bluuebunny/crossref_metadata_2025 huggingface dataset:
INVOLVEMENT OF FACTOR XII (F XII) AND PREKALLIKREIN (PKK) IN THE ACTIVATION OF UROKINASE (UK)-RELATED PROTEINS IN HUMAN PLASMA.
Authors: Binnema D J , Dooijewaard G
Published: August 2018

Recently it has been shown that in human plasma two types of UK-related proteins occur: Type I, plasma UK, with UK-related antigenic determinants directly accessible to anti-UK antibodies and Type II with UK-related antigenic determinants which become accessible only after SDS treatment and separation of polypeptides on PAGE. In this study we compared the molecular and enzymic properties of the two types in: 1. plasma activated by dextran sulphate (DXS) euglobulin precipitation, 2. plasma that was not activated and 3. plasma deficient in F XII, depleted in PKK and subsequently activated by DXS. ACA 34 gel chromatography, SDS PAGE, fibrin underlay zymography and immunoblotting were used. Results:

Conclusions: 1. The UK-related subunits of T1 and TII are active when cleaved, but relatively inactive in the single-chain form. 2. The presence of F XII and PKK is indispensable for activation of TII, but not for that of TI; TII contributes to the F Xll-de-pendent plasminogen activator activity reported earlier, TI to the F Xll-independent part. 3. Activation of TI by DXS with no F XII and PKK present impairs the formation of the 150,000 form. 4. The specific activity of TII is rather low, but its concentration in plasma (not shown) is at least ten times that of TI.
NORMAL APTT IN A PATIENT WITH A PERSISTENT ACQUIRED FACTOR XI INHIBITOR.
Authors: McKenna R , Cole E R , Yuk A , Whisler W
Published: August 2018

A 55 year-old white male (MR) with documented normal hemostatic tests at our institution for 14 years, was admitted for a laminectomy. His pre-operative APTT and prothrombin time activity (Q) were again normal. A prolonged APTT of 58 seconds (21-31 N) and Q of 18% (> . 70% N) was noted on the 14th post-operative day; patient had received I unit PRBC during surgery.

Mixtures of pooled normal plasma (NPP) incubated with MR’ s plasma (301, 37° C) yielded normal APTTs with as little as 20% NPP in these mixtures, suggesting a plasma factor(s) deficiency. Factor XI level was undetectable with normal values of factors Vlll-C, IX and XII. A factor XI level of 7% was found in incubated mixtures (60’ , 37° C) of equal parts of NPP + MR’ s plasma as compared to 70% in NPP + buffer and < 1% in MR’ s native plasma + buffer, indicating the presence of an inhibitor in MR’ s plasma. Infusion of 1540 ml of FFP (22 ml/kg) to the patient resulted in an APTT of 28 seconds and a Q of 36% with a persistent undetectable XI (expected ∿ 45%). The in-vivo lack of response to FFP was also suggestive of an inhibitor.

Additional in-vitro tests showed that the response of MR’ s plasma was no different from that of NPP with the addition of increasing dilutions of human brain thromboplastin, differentiating this inhibitor from the Lupus type anticoagulant. The severely reduced Q was due to severe temporary reductions in factors II (20%), V (34%), Vll/X (2%) and X (20%) without any evidence of hepatocellular dysfunction or vitamin K deficiency. Fibrinogen, thrombin time, FSP, platelet counts and bleeding times remained normal throughout.

There was no external evidence of bleeding after the first operation at any site, despite 1M injections and venepunctures. MR was reoperated on day 15 for a cauda equina hematoma; adequate intra and 72 hour post-operative management was with 1 U FFP Q 6 hourly and Amicar; this was associated with a normal PTT, Q %35% and undetectable XI, but without generalized bleeding. Discontinuation of this regimen resulted in onset of bleeding from drainage tubes only, without bleeding from the rest of the incision. Reinstitution of I U FFP Q 12 hourly and Amicar resulted in a prompt cessation of bleeding. Patient had no evidence for an autoimmune disease, dyspro-teinemia or any other illness. The potential role of the 1 unit of PRBC transfused cannot be discounted. Further data on this inhibitor will be presented.
THROMBOMODULIN INHIBITS THE ACTIVATION OF FACTOR XIII BY THROMBIN.
Authors: Polgar J , Lerant I , Muszbek L , Machovich R
Published: August 2018

The thrombogenic functions of thrombin, studied so far, are diminished or blocked when thrombin is bound to the endothelial cell via its receptor protein thrombomodulin. The thrombomodu-lin-thrombin complex fails to clot fibrinogen, to activate platelet and Factor V, while the activation of the antithrombo-genic protein, protein C is extremely enhanced. Although the activation of Factor XIII (FXIIl) belongs to the thrombogenic functions of thrombin, the effect of thrombomodulin on this process has not been investigated, so far. The aim of this study was to establish whether the presence of thrombomodulin modifies the effect of thrombin on FXIII. The activation was followed by measuring the transglutaminase activity of FXIIIa formed using our UV-kinetic assay (Muszbek et al., Clin. Chem. , 3L, 35, 1985) and by monitoring the amount of activation peptide split off by thrombin from the a subunit. The time dependence of FXIII activation using various thrombin concentrations showed significant difference in the presence and absence of thrombomodulin. Thrombomodulin significantly slowed down the activity of thrombin toward FXIII but did not prevent it completely. The possibility that thrombomodulin influences changes brought about by Ca and noij+the action of thrombin was excluded. When thrombomodulin and Ca were added only after the proteolytic leavage of FXIII had taken place, it had no effect on the Ca induced activation process. The results suggests that thrombomodulin inhibits the rate of conversion of FXIII to its enzymatically active structure but does not influence the amount of FXIIIa formed.
COAGULATION FINDINGS IN ULCERATIVE COLITIS AND THE BENEFICIAL EFFECT OF FACTOR XIII CONCENTRATE SUBSTITUTION.
Authors: Suzuki R , Takamura Y
Published: August 2018

Reports suggest that there is a tendency towards hypercoagulability in ulcerative colitis (UC). However, few reports deal with factor XIII which plays an important role in the wound healing process.

In the present study, 8 coagulation parameters including factor XIII activity were determined in 5 patients with UC at the active stage and in 15 patients at remission. A comparison of the two groups shows that, in the active-stage patients, the levels of factor XIII activity were significantly lower, platelets and fibrinogen higher, and the PT prolonged.

Furthermore, Factor XIII concentrate (Fibrogammin P ) was administered to 4 patients with active-stage UC and abdominal symptoms. Here, the symptoms (i.e. abdominal pain, melaena, diarrhea, etc.) disappeared in accordance with the increase in factor XIII activity. Endoscopyprevealed that treatment with factor XIII concentrate (Fibrogammin P ) had a beneficial effect on mucosal edema, redness and hemorrhage as well as on healing of erosions and ulcars.

The results suggest that the level of factor XIII activity is a remarkably good measure of the severity of UC and that administration of factor XIII concentrate may be useful for treatment.
ON THE DETERMINANTS OF FIBRE THICKNESS AND FIBRIN NETWORK CHARACTERISTICS.
Authors: Nair C H , Shah G A , Dhall D P
Published: August 2018

Spectacular differences are found between characteristics of networks developed in plasma and those developed in pure fibrinogen solution. Networks in plasma have thicker fibres, are more permeable and have lower tensile strength. In this investigation determinants of network structure under "hycio-logical conditions of clotting have been examined in an attempt to account for differences in network structure in plasma and in fibrinogen solution.

Two independent variations of mass-length ratio ( µp and µr-.) from permeability and turbidity respectively were used. Effect of varying fibrinogen and thrombin concentrations and the effect of physiological concentrations of Antithrombin III, fibronectin, albumin, γ-globulin and platelet extract on fibrin network structure was examined.

Fibrinogen and thrombin alter network characteristics through the modification of kinetics of network development.

In these and several other studies it has been found that the kinetics of fibrin formation ultimately determine the final network structure through events preceding the appearance of visible fibrin. In separate experiments it was found that spectacular differences in network structure developed in plasma and fibrinogen were not entirely accounted for by alterations induced in network properties by albumin, y-globulin, fibronectin, ATIII and platelet extract.

It is concluded that the final network structure is determined by kinetics of fibrin fibre growth and is highly responsive to the presence of plasma proteins and platelets.

The findings may have fundamental applications to haemostasis and thrombosis.
SHEAR AND FIBRIN CLOT FORMATION; ROLE OF FPB RELEASE
Authors: Berk H R , Clark H
Published: August 2018

It is well known that flow conditions affect many aspects of hemostasis. These effects range from gross morphological differences of thrombi formed at different flow rates to platelet activation to lysis. In addition, fibrin degradation products can influence blood flow. This investigation concerns itself with the effect of shear on the later stages of fibrin polymerization.

Fibrinogen (human, Kabi) is reacted with thrombin (human, Sigma) under Couette flow conditions (volume-averaged shear 0-250 sec-1) in a pH 6.8, 4mM CaC12 HEPES buffered solution (I.S.-.15). Reaction times are chosen to achieve 30% FPA removal for various IT], Reptilase and Crotalase. Low Reynolds number hydrodynamic theory is used to investigate theoretical effects of shear on fibrils.

The effect of shear on final clot formation is measured in terms of clot mass (%Pt). The effects of shear on orientation, diffusion and coagulation on the protofibril-fibril stage account for the initial decrease in %Pt at low shear. It is determined that the shear at which %Pt reaches a minimum (Gcrit) is thrombin concentration dependent. Lateral aggregation of fibrils is identified as the critical variable, with fibril breakage accounting for the increase in %Pt with increasing shear. The theory that fibrin formation proceeds by oligomer-oligomer addition is supported.

Using hydrodynamic theory, a relationship betv/een axial ratio and Gcrit is obtained (assuming tensional or buckling failure). Using a hypothetical relationship between thrombin concentration and fiber axial ratio, a theoretical Gcrit versus IT] is obtained. This theoretical relationship is similar to that obtained experimentally, supporting the notion of shear induced breakage.

Calcium is shown to increase Gcrit for a given [T]

Experiments with Crotalase and Reptilase implicate FPB removal as an important factor in the minimum position. This provides an important insight into the significance of FPB removal in in vivo clot formation and shear stability.
DIFFERENCES IN PEPTIDE MAPS OF a POLYMERS FROM FIBRIN PRODUCED IN THE PRESENCE AND ABSENCE OF ERYTHROCYTES
Authors: Hoser M , Savidge G F
Published: August 2018

α chain polymerisation during clot formation is accelerated in the presence of erythrocytes. This effect is abrogated if the erythrocytes are obtained from patients with various haemoglo-binopathies. Enzyme digests of the α polymers produced in the presence or absence of erythrocytes were prepared to further define any differences between them.

Clots were produced from citrate/EACA plasma samples or plasma/erythrocyte mixtures by the addition of thrombin and calcium. After five hours, clots were washed iii 8 M urea until traces of haemoglobin were removed. After reduction and alkylation clots were dissolved in 0.5% SDS. a polymers were purified on sephacryl S-300 and protein concentrations were adjusted to 0.5 mg/ml. These were digested with S. Aureus V8 protease (150 mg/ml), papain (50 mg/ml) or chymotrypsin (100 mg/ml) at 37°C at sequential time intervals.

After the addition of 2% SDS samples were analysed on 15% SDS polyacrylamide gels.

In all cases digestion of a polymers from clots formed in the absence of erythrocytes took place more rapidly and contained peptide bands not apparent in other digests.

The observations suggest that α polymers formed in the presence or absence of erythrocytes exhibit differing kinetics of response to proteolytic cleavage and indicate that erythrocytes may influence the primary and/or quartenary structure of the polymers studied.
EARLY a CHAIN CROSSLINKING OF PARTIALLY DEGRADED FIBRIN(OGEN) MOLECULES
Authors: Sobel J H , Thibodeau C A , Canfield R E
Published: August 2018

Previous immunochemical studies have demonstrated that the introduction of a chain crosslinks by Factor XIII begins immediately after fibrin aggregation. While there area data to indicate that regions Aα #241-476 and Aα #518-584 are specifically involved in this early crosslinking process, identification of the exact glutamine and lysine residues that participate in the reaction remains to be determined. In this study monoclonal antibodies, whose specificity for these two regions has been defined (anti-Aα #259-276, F-105; anti-Aα #540-554, F-102), were used to isolate preparations of partially degraded fibrinogen molecules which could then be characterized for C00H-terminal Aα chain heterogeneity and crosslinking capacity to obtain a more precise localization of residues involved in early crosslinking. In order to assess the relative contribution of sites within the COOH-terminal region that includes 1 potential GLN at Aα #565 and 4 potential LYS at Aα #556, 562, 580 and 585, fibrinogen molecules that were missing these residues were isolated from preparations of purified fibrinogen using F-102-Sepharose immuno-affinity adsorption. Characterization of the partially degraded material (i.e., non-binding) by Western Blotting using F-102 and F-105 confirmed the presence of Aα remnants (29K-66K) that shared the structure Aα #1-276 but differed in the extent to which regions between ∼#276 and ∼#559 were preserved. When the crosslinking capacity of these partially degraded molecules was examined, using Western Blotting to monitor the appearance of crosslinked a chains during in vitro clotting, effective formation of early crosslinked species ( ∼100K, ∼ 200K) as well as eventual accumulation of a polymers, could be demonstrated. These findings indicate that at least one early a chain crosslink involves activity on the part of LYS and GLN residues located proximal to Aα #540. While these data do not rule out the existence of additional crosslinking sites situated more distally (i.e., within Aα #540-584), they do indicate that degraded fibrinogen molecules, circulating under pathophysiologic conditions, may undergo adequate fibrin stabilization despite a loss of at least 70 COOH-terminal Aα chain residues.
IMPORTANCE OF CONFORMATIONAL CHANGE OF FIBRINOGEN INDUCED BY SURFACE ADSORPTION
Authors: He Lu , Mirshahi Mc , Soria J , Soria C , Mirshahi M , Thomaidis A , Boucheix C , Caen J P
Published: August 2018

Concerning the problem of blood - foreign surface interaction, it is a great importance to know whether fibrinogen adsorbed on a solid surface is conformationally modified.

Therefore, we tested the reactivities of soluble and immobilized fibrinogen against monoclonal antibodies obtained by mouse immunisation with fibrin derivatives, by an immunoenzymological assay using a monoclonal antibody which recognized an epitope available in the D domain of the molecule, but which is poorly exposed on soluble undegraded fibrinogen, we found that the epitope became accessible to this monoclonal antibody after the binding of fibrinogen to polystyrene. We concluded that fibrinogen undergoes conformational change when absorbed to a solide phase.

Conformational change of fibrinogen induced by immobilization on polystyrene may modify the biological properties of fibrinogen molecule. We have shown that after its adsorption, fibrinogen allows plasminogen activation by tissue type plasminogen activator (tpA) in a similar manner as fragment D and fibrin. Therefore, this modification of fibrinogen structure may allow the exposure of the epitope which is masked on native fibrinogen and demasked on fragment D and fibrin and which is essential for tpA-induced plasminogen activation.

These results might be very useful in the screening of synthetic material used for grafts and extracorporeal circulation.

A similar conformational change occurs when fibrinogen binds to ADP-treated platelets. Further work however will be required to determine whether conformational change is essential for ADP-induced platelet aggregation.
IMMUNO-VISUALIZATION OF FIBRINOGEN Aa-CHAIN HETEROGENEITY IN NORMAL PLASMA COMPARED TO PLASMA FROM PATIENTS WITH DIC
Authors: Grøn B , Brosstad F
Published: August 2018

Citrated plasma from healthy individuals or fran patients with disseminated intravascular coagulation(DIC) was diluted, and elec-trophoresed on 11% SDS-PAG slabs after reduction.

Subsequently, the electrophoretic pattern was Western-blotted onto nitrocellulose and,-after blocking with gelatine-, reacted with either:a)polyclonal antibodies to human fibrinogen,b)polyclonal antibodies to FPA, or c)monoclonal antibody to non-released FPA (gift from Dr.Nieuwenhuizen, Leyden, Holland).

Visualization of fibrinogen-related material was then done with peroxidase-conjugated secondary antibodies.

In normal plasma a heterogeneity of the Aa-chain which was substantially more pronounced than hitherto described was noted. This pattern of fibrinogen Aa-chain heterogeneity was even more noticeable in plasma from patients with DIC. The presence/absense of all appropriate fibrinolytic inhibitors during sampling of blood had no effect upon the Aa-chain heterogenity patterns described above. This picture of heterogeneity was also seen when unreduced plasma was electrophoresed on SDS-PAG(2.5%)/AGAROSE(0.5%) slabs.

When comparing the patterns produced by the three different antibodies used for identification/visualization, it could be deduced that: l)The pattern observed was mainly due to Aa-chain heterogeneity, and that the N-terminus of almost all species of Aa-chains were intact, confirming earlier observations that cell- or plasmin -mediated proteolysis of this chain is mainly C-terminal; 2) The present technique allows extranely sensitive characterization of both reduced and non-reduced plasma fibrinogen in the presence of all other plasma proteins.
ON THE IDENTIFICATION OF POLYMORPHIC SITES IN HUMAN FIBRINOGEN PEPTIDE CHAINS
Authors: Wilhelm S , Henschen A
Published: August 2018

Human fibrinogen has repeatedly been shown to occur in a great number of different molecular forms. Some types of heterogeneity are evident already from variations in solubility properties and in ion-exchange-chromatographic as well as gel-electrophoretic behaviour of the total molecule and of its peptide chain components. The reason for these variations are partly known.

Thus, degradation of the C-terminal parts and phosphorylation of two serine residues gives rise to heterogeneity in the Aα-chain. Differences in the sialylation of the carbohydrate side chain causes heterogeneity both in the Bβ- and the γ-chain. Differences in chain length at the C-terminus of the γ-chain are responsible for additional variation. All these variants are expected to exist in each human being. An other category of human fibrinogen variants may be due to genetic polymorphism within the population, i.e. the presence of inherited, common, normal variants. Seven sites of microheterogeneity have so far been tentatively identified, mainly by disagreements between protein and DNA sequence analyses. Three of the sites are located in the Aα-chain (positions 47, 296 and 312), three in the Bβ-chain (positions 162, 296 and 44-8) and one in the γ-chain (position 88). The aim of the present study was to identify these sites on the proteinchemical level in pooled plasma as well as in plasma from single individuals, especially various members of the same family. For this purpose suitable fibrinogen fragments containing the tentatively microheterogeneous sites were isolated after cleavage of fibrinogen with cyanogen bromide, trypsin and/or chymotrypsin by repeated fractionations by means of conventional and reversed-phase high-performance liquid chromatography and counter-current distribution. The components were characterized by N-terminal sequence and amino acid composition. Polymorphism in human fibrinogen has previously only once been identified by restriction fragment length analysis in a non-transcribed region of the Aα-chain locus but never in the transcribed regions, i.e. the peptide chains.

The present investigation will allow the estimation of the number of peptide chain haplotypes and their possible correlation to other genetic variants of fibrinogen.
THE DYSFIBRINOGEN OF CHILDHOOD NEPHROSIS
Authors: Abshire T , Fink L , Christian J , O'Connell J , Hathaway W
Published: August 2018

An abnormal fibrinogen (Fib) related to increased sialic acid (SA) has been described in adults with liver disease. This dysfibrinogen (Dysfib) seems most like fetal Fib. A review of 11 patients with nephrosis revealed an unexplained prolonged thrombin time (TT) and otherwise normal coagulation studies. Based on these observations, we sought to answer whether the prolonged TT defined a Dysfib and if this abnormal Fib was similar to fetal Fib. Pooled adult, fetal plasma, and the plasma of 3 patients with nephrosis were studied with TT and reptilase times (RT). Fib was measured by functional (Fib-act) and immunologic (Fib-ag) assays. An enzyme linked immunosorbent assay (ELISA) was established using antifibrinogen as the first antibody and either peroxidase conjugated Fib or a lectin (Limulus Polyphemus) specific for SA as the second antibody. The optical density was recorded per μgm Fib for both conjugated antifibrinogen or lectin and the ratio compared in order to estimate SA reactivity. Patient 3 was also studied by: 1) crossed immunoelectrophoresis (CIE) employing lectin in the first dimension and 2) polyacrylamide gel electrophoresis (PAGE) with transfer to nitrocellulose paper using Western Blot technique.

Results of the CIE showed patient 3 and fetal plasma were similar in electrophoretic pattern and different from adult plasma. The PAGE with Western Blot revealed a similar pattern of Fib for patient 3, fetal and adult plasma. We conclude that the prolonged TT and RT, the greater amount of Fib-ag when compared to Fib-act in patients 1-3 and fetal plasma and the absence of evidence for Fib degradation products, support the diagnosis of Dysfib. The similarity of the CIE for patient 3 and fetal plasma and the difference between ELISA lectin/Fib ratio of patients 1-3 and fetal compared with adult plasma suggest that the Dysfib of nephrosis may be similar to fetal Fib.
A NEW THROMBOTIC DYSFIBRINOGENEMIA PRESENT IN SEVERAL MEMBERS OF A VENEZUELAN FAMILY
Authors: Arocha Piñango C L , Torres A , Marchi R , Rodríguez S , Camarillo H , Muller-Soyano A , Bosch N B
Published: August 2018

Up to the present, 16 dysfibrinogenemias have been described with thrombotic symptomatology, of which 3 cases showed low affinity of the fibrin for thrombin.

In this study, we describe a family with an elevated frequency of thrombotic episodes which may be due to an alteration in the fibrinogen molecule causing a defective adsorption of thrombin by the fibrin formed.

Two women, mother and daughter, were admitted to our clinic with a history of repeated pulmonary thromboembolisms. Coagulation studies (which included Antithrombin III, Protein C, etc.) revealed only a prolonged thrombin time with high fibrinogen levels (500 mg/dl) by the clot weight and immunological methods. More detailed studies on fibrinogen function showed:

1) Abnormal monomers aggregation and polymerization rate, ho changes were observed in the latter when induced by reptilase followed by thrombin.

2) Normal fibrinopeptide release

3) Normal cross-linked and uncross-linked fibrin chains.

4) Low affinity of fibrin for thrombin

5) Normal plasmin degradation

6) The electron microscopy showed a normal fibrin net with the characteristic periodic cross-striations pattern but which formed more slowly than normal.

Both patients were treated with oral anticoagulants. The mother has not suffered any thromboembolic episodes in two years of treatment but the daughter has shown clinical signs of minor episodes of pulmonary thromboembolism which were confirmed by perfussion gammagraphy. In the family study, 4 members have died due to either venous or arterial thrombotic accidents. Fibrinogen function studies carried out on 8 members from 3 generations showed a prolonged thrombin time with delayed polymerization in 4 of the 8 (1 adult, 3 children), none of which have suffered any thrombotic manifestations up to the time of the study.

The name of Caracas V is proposed for this new dysfibrino-genemia.
HUMAN ENDOTHELIAL CELLS MODIFICATIONS DURING LONG TERM CULTURE
Authors: Wautier M P , Wautier J L
Published: August 2018

The culture of human endothelial cells is largely used for vascular research. The possibility of developping long term culture of human endothelial cells (EC) raised the question regarding the identity after several passages. To further investigate this aspect we have cultured human umbilical vein EC until the 12th passage on fibronectin coated dishes supplemented with ECGF. We have studied the EC morphology by light and electron microscopy, the reactivity with 51Cr labelled platelets, and prostacyclin synthesis. Until the 6th passage no major change could be noted, except the occurence of rare large EC and a reduction in the doubling time between 2nd and 5th passage. After the 7th passage up to the 10th EC became more elongated and did not grow in strict monolayer. The number of vacuoles and mitochondria increased as well as the doubling time. After the 12th passage the EC were still viable but proliferated very slowly. The adhesion of radiolabelled platelets dramatically increased (150%) and PGI2 production significantly decreased (6 Keto PGF1α : 1st passage 13±2.5 ng; 6th passage 0.33±0.27 ng/106 EC). In our culture conditions EC kept most of their original characteristics up to the 6th passage but then lost some of them. At any passage EC contained Weibel Palade bodies and von Willebrand factor. We can conclude that after the 7th passage EC in culture are different from the original cells and could possibly represent an in vitro model of EC ageing.
THE EFFECTS OF ISCHAEMIC METABOLITES ON THE ENDOCARDIAL ENDOTHELIUM
Authors: Carter G , Gavin J B
Published: August 2018

Although mural thrombosis is an important complication of myocardial infarction, little is known of the mechanisms by which death of underlying myocytes initiates thrombus fooiation cm the endocardiim. The sequence of morphological changes which develop in the endocardiim when exposed to ischaemic metabolites was determined in isolated beating rat hearts perfused with oxygenated Krebs-Henseleit buffer (KHB). Lactate (pH7.4), hydrogen ions (HC1, pH6.4) and lactic acid (pH6.4) were passed through a two-way concentric catheter ligated into the left ventricles for periods of 1,2,4,6 and 8 hours (n=4 in each group). Ventricles were then flushed with KHB, fixed in 2.5% glutaraldehyde and examined by scanning and transmission electron microscopy. The morphological changes observed wene qualitatively similar following the infusion of each solution, and all changes increased in severity and extent from one to four hours exposure. Quantitatively the most severe changes were observed with lactic acid. After one hour exposure to lactic acid 3.16 ± 2.35% of the endothelial cells on the papillary muscle showed separation and 7.85 ± 2.44% of cells had exfoliated exposing the basal lamina and after four hours 6.68 ± 2.0% of cells had separated and 16.05 ± 2.67% had been lost. Some exposed connective tissue fibres were evident after one hour (0.94 ± 1.19% of surface area exposed) and the smooth basal lamina had been lost from 4.67 ± 1.04% of the surface with subsequent exposure of the connective tissue after four hours. In all groups mitochondria of endothelial cells were swollen, their matrix electron-lucent and their cristae distorted and fragmented; the endoplasmic reticulun was diluted; and chromatin of nuclei was marginated and clmped (H+ ions and lactic acid only). Superficial myocytes showed similar changes. Electron dense inclusions were also observed in the mitochondria of myocytes exposed to hydrogen ions and lactic acid. Thus perfusion of the ventricle with ischaemic metabolites, which could diffuse from an infarct in vivo, can cause substantial damage to the endocardial endothelium.

Research supported by the Medical Research Council of New Zealand
INCREASED UPTAKE OF MONOCYTE-TREATED LOW-DENSITY LIPOPROTEINS BY AORTIC ENDOTHELIUM IN VIVO
Authors: Görög P , Kakkar V V
Published: August 2018

A new technique was elaborated for measuring LDL uptake by rat aortic endothelial cells in vivo, using a fluorescent marker (Dil)-labelled LDL and quantifying the fluorescence in cells selectively removed from the aorta. This technique was used to study the endothelial uptake of LDL modified by activated human monocytes (LDL-A) in comparison with native LDL (LDL-N) protected from oxidation by vitamin E during the preparation. Incubation of LDL with activated monocytes increased endothelial uptake in vivo by 9.3-fold (from 410 ± 99 to 3803 ± 580, ng LDL/106 cells, mean ± s.e.m., p < 0.001) and also by a 4.4-fold (from 613 ± 217 to 2718 ± 605, ng LDL/106 cells, p < 0.01) in cultured confluent porcine endothelium. In contrast, only a 1.5-fold increase (p < 0.05) in uptake of LDL-A was observed in sparse cultures. Cytotoxicity of monocyte-altered or native LDL did not differ as measured by the [3H] deoxyglucose-release test on cultured endothelium. Our results suggest that modification of LDL in the circulation by monocytes may make an important contribution to atherogenesis.
MODULATION BY HEPARINS OR PENTOSAN POLYSULFATE OF THE EFFECTS OF ACIDIC AND BASIC HUMAN FIBROBLAST GROWTH FACTORS (aFGF and bFGF) ON THE REPAIR OF A MECHANICAL LESION OF THE ENDOTHELIUM
Authors: Klein-Soyer C , Driot F , Vonesch J L , Beretz A , Maffrand J-P , Cazenave J-p , Wittendorp-Rechenmann E
Published: August 2018

A mechanical injury to endothelium (EC) may be followed by vascular spasm, platelet adhesion to exposed subendothelium, mural thrombosis and atherosclerosis. Heparin (HEP) can protect EC from injury, inhibits smooth muscle cells proliferation and has been proposed in the treatment of atherosclerosis. aFGF and bFGF are potent mitogens for EC in vitro and angiogenic in vivo. HEP increases the growth promoting activity of aFGF in EC culture. We have studied the effects of HEP (Choay), low molecular weight HEP (LMWH, CY216 Choay) or pentosan poly sulfate (PPS, Clin-Midy) in the presence of aFGF or bFGF on the repair process, after a mechanical injury of confluent cultured human EC by application of a 6 mm diameter cellulose polyacetate paper. Time for 50 % regeneration (T50) of the lesion was calculated after measuring the remaining lesion at time intervals. Human aFGF (brain) and bFGF (placenta) were purified by HPLC and identified by N-terminus analysis. They were used at concentrations that did not modify T50 when added to 5 % serum. HEP, LMWH and PPS (100 μg/ml) alone increased T50. Addition of HEP, LMWH, or PPS to aFGF markedly decreased T50. This effect was not present with bFGF, although their inhibitory effect alone was abolished. The FGFs alone or in the presence of HEP produce an increase in EC density. In this model, the repair of the EC lesion involves cell migration and proliferation and responds to the combined action of FGFs and HEP, LMWH or PPS. Thus, these drugs could be used to stimulate the repair of injured EC and prevent blood-vessel wall interactions leading to thrombosis or atherosclerosis.
ADULT HUMAN SAPHENOUS VEIN ENDOTHELIAL CELLS : ASSESMENT OF THEIR REPRODUCTIVE CAPACITY AND FUNCTIONAL INTEGRITY PRIOR TO IMPLANTATION ON A VASCULAR GRAFT
Authors: Lesèche G , Tobelem G , Caen J , Andreassian B
Published: August 2018

Establishment of an intact functioning endothelial monolayer on a graft, at or near the time of implantation might be one of the ultimate requirement to get a biocompatible intravascular prosthesis. Following this hypothesis, endothelial cells from human stripped varicose veins were harvested with collagenase and grown on a human fibronectin matrix. The cultured cells (either in primary culture or throughout their lifespan in culture, 1 to 10 passages) exhibited characteristic cobblestone morphology and consistently displayed immunofluorescent staining for factor VUI-related antigen. Functional integrity of this endothelial cells was assayed by their ability to produce prostacyclin. After 15 minutes stimulation with 1 U/ml thrombin, production of 6 Keto PGF1α determined by Elisa was 17 ± 1.2 ng/106 cells. Proliferation was investigated in defined medium supplemented with various concentrations of serum (5 up Jto 30 %), ECGS (25 up to 150 μg/ml) and heparin (10−8 to 10−5M). Optimal growth required both, 100 μg/ml ECGS and 10−5M heparin, under these conditions cells culture achieved cell densities at confluence of 1.2 105 cells per square centimeter with doubling times of one day. Using I-heparin. a binding was demonstrated with an apparent Kd of 0.35 × 10−6 M. After characterization according to morphological, proliferative and functional criteria., cells were freezed at −80°C and ultimately used to coat polytetrafluoro-ethylene grafts (PTFE) which are currently used for vascular reconstructive surgery . Protein-treated material did allow cell attachment and growth to a confluent monolayer as assayed by light and scanning electron microscopy.These data suggested that i) stripped varicose veins provide a readily available source of adult human endothelial cell, ii) these cells grow vigorously in long-term culture when both ECGS and heparin are added to the culture medium, iii) they can adhere and grow to a confluent monolayer on vascular graft prior to implantation, iv) lining graft materials in vitro may be useful in improving the performance of small caliber vascular grafts according to prostacyclin production and surface bound heparin of these cells.
MODULATION OF ANTITHROMBOTIC EFFECTS OF CULTURED HUMAN ENDOTHELIAL CELLS BY INHIBITORS OF CYCLOOXIGENASE OR PHOSPHODIESTERASE
Authors: Müller T H , Rühr K , Callisen H H , Eisert W G
Published: August 2018

Intact endothelial cells are known to form a non-thrombogenic surface and to actively restrict the extent of thrombus formation on denuded vessel walls via such mechanisms as the binding of thrombin and activation of protein C, or the synthesis and release of prostacyclin. In an in vitro system, we have investigated how platelet inhibitors modulate the antithrombotic effects of human endothelial cells. Human endothelial cells isolated from umbilical veins were plated on one half of a subendothelial matrix (SEM) harvested from bovine cornea endothelial cells. The endothelial cells were preincubated with a drug and then exposed to anticoagulated whole blood from human donors in the presence or absence of the same drug and agitated for 15 min. The number and size of platelets interacting with the SEM were quantified by morphometric analysis.

In our in vitro system, platelet aggregates on SEM that was partially covered with human endothelial cells were significantly smaller than on uncovered SEM. No difference in platelet adhesion was observed. In the absence of endothelial cells, the cyclooxigenase inhibitors acetylsalicylic acid (ASA) and flurbiprofen strongly reduced the size of aggregates formed on the SEM. Pretreatment of only the endothelial cells with ASA increased the size of the aggregates, while ASA treatment of endothelial cells as well as the whole blood did not reduce the mean aggregate size below that of controls. in contrast, the platelet phosphodiesterase inhibitors AHP 719 and UDCG 212 strongly decreased platelet aggregation without reducing platelet adhesion not only in the absence but also in the presence of endothelial cells pretreated with the inhibitors.

Our results demonstrate that this in vitro model of a partially injured vessel wall is well suited to study the effects of endothelial cells on platelet function. Moreover, inhibitors of phosphodiesterase in contrast to ASA have profound antithrombotic effects in this model.
PLATELET ADHESION TO THROMBIN-TREATED HUMAN ENDOTHELIAL CELLS
Authors: Murata M , Araki Y , Murakami H , Sato K , Ogawa T , Yamamoto M , Watanabe K , Ando Y , Ikeda Y
Published: August 2018

Platelets do not, or hardly ever, adhere to normal endothelial cells (EC), although the reason is not completely clear. It is assumed that breakdown of this non-thrombogenic properties of EC may lead to initiation of thrombotic process. In this communication, we have clearly shown that significant number of platelets adhere to thrombin-treated EC.

Platelet adhesion to cultured human umbilical vein EC was quantitatively determined according to the method of Curwen et al. using 3H-adenine-labelled human platelets. When EC were treated with human ±-thrombin (a generous gift of Dr. J.W.Fenton) at various concentrations (0.05-lU/ml) for 10 minutes followed by addition of hirudin, the number of attached platelets increased in a concentration dependent manner. When EC were preincubated with lU/ml α-thrombin for various time intervals (5-100 minutes), maximal adherence of platelets to EC was observed in 5 minutes' incubation. After treatment of EC with ±-thrombin for 60 minutes, the number of attached platelets returned to the basal level. EC were, then, maintained in M199-20%FCS for 24 hours and the same experiment was repeated. Again, α-thrombin caused the alteration of EC, which permitted platelets to adhere.

The DNA content of monolayer cells was unchanged throughout the experiments indicating that no loss of EC occured during the procedures. Scanning electron microscopy showed that platelets tended to attach to the periphery of thrombin-treated EC. Treatment of EC with monoclonal anti-thrombomodulin IgG (a gift of Dr. I.Maruyama) had no inhibitory effect on thrombin-induced pla-telet-EC interaction.

Our results may indicate that α-thrombin causes the temporary loss of non-thrombogenic properties of EC
1-14C-ARACHIDONIC ACID INCORPORATION INTO THE LIPIDS OF RAT AORTA.EFFECT OF EXHAUSTION ON PGI2 PRODUCTION
Authors: Martinez-Sales V , Valles J , Santos M T , Aznar J
Published: August 2018

Prostacyclin (PGI2) production in rat aortic rings incubated in Tris-buffer decreases with time.It has been shown that this decrease is not dependent on the total arachidonate concentration in the arterial lipids, and it has been suggested that it may be due to a partial enzymatic inactivation of cyclooxygenase and / or PGI2-synthethase. However, there is a possibility that although the total l-14C-arachidonate (AA*) incorporation increases with exhaustion, this incorporation into the different lipid fractions may be different before and after the exhaustion process.In this study we evaluate this possibility by assessing the incorporation of AA* into rat aorta lipids in control samples and after 180 min exhaustion in 10mM Tris-buffer saline pH=7.4 or human plasma. In addition 6-keto-PGFl (6K) formation from endogenous AA was also evaluated inα the above mentioned experimental conditions by RIA. The results show that this 6K production is drastically reduced by the exhaustion in Tris-buffer (91%) and to a lower extent in arteries exhausted in plasma (52%). With regard to AA* incorporation into the total lipid fractions, it was found that in control arteries 67% was incorporated in phospholipids (PL),19% in triglycerides (TG) and 4.2% in esterified cholesterol. The AA* incorporation into the different PL was: 31% in phosphatidyl-choline (PC), 28% in P-ethanolamine (PE), 25% in P-serine+P-inositol, 8% sphingomyeline and 6% lisolecitine.When the arteries are exhausted in Tris-buffer the AA* incorporation decreases in TG and increases in total PL when compared to control samples. Additionally, a very significant increase of AA* in PE and a decrease in PC were found. In plasma-exhausted sairples the AA* incorporation into the arterial lipids shows the same trend as in Tris-buffer exhausted samples, although these differences were lower in the former when compared to the control. When rat aortic rings are exhausted in Tris-buffer there is a remarkable reduction in PGI2 production and an alteration in the AA* incorporation into the arterial lipids.Plasma has a protective effect on rat aorta during the exhaustion process.
PROSTACYCLIN AND THROMBOXANE PRODUCTION IN HUMAN VARICOSE SAPHENOUS VEINS
Authors: Biagi G , Coccheri S , Lapilli A , Grauso F , Bizarri M , Zendron R , Piccinni L
Published: August 2018

Inpaired vascular synthesis of prostacyclin (PGI2) has been described in pro-thrombotic states,including recurrent venous thrombosis. As varicosis predisposes to local thrombogenesis, we measured in this study the generation of PGI2 and thromboxane A2 (TXA2) in segnents of human varicose veins. Samples of saphena magna were obtained after surgical stripping performed for varicosis in 20 patients (pts), or on preparation for coronary by-pass in 7 non-varicose subjects (controls). Segments (length - 2 cm, weight 200 mg) were dissected from macroscopically varicose areas in the pts and from correspondjmg zones in the controls. Varicose changes were confirmed by microscopic and histochemical studies. Vascular PGI2 production was measured both by bioassay (supernatant of 40 mg fresh tissue per ml TRIS buffer) on ADP platelet aggregation (PA), and by RIA assay of 6keto-PGFlalpha; TXA2 by RIA as TXB2. Bioassay showed that PGI2-like material production by varicose segments was lower than by control veins (% PA inhibition: 77+/19 vs 11+/6; p<0.001). This pattern was confirmed by the 6Keto-PGFlalpha levels (399+/38 vs 1477+/21 pg/mg tissue; p<0.001). TXB2 production was higher in varicose than in control segnents (36+/13 vs 2+/0.9 pg/mg; p<0.001). Incubation of varicose segnents with arachidonic acid 1μM enhanced generation of both 6Keto-PGFlalpha (p<0.05) and TXB2 levels (p<0.01).These data suggest that varicosis is associated with endothelial metabolic alterations relevant for thrombogenesis. Thrombosis in these veins seems therefore related, besides hemodynamic factors, to the loss of protective properties of the endothelium.
DECREASED PROSTACYCLIN-LIKE ACTIVITY IN HENOCH-SCHONBEIN PURPURA
Authors: Belch J J F , Turi S , MacLaren M , Beattie T J , Forbes C D
Published: August 2018

Henoch-Schoniein purpura (HSP) is the most common type of childhood vasculitis. The skin lesions are the most obvious sign but visceral involvement carries a more serious prognosis. Increasing amounts of data are available regarding the possible role of prostacyclin (PGI2) in the pathogenesis of other vasculo-pathies, but no-one has previously investigated HSP. 17 HSP patients (mean age (SD) 5.7 (2.5) years) and 17 matched controls were studied. The ability of plasma (patient and control) to support PGI2 -like activity was tested. Human umbilical arteries were chopped into rings and incubated in buffer at 37° C. Aliquots of the supernatant were added to platelet rich plasma (PRP) and platelet aggregation measured (Malin's aggregometer). Repeated washings of the rings then depleted PGI. production. The rings were then incubated with test platelet poor plasma (PPP) at 37° C. PGI2 like activity was assessed as before and the results expressed as a percentage of the aggregation obtained by the same exhausted ring before addition of PPP. The results show that the ability of test PPP to support PGI2 like activity was reduced in 13/17 HSP patients. The difference between patients (21.3 (SD 20.5%) and controls (57.2 (SD 12.2%) was significant (p<0.001). Patients with renal and gastro-intestinal involvement (7.5 (SD 18.1%) were significantly different from those patients with only skin involvement (32.3 (SD 23.5%) (p<0.01). Plasma from six patients in which PGI^like activity was undetectable using the above method were further studied to detect PGI2 -inhibitory activity. Unexhausted umbilical rings were incubated at 37°C for 5mins. The baseline PGI2 like activity of the supernatant was compared with that produced after 5mins. incubation with test PPP (o patients, 6 controls). Less like activity was produced by the unexhausted rings when incubated with PPP from the HSP patients than with either control buffer or control PPP (both p<0.01). It seems likely that these abnormalities, having been demonstrated in yet another vasculitic syndrome, are secondary to endothelial damage. The disturbances, however, may well be of importance in extending the primary microvascular insult
EFFECTS OF I.V. AND I.A. PGE1-INFUSION IN DIFFERENT DOSES ON FIBRINOLYTIC ACTIVITY, PLATELET FUNCTION AND STABLE PGE1 METABOLITES
Authors: Fitscha P , Simmet T , Peskar B A , Tilsner V , Rogatti Waltraud , Sinzinger H
Published: August 2018

As it is well known that PGE1 is degraded during lung passage to a major extent, we examined what the optimal route and dosage concerning platelet function and plasmatic coagulation would be.

6 patients with peripheral vascular disease (PVD) stage lib according to Fontaine were treated either i.a. (1,25 or 2,50 ng PGE1 [Prostavasin]/kg/min) or i.v. (5 or 10 ng/PGEl/kg/min) for 120 minutes.Various parameters reflecting platelet function (βTG, PF4, serum TXB2, ADP-induced aggregation, platelet sensitivity to PGI2, intraplatelet cAMP) plasmatic coagulation and fibrinolytic activity were measured. Blood was drawn from the contralateral cubital vein avoiding occlusion before and 5,30,120,125 and 185 minutes after starting PGE1. Fibrinopeptide A (1,03±0,38 → 2,57±1,32; p<0,01), plasminogen (108,5+10,7 → 1,24±11,5; p<0,01), plasmin changed, whereas α2-anti-plasmin tissue plasminogen activator (72,7±34,7 → 94,0±32,5) and urokinase did not change (1,00±.0,41 → 1,30±0,95) significantly. The long-lasting influence on the fibrinolytic activity can not be explained by a direct action of PGE1 or 15-keto-13,14 DH-PGE1. RIA determination of 15-keto-13,14 DH-PGE1 exhibited a certain dose-dependent effect of PGE1 on the plasma levels of the immunoreactive substance. The findings are suggestive for a process persisting even after complete disappearance of the causative agent, or for a biological capacity of a so far unknown long-lived PGEl-metabolite. PGE1 did not significantly affect the platelet function parameters.