Factors Influencing The Structure Of Terminal Plasmin Degradation Products Of Human Fibrinogen And Fibrin

1981 ◽  
Author(s):  
W Nieuwenhuizen ◽  
A Vermond ◽  
F Haverkate
Keyword(s):  
Protective Effect ◽  
Calcium Ions ◽  
Degradation Products ◽  
Iminodiacetic Acid ◽  
Human Fibrinogen ◽  
Factors Influencing ◽  
Separate Effect ◽  
The Media ◽  
Carboxy Terminal ◽  
D Dimer

Experiments have been carried out with fibrinogen and with purified degradation products of fibrinogen and fibrin which demonstrate that the structure of D fragments obtained after prolonged plasmin digestion is influenced by several factors in the media.The previously described protective effect of calcium ions on the γ-chain carboxy-terminals of fibrinogen against plasmin attack is rather independent of the composition of the media (e.g., also observed in 2 M urea and/or high plasmin activities).Several compounds such as EDTA, EGTA, citrate and iminodiacetic acid appear to have a separate effect, which is best observed at low plasmin concentrations and in the absence of Ca2+ . Under these conditions, these compounds appear to make the γ-chain carboxy-terminal ends of the D- and D-dimer fragments more susceptible to plasmin digestion.Finally, as demonstrated by experiments with purified D;E complexes from fibrinogen and with whole fibrinogen digests, the E-moiety of the D:E complexes appears to be capable of protecting the D-moiety against low plasmin concentrations also in the absence of calcium ions.

Anticoagulant And Calcium-Binding Properties Of High Molecular Weight Derivatives Of Human Fibrinogen, Produced By Plasmin (Fragments X)

1981 ◽  
Author(s):  
W Nieuwenhuizen ◽  
M Gravesen
Keyword(s):  
Calcium Ions ◽  
Calcium Binding ◽  
Degradation Products ◽  
Amino Acid Sequences ◽  
Binding Properties ◽  
Human Fibrinogen ◽  
Molecular Weights ◽  
Amino Terminal ◽  
Terminal Amino ◽  
Carboxy Terminal

Early plasmin degradation products (fragments X) of human fibrinogen were prepared and purified on Sepharose 6B-CL. X-fragments were characterized with respect to amino-terminal amino acid sequences, polypeptide-chain composition, anticlotting properties and calcium-binding. Amino-terminal amino acids were alanine and tyrosine. The molecular weights of the chains were about 26,000, 58,000 and 48,000 for Aα-, Bβ- and γ-chains, respectively. Fragments X were about 6 times as potent in anticlotting behaviour as D-fragments prepared in the presence of calcium ions. Calcium-binding properties were similar to those of fibrinogen: No differences were observed between fragments X prepared in the presence of calcium ions and those prepared in the presence of EGTA. Results indicate that the carboxy-terminal parts of the Aα-chains of fibrinogen are not involved in calcium-binding and that differences in chain-remnants as observed in late plasmic degradation products (which depend on the presence of calcium ions or EGTA in the incubation medium) are introduced beyond the stage of fragment X-formation.

Human Fibrinogen Binds Edta And Citrate

1981 ◽  
Author(s):  
W Nieuwenhuizen ◽  
A Vermond ◽  
J Hermans
Keyword(s):  
Calcium Ions ◽  
Equilibrium Dialysis ◽  
Strong Dependence ◽  
Direct Interaction ◽  
Heat Stability ◽  
Binding Studies ◽  
Human Fibrinogen ◽  
Direct Binding ◽  
Carboxy Terminal ◽  
Fibrinogen Molecule

It was shown that EDTA and citrate make the carboxy-terminal parts of the γ-chains of fibrinogen more susceptible to plasmin degradation. This is an effect independent of the calcium-chelating properties of those compounds. Furthermore, EDTA prolongs the thrombin times decreases the heat stability and causes the formation of abnormal clots.This suggests a direct interaction of EDTA (and possibly also of citrate) with the fibrinogen molecule thereby causing a conformational change. The interaction of citrate and EDTA with fibrinogen was measured by direct binding studies (equilibrium dialysis).It was found that at pH 7.5 human fibrinogen binds 3.4 molecules of citrate with Kd = 1.4 x 10-4M or 0.4 molecules of EDTA with Kd = 2.2 x 10-5M. Citrate and EDTA compete for the same binding site(s). No binding of acetate could be demonstrated.The binding of EDTA and citrate shows a strong ^-dependence suggesting a (partly) ionogenic binding between charged areas on the fibrinogen molecule and charged groups on EDTA or citrateOur results support the idea that the binding of EDTA and citrate and the concomitant effect on the plasmin attack are independent of the effect of calcium ions

Binding Properties of Monoclonal Antibodies against Human Fragment D-Dimer of Cross-Linked Fibrin to Human Plasma Clots in an In Vivo Model in Rabbits

1989 ◽  
Vol 61 (02) ◽  
pp. 307-313 ◽  
Author(s):  
P Holvoet ◽  
J M Stassen ◽  
Y Hashimoto ◽  
D Spriggs ◽  
P Devos ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Human Plasma ◽  
Jugular Vein ◽  
Degradation Products ◽  
Control Group ◽  
Human Fibrinogen ◽  
Fab Fragments ◽  
Vein Segment ◽  
D Dimer

SummaryTwo (MA-15C5 and MA-8D3) out of approximately 500 monoclonal antibodies, obtained by fusion of P3X63-Ag8-6.5.3 myeloma cells with spleen cells of mice immunized with purified fragment D-dimer from human fibrin, demonstrated a more than 1,000-fold higher affinity for fragment D-dimer than for native fibrinogen. MA-15C5 was directed against a neoantigenic determinant only expressed in fragment D-dimer. MA-8D3 reacted equally well with fragment D-dimer of crosslinked fibrin and with fragment D of non-crosslinked fibrin but not with fragment D of fibrinogen. Both monoclonal antibodies did not crossreact with rabbit fibrin and its degradation products.The binding of 125I-labeled Fab fragments to human plasma clots, introduced and aged for 1 hr in the jugular vein of heparinized rabbits was studied. Following injection of an equimolar mixture of Fab fragments derived from MA-15C5 and MA-8D3, the clot to blood ratios of radioactivity increased from 3.2 ± 1.2(mean ± SD) at 4 hr to 7.2 ± 1.4 at 17 hr. The binding of Fab fragments of MA-15C5 and MA-8D3 was independent of the age (1 to 72hrs) of the clot and of heparin anticoagulation and was only slightly decreased (by 20%) in the presence of circulating human fibrinogen (90 mg/kg body weight) and of human crosslinked fibrin degradation products at a plasma concentration of 10 pg/ml. The binding of Fab fragments of MA-15C5 and MA-8D3 to occlusive human plasma clots in the femoral artery of rabbits was comparable to that of the non-occlusive human plasma clots in the jugular vein. The Fab fragments of MA-15C5 and MA-8D3, labeled with 123I to a specific activity of 10 μCi/pg were injected intravenously (3 μg/kg) in 72 rabbits with a nonocclusive 0.2 ml human plasma clot in the jugular vein and in 7 control rabbits that underwent the surgical procedure without clot formation. Total body scans performed at hourly intervals revealed a higher relative increase in gamma counts over the thrombus region in the group with thrombus as compared to controls: at 6 hr 54 ± 18 vs 16 ± 13% (mean ± SEM, p <0.1) and at12 hrs 35 ± 11 vs –7 ± 12 (p <0.05). The vein segment to blood ratios of 123I at 24 hrs were 6.6 ± 2.4 in the group with clot and 1.5 to 0.7 in the control group (p <0.01). We conclude that these Fab fragments may have a sufficiently high fibrin-affinity to allow in vivo thrombus localization by external scanning.

Antigenic Properties Of Human Fibrin Plasmic Fragment D-Dimer And Its γ-γ Chain Remnant

1981 ◽  
Author(s):  
C S Cierniewski ◽  
P Nowak ◽  
T Krajewski
Keyword(s):  
Ion Exchange ◽  
Competitive Inhibition ◽  
Degradation Products ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Antigenic Determinants ◽  
Human Fibrinogen ◽  
Antigenic Properties ◽  
D Dimer

Immunochemical analyses of human D-dimer and its γ-γ chain remnant were performed to identify their antigenic markers which would be of value in distinguishing between disseminated intravascular coagulation and primary fibrinogenolysis. Cross-linked fibrin was obtained by direct clotting of the fresh citrated plasma after adding excess CaCl2. The plasmic digests of the cross-linked fibrin were fractionated by ion-exchange chromatography and gel filtration. Thus, high molecular weight D-dimer was purified. The γ-γ peptide remnant was purified by ion-exchange chromatography on CM-52 cellulose from the reduced D- dimer. Purity of both polypeptide fragments was determined by standard techniques. They were used to immunize rabbits. Antisera to D-dimer and γ-γ chain remnant were characterized in binding assays and in equilibrium competitive inhibition assays in order to analyze the expression of their antigenic determinants in intact fibrinogen and its plasmic degradation products. Antisera to D-dimer preferentially bound D-dimer and γ-γ chain remnant and to a lesser extent fragment D and fibrinogen. Similarly, antisera to γ-γ chain remnant bound mostly γ-γ , D-dimer as well as γ poiypeptide chain. There was no binding of the intact human fibrinogen. The results obtained in the competitive inhibition assays employing antisera to D-dimer and γ-γ chain remnant, before and after adsorption with fragment D or γ polypeptide chain respectively, as well as fibrin fragments are discussed in this report.

Localization of the α-Chain Cross-Linking Sites in Human Fibrinogen

1979 ◽  
Author(s):  
B. Hessel ◽  
G. Savidge ◽  
B. Blombäck
Keyword(s):  
Experimental Data ◽  
Calcium Ions ◽  
Whole Blood ◽  
Cyanogen Bromide ◽  
Degradation Products ◽  
Factor Xiiia ◽  
Antigenic Determinants ◽  
Cross Linking ◽  
Human Fibrinogen ◽  
The Cross

Antisera were raised in rabbits against the fragments Hi2-DSK and N-DSK obtained from cyanogen bromide treatment of human fibrinogen. Sensitive radioimmunoassay techniques were developed to quantitate substances sharing the same antigenic determinants as these fragments. We have found that the cross-linking process in fibrin formation involves the Hi2-DSK region which has its origin in the Aα-chain of fibrinogen. This relationship is based upon the following experimental data: - Fibrin samples produced in the presence and absence of Factor Xllla and Ca++ and thus cross-Linked and non cross-linked respectively were treated with cyanogen bromide. The resulting degradation products were chroraatographed on Sephadex G-100, and radioimmunoassays for Hi2-DSK and N-DSK were performed on the eluates. The elution profiles of Hi2-DSK and N-DSK antigenic determinants demonstrated that Hi2-DSK eluated after N-DSK in non cross-linked fibrin samples but in the cross-linked samples Hi2-DSK eluated before N-DSK. These results demonstrate that the Hi2-DSK region is involved in the cross-linking process in the presence of Factor XIIIa and calcium ions. Fibrin samples produced in the coagulation of whole blood showed a similar cross-linking profile.Supported by The Bank of Sweden Tercentenary Foundation.

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.

Permeability Enhancing and Chemotactic Activities of Lower Molecular Weight Degradation Products of Human Fibrinogen

1981 ◽  
Vol 45 (01) ◽  
pp. 090-094 ◽  
Author(s):  
Katsuo Sueishi ◽  
Shigeru Nanno ◽  
Kenzo Tanaka
Keyword(s):  
Molecular Weight ◽  
Degradation Products ◽  
Electron Microscopic Observation ◽  
Chemotactic Activity ◽  
Lower Molecular Weight ◽  
Electron Microscopic ◽  
Human Fibrinogen ◽  
Rabbit Skin ◽  
Molecular Weights ◽  
Active Fragments

SummaryFibrinogen degradation products were investigated for leukocyte chemotactic activity and for enhancement of vascular permeability. Both activities increased progressively with plasmin digestion of fibrinogen. Active fragments were partially purified from 24 hr-plasmin digests. Molecular weights of the permeability increasing and chemotactic activity fractions were 25,000-15,000 and 25,000 respectively. Both fractions had much higher activities than the fragment X, Y, D or E. Electron microscopic observation of the small blood vessels in rabbit skin correlated increased permeability with the formation of characteristic gaps between adjoining endothelial cells and their contraction.These findings suggest that lower molecular weight degradation products of fibrinogen may be influential in contributing to granulocytic infiltration and enhanced permeability in lesions characterized by deposits of fibrin and/or fibrinogen.

Fibrinolytic Activity of the Arterial Wall

1969 ◽  
Vol 21 (01) ◽  
pp. 001-011 ◽  
Author(s):  
K Onoyama ◽  
K Tanaka
Keyword(s):  
Carotid Artery ◽  
Fibrinolytic Activity ◽  
Arterial Wall ◽  
Aortic Wall ◽  
Internal Carotid ◽  
Atheromatous Plaque ◽  
Human Fibrinogen ◽  
The Media ◽  
The Common ◽  
Almost All

SummaryThe tissue fibrinolysis was studied in 550 specimens of 7 kinds of arteries from 80 fresh cadavers, using Astrup’s biochemical method and Todd’s histochemical method with human fibrinogen.In the microscopically normal aortic wall, almost all specimens had the fibrinolytic activity which was the strongest in the adventitia and the weakest in the media.The fibrinolytic activity seemed to be localized in the endothelium.The stronger activity lay in the adventitia of the aorta and the pulmonary artery and all layers of the cerebral artery.The activity of the intima and media of the macroscopically normal areas seemed to be stronger in the internal carotid artery than in the common carotid artery.Mean fibrinolytic activity of the macroscopically normal areas seemed to decrease with age in the intima and the media of the thoracic aorta and seemed to be low in the cases with a high atherosclerotic index.The fibrinolytic activities of all three layers of the fibrous thickened aorta seemed to decrease, and those of the media and the adventitia of the atheromatous plaque to increase.The fibrinolytic activity of the arterial wall might play some role in the progress of atherosclerosis.

A Novel Method for the Rapid Purification of Human and Rat Fibrin(OGEN) Degradation Products in High Yields

1979 ◽  
Author(s):  
W. Nieuwenhuizen ◽  
I. A. M. van Ruijven-Vermeer ◽  
F. Haverkate ◽  
G. Timan
Keyword(s):  
Degradation Products ◽  
Complete Separation ◽  
Purification Method ◽  
Amino Terminal ◽  
Novel Method ◽  
Rapid Purification ◽  
The One ◽  
High Yields ◽  
Size Heterogeneity ◽  
D Dimer

A novel method will be described for the preparation and purification of fibrin(ogen) degradation products in high yields. The high yields are due to two factors. on the one hand an improved preparation method in which the size heterogeneity of the degradation products D is strongly reduced by plasmin digestion at well-controlled calcium concentrations. At calcium concentrations of 2mM exclusively D fragments, M.W.= 93-000 (Dcate) were formed; in the presence of 1OmM EGTA only fragments M.W.= 80.000 (D EGTA) were formed as described. on the other hand a new purification method, which includes Sephadex G-200 filtration to purify the D:E complexes and separation of the D and E fragments by a 16 hrs. preparative isoelectric focussing. The latter step gives a complete separation of D (fragments) (pH = 6.5) and E fragments (at pH = 4.5) without any overlap, thus allowing a nearly 100% recovery in this step. The overall recoveries are around 75% of the theoretical values. These recoveries are superior to those of existing procedures. Moreover the conditions of this purification procedure are very mild and probably do not affect the native configuration of the products. Amino-terminal amino acids of human Dcate, D EGTA and D-dimer are identical i.e. val, asx and ser. in the ratgly, asx and ser were found. E 1% for rat Dcate=17-8 for rat D EGTA=16.2 and for rat D- dimer=l8.3. for the corresponding human fragments, these values were all 20.0 ± 0.2.

Sign in / Sign up

Export Citation Format

Share Document