analyses in plasma from 33 patients with cirrhosis presenting various degrees of impairment of liver function. Thirty-three healthy subjects served as normal controls. Patients with cirrhosis had significantly lower plasma levels of HK (median 49 μg/ml [range 22-99 μg/ml]) and LK (58 μg/ml [15-100 μg/ml]) than normal subjects (HK 83 μg/ml [65-115 μg/ml]; LK 80 μg/ml [45-120 μg/ml]) (p < 0.0001). The plasma concentrations of HK and LK were directly related to plasma levels of cholinesterase (P < 0.0001) and albumin (P < 0.0001 and P < 0.001) and inversely to the Child-Pugh score (P < 0.0001) and to prothrombin time ratio (P < 0.0001) (reflecting the clinical and laboratory abnormalities in liver disease). Similar to normal individuals, in patients with cirrhosis, plasma HK and LK levels paralleled one another, suggesting that a coordinate regulation of those proteins persists in liver disease. SDS PAGE and immunoblotting analyses of kininogens in cirrhotic plasma showed a pattern similar to that observed in normal controls for LK (a single band at 66 kDa) with some lower molecular weight forms noted in cirrhotic plasma. A slight increase of cleavage of HK (a major band at 130 kDa and a faint but increased band at 107 kDa) was evident. The increased cleavage of HK was confirmed by the lower cleaved kininogen index (CKI), as compared to normal controls. These data suggest a defect in hepatic synthesis as well as increased destructive cleavage of both kininogens in plasma from patients with cirrhosis. The decrease of important regulatory proteins like kininogens may contribute to the imbalance in coagulation and fibrinolytic systems, which frequently occurs in cirrhotic patients.

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Heterozygous Abnormal Fibrinogen Osaka III with the Replacement of γ Arginine-275 by Histidine Has an Apparently Higher Molecular Weight γ-Chain Variant

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646313 ◽

1992 ◽

Vol 68 (05) ◽

pp. 534-538 ◽

Cited By ~ 5

Author(s):

Nobuhiko Yoshida ◽

Shingi Imaoka ◽

Hajime Hirata ◽

Michio Matsuda ◽

Shinji Asakura

Keyword(s):

Molecular Weight ◽

Sodium Dodecyl Sulfate ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Tetraacetic Acid ◽

Fibrin Monomer ◽

Sds Page ◽

Thrombotic Tendency ◽

Chain Variant

SummaryCongenitally abnormal fibrinogen Osaka III with the replacement of γ Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of α- and γ-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal γ-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 γ remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethy-lenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

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Isolation and Structural Charaderization of a Potent Inhibitor of Coagulation Factor Xa from the Leech Haementeria ghilianii

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646610 ◽

1989 ◽

Vol 61 (03) ◽

pp. 437-441 ◽

Cited By ~ 28

Author(s):

Cindra Condra ◽

Elka Nutt ◽

Christopher J Petroski ◽

Ellen Simpson ◽

P A Friedman ◽

...

Keyword(s):

Molecular Weight ◽

Salivary Gland ◽

Amino Acid ◽

Amino Acid Sequence ◽

Western Blot ◽

Potent Inhibitor ◽

Factor Xa ◽

Coagulation Factor ◽

Sds Page ◽

Bovine Factor

SummaryThe present work reports the discovery and charactenzation of an anticoagulant protein in the salivary gland of the giant bloodsucking leech, H. ghilianii, which is a specific and potent inhibitor of coagulation factor Xa. The inhibitor, purified to homogeneity, displayed subnanomolar inhibition of bovine factor Xa and had a molecular weight of approximately 15,000 as deduced by denaturing SDS-PAGE. The amino acid sequence of the first 43 residues of the H. ghilianii derived inhibitor displayed a striking homology to antistasin, the recently described subnanomolar inhibitor of factor Xa isolated from the Mexican leech, H. officinalis. Antisera prepared to antistasin cross-reacted with the H. ghilianii protein in Western Blot analysis. These data indicate that the giant Amazonian leech, H. ghilianii, and the smaller Mexican leech, H. officinalrs, have similar proteins which disrupt the normal hemostatic clotting mechanisms in their mammalian host’s blood.

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Chrono-Pharmacological Study of Once Daily Curative Dose of a Low Molecular Weight Heparin (200IU antiXa/kg of Dalteparin) in Ten Healthy Volunteers

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649794 ◽

1995 ◽

Vol 74 (02) ◽

pp. 660-666 ◽

Cited By ~ 15

Author(s):

P Mismetti ◽

J Reynaud ◽

B Tardy-Poncet ◽

S Laporte-Simitsidis ◽

M Scully ◽

...

Keyword(s):

Molecular Weight ◽

Healthy Volunteers ◽

Low Molecular Weight Heparin ◽

Pharmacological Study ◽

Area Under The Curve ◽

Factor Xa ◽

Similar Efficacy ◽

Low Molecular Weight ◽

Thrombin Time ◽

Once Daily

SummaryLow molecular weight heparin (LMWH) is currently prescribed for the treatment of deep vein thrombosis at the dose of 100 IU antiXa/kg twice daily or at a dose of 175 IU antiXa/kg once daily with a similar efficacy. We decided to study the chrono-pharmacology of curative dose of LMWH once daily administrated according to the one previously described with unfractionated heparin (UFH).Ten healthy volunteers participated in an open three-period crossover study according to three 24 h cycles, separated by a wash-out interval lasting 7 days: one control cycle without injection, two cycles with subcutaneous injection of 200 IU antiXa/kg of Dalteparin (Fragmin®) at 8 a.m. or at 8 p.m. Parameters of heparin activity were analysed as maximal values and area under the curve.Activated partial thromboplastin time (APTT), thrombin time (TT), prothrombin time (PT) and tissue factor pathway inhibitor (TFPI) were higher after 8 p.m. injection than after 8 a.m. injection (p <0.05) while no chrono-pharmacological variation of anti factor Xa (AXa) activity was observed. Thus the biological anticoagulant effect of 200 IU antiXa/kg of Dalteparin seems to be higher after an evening injection than after a morning injection.A chrono-therapeutic approach with LMWH, as prescribed once daily, deserves further investigation since our results suggest that a preferential injection time may optimise the clinical efficacy of these LMWH.

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