Studies on Fibrin Formation and Effects of Dextran

1972 ◽  
Vol 28 (02) ◽  
pp. 244-256 ◽  
Author(s):  
T. Z Muzaffar ◽  
G. G Youngson ◽  
W. A. J Bryce ◽  
D. P Dhall
Keyword(s):  
Molecular Weight ◽  
Enzymatic Activity ◽  
Human Platelet ◽  
Clot Formation ◽  
Human Fibrinogen ◽  
Platelet Poor Plasma ◽  
Fibrin Formation ◽  
Fibrin Monomer ◽  
Kinetics Of ◽  
Three Phases

SummaryIn this study highly reproducible turbidimetric techniques are used to investigate clotting of human platelet-poor plasma and purified human fibrinogen solutions by thrombin. Three phases are clearly distinguishable turbidimetrically during clotting. All the three phases are altered by dextran. This effect of dextran is not mediated by any action on the enzymatic activity of thrombin. Further, it is independent of the molecular weight of dextran, but is found related to the final dextran, thrombin and fibrinogen concentrations. Evidence is presented to show that dextran accelerates the polymerisation of fibrin monomer. However, it seems clear that dextran has an additional action quite apart from this effect. These actions of dextran are discussed in relation to kinetics of clot formation.

The Association Of Thrombin With Fibrin And Its Solubilization During Plasmic Digestion

1981 ◽  
Author(s):  
Charles W Francis ◽  
Victor J Marder ◽  
Grant H Barlow ◽  
S Eric Martin
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Enzymatic Activity ◽  
Free Enzyme ◽  
Active Enzyme ◽  
Clot Formation ◽  
Human Fibrinogen ◽  
Soluble Fibrin ◽  
Human Thrombin ◽  
Fibrin Clots

It has previously been demonstrated that thrombin binds to fibrin during clot formation. We have studied the nature of this association and the fate of bound thrombin during fibrin degradation by plasmin. Approximately 10% of human thrombin used to clot human fibrinogen bound to the fibrin and could not be removed by washing in buffers of physiologic pH and ionic strength. Plasmic digestion released thrombin into the lysate where it retained enzymatic activity, as measured by chromogenic and clotting assays. The fibrin was degraded to a group of unique degradation complexes of molecular weight between 230,000 and greater than 800,000. The active thrombin was present in the lysate both as free enzyme and bound to certain of these complexes, primarily those larger than 230,000 daltons. At low initial thrombin concentrations, most of the active enzyme released during plasmic digestion was bound to fibrin derivatives, while at higher initial thrombin concentrations, most was released as free enzyme. This suggests that there are at least two types and/or sites of association between thrombin and fibrin. The presence of thrombin on fibrin clots and bound to soluble fibrin derivatives may have pathophysiologic importance in the propagation of thrombi and in the development of hypercoagulable states.

scholarly journals Fibrin formation: effect of calcium ions

Blood ◽  
1978 ◽  
Vol 52 (4) ◽  
pp. 654-658 ◽  
Author(s):  
EP Brass ◽  
WB Forman ◽  
RV Edwards ◽  
O Lindan
Keyword(s):  
Particle Size ◽  
Calcium Ions ◽  
Fibrin Clot ◽  
Clot Formation ◽  
Size Change ◽  
Fibrin Formation ◽  
Fibrin Monomer ◽  
Small Change ◽  
Time Required ◽  
Kinetics Of

Abstract Using laser fluctuation spectroscopy, a technique that measures particle size change in solution, the kinetics of fibrin clot formation from fibrinogen can be studied. With this technique the effect of calcium on the three distinguishable phases of clot formation, (1) proteolysis of fibrinogen, (2) fibrinogen-fibrin monomer complex formation, and (3) fibrin monomer polymerization, were investigated. Only a small change in the length of the induction period that results from the fibrinogen-fibrin monomer interactions was observed. However, there was a marked increase in the rate of fibrin monomer polymerization in the presence of calcium ions. These data show that calcium decreases the time required for fibrin formation from fibrinogen by markedly accelerating the phase of fibrin monomer polymerization.

scholarly journals Fibrin formation: effect of calcium ions

Blood ◽  
1978 ◽  
Vol 52 (4) ◽  
pp. 654-658 ◽  
Author(s):  
EP Brass ◽  
WB Forman ◽  
RV Edwards ◽  
O Lindan
Keyword(s):  
Particle Size ◽  
Calcium Ions ◽  
Fibrin Clot ◽  
Clot Formation ◽  
Size Change ◽  
Fibrin Formation ◽  
Fibrin Monomer ◽  
Small Change ◽  
Time Required ◽  
Kinetics Of

Using laser fluctuation spectroscopy, a technique that measures particle size change in solution, the kinetics of fibrin clot formation from fibrinogen can be studied. With this technique the effect of calcium on the three distinguishable phases of clot formation, (1) proteolysis of fibrinogen, (2) fibrinogen-fibrin monomer complex formation, and (3) fibrin monomer polymerization, were investigated. Only a small change in the length of the induction period that results from the fibrinogen-fibrin monomer interactions was observed. However, there was a marked increase in the rate of fibrin monomer polymerization in the presence of calcium ions. These data show that calcium decreases the time required for fibrin formation from fibrinogen by markedly accelerating the phase of fibrin monomer polymerization.

Fibrin Formation: The Role of the Fibrinogen-Fibrin Monomer Complex

1976 ◽  
Vol 36 (01) ◽  
pp. 037-048 ◽  
Author(s):  
Eric P. Brass ◽  
Walter B. Forman ◽  
Robert V. Edwards ◽  
Olgierd Lindan
Keyword(s):  
Particle Size ◽  
Induction Period ◽  
Fibrin Clot ◽  
Clot Formation ◽  
Appreciable Amount ◽  
Buffer System ◽  
Homeostatic Mechanism ◽  
Fibrin Formation ◽  
Fibrin Monomer

SummaryThe process of fibrin formation using highly purified fibrinogen and thrombin was studied using laser fluctuation spectroscopy, a method that rapidly determines particle size in a solution. Two periods in fibrin clot formation were noted: an induction period during which no fibrin polymerization occurred and a period of rapid increase in particle size. Direct measurement of fibrin monomer polymerization and fibrinopeptide release showed no evidence of an induction period. These observations were best explained by a kinetic model for fibrin clot formation incorporating a reversible fibrinogen-fibrin monomer complex. In this model, the complex serves as a buffer system during the earliest phase of fibrin formation. This prevents the accumulation of free polymerizable fibrin monomer until an appreciable amount of fibrinogen has reacted with thrombin, at which point the fibrin monomer level rises rapidly and polymerization proceeds. Clinically, the complex may be a homeostatic mechanism preventing pathological clotting during periods of elevated fibrinogen.

The Presence of Antithrombin Activity in Platelets

1979 ◽  
Author(s):  
E. E. Czapek ◽  
H. C. Kwaan
Keyword(s):  
Antithrombin Iii ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Clot Formation ◽  
Chromogenic Substrate ◽  
Antithrombotic Activity ◽  
Platelet Poor Plasma ◽  
Antithrombin Activity ◽  
At Iii ◽  
Platelet Pellet

The platelet has been implicated as the nidus around which clot formation occurs. We therefore decided to investigate whether any antithrombotic activity was associated with platelets. Thrombin activity was measured using the chromogenic substrate S-2238. Freshly prepared human platelet rich plasma (PRP) and platelet poor plasma (PPP) were found to have similar amounts of antithrombin activity. This antithrombin activity could be easily removed from a platelet pellet by repeated washing. However, sonicating PRP resulted in the appearance of additional antithrombin activity. After 6 minutes of sonication, the antithrombin activity of PRP decreased to 35% of presonication levels, whereas the antithrombin activity of PPP remained 100%. When subjected to Laurell Immunoelectrophoresis using an antibody to antithrombin III (AT-III), a greater amount of antigen was detected in PRP than in PPP. We conclude that platelets contain antithrombin material which is probably AT-III.

Dose Finding Study of a Low Molecular Weight Heparin, Innohep, in Haemodialysis

1991 ◽  
Vol 66 (03) ◽  
pp. 277-282 ◽  
Author(s):  
K E Ryan ◽  
D A Lane ◽  
A Flynn ◽  
J Shepperd ◽  
H A Ireland ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Dose Finding ◽  
Clot Formation ◽  
Low Molecular Weight ◽  
Maintenance Haemodialysis ◽  
Fibrin Formation ◽  
Significant Difference ◽  
Finding Study ◽  
Pilot Investigation ◽  
Dose Finding Study

SummaryA pilot investigation was performed with Innohep, a low molecular weight (LMWH) preparation (peak maximum molecular mass 3,000-6,000), to determine possible dose regimens for patients undergoing regular maintenance haemodialysis for chronic renal failure. Results from this study suggested that suppression of macroscopic clot formation and fibrinopeptide A (FPA), a marker of fibrin formation, could be achieved following bolus injections rather than bolus injections and an infusion. On the basis of these preliminary findings, a randomised crossover study was performed in eight patients undergoing regular maintenance haemodialysis for 5-7 h to determine the effective antithrombotic dose of this LMWH. Single i.v. bolus doses of 1,250 AFXa u, 2,500 AFXa u and 5,000 AFXa u (n = 7-8) were compared to an UFH regime of 5,000 iu + 1,500 iu/h. Excessive clot formation in the dialyser bubble trap, necessitating additional UFH to enable completion of a prolonged (up to 7 h) dialysis, was observed in all patients on the 1,250 AFXa u dose (mean duration of dialysis prior to UFH, 3 h) but in a single patient only receiving the other LMWH doses. A dose-related response in the AFXa activity, measured by chromogenic substrate (CS) assay was seen in the three LMWH groups, with levels declining significantly (p <0.05) from 1-7 h. This contrasted with the constant levels maintained during dialysis with UFH. FPA levels were significantly elevated after 2 h following the 1,250 AFXa u bolus and after 4 h following the 2,500 AFXa u bolus. There was no significant difference in FPA levels between the 5,000 AFXa u bolus and UFH. β-thromboglobulin (pTG) levels rose significantly towards the end of dialysis in all LMWH groups and, at 5 h, were significantly increased following the 5,000 AFXa u and 2,500 AFXa u doses when compared to the UFH regime. AFXa levels correlated negatively with FPA levels (r = -0.62; p <0.01). In conclusion, for administration of Innohep, a bolus dose of 2,500 AFXa u would appear to be sufficient for dialyses of short duration (up to 4 h), whilst a 5,000 AFXa u bolus is as effective as UFH for a 6 h dialysis. AFXa activity measured by CS assay is related to inhibition of fibrin formation and can be used for monitoring purposes.

Isolation And Characterization Of Procoagulant Substances From Human Ascites

1981 ◽  
Author(s):  
M F M Johnston ◽  
J Vargo ◽  
J H Joist
Keyword(s):  
Molecular Weight ◽  
Ascitic Fluid ◽  
Human Platelet ◽  
Deae Cellulose ◽  
Procoagulant Activity ◽  
Ph Change ◽  
Human Fibrinogen ◽  
Isolation And Characterization ◽  
Wide Range

The treatment of massive, medically intractable ascites by a peritoneovenous shunt (PVS) is associated with variably severe disseminated intravascular coagulation (DIC). Ascitic fluid obtained from cirrhotic patients at the time of placement of a PVS was found to shorten the partial thromboplastin time (PTT) of normal human platelet-poor plasma. This procoagulant activity which was found to reside in the cell-free fraction of ascitic fluid was heat stable and insensitive to pH change over a wide range. Chromatography on Bio-Rad Agarose 1.5 yielded one major high molecular weight component and several smaller fractions of lower molecular weight exhibiting procoagulant activity as determined by the PTT assay. The activity in the major fraction (80% total activity) coprecipitated with human fibrinogen but could be separated from fibrinogen by affinity chromatography using anti-human fibrinogen. This procoagulant did not hydrolyze chromogenic substrates S- 2222 and S-2238 and was not inhibited by diisopropylfluoro-phosphate (DFP). Purification of a minor procoagulant fraction by aluminum hydroxide (Al (OH)3) adsorption and chromatography on Sephadex G-200 yielded a fraction that clotted both citrated normal plasma (without the addition of calcium chloride) and purified human fibrinogen and induced platelet aggregation in citrated human platelet- rich plasma. Chromatography on DEAE cellulose yielded two peaks with procoagulant activity (PTT), one of which hydrolyzed S-2238 and was DFP-sensitive. These studies indicate that at least three distinct, clotting active substances are present in ascitic fluid some or all of which may be responsible for PVS-induced DIC. Further studies on purification and characterization of these ascitic procoagulants are in progress.

The Presence of Antithrombin Activity in Platelets

1979 ◽  
Author(s):  
E.E. Czapek ◽  
H.C. Kwaan
Keyword(s):  
Antithrombin Iii ◽  
Human Platelet ◽  
Platelet Rich Plasma ◽  
Clot Formation ◽  
Chromogenic Substrate ◽  
Antithrombotic Activity ◽  
Platelet Poor Plasma ◽  
Antithrombin Activity ◽  
At Iii ◽  
Platelet Pellet

The platelet has been implicated as the nidus around which clot formation occurs. We therefore decided to investigate whether any antithrombotic activity was associated with platelets. Thrombin activity was measured using the chromogenic substrate S-2238. Kreshly prepared human platelet rich plasma (PRP) and platelet poor plasma (PPP) were found to have similar amounts of antithrombin activity. This antithrombin activity could be easily removed from a platelet pellet by repeated washing. However, sonicating PRP resulted in the appearance of additional antithrombin activity. After 6 minutes of sonlcation, the antithrombin activity of PRP decreased to 35% of presonication levels, whereas the antithrombin activity of PPP remained 100%. When subjected to Laurell immunoelectrophoresis using an antibody to antithrombin III(AT-III), a greater amount of antigen was detected in PRP than in PPP. We conclude that platelets contain antithrombin material which is probably AT-III.

High Molecular Weight Complexes Derived From Fibrinogen

1981 ◽  
Author(s):  
N Alkjaersig ◽  
A Fletcher
Keyword(s):  
Molecular Weight ◽  
Complex Formation ◽  
High Molecular Weight ◽  
Molecular Size ◽  
Gel Permeation Chromatography ◽  
Linear Increase ◽  
Clot Formation ◽  
High Molecular Weight Complex ◽  
Fibrin Monomer ◽  
Gel Permeation

Fibrinogen, 3×10-5 M, incubated at 25°C with thrombin 10-10 M, is slowly transformed to fibrin. Fibrinopeptide A (FPA) is released at a rate of approximately 1 nM/ml per min, and after 30 min a clot is formed. Prior to clot formation serially timed solution aliquots were examined by gel permeation chromatography on a Bio Gel 5M column (void volume 140 ml). High molecular weight complex concentration and molecular size increased with incubation time. Fibrinogen antigen and FPA concentration was determined for each effluent fraction and relative FPA content (ng FPA/μg fibrinogen equivalent) was linearly related to chromatographic effluent volume between 160 and 260 ml (Ve for fibrinogen 255 ml). Computer analysis of the effluent profiles indicated a linear fall in monomeric fibrinogen to 20% of original concentration, a linear increase in “fibrin” dimer (2 fibrinogen equivalents with 2 FPA) to 30% and in trimer (3 fibrinogen equivalents with 2 FPA) to 20% of original concentration with higher polymers accounting for the remaining protein. These complexes were stable and remained in solution after fractionation. Fibrin monomer, in 3 M urea and devoid of FPA, when added to fibrinogen resulted in clot formation, equivalent to 66% of added monomer, but minimal complex formation, 5% of total fibrinogen equivalents. Thus intermediate fibrin polymers which contain residual FPA are stable soluble molecular entities in contradistinction to fibrin monomer lacking FPA. Fibrin proteolysis products added to fibrinogen also show minimal complex formation with fibrinogen if the products are free of thrombin and FPA. Patient plasma samples can be similarly assayed and distinction can be made between high molecular weight complexes derived from fibrinogen through thrombin action or from fibrin through plasmin action.

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

1992 ◽  
Vol 68 (05) ◽  
pp. 534-538 ◽  
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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