bluuebunny/crossref_metadata_2025 huggingface dataset:
A NEW VARIANT OF FACTOR X DEFICIENCY (FACTOR X ROMA).
Authors: De Stefano V , Leone G , Ferrelli R , Bizzi B
Published: August 2018

A 13 years-old-girl was admitted in our Hospital for a large muscle hematoma of left psoas. At age 3 she had a severe tonsillar bleeding following angina. Afterwards she suffered from easy bruisability and recurrent epistaxis. Prothrombin time (PT) was slightly prolonged (14.7 sec, control 12 sec, INR 1.5), while a more marked prolongation in aPTT was noticed (54.7 sec, control 30.3 sec). All clotting factors resulted within the normal range but Factor X. Factor X antigen was 0.95 U/ml (Laurell), whereas Factor X activity was 0.52 U/ml by extrinsic system assay (rabbit brain and lung thromboplastin), 0.06 U/ml by intrinsic system assay (rabbit brain cephalin) and 1.15 U/ml by activation by RW-cephalin. Chromogenic assays (S-2222 and CBS 3139) perfor med after activation with RW gave 1.02 U/ml and 1.00 U/ml, respectively. The patient plasma had no inhibitory activity against Factor X. The parents of the proposita (first cousins) had Factor X antigen levels and RW functional activity around 1.00 U/ ml, whereas Factor X activity tested by extrinsic and intrinsic system assay was about 0.60 U/ml and 0.50 U/ml, respectively. This Factor X variant seems different from the other ones previously described, showing normal antigen levels and RW activation, a severe defect in the intrinsic activation and only a partial defect in the extrinsic activation. In particular the two defects more close to it (Factor X Friuli and Factor X Melbourne), both found in patients with Italian ancestry, were dif ferent because of a very prolonged PT (Factor X Friuli) or a normal PT (Factor X Melbourne).
REDUCED β-FXIIa-INHIBITION CAPACITY IN KEPARINIZED PLASMA SAMPLES.
Authors: Fuhrer G , Gallimore M J , Heller W , Hoffmeister H E
Published: August 2018

It has been shown by our group that B-FXIIa-inhibi-tion was reduced in platelet rich plasma. To investigate the effect of heparin on B-FXIIa-inhibition, kallikrein inhibition and C1-esterase inhibition high and low molecular weight heparin preparations were added to plasma samples. Using high molecular weight heparin from 0 to 5 anti Xa/ml a reduction in B-FXIIa-inhibition was seen from 103% to 59%.These effects were abolished by the addition of protamine chloride. No changes of kallikrein inhibition and C1-esterase-inhibition were observed in heparinized plasma samples After the addition of a low molecular heparin preparation to plasma samples, the reduction of B-FXIIa-inhibition was markedly decreased. The levels of the Hage-man fragment inhibitor capacity were lowered to 22% compared to initial values of 91%. The antagonization of heparin normalized these values. No reduction in kallikrein inhibition and C1-esterase inhibition were found in these samples because in all three assays C1-inhibitor activity is analysed, purified C1-inhibitor was added to buffer containing several heparin concentration ( see table )

These results suggest that the inhibition of B-FXIIa is markedly affected by heparin whilst no changes were seen in the inhibition capacity for C1-esterase and kallikrein.
HAGEMAN TRAIT INVESTIGATED BY FACTOR XII cDNA PROBES
Authors: Volinia S , Patracchini P , Vannini F , Felloni L , Panicucci F , Beranardi F
Published: August 2018

The presence of gene lesions and of restriction fragment length polymorphisms (RFLPs) has been investigated by means of cDNA probes for the coagulation factor XII (FXII).

A TaqI additional fragment (2.1Kb) has been found in two brothers with Hageman trait and in 11 members of their paternal lineage. Digestions with different enzymes exclude that FXII gene deletion is responsible for Hageman trait in this family. A point mutation originating an additional TaqI site is likely.

The abnormal pattern (not present in 40 normal subjects) is correlated with a reduced FXII activity and identifies the heterozygous subjects in the paternal lineage. The presence of two different gene lesions causing Hageman trait in this family can be inferred.

The TaqI additional site has been mapped within the 5 portion of the gene.

Data suggest the presence of one FXII gene per aploid genome and disagree with previous localization of FXII gene on chromosome 6.

Work supported by P.F. Ing. Gen. e Basi Mol. Mai. Ered. contratto CNR N.8400877
PLASMA FACTOR XIII AND ACTIVATION PROCESS ANALYZED BY ELISA METHOD FOR A2B2 COMPLEX.
Authors: Koike K , Hada M , Yorifuji H , Ikematsu S , Fukutake K , Fujimaki M
Published: August 2018

Factor XIII induces the crosslinking of fibrin at terminal stage of blood coagulation. New ELISA methods of a-subunit, b-subunit and a2b2 complex of factor XIII were developed by the author and the following results concerning the activation process of factor XIII were obtained. New ELISA methods for a-subunit, b-subunit and a2b2 complex of factor XIII were specific with high sensitivities on each items and indicated the measurement capacity for simultaneous quantitation of many samples, more over we looked upon the dissociation of a2b2 complex with these methods to able to analyze at the subunit levels of factor XIII in details. When a-subunit dimer of platelets was discharged into the plasma by the use of freezing and thawing method on PRP, it was more easily affected to lose their antigeniety than that of a2b2 complex, a-subunit levels in the plasma of congenital factor XIII deficiencies were measured in very low concentration or below the measurement sensitivity of this ELISA, b-subunit levels in the same plasma were indicated around the half of normal levels. These results were as same as another immunological method. It was suggested that the molecular conformation of a-subunit could be changed by the addition of thrombin in high concentration and consequently a-subunit with thrombin was modifyed to show high antigeniety. It was observed that a2b2 complex was dissociated by the addition of thrombin without calcium ion, and the process of this dissociation of a2b2 complex was remarkably accelerated by the addition of calcium ion. Because a-subunit and b-subunit were adsorved on fibrin clot, it might be presumed that the crosslinking of fibrin molecules could be accelerated locally on the surface of fibrin clot.
POST-OPERATIVE CHANGES IN FACTOR XIII and FIBRCNETITN
Authors: Reintz M , Sauer H , Witzke G , Neher M
Published: August 2018

Wound healing processes after surgery are not restricted to certain areas tut affect the vhole body; the ooagulaticn systan in particular is involved. We studied Factor XIII and fibrcnectin in the blood plasma of 16 patients before surgery, after surgery and cn the 1st, 3rd and 7th days after surgery; fibrcnectin was determined using IC-Partigen iimunodiffusicn and Factor XIII by Behringwerke rapid test. In 11 patients normal wound healing was observed (group A), while in 5 patients explications oocured (group B). Factor XIII: normal group (A): fall in concentration oenpared with the precpera-tive value up to the 7th day after surgery; group with explications (B): fall in concentration more pronounced than in the normal group up o the 7th day after surgery.

Fibrcnectin : Normal group (A): Fall in concentration ocpared with the precperative value up to the 1st day after surgery, followed by a rise in concentration; group with ocplicaticns (8): fall in concentration more marked than in the normal group, less marked rise in concentration than in the normal group. Duplications in wound healing were characterized by an increased fall in Factor XIII and fibrcnectin.
QUANTIFICATION OF FACTOR XIIIa-MEDIATED FIBRIN CROSSLINKING USING HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY-BASED IDENTIFICATION OF CROSSLINKED γ-CHAINS AND γ-CHAIN COMPONENTS.
Authors: Kiso U , Kaudewitz H , Henschen A
Published: August 2018

Factor XIIIa catalysis the formation of isopeptide bonds in fibrin vhereby first and quickly the C-termini of two γ-chains in adjacent molecules are crosslinked and then much more slowly several a-chains. The crosslinking sites of the γ-chain are well-known, but those of the a-chain still only tentatively and partially identified. The crosslinking reaction has previously usually been monitored by sodium dodecylsulfat-polyacrylamide gelelectrophoresis (SDS-PAGE) of the mercaptolysed fibrin or fibrin degradation products. More recently, specific antibodies against the crosslinked γ-chain region have been produced which allow immunological assays for crosslinking products, i.e. for D-dimer.

The present study deals with novel, high-performance liquid chromatography (HPLC)-based procedures for the identification and quantification of crosslinked γ-chains or γ-chain products. The degree of crosslinking was determined by quantifying the dimer either of the total γ-chain or of its C-terminai cyanogen bromide fragment. For this propose factor Xllla-containing fibrinogen was incubated with thrombin in the presence of calcium ions and cysteine. The reaction was interrupted by the addition of a urea-mercaptoethanol solution after various periods of time and the samples analysed in parallel by reversed-phase HPLC and SDS-PAGE. In both systems the steady decrease first in γ- and then in a-chain and simultaneous increase in γ-chain dimer was observed. The dimeric γ-chain appeared as a well separated and defined peak on HPLC. In the alternative approach crosslinked fibrin, fibrin degradation products or γ-chain were first cleaved by cyanogen bromide and then the resulting fragments were analysed by reversed-phase HPLC. Also here a characteristic component appeared which was identified by sequence analysis as the dimeric C-terminal fragment of the γ-chain and which only was present in crosslinked material.
POLYMERIZATION OF FIBRINOGEN AND FIBRONECTIN CATALYZED BY FACTOR XIII.
Authors: Procyk R , Block M , Blomback B
Published: August 2018

Factor XIII catalyzed the formation of gels in solutions containing physiological concentrations of fibrinogen and fibro-nectin. Oligomeric intermediates were isolated from reaction mixtures at early times prior to gel formation by chromatography on gelatin-Sepharose and by FPLC using Superose 6 columns. The products of two simultaneous polymerization reactions were characterized: fibrinogen oligomers (fibrinogenin) from the poly merization of fibrinogen, and conjugates of fibrinogen-fibro-nectin (heteronectin) from heteropolymer formation involving the two proteins.

At a constant concentration of fibrinogen (2.5 mg/mL) and factor XIII (0.4 U/mL), the appearance of different sizes of fibrinogen polymers depended on the concentration of fibronectin added to the reaction mixture. At fibronectin concentrations in the range of the normal plasma level of 0.3 mg/mL, fibrinogen formed oligomers of various sizes up to heptamer before incorporating a molecule of fibronectin. At a high fibronectin concentration (3.2 mg/mL) most of the fibrinogen reacted with the fibronectin at the monomer stage, although small amounts of fibrinogen dimers and trimers were also formed.

Heteronectin formation coincided with the appearance of filamentous and particulate matter. This material became incorporated into a gel structure if sufficient fibrinogen was present in the reaction mixture (about 0.5 mg/mL). If these factor XIII catalyzed polymerization reactions occur in the microvasculature under conditions where the fibrinogen concentration might be significantly lowered, the production of fibrinogen-fibronectin polymeric material without gel formation would be favored.
FACTOR XIII CONCENTRATE FOR PROPHYLAXIS OF REBLEEDING IN SUBARACHNOID HEMORRHAGE (SAH) - RESULTS OF A PROSPER TIVE MULTICENTER PILOT STUDY
Authors: Thie A , Henze T h , Deggar D , Obering M , Clemers R , Kleinz H J , Lombard G F
Published: August 2018

Rebleeding occurs in subarachnoid hemorrhage (SAH) in 20 - 25 % of patients, with a mortality rate being above 50 %. The cause of rebleeding is considered to be premature fibrinolysis of the fibrin clot surrounding the site of rupture. Since the stability of the fibrin clot is influenced by the activity of coagulation factor XIII, and moreover, a factor XIII deficiency has been reported in SAH patients, the question arises as to whether the incidence of rebleeding can be influenced by the administration of F XIII concentrate.

During a period of 6 months, 69 patients with acute SAH were enlisted in an open, prospective, multicenter study. On admission, 5 patients were classified as stage I (7.2%) according to the Hunt and Hess scale, 22 as stage III (31.9%), 11 as stage IV (16%) and 9 as stage V (13%). Aneurysm was confirmed by angiography in 52 patients (75%). All the patients received 10 x 1250 U F XIII concentrate during the first 15 days after the initial hemorrhage. Surgery on the aneurysm was performed between day 3 and 32 (median: day 13) in 35 patients.

A total of 7 rebleedings occurred in 6 patients (8.7%), of whom 2 were stage I - II and 4 were stage III - V cases. Cerebral infarction was observed in 10 patients (14.5%), and hydrocephalus requiring shunting occurred in 1 patient. There were no cases of peripheral thromboses or embolisms. After 4 weeks, the overall mortality rate was 26%. (stage I - II: 11.1%, stage III - V: 37.5%).

The conventional approach in the prophylaxis of rebleeding in SAH is an early operation or intravenous administration of antifibrinolytics. However, as none of these measures significantly reduce overall mortality, the present pilot study investigated a new, therapeutic approach in which F XIII concentrates were administered in order to stabilize the fibrin clots and prevent premature fibrinolysis. The data so far show that Fibrogammin P is an effective and well tolerated agent for the prophylaxis of post-SAH rebleeding. In order to statistically confirm the results of the pilot study, we have, in the meantime, started a prospective, randomized, placebo-controlled, multicenter double-blind study, which will involve 750 patients over a period of 2 years.
FIERIN POTENTIATING THE ACTIVATION OF FIBRINOLYTIC SYSTEM ON THE ENDOTHELIAL CELL SURFACE: FORMATION OF THE SURFACE-BOUND TRIMOLECULAR COMPLEX OF FIBRIN, PLASMINOGEN, AND PLASMINOGEN ACTIVATOR
Authors: Liu Chung Y , Wallen Per , Handley Dean , Smith Jena
Published: August 2018

Previous reports have shown that f ibrin (Fn), plasminogen (Pig), and tissue plasminogen activator (tPA) can bind to bovine aortic endothelial cell (BAEC) respectively. The present studies are to examine the formation of the trimolecular complex of Fn, Pig, and tPA on the BAEC surface. BAEC monolayers were first incubated with one of the three components in buffer (pH 7.5, 25 C) and washed, and then incubated with the second component and washed. Finally, the BAEC monolayers after the first and second incubations were incubated with the third component in the presence of plasmin substrate S-2251. Plasminogen activation rates (PAR)(pM/min) were measured. The results (TABLE) show that Pig and tPA retain their respective activities after their binding to BAEC surface, either Pig or tPA as the first bound component. These results suggest that the formation of Plg-tPA complex can occur after their binding to the BAEC surface. With Fn as the second binding component, BAEC monolayers show higher PAR values (138 & 187, pM/imin) than those without Fn (34.5 & 59.1 pM/min), suggesting Fn potentiation of tPA-induced Pig activation on the BAEC surface. Since the reaction of Fn with the Plg-tPA complex is required for the expression of fibrin potentiation, the present results suggest that the formation of trimolecular complex can occur on the BAEC surface. Further, when Fn was the first bound component and Pig or tPA was the second or third bound component, the BAEC rmonolayers show the highest PAR values (353 & 331 nVrmin), suqqestinq that both Plq and tPA can bind not only to BAEC surface but also to Fn which was already first bound to BAEC surface. Thus, trimolecular complex may have multiple binding sites on BAEC surface and anyone of the three binding sites (Fn, Pig, and tPA) may act as the binding site for the trimolecular couplex on the BAEC surface.
MODIFICATION OF FIBRIN POLYMERIZATION INDUCED BY MONOCLONAL ANTIBODIES AGAINST FRAGMENT D DOMAIN OF FIBRIN/OGEN DEGRADATION PRODUCTS
Authors: Mirshahi M , Soria J , Soria C , Mirshaho S , Perrot J Y , Boucheix C , Bernadou A
Published: August 2018

Monoclonal antibodies (McAb) are often used to detect the domains of a molecule involved in an important function of a biological system.

In this work, the effect of 20 McAb directed against the D domain of fibrin/ogen degradation products (FbDP) were analyzed on fibrin polymerization. Two McAb which react with fragment D1 but not with fragment D3 inhibited both thrombin and reptilase-induced fibrin formation. It is therefore suggested that these 2 McAb recognize an epitope in the vicinity of the polymerization site "a" located in the JT 374-396 domain in fibrin/ogen molecule. (This sequence is only present in D1 and not in D3.

An inhibitory effect on thrombin and reptilase-induced fibrin formation was also induced by 2 other anti D McAb which recognize an epitope present in both D1 and D3 fibrinogen-degradation products. Therefore, these 2 McAb do not react directly with the polymerizing site "a". A possible mechanism of this inhibition could be a conformational change induced by the binding of these McAb, leading to the masking of the polymerizing site "a". Another possible mechanism of the inhibitory effect is that thrombin and reptilase-induced fibrin formation may involve other sites on the fibrinogen molecule. It should therefore be suggested that unknown domains could be important for fibrin polymerization. These domains may act either directly or indirectly by inducing a three dimensional structure of the native molecule required for the expression of polymerizing domains.
IMMUNO-VISUALIZATION OF FIBRINOGEN AND FIBRIN IN GELS PRDUCED BY GELATION OF PLASMA WITH ETHANOL
Authors: Grøn B , Brosstad F
Published: August 2018

The Ethanol Gelation Test(=EGT) is a well-documented simple,specific and frequently used test to detect plasma soluble fibrin(Godal & Abildgaard:Gelation of soluble fibrin in plasma by ethanol.Scand.

J.Haanat.3,342,1966) .If soluble fibrin present in plasma amounts to 1% or more of the plasma-f ibrinogen conc.,the admixing of 0.15 ml 50% ethanol to 0.5 ml plasma in a test tube will-(subsequent to incubation for 10 min at 20°C)-upon tilting the test tube semi-horizontally produce a characteristic,(upwardly) convex gel. Although earlier studies have confirmed the validity and specificity of EGT as a means to detect soluble fibrin,we found it of interest to see to which degree such soluble fibrin is FXIII-stabilized EGT-positive(from patients with Disseminated Intravascular Coagula-tion(DIC) and EGT-negative plasma was studied as follows: The EGT test was performed as above,and the entire content of the test tube emptied upon a nylon micro-meshed membrane.Applying slight suction underneath the nylon manbrane the fluid was removed,leaving the ethanol precipitated material which was immediately dissolved in SDS (1%)-urea(5M)-Tris-HCl (0.15M,pH8.6). After incubation at 100°C for 1 min the material was SDS-electrophoresed on flat-bed agarose(2%) Subsequent to Western-blotting onto nitrocellulose and gelatine-blocking, the fibrin(ogen)-related pattern was reacted with either: a)polyclonal antibodies to fibrinogen,b)plyclonal antibodies to FPA or c)monoclonal antibody to FPA(gift from Dr.Nieuwenhuizen, Leyden, Holland) .Then,the fibrin(ogen)related pattern was developed using peroxidase-conjugated secondary antibodies.From the specificity of the primary antibodies used,it could be deduced that:1)A substantial amount of the soluble fibrin content of DIC-plasma was present in an oligomeric form(up to 6-mers) .2)These oligomers contained fibrinogen, i.e. thus representing FXIII-1 inked fibrinogen/fibrin hybride molecules .3) Even normal plasma contained some detectable oli-gomers(up to 3-mers) .4) Col lecting blood with all appropriate thrombin- and FXIII-inhibitors did not change the patterns obtained and described above.It may be concluded that soluble fibrin occurs mainly in a FXIII-stabilized,oligomeric form which contains fibrinogen Due to the nature of the polymerization process,the fibrinogen moiety of these hybride molecules must be end-located representing a physiological means to inhibit further polymer growth.
HUMAN PLASMA FIBRINOGEN MOLECULAR WEIGHT VARIANTS:CHARACTERIZATION BY HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY AND IDENTIFICATION BY SEQUENCE ANALYSIS
Authors: Müller E , Henschen A
Published: August 2018

Human plasma has been shown to contain several fibrinogen variants which differ from each other in molecular weight. The three most common forms, with molecular weights of 340 kD, 305 kD and 270 kD, have been reported to change considerably in their relative amounts during certain pathophysiological processes such as acute phase reactions, disseminated intravascular coagulation, fibrinolytic disorders, liver disease and cancer. Though it has been suggested that the differences in molecular weight are due to degradation of one or both, respectively, of the carboxy-terminal parts of the Aα-chains, the removed or altered segments have so far never been precisely determined. Consequently, it has only been possible to speculate about the origin of the variants.

The aim of this study was to isolate the various molecular weight variants from plasma and to characterize their peptide chain components proteinchemically. Fibrinogen was first isolated from plasma by glycin precipitation and the variants were then separated by stepwise ammonium sulfate precipitation, taking advantage of their different solubility.

The peptide chain components of the various fractions were isolated by reversed-phase high-performance liquid chromatography (HPLC). The N-termini were identified by direct sequence analysis. Chemical and enzymatic cleavages of the peptide chains resulted in fragment mixtures which were compared with the corresponding mixtures obtained from commercial fibrinogen by HPLC fingerprinting. Finally, the fragments which differed were identified by N-terminal sequence and amino acid analysis so that the exact C-termini of the peptide chains,especially in the lower molecular weight variants,could be determined. With the help of this information conclusions may also be drawn about the origin of the lower molecular weight variants and about the mechanism by which they may he formed.
STRUCTURE AND FUNCTIONAL PROPERTIES OF A FIBRINOGEN EERIVATIVE FORMED BY LIMITED PROTEOLYSIS WITH RED BLOOD CELL
Authors: Lipinski B , Ewaskiewicz J , Niewiarowski S , Budzynski A Z
Published: August 2018

Normal human plasma contains, in addition to fibrinogen (Mr 340,000), two major thranbin-coagulable fibrinogen derivative of Mr 320,000 and 300,000. Previous investigations demonstrated that these derivatives do not originate from plasmic cleavages, however, no enzyme(s) responsible for the cleavages have been identified. The purpose of this study was to investigate a possible role of red blood cells (RBC) in the formation of these physiologic fibrinogen derivatives. RBC ghosts were prepared from ACD blood obtained from normal donors after removal of leukocytes and platelets. Purified human fibrinogen, that contained only one band of Mr 340,000 as judged by SD6-PAGE, was incubated with RBC ghosts at 37°C. SDS-PAGE of the supemate revealed the presence of a single band of approximate Mr 275,000 after one hour of incubation. Proteolytic degradation of fibrinogen by RBC ghosts was inhibited by STI but not by trasylol, leupeptin or EDTA suggesting that putative enzyme is distinct frcm plasmin and calpain. Polypetide chain ánalysis of Mr 275,000 derivative demonstrated degradation of its A alpha chain. The derivative shewed an impaired binding to AEP-activated platelets as shown by decreased number of fibrinogen binding sites calculated frcm Scatchard plots.

Study of polymerization reaction of Mr 275,000 derivative catalyzed by thrombin revealed significantly impaired kinetics.

The maximum polymerization rate of Mr 275,000 monomers was 51% of that of intact fibrinogen. The data suggest that RBC ghosts contain enzyme (s) that are capable to induce a limited proteolytic degradation of fibrinogen that may contribute to alteration of its biologic functions inportant in hemostasis and thrombosis.
COMPLEX FORMATION,BETWEEN THROMBIN AND FIBRINOGEN OR FIBRINOGEN DEGRADATION PRODUCTS (FDP)
Authors: Kaczmarek E , McDonagh J
Published: August 2018

To identify the part of the fibrinogen molecule which interacts with thrombin binding of human thrombin to plasmic FDP was analyzed.125I-thrombin was incubated with FDP, purified fibrinogen fragment D or fragment E in the presence of 0.2% glutaraldehyde. Incubation mixtures were analyzed by SDS-PAGE and autoradiography. Under non-reducing conditions, the autoradiogram from the thrombin and fibrinogen fragment D incubation showed only one dark band, the molecular weight (Mr) of which was identical to that of thrombin, indicating no complex formation between thrombin and fragment D. With thrombin and fibrinogen fragment E, two dark bands were observed: the electrophoretic mobility of the first was the same as that of thrombin and the Mr of the second was equal to the sum of the Mr of thrombin and fragment E. This shows that human thrombin Forms a complex with fibrinogen fragment E. Hence, we can conclude that only the N-terminal part of the fibrinogen molecule is necessary for interaction with thrombin. Under reducing conditions, the complex of thrombin with fragment E produced four bands on gel electrophoresis. One was thrombin; the remaining three were complexes of thrombin with fragment E chain remnants. To investigate this further, carboxymethylated human fibrinogen chains Aα, Bβ and γ were purified and coupled to Sepharose 4B. 125I-thrombin was applied on the three columns. Nearly all radioactivity was bound to the three affinity columns and was eluted with higher NaCl concentration. We can infer that complex formation between thrombin and fibrinogen requires interaction between thrombin and all three fibrinogen chains. To find which thrombin amino acid residues are responsible for interaction with fibrinogen, human thrombin was coupled to Affi-Gel 102 and Affi-Gel 202 through thrombin's carboxyl and amino groups, respectively. We observed binding of fibrinogen and fibrinogen fragment E only to Affi-Gel 102 column, indicating that lysine residues and perhaps the N-terminal of the thrombin molecule interact with fibrinogen. When thrombin was bound to the gel through its amino groups, there was no interaction between thrombin and fibrinogen or fragment E.
CALCIUM-INDUCED FORMATION OF FIBRINOGEN -- FIBRIN COMPLEXES
Authors: Kaminski M , McDonagh J
Published: August 2018

Presence of calcium chloride (8.3 mM) at u = 0.1 enhanced precipitate formation in several different fibrinogen preparations. The precipitate had gel like properties and demonstrated syneresls when disturbed. Precipitation proceeded in the time dependent manner with a plateau after 30 min. The amount of precipitate ranged from 4-24% of total protein content of different preparations. Precipitates did form at u = 0.1 without calcium ion; however, the amount of protein was 3 to 7 fold less than when calcium ion was present.

SOS gel electrophoresis of calcium precipitates demonstrated cross-linking of gamma chains. Cross-linking, but not precipitate formation was blocked by parachloromercurie-benzoate (PCMB). The precipitates were redissolved in 2M urea, treated with thrombin, and analyzed for flbrinopeptide content by HLPC. Precipitate formed in absence of calcium ion contained 19% of FPA and 89% of FPB. Precipitates formed in the presence of calcium ion contained 49% of the expected amount of FPA and 98% of expected FPB. The precipitation phenomenon could be reproduced after fibrin monomer-free fibrinogen (prepared by dialysis of calcium supernatants against calcium free buffer) was treated briefly with thrombin.

The above finding suggests that calcium ion enhances polymerization of Intact fibrinogen and molecules missing one or two FPA's.
PROPERTIES OF A HUMAN FIBRINOGEN A α-CHAIN DEGRADATION FRAGMENT
Authors: LAU H K F
Published: August 2018

A fragment of human fibrinogen was obtained by a 30-min digest of fibrinogen with plasmin. The reaction was terminated by 10 mM diisopropyl fluorophosphate and the fragment obtained by sequential gel filtrations on Sephacryl S-200 and Bio Gel P-30. This fragment displayed a single band on both reduced and nonreduced SDS polyacrylamide electrophoreses with a relative Mr of ∼25,000. It did not crossreact with antisera against fibrinogen, fragment D or fragment E and suggested its sequestered location in native fibrinogen. An antiserum raised against this fragment reacted with the fragment itself as well as fibrinogen. Its amino acid composition and other properties suggested that it was derived from the Aa -chains of fibrinogen. The fragment had anticoagulant activity as it was able to inhibit thrombin time in a dose-dependent fashion and it could also inhibit the polymerization of fibrin monomers. However, radioactively labelled fragment was not able to bind calcium ions in equilibrium dialysis experiments, nor was it able to bind plasma fibrin clot, fibrinogen- or fibrin-bound Sepharose columns. The activation of plasminogen by streptokinase was inhibited by the fragment. On the other hand plasminogen activation by melanoma tissue plasminogen activator and urokinase were both enhanced in the presence of the fragment.
FIBRINOGEN GENOVA III: A NEW CONGENITAL DYSFIBRINOGENEMIA WITH BLEEDING DIATHESIS AND DEFECTIVE LYSIS BY PLASMIN
Authors: Castaman G , Rodeghiero F , Galletti A , Barone E , Gastaldi G
Published: August 2018

A new abnormal fibrinogen was recognized in a woman with lifelong bleeding Symptoms. Patient's plasma exhibited prolonged thrombin and reptilase times. Very low fibrinogen level was obtained by functional assay whereas heat precipitation and immunological methods gave normal fibrinogen values. This pattern was also observed in her daughter. Patient's plasma prolonged thrombin time of normal plasma. Fibrin monomer polymerization of purified fibrinogen was severely impaired, whereas fibrinopeptide A release by thrombin occured at rate and extent indistinguishable from normal. Sialic acid content was normal. SDS-PAGE of purified molecule revealed normal pattern and factor XIII-dependent crosslinking occurred as in normal controls. Lysis by plasmin of fibrinogen examined on SDS-PAGE showed an abnormal degradation profile, only minimal traces of fragments D and E being detectable after prolonged digestion in comparison with normal. Binding of plasminogen and alfa-2-antiplasmin to fibrin was normal. This abnormal fibrinogen has been tentatively called Genova III.
ROLE OF ANTITHROMBIN FOR INACTIVATION OF THROMBIN ON ENDOTHELIUM
Authors: Swedenborg J , Frebelius S , Nydahl S , Olsson P
Published: August 2018

Inactivation of thrombin which occurs mainly on the endothelium requires binding of the enzyme to receptors on the endothelial cells. One receptor for thrombin is glycosaminoglycans (GAG). Antithrombin (AT), however, also binds to GAG and may be involved in endothelial inactivation of thrombin.

The purpose of the present study was to study the role of AT for the inhibition of thrombin on endothelium. The experiments were performed on rabbit aorta segment in vitro. Thrombin in solution and on the endothelial surface was functionally assayed with aid of a synthetic chromogenic substrate (thrombinS) or fibrinogen (thrombinF). In the latter case liberation of fibrinopeptide A (FPA) was measured. Inactivation of thrombin was estimated by the difference between loss of thrombin from the solution and recovery of thrombin on the endothelial surface during the incubation with thrombin.

Preincubation of the endothelium with AT or plasma increased the inactivation of both thrombinS and thrombinF but AT-free plasma had no such effect. Preincubation with heparin (5 IU ml) decreas-the inactivation of thrombin on the endothelium. Endothelial segments preincubated with AT caused a much more rapid inactivation of thrombin over time as compared to control segments. Aortic segments preincubated with heparin caused a slower inactivation of thrombin on the sruface as compared to control segments.

It is concluded, that preincubation with AT enhances the inactivation of thrombin on the endothelium whereas preincubation with heparin has the opposite effect. Heparin causes liberation of endogenous AT from the endothelial surface which may explain the decreased inactivation on the surface in the latter case.
THE BINDING OF ANTITHROMBIN TO CAPILLARY ENDOTHELIAL CELLS GROWN IN VITRO
Authors: de Agostini A , Marcum J , Rosenberg R
Published: August 2018

Cloned endothelial cells from rat epididymal fat pads synthesize anticoagulantly active heparan sulfate proteoglycans containing the disaccharide, GlcA→ AMN-3,6-O-SO3, which is a marker for the antithrombin-binding domain of heparin. To demonstrate that antithrombin (AT) binds to cell surface heparan sulfate, a binding assay employing 125I-AT and cell monolayers has been developed. Post-confluent endothelial cells (7 days) were incubated with radiolabeled AT for 1 h at 4° and washed with PBS. Bound radioactivity was quantitated after solubilizing whole cells. Under these conditions, ∼1% (2174±50 cpm/5x104 cells) of the 125I-AT bound to the endothelial cell monolayer, whereas none of the radiolabeled protein bound to CHO cells or bovine smooth muscle cells. Utilization of unlabeled AT (1 μM) in experiments conducted as described above resulted in a reduction (73%) of the binding of the labeled species to endothelial cells. To assess whether heparan sulfate was responsible for AT binding, cell monolayers were incubated for 1 h at 37° with purified Flavobacterium heparinase (0.2 units). Over 90% of 125I-AT binding to these cellular elements was suppressed with the bacterial enzyme. Internalization of radiolabeled AT by endothelial cells was examined by incubating the protease inhibitor and cells at 4° and 37 . An initial rapid binding was observed at both temperatures. At 4° AT binding plateaued within 15 min, whereas at 37° binding did not plateau until 60 min and was 30% greater than that observed at 4. These data suggest that surface-associated AT can be internalized by endothelial cells. In addition, AT binding was shown to increase with the length of endothelial cell postconfluence, indicating an accumulation of heparan sulfate by these cells during quiescence. In conclusion, our studies support the hypothesis that the vascular endothelium is coated with heparan sulfate-bound AT, which is responsible for the antithrombotic properties of these natural surfaces.
ALTERED INTERACTION OF THROMBIN AND ANTITHROMBIN III WITH THE VASCULAR WALL
Authors: Bashkov G , Kalishevekaya T , Strukova S
Published: August 2018

The role of the endothelial injury in the development of the thrombophylic state was studied in rats with nephrotic syndrome (NS,Heymann nephritis).There were a 6-fold increase of the soluble fibrin concentration and a 30% decrease of plasma antithrombin III (AT) activity in the NSIt was found that the plasma half-life of 125 J-labelled α-thrombin (10-7 M) is 3,0 ± 0,6 min in control animals and 4,0 ± 0,1 min in NS rats. At 20 min following the administration of bovine 125J-thrombin it was observed that in normal animals 84% of the radiolabelled enzyme was bound with vessel wall.while in NS rats the figure was only 63% (p< 0,05). The alteration of thrombin binding to the vascular wall lead to an increase in the amount of soluble fibrin-monomer and AT-proteinase complexes.AT-thrombin complexes and a proteolytically modified form of AT (Mr<68 kDa) were isolated from NS rats plasma by affinity chromatography on heparin-sepharose and chromatofocusing.At 3 min following injection of a 100-fold molar excess of bovine AT (1,7 .10-5 M) it was observed that 35% of thrombin reversibly bound to the endothelium could be detected in the circulation of normal rats. The same excess of AT induced only a 10% (p<0,001) release of 125J-thrombin to the blood stream in the NS rats through the formation of 125 J-thrombin complexes with Mr≥100 kDa.It is being proposed that injury of the vascular wall in the NS animals facilitated the interaction of the enzyme with the substrate (fibrinogen) and inhibitor (AT), and leads to ineffective inactivation of thrombin bound to the endothelium by AT.
VASCULAR DAMAGE IN PATIENTS ON CHRONIC HEMODIALYSIS
Authors: Izumi K , Noguchi M , Iwasaki E , Deguchi K , Shirakawa S
Published: August 2018

Calcification in the abdominal artery and endothelial cell function of the peripheral veins were studied in the patients recieving maintenance hemodialysis for the chronic renal failure.

The grade of calcification was expressed by the aortic calcification index (ACI) calculation from the abdominal CT scan films. The endothelial cell function was estimated from the release capasity for tissue plasminogen activator (t-PA) and von Willebrand factor (vWF), and the fibrinolytic capacity (by ELT and fibrin plate lysis area) during 10-min venous occlusion (VO) of the cubital vein. The dialysed patients were divided into 4 groups according to the duration of hemodialysis : group A ; under 1 year (n=7), group B ; 1-4 years (n=14), group C ; 4-7 years (n=15) and group D ; 7-10 years (n=8).

The level (X + SD) of vWF : Ag, vWF act. (RCoF) and t-PA in patients before VO were 439 ± 255(%),171 ± 42(%), 4.2 ± 0.6(ng/ml) in group A, 292 ± 157, 151 ± 35, 4.1 ± 1.2 in group B, 174 ± 114, 133 ± 47, 3.7 ± 0.8 in group C and 130 + 27, 66 ± 21, 3.1 ± 0.7 in group D, respectively. With the increase in the duration of hemodialysis, the release capacity for vWF and t-PA, and the fibrinolytic capacity during VO decreased regardless of the aggravation of fibrinolytic activity before VO, When the activity was estimated by ELT, Cl-inactivator resistant fibrinolytic activity and fibrin lysis area. These findings are thought to reflect the exhaustion of endotherial cells. In elder patients (60-80 years old), the release capacity for vWF and t-PA during VO decreased with the increase in the ACI level.

It is conclude that the patients on chronic hemodialysis have a greater incidence of calcification of abdominal artery and lower function of endothelial cells in the cubital vein than non-dialysed control.
HUMAN HEPATIC ENDOTHELIAL CELLS AND HEPATOCYTES IN CULTURE: MORPHOLOGICAL FEATURES, AND PRODUCTION OF VON WILLEBRAND FACTOR AND FIBRINOGEN
Authors: Harrison R
Published: August 2018

Liver cells were derived from cadaveric organ donors. Pieces of human liver 5 to 50 grams were minced, washed, and incubated in collagenase at 37 degrees C. After washing, the cell suspension was plated into culture vessels that had been briefly pre-treated with an extract derived from human liver. A mixed population of liver cells, including endothelial cells, hepatocytes, and Kupffer cells, attached within hours. At the end of 2 to 3 weeks there developed clusters of densely packed cells of two types. The most numerous cells were initially fusiform but grew as a monolayer even when densely packed. As density increased they assumed a polygonal form; cells with this morphological appearance stained immunocytochemically for von Willebrand factor antigen. They were relatively small and resembled cells derived from human umbilical vein except that the cytoplasm was more filmy in appearance. The second prominent cell type was significantly larger and likewise replicated to form clusters. These large cells sometimes contained multiple nuclei, exhibited a relatively low nuclear to cytoplasmic ratio, and immunocytochemically stained for human fibrinogen. A more distinct nuclear membrane and prominent nucleoli were characteristics of hepatocytes that were useful light microscopically in distinguishing these cells from sinusoidal endothelial cells. Ultrastructurally, endothelial cells were characterized by small size, holes in and among the cells that probably were the in vitro analogue of fenestrae, and numerous Weibel-Palade bodies in the cytoplasm, which otherwise was relatively bland. Hepatocytes, by contrast, had an active appearing cytoplasm containing more organelles. Canaliculi and typical tight junctions formed between adjacent hepatocytes. Levels of vWF and fibrinogen increased in a time dependent manner in media overlying this mixed population of cells. Human factor VIII has not yet been detected in the media overlying these mixed cells derived from human liver, and factor VIII antigen has not yet been demonstrable immunocytochemically in either cell type.
PERMSELECTIVITY OF CULTURED ENDOTHELIAL MONOLAYERS: EFFECT OF SIZE AND CHARGE OF THE TRANSPORTED MOLECULES
Authors: Bizios R , Blumenstock F A , Del Vecchio P J , Malik A B
Published: August 2018

Known molecular size neutral dextrans (molecular weight (MW) range ∼6,000-∼500,000), anionic (dextran sulfate, ∼500,000 MW), and cationic (DEAE-dextran, ∼500,000 MW) were used to determine the permselectivity characteristics of bovine pulmonary arterial endothelial monolayers. The experimental system consisted of two compartments separated by a gelatinized polycarbonate membrane (0.8 μm pore size) on one side of which endothelial monolayers were grown to confluence. Dextran solutions (1 gram %) were prepared in phosphate buffered saline buffer (containing 0.5 gram % serum albumin) and placed on the luminal side. Transendothelial dextran transport at 37°C was studied as a function of time. Dextran concentrations were determined spectrophotometrically using the anthrone method. The results (mean ± S.E.) of the dextran concentrations in the abluminal compartment at 45 minutes are given in the table.

The endothelial monolayers markedly restricted dextran transport compared to transport across gelatinized, polycarbonate membranes alone (controls). Furthermore, the results indicate that size and charge of the transported molecules determine the sieving mechanism responsible for their passage across the cultured endothelial monolayer. (Supported by HL32418.)
FACTOR VIII IN VIRUS INFECTED CELLS: A LIGHT MICROSCOPIC OBSERVATION
Authors: Bruggeman C A , Debie W H M , van Dam-Mieras M C E
Published: August 2018

Viruses may be important in atherosclerosis as inciters of arterial injury or as modulators of the metabolism of the infected cells. Cytomegalovirus (CMV) is one of the members of the herpesvirus family ;that infect human beings. Virus was detected in arterial walls of patients with atherosclerosis (1). The effect of CMV on endothelial cells and on the metabolism of these cells was subject of this study.

Human endothelial cells monolayers were established from cells obtained by collagenase treatment of human umbilical cord veins and arteries. The cells were grown in medium 199 containing growth factor and 20% fetal calf serum. All monolayers were near confluency at the time of viral infection. The cells were purified by FACS cell sorting using dilacLDL as marker (2). Infection of endothelial cells was performed using CMV laboratory strains ADI69 and Kerr and wild strains isolated from immunosuppressed patients (obtained by Dr. H.T. Wei land, Dept. Virology, University of Leiden, The Netherlands). Intracytoplasmic and intra-nuclear viral antigens were detected in 10-20% of the endothelial cells by fluorescence microscppy using TRITC-labeled monoclonal antibodies against CMV. Centrifugation of the cell monolayer during virus incubation resulted in an increase in the percentage of virus positive cells. The effect of virus infection on presence ot Tacxor VIII in the cells was studied using fluorescence microscopic examination of cell monolayers using a double staining technique. The presence of factor VIII in the cytoplasm of the cells was demonstrated using FITC-labeled antibodies and viral antigens were demonstrated by TRITC-labeled antibodies The results of these experiments showed that HCMV-infection of endothelial cells resulted in the disappearance of factor VIII from the cytoplasm of the endothelial cells at 48-72 hrs post infection without affecting the viability of the infected cells. The importance of this finding in relation to the function of the endothelial cells will be discussed.

References:

(1) Melnick, J.L. et al. The Lancet (1983): 644-647.

(2) Voyta, J.C. et al. J. Cell Biol. 99 (1984): 2034-2040.
DISTRIBUTION OF ENDOTHELIAL MARKERS ALONG THE VASCULAR TREE
Authors: Baudin B , Drouet L , Carrier J L , Bérard M , Wu Q Y
Published: August 2018

Few specific markers of the endothelial cells are available. Von Willebrand factor has been recognized as the most specific but we have shown that its intracellular content and extracellular release varies largely along the vascular tree of the pig. Synthesis is maximal in capillaries and in pulmonary artery endothelial cells. Synthesis is almost nil in the aorta. Intermediary values are encountered in the inferior vena cava. We studied the distribution of angiotensin converting enzyme (ACE) in endothelial cells along the vascular tree, as synthesis, storage, membrane expression and release of this enzyme is totally different from that of von Willebrand factor. Primary culture of endothelial cells from various origin of the vascular tree were studied. ACE was assessed by a functional radiometric assay using a specific substrate, cellular expression depends : 1 - on culture conditions (medium with serum, adult pig serum, foetal calf serum, medium without serum), 2 - on time in culture and 3 - on the degree of cellular confluency. ACE is easily measured both in cellular extract and in the medium. After cellular fractionation, ACE is concentrated in membrane fractions. Changes were found between the arterial and venous endothelial cells but no significant variation in the distribution between intracellular and released ACE were noted. Arterial endothelium (aortic and pulmonary artery) behaved similarly while in venous endothelium (inferior vena cava) both the cellular and the releasable ACE decreased. A specific antibody to the cellular form of the porcine ACE is being raised to complete these functional studies by antigenic dosages and immunolocalization.
ELASTASE TYPE ACTIVITY OF ENDOTHELIAL CELLS
Authors: Menasni S , Hornebeck W , Robert L , Legrand Y
Published: August 2018

Elastin degrading enzymes have been reported in the vessel wall and both fibroblasts and smooth muscle cells have been shown to produce elastase type enzymes in culture. Data is presented here showing that porcine aortic endothelial cells produce enzyme activities hydrolyzing elastin and synthetic substrates I Sue Ala Ala Ala nitroanilide, SAPNAI considered specific for elastase. Enzyme activity against the SAPNA but not against H-elastin was found to be associated with the cells after triton lysis .This activity was not secreted into the culture medium . The elastolytic activity has been partially characterized in relation to the kinetic of hydrolysis, pH optimum and susceptibility to different inhibitors. These studies revealed the presence of at least two enzymes: a metalo-protease with a pH optimum of 7.5 which accounts for approx. 80% of the total activity, and a serine protease with pH optimum of 8.0 which accounts for the remaining 20% . When the conditioned culture medium was studied, virtually no proteolytic activity could be detected even after activation with an organomercurial agent. However fractionation of the culture medium by gel filtration on HPLC resulted in elastolytic activity both against H-elastin and SAPNA. Proteolytic activity against casein could also be revealed after separation on SDS-PAGE. It is likely that these separation techniques remove an inhibitor also produced by the endothelial cells and allow the expression of proteolytic activity. That the elastolytic activity and the caseinolytic activity revealed by HPLC and PAGE respectively represent the activity of the same enzyme hase not yet been determined, and its relationship to the Stromelysin described by Herron et al(J. Biol. Chem. , 1986, 261. 2810-2813) in rabbit brain capillary endothelial cells is being investigated.