bluuebunny/crossref_metadata_2025 huggingface dataset:
The Trinodular Structure of Fibrinogen: Confirmation by both Shadowing and Negative Stain Electron Microscopy
Authors: Ericksen H.P. , Fowler W.P.
Published: May 2019

Fibrinogen molecules sprayed on mica, dried in vacuo and shadowed with platinum appear as trinodular rods. We describe here the flotation technique of negative staining by which we were able to visualize individual fibrinogen molecules. The fibrinogen molecules in our negatively stained preparations have a trinodular structure identical to that of shadowed molecules. The molecules are 45 nm long, the two outer nodules are 6-7 nm in diameter, and the middle nodule is 4-5 nm in diameter. We believe that the 20 nm diameter globules seen in previous negative staining studies are aggregates of these molecules.
Histochemical and Ultrastructural study of the Platelet-Arterial Subendothelium Interaction
Authors: Legrand Y.J. , Bariety J. , Birembaut P. , Michel H. , Fauvel F. , Caen J.P.
Published: May 2019

In order to characterize the subendothelial macromolecules able to interact with platelets rabbit aortas were de-endothelialized and incubated either with chymotrypsin (24 h at 37° C), with a highly purified bacterial collagenase (2 h at 37° C), or with chymotrypsin followed by collagenase or the reverse. Histochemical ultrastructural studies were performed before and after blood perfusion, using three different staining procedures: tannic acid (for the microfibrillar structures and elastin), ruthenium red (for the polyanions) and two peroxidase-labelled lectins (concanavalin A and ricinus communis (for the detection of saccharidic determinants)). After chymotrypsin, the tannic acid +ve, ruthenium red +ve and lectin +ve microfibrils had been digested; platelet adherence to the remaining sparse collagen fibrils persisted. After collagenase, these microfibrils are preserved, collagen has been digested, but the platelets can still adhere. After combined incubation with both enzymes, elastin is the only remaining constituent and no platelet adhesion can be observed.

These results show that in the rabbit aorta subendothelium besides collagen, microfibrillar glycoproteins are able to interact with human platelets.
Inhibition of Human Platelet Function Induced by Sulphinpyrazone
Authors: Maquire E.D. , Pay G.F. , Turney J. , Wallis R.B. , Weston M.J. , White A.M. , Williams L. , Woods H.F.
Published: May 2019

A single 400 mg dose of sulphinpyrazone (S) administered orally to 5 volunteers inhibited arachidonic acid (AA)-induced platelet aggregation and platelet MDA production ex vivo biphasically. The initial inhibitory phase coincided with the maximum plasma concentration of S but the second phase occurred 24 h after dosing when no plasma S was detectable. At 2 h the concentration of AA inducing half-maximal aggregation was increased 1.95 times and at 24 h by 1.61 times. Dosing 7 volunteers with 200 mg q.d.s. for 7 days and 5 volunteers with 400 mg b.d.s. for 5 days also led to inhibitory activity against AA-induced platelet aggregation and MDA production which declined to zero 48 h after the last dose. Platelet count, fibrinogen, F VIII; ß-TG and AT III were not influenced by the longer term treatment but bleeding time was slightly extended in all but one subject.
An Assessment of Red Cell Deformability Using a Simple Filtration Method
Authors: Drummond M. M. , Lowe G.D.O. , Belch J.J.F. , Forbes C.D. , Barbenel J.C.
Published: May 2019

We investigated the reproducibility and validity of a simple method of measuring red cell deformability (filtration of whole blood through 5 μ sieves) and its relationship to haematocrit, blood viscosity, fibrinogen, white cell count, sex and smoking. The mean coefficient of variation in normals was 3. 7%. Tanned red cells showed marked loss of deformability. Blood filtration rate correlated with haematocrit (r = 0. 99 on dilution of samples, r = 0. 7 in 120 normals and patients). After correction for haematocrit, deformability correlated with high shear viscosity, but not low shear viscosity, fibrinogen or white cell count. In 60 normals there was no significant difference between males and females, or smokers and non-smokers, but in 11 smokers there was an acute fall in deformability after smoking 3 cigarettes (p〈0. 05). Reduced deformability was found in acute myocardial infarction (n = 15, p〈0. 01) and chronic peripheral arterial disease (n = 15, p〈0. 01). The technique is reproducible, detects rigid cells and appears useful in the study of vascular disease.
Viscosity and Red Cell Deformity in Arterial Disease
Authors: Cella G. , de Haas H.A. , Rampling M. , Kakkar V.V.
Published: May 2019

Haemorrheological factors have been shown to be affected in many kings of vascular disease. The present study was undertaken to correlate these factors in normal subjects and patients suffering from peripheral arterial disease. Twenty-two patients were investigated; they had moderate or severe intermittent claudication, extent of disease being confirmed by aorto-arteriography and ankle-systolic pressure studies. Twenty-five controls with no symptoms or signs of arterial disease were selected withcomparable age and sex distribution. Whole blood viscosity was measured at shear rates of 230 secs-1 and 23 sees-1 at 37°C using a Weils Brookfield cone plate microvisco meter. Plasma viscosity was also measured in an identical manner. Erythrocyte flexibility was measured by centrifuge technique and fibrinogen concentration as well as haematocrit by standard techniques. The fibrinogen concentration appeared to be the only significant parameter; the mean concentration in patients with peripheral vascular disease of 463 ± 73mg/100ml in the control group ( 〈 0.05). Although whole blood viscosity was high in patients, when corrected t. a common haematocrit, there was no significant difference between patients and controls The same megative correlation was found for plasma viscosity. The red cell flexibility was found to be increased in patients as compared to the control group, but this effect appeared to be simply proportional to the fibrinogen concentration.
Comparisons of Biochemical and Pharmacological Properties of Prostacyclins with Modified ω-Side Chain
Authors: Weithmann K.U. , Bartmann W. , Beck G. , Lerch U. , Konz E. , Schölkens B.A.
Published: May 2019

PGI2-analogs in which the n-pentyl moiety in position IS was replaced by several residues, were subjected to structure-activity studies. Some of the modified PGI2’s showed remarkable potency in regard to antiplatelet and vasodilator actions. In order to evaluate these potencies the inhibition of arachidonic acid induced platelet aggregation in human platelet rich plasma, relaxation of bovine coronary artery (BCA) and systemic blood pressure (BP) in the anesthetized rat were determined. Platelet aggregation was inhibited by PGI2, with an IC50 approx. 3.10-9, BP was decreased in a dose dependent manner with an ED25 of 0.23 ug/kg i.v. and a marked relaxation of BCA was observed. Similar results were obtained with PGK-methylester. Substitution of the n-pentyl moiety by cyclohexyl, 3-furyl-2-ethyl or 3-thienyl-oxymethyl resulted in PGI2, and PGK2-methyl-ester-analogs with high biological activity, whereas substitution by 16, 16-dimethyl-18-oxa-alkyl or phcnoxymethyl caused a loss of activity in the models used. The potency of the prostacyclins in the platelet model seem to depend upon their ability to elevate the platelet cyclo-AMP level. Thus the antiplatelet potency of modified PGI2’s may reflect their ability to affect the adenylate cyclase system.
Purification and Properties of RAT α2 Acute-Phase Macroglobulin (α2M)
Authors: Nieuwenhuizen W. , Emeis J.J. , Hemmink J.
Published: May 2019

For our studies on the relation between blood fibrinolytic activity and repair of mechanically damaged arteries in our rat mode 1 we need a specific and sensitive assay for α2M, In the rat α2M is an acute-phase protein of which the level in blood is normally near zero but increases as a result of the damage. Moreover α2M is known to inhibit proteases involved in the fibrinolytic system. We developed a new purification procedure in which, conditions known to be harmful to the functionality of α2M were avoided. α2M was purified from plasma of turpentine-treated rats and proteolytic activities were suppressed throughout the purification procedure. The purification scheme successively involves: rivanol precipitation, Con A-Sepharose chromatography and DEAE-ce1 Iulose chromatography. Thusmg of α2M was obtained from 100 ml rat plasma i.e, 20% recovery. The preparations were biochemically and immunologically pure, Amino acid and carbohydrate compositions were determined. The molecular weight is 760.000, The molecule consists of 4 subunits, M.W. = 190.000. A 1% 1cm = 8.8 and pl = 4.8. It binds 1 mole of trypsin or plasmin per mole. Bound proteases were only active on low molecular weight substrates such as BAEE and BOC-L-va1-gIy-L-arg ßNA. Kinetic data of the bound enzymes (pH-optimas, Km and Vmax) indicate that factors other than steric hindrance are involved in the inhibitory act ion of α2M.
Heparin Catabolism Under Extracorporeal Circulation in Cardiac Surgery: Experimental Study
Authors: Simonuff M. , Doutremepuich Ch. , Chauve A. , Nicod J. , Fontan F.
Published: May 2019

Extracorporeal circulation E. C. C. was performed on 13 Beagle dogs to deter mine heparin catabolism. Three types of heparin were used: a cold heparin and two radio-active(R. A.) heparins (S-35 and H-3) The E.C.C. was performed for 70 minutes (mn.) in all dogs, at a temperature of 37°C. in 4 dogs, 28°C in 4 and 20°C. in 5, (duration of hypothermia: 30 mn.).

1) Plasmatic heparin elimination was related to the E. C. C. temperature: 42.6% at 37°C., 30 7% at 28 ° C. and 14.6% at 20°C. The biological half-lives were respectively 60 + 10 mn, 80 + 10 mn, and 200 + 20 mn.

2) Urinary heparin elimination was delayed with respect to the E. C. C. temperature and independant of the type of heparin used. At the same time, the relation of heparin in plasmatic R.A, to urinary R.A. was constant(≈ 10) during the entire E. C. C.

3) The study of the gastric fluid in 8 dogs showed an heparin elimination increased to a maximum at the end of the E.C.C.; at that time, the plasmatic R. A. to gastric R.A.heparin relation, in 5 dogs, was equal to 1.

The study indicates that a)there is a correlation between the dosage made, try classical and radta-active melhods, b) the lowering of E. C. C. temperature diminfshes the catabolism of heparin, and c) a gastric heparin elimination during E. C. C.
Interactions of Human Fibrinogen with Glass Surface
Authors: Chan B. , Brash J.L.
Published: May 2019

Plasma protein adsorption is an important effect in thrombosis at artificial surfaces. A study was made of interactions between human fibrinogen and glass. Three types of information were developed: (1) rates of adsorption and equilibrium surface conc. (mass/area). (2) reversibility properties of the adsorbed layer, e.g. exchange between surface and solution; (3) molecular changes in fibrinogen after desorption. Kinetics of adsorption, exchange and desorption were studied in tris buffer, using Kabi fibrinogen labelled with iodine (125 or 131). Changes in desorbed fibrinogen were assessed by circular dichroism and SDS-acrylamide gel electrophoresis. It is found that adsorption is limited to a monolayer over conc, range 50 to 300mg%. Desorption into original buffer does not occur, but rapide exchange between solution and surface is observed. Exchange increases in rate and extent with increasing protein conc, but a residue of 20% of the layer remains unexchangeable. Adsorption decreases with increase in conc, of tris at constant pH. Also treatment of adsorbed layers with tris of higher conc, than was used for adsorption results in desorption, with 20% remaining nondesorbable. Desorbed fibrinogen has a greatly reduced α-helix content and exhibits only one band (compared to 3 originally) in SDS-acrylamide gels, intermediate between Bβ and γ bands. Since 20% of bound radioactivity resides in v-chains these data may indicate that fibrinogen adsorption on glass involves preferential binding of γ-chains. Solution-surface exchange may analogously involve subchains as opposed to whole molecules. Such phenomena may play a role in blood-surface contact situations.
Plasma β-thromboglobulin and Platelet Factor 4 Levels in Cardiac Valve Prosthesis
Authors: Cella G. , Lane D.A. , Kakkar V.V. , Donato A. , Dalla Volta S.
Published: May 2019

Thromboembolic complications occur frequently in patients who have undergone cardiac valve replacement and platelets are known to be actively involved in the pathogenesis of such thrombi, A method of in-vivo assessment of platelet function would be useful in the management of such patients. βTC and PF4 Levels, the indicators of platelet release reaction in-vivo were measured in 100 patients who had cardiac valve replacement at least six months previously) Group A-54 patients had porcine heterograft, Group B-32 patients had disc valves and Group C-14 patients had double valve replacement. Findings of these patients were compared with 50 controls. In the control group, the mean βTG was 27.6ng/ml, and PF4 9.0 ng/ml. In each of the three groups of operated patients, both mean βTC and PF 4 levels were significantly raised (p < 0.05). Group A 61.5 ng/ml and 26.5 ng/ml, Grout B 57.0 ng/ml and 19.6 ng/ml and Group C 63.0 ng/ml and 36.6 ng/ml respectively. Of the 100 cases studied, 57 involved mitral, 29 aortic and 14 multiple valve replacement. No significant differences were observed in βTC or PF4 plasma levels with the different type of valve involvement. The clinical implications of these results will be discussed.
Platelet Function Studies in Normal Volunteers and Patients with Angina Pectoris
Authors: Jonker J.J.C. , Ottolander den G.J.H.
Published: May 2019

In 30 normal subjects (group I) and in 89 patients with angina pectoris we studied: the platelet survival time (PST), the platelet aggregation test I (PAT I) acc. to Breddin, the platelet aggregation ratio (PAR) acc. to Wu and Hoak and the Filtragometer log TA acc. to Hornstra. The patients were divided in two groups: 46 patients had already been treated for 6 months with Clofibrate (group II) and 43 patients with placebo (group III) in a double blind trial. The average PST (T½) was within the normal range (group I 99 hrs. group II 105,7 hrs.; group III 102,0 hrs.). About 20% of patients of group II and III had abnormally shortened T½. The PAT I was on average abnormal in group II and III (PAT I in group II 2,3; group III 2,7), but group II normalized after 12 months treatment (PAT I 1,85). The PAR was abnormal in group III, while group II was within the normal range (group I 0,87; group II 0,82; group III 0,69). The log TA results were abnormal in group II and III (group I 2, 45, group II 2,1; group III 2, 1), after 12 months treatment the patient group remained abnormal (group II 2,2; group III 2,1). We failed to find a correlation between the four platelet function tests, nor with these tests and basic laboratory values. The PAT I, the PAR and the Filtragometer seems to be valuable in the detection of abnormal platelet behavior in vitro, but it does not mean than an abnormal platelet survival in vivo occurs in the same individuals.
Aspirin (ASA) Inhibits both Prostacyclin and Thromboxane Formation in Man
Authors: Pareti F.I. , Smith J.B. , D’Angelo A. , Mannucci P.M.
Published: May 2019

The effects of two different doses (0,3-1,5 g) of ASA on platelet thromboxane (Tx) and endothelial prostacyclin (PCI2) formation have been investigated in 4 healthy adults. Both doses gave a complete suppression of TxB2 formation as measured by radioimmunoassay (RIA) of serum obtained 2 hours after ASA. PGI2 production was assessed with a bioassay based on the inhibition of ADP-induced platelet aggregation. The formation of PGI2-like inhibitory activity in vein specimens obtained at biopsy was abolished by either dose of ASA. In two subjects taking 0,3 and 1,5 g of ASA respectively, inhibition of PGI2 formation was confirmed by measuring 6-keto-PCF1α, the stable hydrolysis product of PGI2, by RIA. We have also studied a patient with an “aspirin-like” syndrome characterized by a mild congenital bleeding tendency, defective platelet release reaction, lack of aggregation by arachidonic acid and no TxB2 formation after thrombin. Venous biopsy specimens failed to produce PGI2 as measured by both bioassay and RIA. These studies show that the cyclo-oxygenase enzyme systems of human platelets and endothelial cells are both susceptible to Inhibition by ASA in vivo; and no difference was noted in the degree of inhibition induced with the two dosages. The congenital deficiency of both PCI2 and Tx formation in one patient was associated with a mild bleeding diathesis but no thrombosis tendency.
Platelet Collagen Activation by Basement Membrane Collagens
Authors: Balleisen L. , Rauterberg J.
Published: May 2019

The collagen component of the vessel intima consists of collagen type I, III and basement membrane collagens. For type III a particularly potent platelet aggregation activity was found earlier.

Three different basement membrane collagens were isolated from call placenta. Occurence of type IV and V(A/B collagen) in the intimal and medial layer of vessel walls has been shown by chemical and immunhistological means(1). The third basement membrane collagen is probably isentical with the 55000 molecular weight component described by CHUNG et al. (2).

Platelets from normal subjects were tested for aggregation and spreading on Zaponlack with type IV, V, the separated A and B chains of type V and the short chain collagen. Complete aggregation was obtained only with native type V collagen. Spreading of platelets was induced only by collagen type IV, the A chain of type V and the short chain collagen.

The data indicate that all basement membrane collagens activate platelets but in different respects.

1. Rauterberg J. et al.: Coll. Int. Cent. Nat. Rech. Sc. 287:220(1978)

2. Chung E. et al.: Bioehem. Biophys. Res. Comm. 71: 1167(1976)
Direct Activation of Factor X by Some Cancer Cells: Evaluation by an Amidolytic Assay
Authors: Colucci M. , Curatolo L. , Donati M.B. , Semeraro N.
Published: May 2019

The procoagulant activity of cells from some experimental tumors isolated in culture or as cell suspensions fron ascitic fluid was investigated. Cells from Lewis Lung carcinoma (primary and metastasis), Ehrlich carcinoma ascites and JK sarcoma ascites markedly shortened the recalcification time of normal, F.VIII and F.VII-deficient, not of F.X-deficient human plasma. This suggests a direct activation of coagulation F.X. To verify this assumption, cells were incubated with a BaS04-eluate of normal (N.E.) or F.VII-deficient (VII-D.E.) human serum (as a source of F.X) and CaCl2. The chromogenic substrate S-2222 was used for monitoring F. Xa generation. Substrate cleavage occurred to different degrees with all cell types; it was similar both with VII-D.F. and N.F., proportional to cell number and incubation period and was not influenced by hirudin. When tested separately, neither the eluate nor the cells had any amidolytic activity. Our assay readily discriminated between direct and F.VII-mediated activation of F.X. Indeed when cells with thrombopla-stin-like activity (mouse embryo fibroblasts) were tested, the rate of S-2222 cleavage was markedly lower with VII-D.E. than with N.E. Cells from sarcoma 180 ascites were completely inactive in both plasma recalcification time and amidolytic assay. It is concluded that cells from some experimental tumors directly activate F.X.(Supported by CNR (Italy), and NIK, NCI (USA).
Isolation of Antithrombin III Without Interfering with Ethanol Fractionation System
Authors: Wickerhauser M. , Williams C.
Published: May 2019

We described previously the isolation of antithrombin III (AT III) from the 20% polyethylene glycol (PEG 4000) supernatant of plasma or of Cohn Fraction IV-1 (Vox Sang., in press). The first of these two methods gives good recoveries of AT III but cannot be integrated with the conventional ethanol fractionation system due to the presence of PEG in the remaining plasma fraction, while Cohn Fr action IV-1, a byproduct of routine fractionation, is a poor source of AT III in terms of yield. Our modified’method involves batchwise adsorption of AT III from plasma (cryosup-ernatant) with heparin-Sepharose, using one volume of gel for each 50 volumes of plasma. The unadsorbed plasma can be used for ethanol fractionation. The AT III eluate is further purified by precipitation of some impurities including HB Ag, if present, with 20% PEG. Final purification of AT III and removal of PEG is achieved by a second adsorption-elution step on heparin-Sepharose. This method is economical and suitable for large scale application. Recovery of a highly purified AT III was 25%.
The Thrombin Protecting Function of a Complement Inhibitor in Human Plasma
Authors: Podack E.R. , Curd J.G. , Griffin J.H. , Müller-Eberherd H.J.
Published: May 2019

S-protein (S) is a newly discovered 80,000 MW plosma glycoprotein. It functions as an inhibitor of the membrane attack complex of complement. We now wish to report that S also functions as thrombin protecting factor in coagulation; S forms a reversible complex with thrombin which is more resistant to inactivation by antithrombin III (AT III) than thrombin alone. An S-thrombin complex and on S-throm-bin-AT III complex were formed in clotted plasma and with isolated proteins as demonstrated by two dimensional Immunoelectrophoresis. Functional studies measuring the esterolytic or clotting activity of thrombin showed that S in the presence and absence of heparin decreased the rate of inactivation of thrombin by AT III. Similar results were observed using plasma. For example, in the presence of 0.04 u/ml heparin and 1.6 u/ml thrombin, the thrombin time of plasma depleted in S was 150 sec. as opposed to 15 sec. when the plasma was reconstituted with purified S. That this effect of S was due to a decreased inactivation of thrombin by AT III was demonstrated directly by SDS-PAGE analysis of plasma containing 125l-thrombin. In the presence of S the rate of formation of the 95,000 dalton 125I-thrombin-AT III complex was markedly decreased compared to the rate of complex formation in the S-depleted plasma. These data suggest that S may modulate the interactions of thrombin and AT III.
Rapid binding of ADP to a small population of platelet receptor sites
Authors: Legrand C. , Bauvois B. , Dubernard V. , Caen J.P.
Published: May 2019

By studying binding of ADP to isolated human plasma membranes, we have found that ADP binds with different affinities and/or different kinetics to several types of receptor sites whose nature and localization have been assessed by subjecting the membranes to various treatments such as proteolytic digestion or alkaline extraction. From kinetic results, two populations of receptor sites were distinguished, with t 1/2 = IE-30 secor (approximately 20 % of the sites) and with t 1/2 = 4-6 minutes (80 % of the sites). The binding of AMP was studied to determine whether, as expected from its inhibition of ADP-induced platelet shape change and aggregation, it could react with ADP receptor sit We found that AMP bound rapidly to the platelet membrane (t 1/2 =15-30 seconds) ; up to approximately 100 picomoles of AMP could bind to One milligram of membrane protein, which represented 20-30% of the total ADP binding capacity, with an affinity constat (Ka =0.6 × 106M-1) higher than that of ADP (Ka=0.4 × 106M-1). AMP partially and conspectitively inhibited the binding of ADP. It was shown mainly to inhibit those molecules of ADP which bind to the platelet membrane during the first 90 seconds of incubation and which represent approximately 20 - 30 % of the total ADP binding.

From this study, it is concluded that a population of receptor sites showirg high react vity towards ADP and AMP is present on the platelet membrane.
Abnormalities in Fibrin Formation in Hepatic Disease. Consequences of Acquired Dysfibrinogenemia on Fibrin Formation and on Platelet Aggregation Cofactor Activity
Authors: Soria J. , Soria C. , Ryckewaert J.J. , Samama M.
Published: May 2019

Different causes of abnormal fibrin formation can be identified in severe hepatic diseases.

A decrease in the level of Aα band of isolated fibrinogen can be related to increased fibrinolysis.

Soluble complexes detected by the haemagglutination assay of red blood cells sensitized with fibrin monomers are present in cases with associated intravascular coagulation. An abnormal fibrin monomers aggregation can be due either to a plasma inhibitor or to dysfibrinogenemia. The anomaly is only present in plasma in the former case and in both plasma and isolated fibrinogen in the latter case.

In acquired dysfibrinogenemia an increase in sialic acid content in the fibrinogen molecule can be demonstrated and is associated with an increase of ADP platelet aggregation cofactor activity. The effect of desialidation was also studied. These results are in good correlation with the hypothesis that oreviously isolated fibrinogen glycopeptides might play an important, roie in platelet aggregation.
Effect of in vivo Administration of FeibaR on Clotting Tests: Dose-Response Relationship
Authors: Nowotny Chr. , Lechner K. , Niessner H. , Thaler E.
Published: May 2019

FEIBA causes in vitro a concentration-dependent shortening of APTT of hemophilic plasma containing antibody to factor VIII. Since the potency of preparation is based on this in vitro test, it was of interest to examine whether a dose-dependent shortening of APTT and clotting time is also observed under in vivo conditions, APTT, whole blood glass clotting time and reaction time (=R value of thrombelastogramm) were determined before and 30 to 60 min. after 91 infusions of FE IBA (dose range 6 - 100 U/kg) to 10 hemophiliacs with antibody. Although there was a considerable scatter, a significant inverse correlation between the dose of FE IBA and postinfusion APTT and R-value was observedThe dose response curve was, however, rather flat. No significant correlation existed between FE IBA dose and glass clotting time. The effect of FE IBA on the APTT was not dependent on the antibody level, both, in vitro and in vivo. There was only a slight variant ot the effect among different patients, but a marked variation among different lots ot tne preparation, FE IBA seems to have a weak clinical hemostatic effect. A clear relationship between dose and clinical effect could not be established but it appears that doses higher than 50 U/kg are not more effective than smaller doses.
Morphilogy and Ultrastructure of in Vitro Cultures of Aortic Endothelial Cells Interacting with Antibodies
Authors: Korach S. , Ngo D.
Published: May 2019

Adult pig aortas, sectioned longitudinally, were incubated in 0.1% collagenase-PBS (15 mn, 37°C). Gentle scraping of the lumenal surface resulted in high yields (3-4 x 106 cell/aorta) of viable endothelial cells, essentially devoid of other cell types by morphological and immunochemical (F VIII-antigen) criteria. Confluent monolayers were incubated for various times (5 mn to 1 wk) with decomplemented rabbit antisera raised against pig endothelial cells. Changes in cell morphology appeared to depend on antibody concentration rather than on duration of contact with antiserum. High concentrations of antiserum (5 to 20%) led to cytoplasmic shredding, bulging of cells and extensive vacuolization, whereas at lower concentrations, cells appeared almost normal. Transmission EM studies by the indirect immunoperoxydase method showed antibodies reacting with unfixed cells to be distributed all over the upper cell surface, in the outer parts of intercellular junctions, and within numerous pinocytotic vesicles. Much weaker reactions could also be seen at the lower cell surface. When viewed under the Scanning EM, antiserum-treated endothelial cells also disclosed antibody concentration-dependent bulging and release of cells from their substrate. In vitro studies of gradual modifications of vascular endothelial cells acted upon by antibodies should provide a better understanding of the structural and biochemical processes underlying endothelial damage and detachment.
Thrombin induces Contraction And increased 51-Cr Release from Cultured Human Endothelial Cells
Authors: Galdal K.S. , Evensen S.A.
Published: May 2019

Injury to human endothelial cells(EC) in primary culture was evaluated by a 51-Cr release assay, phase contrast microscopy and the trypan blue exclusion test. Normal integrity of EC, was maintained and 51-Cr release showed a slow linear increase during 24h incubation with either RPMI 1640 supplemented with 20% foetal calf serum, glutamine and antibiotics(SCM) or normal human serum(NHS). Thrombin in a concentration as low as 0.1 IU/ml induced obvious contraction of EC incubated in SCM, but the cells remained fixed to the bottom of the wells. Cell contraction was maximal after 15min and disappeared within 4h. 51-Cr release increased 2-3 fold within a few minutes and remained increased during an incubation period of 4h. The injurious effect of thrombin was inhibited in SCM containing hirudin(l.7 u/ml) or in NHS. ADP(10-5 M), endotoxin (0.1mg/ml) and 5 vasoactive agents adrenalin 5.5 10-5, noradrenalin 5.9 10-5 M, histamine 9.10-4 M, bradykinin 7.5 10-7 M and serotonin 10-5 M) did not cause cellular injury. Cultured human EC are injured by thrombin. The other tested agents do not appear to induce direct injury in vitro, but may interact with cellular elements in the blood to produce the endothelial injury previously observed in vivo.
Thrombin-Induced Endothalial Cell Retraction
Authors: Lerner R.G. , Cheong L.C. , Nelson J.C.
Published: May 2019

The interaction of vascular endothelial cells with various components of the coagulation mechanism could provide insight into the process of intravascular thrombosis. Thrombin has been reported to bind to endothelial cells and cause several functional changes in the endothelial cells, including shape change and cytogel extrusion. In our experiments, Chromatographically pure human α-thrombin was added to human umbilical vein endothelial cells growing in monolayer. Thrombin caused a pronounced retraction of endothelial cells into heaped up ridges beginning at about 20 minutes, peaking at 1-2 hours and spontaneously reversing overnight. The retraction was dose dependent and prevented by heparinized plasma. The retraction caused loss of culture confluency, loss of areas of cell-cell contact and exposure of subendothelial surface. However, there was no sloughing of cells into the medium or killing of endothelial cells as determined by fluorochromatic cytotoxicity assay using ethidium bromide. The exposure of a potentially reactive subendothelial surface by thrombin could be important in the process of intravascular thrombosis by providing a site for thrombus attachment. Heparin may help stop further thrombosis by potentiating Antithrombin III and preventing exposure of subendo-thelium to circulating blood.
Advantages of a Sequential Compression Device in Prevention of Deep Vein Thrombosis
Authors: Nicolaides A.N. , Fernandas J. , Pollock A.V.
Published: May 2019

A sequential compression device (SCD) (6 chambers) compressing the ankle, calf end thigh sequentially at 35, 30 and 20 mm Hg for 12 seconds in every minute produced a 240% increase in peak velocity in the femoral vein. A non-sequential device (NSD) inflated to 25 mm Hg with a similar time cycle produced only an 180% increase in blood velocity.

The two devices were tested clinically; 250 surgical patients were randomised to 3 groups scanned with the 125 I-fibrinogen test. Group A: 7 days subcutaneous heparin. Group B: NSD for 24 hours. Group C: SCD for 24 hours. The SCD was found to be as effective as heparin during the period it was used and more effective than NSD in preventing deep vein thrombosis proximal to the calf.
Structure - Function Relationships of Human High MW Kininogen(HMWK), A Cofactor in Contact Activation of Human Plasma
Authors: Kerbiriou D. M. , Griffin J.H.
Published: May 2019

HMWK has been isolated from human plasma as a single polypeptide chain of apparent 110,000 MW on SDS gels. Purified plasma kallikrein cleaved HMWK to produce kinin and a kinin-free molecule made up of two disulfide-linked polypeptide chains with apparent MW of 65,000 and 44,000. Following reduction and carboxymethylation of the two-chain, kinin-free molecule, a histidine-rich light chain was isolated that quantitatively retained the full procoagulant activity of the native molecule. HMWK was digested with a bacterial protease to produce kinin and polypeptides that have no procoagulant activity. From this digest, cationic fragments were isolated which possess a high histidine (36%) and glycine (36%) content that is similar to the composition of Fragment 1.2 cleaved from bovine HMWK by bovine plasma kallikrein. When kaolin was added to normal human plasma containing 125I-HMWK and 131I-Factor XII , the HMWK was rapidly cleaved with a half-time of cleavage of 40 ± 10 sec which was similar to the kinetics of cleavage of Factor XII. This surface-initiated cleavage of 125I-HMWK in plasma did not occur in plasmas deficient in either prekallikrein or Factor XII, but occurred when purified kallikrein was added. These observations suggest that when kaolin is added to plasma, kallikrein cleaved HMWK to release kinin and to give rise to a two-chain, kinin-free form of HMWK whose ability to correct the clotting deficiency of HMWK-deficient plasma resides totally in the histidine-rich light chain of the molecule.
Structure-Function Studies on Native and Synthetic Permeability Increasing Peptide Derived from Fibrinogen or Fibrin
Authors: Belew M. , Gerdin B. , Larsson L.-E. , Lindberg G. , Porath J. , Ragnarsson U. , Saldeen T. , Wallin R.
Published: May 2019

Vasoactive peptides liberated from fibrin microemboli have been suggested to be responsible for the increased microvascular permeability in the delayed microembolism syndrome. Two such peptides were isolated from fibrin or fibrinogen lysate after chromatography on Bio Gel P-6 and column zone electrophoresis. These peptides designated as 6A and 6D show a strong effect on microvascular permeability in the rat skin after injection of as little as 5 μg. Amino acid sequence: Ala-Arg-Pro-Ala-Lys for 6A and Ser-Gln-Leu-Gln-Lys-Val-Pro-Pro-Glu-Trp-Lys for 6D. Synthetic counterparts were achieved by the Merrifield solid phase method. Structure-function studies revealed 1) Removal of the C-terminal Lys by carboxypeptidase B resulted in complete loss of activity. 2) Replacement of the C-terminal Lys with Arg in the pentapeptide did not affect its potency whereas substitution of the N-terminal Ala with a propionyl group resulted in an inactive product. 3) Cleavage of 6D with trypsin gave two inactive fragments. 4) Formylation of the Trp-residue in 6D gava product which was about 50% more active than the parent molecule

These studies show that the N-terminal amino-group, the C-terminal Lys (or Arg) as well as Trp (in 6D) are apparently essential for the permeability increasing effect of these peptides.
Presence of Activated Factor VII in Factor IX Concentrates and its Persistence in the Circulation After Infusion
Authors: Seligsohn U. , Kasper C.K. , Østerud B. , Rapaport S.I.
Published: May 2019

Six brands of Factor IX concentrates were evaluated for their Factor VII content and for the presence of activated Factor VII through use of a coupled amidolytic assay, insensitive to activated Factor VII, and a clotting assay, sensitive to activated Factor VII. The Factor VII content of the concentrates studied (except for one concentrate purposely produced to exclude Factor VII) varied between 33 to 621 U per vial. All concentrates contained activated Factor VII, as indicated by ratios of Factor VII clotting activity to Factor VII amidolytic activity of from 1.6 to 21.5. Higher ratios were found in two brands of activated concentrates than in non-activated concentrates. In 10 patients infused with Factor IX concentrates, plasma Factor VII activity rose strikingly in the clotting assay but not in the amidolytic assay. Thus, the elevated Factor VII levels by the clotting assay after infusion of Factor IX concentrates stem from circulating activated Factor VII. A mean intravascular half-disappearance time of 144 min was found for activated Factor VII. Its persistence in the circulation makes it important to evaluate the possible role of activated Factor VII in the thrombogenicity of Factor IX concentrates and in their reported effectiveness in treating bleeding in Hemophilia A patients with inhibitors.
On the Relationship Between Low Affinity PF4 and ß-Thromboglobulin
Authors: Holt J. C. , Niewiarowski S.
Published: May 2019

Platelets stimulated by thrombin or Ca-ionophore A 23187 release a number of proteins which bind specifically to heparin. Those with low affinity (displacement by 0.5M NaCl) include low affinity PF4 (LA-PF4) and β-thromboglobulin (βTG). LA-PF4 and βTG are closely related as shown by their comparable affinity for heparin, immunological cross-reactivity and the similarity of the limited amino acid sequences which have been compared. The latter suggest that LA-PF4 differs from βTG only by a 4-residue NH2-terminal sequence (asn-leu-ala-lys-) unique to LA-PF4. Isoelectric focussing and disc gel electrophoresis, which distinguish the two proteins, were used to characterise preparations from various laboratories. That different isolation procedures yielded predominantly one or other species implies that βTG may be derived from the larger LA-PF4 by proteolysis. Direct evidence was sought by exposing pure LA-PF4 to thrombin and plasmin for 1 h at 37°. While thrombin (40 u/ml) had no effect, highly purified plasmin (2 u/ml) converted LA-PF4 quantitatively to a component with mobility and isoelectric point close to βTG. The alteration in charge resulted only from the cleavage of terminal residues since the mobility on SDS-gels was unchanged. Our evidence therefore suggests that βTG is a stable proteolytic fragment of LA-PF4. Whether this conversion occurs in vivo, and involves platelet-associated proteases, is under investigation.
Synthesis of an Arginine Analog of the Fragment 56-68 of Human Platelet Factor 4 Having Significant Antiheparin Activity
Authors: Bajusz S. , Fauszt I. , Barabáa É. , Bagdy D.
Published: May 2019

Of the 70-residue polypeptide human platelet factor 4, which binds heparin stoichiometrlcally, the C-terminal fragment 50-70 /Tc-3/ showed a reduction of the heparin induced prolongation of thrombin time /Deuel et al., Proc. Natl. Acad. Sei. USA., 74:2256, 1977/.

The arginine analog of the fragment 58-68 of Tc-3, which comprises the unique lysine rich region /Lys-Lys-Ile-Ile-Lys-Lys/, was prepared; Pro-Leu-Tyr-Arg-Arg-Ile-Ile-Arg-Arg-Leu-Leu-NH2. The aim of replacing of lyslne residues of the native molecule by arginine was to increase the interactions between the basic side chains of the peptide and SO3
- and/or COO- groups of heparin by introducing additional H-bridges and strengthening the ionic bonds. The new undecapeptide was synthesized by the standard solid phase method using Boc amino acids and dicyclo-hexylcarbodiimide condensation.

1 nmole /1.8/ug/ of synthetic peptide completely inhibits the action of 0.3 units of heparin which corresponde a heparin/peptide molecular ration of about 1:6.
Purification and Properties of a Low Molecular Weight Protein from Human Platelets and its Relationship to Beta-Thromboglobulin
Authors: Walz Daniel A. , Niewiarowski Stefan
Published: May 2019

Activated human platelets secrete specific intracellular proteins which have anti-heparin activity. These proteins include platelet factor 4 (PF-4), beta-thromboglobulin (β-TG) low-affinity platelet factor 4 (LA-PF-4), and platelet growth factor. Outdated platelet concentrates were initially heat-treated for 10 minutes at 70°C, centrifuged, and the supernatant fractionated on Sephadex C-50. The fraction eluted with 0.5M NaCl contained the majority of the anti-heparin activity. Subsequent gel filtration resulted in a final product which was homogeneous on SDS electrophoresis (9,000 daltons). Amino terminal analysis of this fraction gave the sequence: Asn-Leu-Ala-Lys-Gly-Lys-Glu-Glu-Ser-. Residues 5-21 were identical to residues 1-16 of β-TG. This protein shared common antigenic determinants with β-TG in a radioimmunoassay. Isoelectric focusing of this material resulted in the majority of protein recovered at an isoelectric point of 8.0 (β-TG = 7.0). This protein fraction possessed four times the specific molar activity of insulin in stimulating the mitogenic expression of Balb 3T3 cells; however, additional protein fractions from the gel filtration procedure were far more potent, though less homogeneous. This protein, therefore, appears identical to LA-PF-4, and is apparently the precursor of β-TG; it is not the most potent of platelet growth factors.(Support: MI Heart; NIH 14217).
Biological Findings During Sequential Thrombolytic Treatments With Defibrase, Lysyl-Plasxinogen and Urokinase
Authors: Griguer P. , Dusson PH. , Brochier Mireille
Published: May 2019

Biological changes induced by a snake venom (Defibrase - DEF) infused prior to a thrombolytic treatment are studied in a series of 35 pulmonary embolism (P. E) and/or deep vein thrombosis (D. V. T). Group I (n : 13) includes old PE and or D.V.T (14 ± 6 days old) in which DEF infusion is performed prior to thrombolytic treatment ; Group II (n : 22) includes recent P. E and/or D.V.T. A first treatment with Lysyl-Plasminogen (Lys-Plg) and Urokinase (UK) with poor or insufficient results is followed by DEF infusions just before a second thrombolytic treatment. The results of these treatments controlled angiographically (before and 24 hours after the end of the last UK infusion) have shown that a better revascularisation could be obtained even in old PE, when Defibrase is given prior to thrombolytic treatment. During the DSF infusions, we observed a decrease of fibrinogen m less than 36 hours (to a mean level of 0,20 to 0,30 g/l). The % of circulating plasminogen consumption is 48.22 % ± 22, and a rapid drop of the fast reacting antiplasmin level (F.R. antipl) occurs (from 77,5 % ± 30 to 12,9 % ± 7,6) During the thrombolytic treatment following DEF, we observed an enhancement of fibrinolytic activity. From these data we may conclude that DEF is able to determine plasminantiplasmin complexes and a large drop of the fast antiplasmin level, in the blood
Plasma Fibrinogen and Factor XIII During Intermittent Thrombolytic Therapy
Authors: Dubiel M.K. , Scully M.F. , Kakkar V.V.
Published: May 2019

The precursor components of the fibrin crosslinking system, viz, fibrinogen, and Factor XIII have beer, studied in plasma from patients undergoing intermittent thrombolytic therapy. Fibrinogen was isolated as fibrin monomer in the presence of EDTA and Trasylol and the level of degradation of the subunits quantitated by densitometric scans after separation on SDS gels Plasma samples were also clotted in the presence of calcium and the crosslinked subunits separated on SDS gels. Gels were radioiodinatedusing a chloramine T method, then sliced and counted to determine the amounts of γ dimer and α polymers. Daily single infusion of streptokinase caused degradation of circulatory clottable fibrinogen such that about 80% contained degraded chains MW 25,000. In crosslinked fibrin prepared from the plasma there was no diminution of the relative amount of γ dimer formed but tha of the high molecular weight α polymers was greatly reduced. In the interval between infusion of streptokinse (12 hours) the fraction of undegraded fibrinogen returned to near normal which was associated with an increased capacity to form α polymers, Three methods were used to determine Factor XIII Levels during therapy, viz, dansyl cadaverine incorporation, immunoelectrophoresis (a and b subunits) and the rate of formation of γ dimers and α polymers using 125I fibrinogen free of Factor XIII. Over the course of the therapy Levels fell by 30% of the pretreatment value using the first two methods, slightly great er losses being observed using the third procedure. The daily fluctuation of plasma levels observed with fibrinogen, pre and post streptokinase infusion did not occur with Factor XIII.
Hypothetical Role of Heparan Sulphate (HS) in Atherogenesis
Authors: Teien A.N.
Published: May 2019

Evidence indicates that the following factors are concerned with atherogenesis: Blood flow, thrombosis, endothelial injury, lipid and protein accumulation in the intima. The initiating mechanism is unknown. Several investigators have observed thrombus formation on structurally normal intima. The changes in the intima starting the process may, however, be biochemical rather than structural, HS is a weak heparin-like anticoagulant attached to the external endothelial cell surface. HS may be liberated by a platelet endoglycosidase, and may be inactivated by platelet factor 4 or beta-lipo-proteins. Assuming platelet damage because of the flow pattern, platelet aggregation and release reaction may result in inactivation of endothelial bound HS, thus favouring local thromboeia. High levels of beta-lipo-proteins in the circulating blood may add to this effect. Released platelet substances may increase vascular permeability counting for the plasma components found in atherosclerotic lesions. Exogeneous heparin binds to the intima, probably by substituting HS at the endothelial cell surface. Long term heparin prophylaxis may hypothetically prevent or retard advancing atherosclerosis. Heparin fractions, possibly with less side effects, could be of value.
Conditions Affecting the Molecular Weight of Factor VIII.
Authors: Rock G. , Tackaberry E. , Palmer D.
Published: May 2019

By purifying Factor VIII while maintaining physiological concentrations of calcium we have recently demonstrated that about 50% of the procoagulant activity is in a very low molecular weight (VLHW) form not associated with the carrier (VIII: RAG). The remainder is carrier associated and elutes at Vo as a high molecular weight (HMW) compound upon Sepharose 6B chromatography. Reduction of the calcium concentration by increasing the amount of citrate added to heparin results in decreasing the ratio of VLMW:HMW from 1:1 in pure heparin to 1:5 in pure citrate. If citrate is replaced with the more strongly chelating EDTA no VLMW is detectable in the plasma. It has also been found that most of the biochemical purification techniques which have been previously used to prepare Factor VIII for study actually result in the aggregation of this VLMW with the carrier to produce the high molecular weight form. This includes: cryoprecipitation, precipitation by polyethylene glycol and storage -80°C. As well, the VLMW material will self-associate upon freezing to produce an aggregate with a molecular weight of 106. However, this material does not cross-react with rabbit antibody directed against VIII: RAG. The data indicate that many of the previously reported biochemical characteristics, including molecular weights, actually describe species which are artifacts of the isolation process rather than those of the physiologically occuring Factor VIII.
Early Changes of the Endothelial Antithrombotic Properties in Cholesterol Fed Rabbits. III-Decreased PGI2 Production by Aortic Wall
Authors: Masotti G. , Galanti G. , Poggesi L. , Curcio A. , Neri Serneri G.G.
Published: May 2019

In rabbits PGI2 activity of aortic rings was investigated both by Vane superfusion bioassay and by measuring the capacity to inhibit platelet aggregation according to Moneada et al. (1976). In cholesterol (lg/die) fed rabbits PGI2 already after one week treatment resulted reduced by comparing to controls (P 0.01) ana is still reduced after two and four weeks of treatment. PGI2 reduction results also by assaying the inhibiting activity of aortic rings on platelet aggregation. These results indicate that after cholesterol diet the antithrombotic mechanisms of endothelial wall is impaired very early in a period when the aortic lesions are minimal or absent.