Effect of dextran on clotting time: Steric exclusion acclerates fibrin monomer polymerisation

1976 ◽  
Vol 9 (4) ◽  
pp. 379-386 ◽  
Author(s):  
M.W. Rampling ◽  
D.A. Lane ◽  
V.V. Kakkar
Keyword(s):  
Clotting Time ◽  
Fibrin Monomer ◽  
Steric Exclusion

Polymerization Sties Of Desialated Fibrinogen

1981 ◽  
Author(s):  
P M Allison ◽  
N U Bang
Keyword(s):  
Sialic Acid ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Clotting Time ◽  
Mean Values ◽  
Fibrin Monomer ◽  
Sds Page

It has long been known (Laki and Chandrasekhar, Nature, 197:1267,1963) that the enzymatic removal of sialic acid (SA) frcm fibrinogen (F) shortens the thrccabin clotting time. Martinez et al. (J. Lab. Clin. Med., 89:367, 1977) demonstrated that this phenomenon is due to enhanced fibrin monomer polymerization. To more accurately identify the mechanisms involved we examined the binding of normal F, V. cholerae neuraminidase - treated F (NF) and their plasmic digests. Mean SA content of F of 4.5 residues / molecule decreased to unmeasurable levels in NF with a concomitant 10s shortening of the thrombin clotting time. F, NF, and plasmin digests of F (FP) and NF (NFP) were subjected to fibrin-monomer-Sepharose chromatography (FMSC) essentially according to Kudryk and Blcmback (J.Biol. Chan., 249:3322, 1974) and effluent fractions analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) under nonreducing conditions. Mean values for F retained on FMSC and eluted with acid urea buffer was 61%, for NF 86%, for for FP 65%, for NFP 78%. In NFP and FP 3 fragments D (D1, D2 D3) of Mr 103K, 89K, and 83K were discerned by SDS-PAGE. D1 in FP and NFP was almost quantitatively retained on FMSC, whereas little, if any, D3 was retained frcm either digest. FMSC resulted in poor retention of D2 for FP and virtually complete retention of this fragment from NFP. Fragments E were not retained on FMSC of either FP or NFP. The data suggests that desialation of F exposes additional polymerization sites present in D2 but not D3 of F.

Zinc Alters Fibrin Ultrastructure

1987 ◽  
Vol 57 (01) ◽  
pp. 073-076 ◽  
Author(s):  
Gerard Marx ◽  
Pierre Hopmeier ◽  
Dorit Gurfel
Keyword(s):  
Cross Section ◽  
Electron Micrographs ◽  
Fibre Diameter ◽  
Fold Increase ◽  
Fibrin Clot ◽  
Dependent Manner ◽  
Clotting Time ◽  
Concentration Dependent Manner ◽  
Sem Micrographs ◽  
Fibrin Monomer

SummaryTurbidimetric studies indicate that Zn(II) accelerates fibrin gelation [decreases clotting time (CT)] and increases maximal fibrin clot turbidity. For any given level of fibrinogen (0.2-2.6 mg/ ml), the relative fibrin turbidity of thrombin-induced clots increases with Zn(II) in a concentration dependent manner. Zinc-associated turbidity increases are also observed in the presence of 2 mM Ca(II). With citrate, similar turbidity increases are observed, though at higher cation levels. Thus, turbidimetry indicates that the gel formed with Zn(II) is coarser, or has thicker fibre strands. SEM micrographs confirm that fibre thickness ranges from 260 Å to 2600 Å, when Zn(II) levels range from 0-50 uM. With citrate, TEM micrographs reveal amore than 20 x fold increase in fibre diameter (100 Å->2000 Å) with higher Zn(II) (<1 mM) levels. Based on a fibrin monomer cross-section of ~60 Å, the electron micrographs indicate that depending on the Zn(II) levels, fibrin strands are composed of between 2 to 40 monomeric fibrin molecules. Thus, at physiologically relevant levels, Zn(II) can drastically modulate fibrin ultrastructure.

Proteolytic and polymerase activity of thrombin

1960 ◽  
Vol 198 (1) ◽  
pp. 173-179 ◽  
Author(s):  
Ricardo H. Landaburu ◽  
Walter H. Seegers
Keyword(s):  
Fibrin Clot ◽  
Polymerase Activity ◽  
Urea Solution ◽  
Polymerization Time ◽  
Reaction Products ◽  
Clotting Time ◽  
Fibrin Monomer ◽  
As If

In addition to splitting peptides from fibrinogen, thrombin is necessary for the polymerization of fibrin monomer. Thrombin also uses fibrin as a substrate and eventually the clot may lyse. On the basis of these views, previous equations used to outline the events when thrombin and fibrinogen are mixed can be revised as follows: Proteolysis: F + T ⇆ f + P + T (1) Polymerization: Nf + T ⇆ NfT (2) (fibrin clot) Proteolysis: NfT → Inf + T + P2 (3) F is fibrinogen, T is thrombin, f is fibrin monomer, P is peptides and carbohydrates, N is any number, Inf is inactive fibrin monomer, and P2 is peptides or any reaction products. As the thrombin concentration increases the polymerization time becomes shorter. TAMe retards the polymerization time. For a given thrombin concentration the clotting time of fibrinogen may be inversely proportional to the concentration of fibrinogen. Most of the clotting time is accounted for as polymerization time, but the amount of thrombin required for the latter is relatively small. In 3 m urea solution thrombin may irreversibly lose its activity. When protected by a substrate such as TAMe, fibrinogen or fibrin, thrombin is active in urea solution. The activity then depends upon TAMe concentration under conditions when the enzyme behaves as if saturated with the substrate. To have this activity in urea solution the substrate must be in the urea before the thrombin is added. Esterase thrombin also uses fibrin as a substrate; it is, however, not a polymerase. It does not use fibrinogen as a substrate.

Un nouveau Cas d’Anomalie constitutionnelle et familiale du Fibrinogène sans Diathese hémorragique

1971 ◽  
Vol 26 (03) ◽  
pp. 565-576 ◽  
Author(s):  
F Streiff ◽  
P Alexandre ◽  
C Vigneron ◽  
J Soria ◽  
C Soria ◽  
...  
Keyword(s):  
Sialic Acid ◽  
Electrophoretic Mobility ◽  
Functional Characterization ◽  
Bleeding Tendency ◽  
Clotting Time ◽  
New Family ◽  
Fibrin Monomer ◽  
Normal Plasma ◽  
High Level

SummaryA new family with congenital dysfibrinogenemia without bleeding tendency has been discovered in Nancy. Identical laboratory abnormalities were found in the mother and son plasma: prolongation of thrombin and reptilase clotting time.The abnormal fibrinogen affects, only a little, the normal plasma fibrinoformation.The functional characterization of the defect is localized in fibrin monomer aggregation. The release of fibrinopeptides is normal.The abnormal fibrinogen showsa) an alteration in glucidic moiety of the molecule with a high level of sialic acid,b) an disturbance of chromatography on D. E. A. E. cellulose andc) of the electrophoretic mobility.

THROMBIN BINDING FRAGMENT E GENERATED DURING FIBRINOGENOLYSIS

1987 ◽  
Author(s):  
C A Goodwin ◽  
M F Scully ◽  
V Ellis ◽  
V V Kakkar
Keyword(s):  
Heat Treatment ◽  
Binding Activity ◽  
Gel Chromatography ◽  
Slow Inactivation ◽  
Carboxy Terminus ◽  
Clotting Time ◽  
Amidolytic Activity ◽  
Early Stages ◽  
Fibrin Monomer ◽  
Insight Into

Binding of fibrinogen by thrombin was measured by inhibition of amidolysis of S2238 and found to be 12 μM. Upon digestion of fibrinogen with plasmin (0.16ugs/mg fibrinogen) for 4 hours at 37°C, thrombin binding activity remained in the supernatants upon heat treatment. The thrombin binding activity in the dialyzed supernatant reached a maximum after two hours coinciding with maximal release of B 1-42 and 45-39 KDa chain fragments. Measured immunologically, levels of fragment E at this time were 45% of the maximum generated after 4 hours digestion. FPA levels in the dialyzed supernatant (measured by RIA and HPLC) after thrombin treatment, were zero and did not increase until 1½ hours after the beginning of digestion, reaching a maximum at 4 hours. The thrombin binding activity generated was stable to further plasmin action. Upon gel chromatography of 2 and 4 hour supernatants, thrombin binding activity coincided closely with fragment E, measured immunologically. Further purification showed the fragment to have Ki for thrombin amidolytic activity of 0.5μM. The fragment also inhibited the thrombin clotting time of plasma but did not affect fibrin monomer polymerization.The fragment was susceptible to very slow inactivation by thrombin but not arvin (though it did inhibit arvin amidolytic activity). A thrombin binding (thrombin inhibitory) fragment is therefore generated during the early stages of f ibrinogenolysis and may be the result of protection by 45 and 39 KDa A α carboxy terminus fragments since E fragments generated in later stages (in the presence of 29 and 25 KDa fragments) do not have this property. These findings may give interesting new insight into thrombin/fibrinogen interaction.

Fibrinogen Bastia (γ 318 Asp → Tyr) a Novel Abnormal Fibrinogen Characterized by Defective Fibrin Polymerization

1999 ◽  
Vol 82 (12) ◽  
pp. 1639-1643 ◽  
Author(s):  
Karim Chabane Lounes ◽  
Claudine Soria ◽  
Antoine Valognes ◽  
Marie France Turchini ◽  
Jaap Koopman ◽  
...  
Keyword(s):  
Calcium Ions ◽  
Clinical Symptoms ◽  
Degradation Products ◽  
Base Substitution ◽  
Chain Gene ◽  
Fibrinogen Concentration ◽  
Clotting Time ◽  
Terminal Part ◽  
Fibrin Polymerization ◽  
Chain Sequence

SummaryA new congenital dysfibrinogen, Fibrinogen Bastia, was discovered in a 20-year-old woman with no clinical symptoms. The plasma thrombin-clotting time was severely prolonged. The functional plasma fibrinogen concentration was low (0.2 mg/ml), whereas the immunological concentration was normal (2.9 mg/ml). Purified fibrinogen Bastia displayed a markedly prolonged thrombin-clotting time related to a delayed thrombin-induced fibrin polymerization. Both the thrombin-clotting time and the fibrin polymerization were partially corrected by the addition of calcium ions. The anomaly of fibrinogen Bastia was found to be located in the γ-chain since by SDS-PAGE performed according to the method of Laemmli two γ-chains were detected, one normal and one with an apparently lower molecular weight. Furthermore, analysis of plasmin degradation products demonstrated that calcium ions only partially protect fibrinogen Bastia γ-chain against plasmin digestion, suggesting that the anomaly is located in the C-terminal part of the γ-chain. Sequence analysis of PCR-amplified genomic DNA fragments of the propositus demonstrated a single base substitution (G → T) in the exon VIII of the γ chain gene, resulting in the amino acid substitution 318 Asp (GAC) → Tyr (TAC). The PCR clones were recloned and 50% of them contained the mutation, indicating that the patient was heterozygous. These data indicate that residue Asp 318 is important for normal fibrin polymerization and the protective effect of calcium ions against plasmin degradation of the C-terminal part of the γ-chain.

The uncalibrated prothrombinase-induced clotting time test

2010 ◽  
Vol 30 (04) ◽  
pp. 212-216 ◽  
Author(s):  
R. Jovic ◽  
M. Hollenstein ◽  
P. Degiacomi ◽  
M. Gautschi ◽  
A. Ferrández ◽  
...  
Keyword(s):  
Factor Xa ◽  
Heparin Therapy ◽  
Thrombin Inhibitors ◽  
Coagulation Cascade ◽  
Diagnostic Tools ◽  
Direct Thrombin Inhibitors ◽  
Functional Assay ◽  
Clotting Time ◽  
Coagulation Time ◽  
Time Test

SummaryThe activated partial thromboplastin time test (aPTT) represents one of the most commonly used diagnostic tools in order to monitor patients undergoing heparin therapy. Expression of aPTT coagulation time in seconds represents common practice in order to evaluate the integrity of the coagulation cascade. The prolongation of the aPTT thus can indicate whether or not the heparin level is likely to be within therapeutic range. Unfortunately aPTT results are highly variable depending on patient properties, manufacturer, different reagents and instruments among others but most importantly aPTT’s dose response curve to heparin often lacks linearity. Furthermore, aPTT assays are insensitive to drugs such as, for example, low molecular weight heparin (LMWH) and direct factor Xa (FXa) inhibitors among others. On the other hand, the protrombinase-induced clotting time assay (PiCT®) has been show to be a reliable functional assay sensitive to all heparinoids as well as direct thrombin inhibitors (DTIs). So far, the commercially available PiCT assay (Pefakit®-PiCT®, DSM Nutritional Products Ltd. Branch Pentapharm, Basel, Switzerland) is designed to express results in terms of units with the help of specific calibrators, while aPTT results are most commonly expressed as coagulation time in seconds. In this report, we describe the results of a pilot study indicating that the Pefakit PiCT UC assay is superior to the aPTT for the efficient monitoring of patients undergoing UFH therapy; it is also suitable to determine and quantitate the effect of LMWH therapy. This indicates a distinct benefit when using this new approach over the use of aPPT for heparin monitoring.

Comparison of the Anticoagulant and Antithrombotic Effects of Synthetic Thrombin and Factor Xa Inhibitors

1990 ◽  
Vol 63 (02) ◽  
pp. 220-223 ◽  
Author(s):  
J Hauptmann ◽  
B Kaiser ◽  
G Nowak ◽  
J Stürzebecher ◽  
F Markwardt
Keyword(s):  
Factor Xa ◽  
Thrombin Inhibitors ◽  
Factor Xa Inhibitors ◽  
Venous Stasis ◽  
Clotting Time ◽  
Thrombosis Model ◽  
Activity Factor ◽  
Antithrombotic Effects

SummaryThe anticoagulant effect of selected synthetic inhibitors of thrombin and factor Xa was studied in vitro in commonly used clotting assays. The concentrations of the compounds doubling the clotting time in the various assays were mainly dependent on their thrombin inhibitory activity. Factor Xa inhibitors were somewhat more effective in prolonging the prothrombin time compared to the activated partial thromboplastin time, whereas the opposite was true of thrombin inhibitors.In vivo, in a venous stasis thrombosis model and a thromboplastin-induced microthrombosis model in rats the thrombin inhibitors were effective antithrombotically whereas factor Xa inhibitors of numerically similar IQ value for the respective enzyme were not effective at equimolar dosageThe results are discussed in the light of the different prelequisiles and conditions for inhibition of thrombin and factor Xa in the course of blood clotting.

Fibrinogen Montreal

1973 ◽  
Vol 29 (03) ◽  
pp. 536-546 ◽  
Author(s):  
M Lacombe ◽  
J Soria ◽  
C Soria ◽  
G d’Angelo ◽  
R Lavallee ◽  
...  
Keyword(s):  
Optical Density ◽  
Polyacrylamide Gel ◽  
Clotting Time ◽  
Functional Defect ◽  
A Chain ◽  
Fibrin Monomers

SummaryA new case of congenital dysfibrinogenemia characterized by a prolonged thrombin clotting time and a low optical density of the polymerization curve has been discovered in Montreal. The functional defect is due to an abnormal aggregation of fibrin monomers.The characteristics of this abnormal fibrinogen are serum gélification (Paracoagulation) at 37°, 22° and 4° C, a normal immuno-electrophoretic and electrofocusing pattern, a slight increase in the mobility in the α (A) chain by electrophoresis of the dissociated chains in polyacrylamide gel. However, no abnormality was found in the α (A) chain of the disulphide knot.

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