bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role of Calcium ION in Platelet Aggregation
Authors: Deguchi N. , Ikeda Y. , Toyama K. , Hirano T. , Watanabe K. , Ando Y.
Published: May 2019

Calcium is required for platelet aggregation but the precise role of Ca++ is not yet known. We have investigated the role of Ca++ in platelet aggregation, performing an ultra-structural and biochemical analysis of Ca++ treated platelet membrane vesicles. Human platelet membrane vesicles isolated by glycerol lysis technique according to the method of Barber and Jamieson, were suspended in 10 mM tris buffered saline, pH 7.4. Aggregation of platelet membrane vesicles was clearly observed under the phase-contrast microscopy in the presence of 5 mM CaCl2. Aggregated membranes were solubilized in 1Ï SDS and 1% 2-mer-captoethanol, and electfophorcsed on 5% SDS-polyacrylamide gels. Comparison of polypeptides of aggregated and native membranes revealed that the polypeptide of molecular weight 200,000 Daltons disappeared, instead, coomassie blue staining became visualized at the top of the gel in aggregated membranes. Ultrastructural observations were performed after fixation of membrane vesicles with 2% glutaraldehyde for both thin-section and freeze-fracturing. Freeze-fractured image of native membranes exhibit a smooth surface with a random distribution of intramembranous particles (IMP). Membrane vesicles treated with 5 mM CaCl2 possess round depressions suggestive of areas of mutual contact. These circular attachment sites are free of IMP, which accumulated at the periphery. It is suggested that these ultrastructural changes induced by Ca++ may be regarded as crucial events in platelet aggregation.
Electron Microscopic Analysis of Subcellular Distribution and Translocation of Ca ION in Human Platelet
Authors: Kazama M. , Daimon T. , Nakamura K. , Matsuda J. , Naito I. , Abe T.
Published: May 2019

For the analysis of the subcellular distribution of Ca ion in human platelet, the washed platelet suspension was fixed in the potassium antimonate-OsO4 method or in the potassium oxalate-glutaraldehyde method without post-fixation with OSO4. The STEM-images were observed with scanning image system fitted with the transmission electron microscope without electron staining.

The dense deposition of antimonate or oxalate was found in plasmalemma, the membrane of open canalicular system, mitochondria, α-granules and dense bodies. It was revealed with the energy dispersive type electron probe x-ray microanalyzer system that the deposition of oxalate was exclusively composed of Ca.

In the process of platelet aggregation with various agents, α-granules and dense bodies were expelled out together with Ca, which suggested the least role of these ion depositions in the aggregation and clot retraction. The translocation of antimonate deposits was not observed in the Verapamil-treated platelets even after the addition of aggregation agents. This phenomenon indicated that this drug inhibited the release of Ca from the Ca-storing organelles. The distribution of Ca was identical in the platelets of a case of thrombasthenia and the translocation of Ca was not observed with the addition of various aggregating agents.
Effect of Heparin on Lysis of Fibrinogen/Fibrin
Authors: Chakrabarti R.
Published: May 2019

The incubation of citrated human plasma with small doses of streptokinase or urokinase for 30 minutes does not result in any appreciable loss of recoverable fibrinogen in the plasma. However, the addition of heparin before incubation causes a loss of fibrinogen which is dependent on the dose of heparin added. This in vitro effect has been confirmed in plasma from patients before and after receiving 5-7, 500 units of heparin intravenously. It is possible that the loss of fibrinogen is due to the interaction of heparin with the inhibitors of fibrinolysis. To study this, in vitro tests were performed in a purified system (i.e. without inhibitors), using plasminogen-rich human fibrinogen and urokinase. These tests revealed that the addition of heparin to the fibrinogen before it wae clotted with thrombin in the presence of urokinase shortened clot lysis times. On the other hand, the addition of heparin prolonged the lysis time of a preforaed clot by urokinase. These results imply that the loss of fibrinogen originally observed was not due to an interaction of heparin with inhibitors. The results indicate that heparin augments or retards fibrinolysis by urokinase depending on whether the heparin is present before or after fibrinogen is converted to fibrin. If opposing effects of heparin on fibrinolysis are confirmed in vivo, it will be necessary to distinguish carefully between its use prophylactically and therapeutically, especially if used in association with a thrombolytic agent.
Contact Activation of Plasma Prekallikrein and Fibrinolysis by Heparinoids and Heparin
Authors: Soulier J.P.
Published: May 2019

Contact activation of plasma prekallikrein (PPK), using a tripeptide chromogenic substrate, and of surface mediated fibrinolysis using the euglobulin test (both tests insensitive to the anticoagulant effect of heparin and heparinoids) were studied. The following substances were tested : (1) Dextrans (different commercial therapeutic preparations) and dextran sulphate of different molecular weights. (2) Heparinoids such as: pentosane, mannuronate and galactose sulfuric polyester and liquoid. (3) Twelve different commercial therapeutic preparations of heparin.

Results: XII, PPK and HMWK are necessary for the full activation. Heparinoids have a strong activation capacity on PPK and fibrinolysis, dextran sulphate (500 000) being the most active as shown by Kluft. Dextran solutions as well as different brands of heparin have a slight but definite surface activation capacity (still unnoticed, being masked by the anticoagulant activity). This explains some side effects of heparinoids and why heparin is unable to prevent contact activation, in vitro and in vivo.
Enhancement by Heparin of Affinity between Cold-Insoluble Globulin and Native Collagen Type III
Authors: Hörmann H. , Jilek F.
Published: May 2019

Affinity between collagen and cold-insoluble globulin was measured by complexing soluble 125-J labelled collagen preparations with the globulin. Precipitates containing considerable activity were formed at 4°C and 22°C by denatured soluble collagen, type I and type III, but only little by native soluble collagen. The precipitation of native collagen, type III, by cold-insoluble globulin was enhanced by heparin. Under optimal conditions at a weight ratio or heparin and cold-insoluble globulin of about 1:1 up to 60% of the collagen applied was insolubilized. Native collagen, type I, was complexed far less effectively even in presence of heparin. Electronmicroscopic and precipitation experiments using 125-J labelled cold-insoluble globulin indicated that heparin might induce a partial conversion of cold-insoluble globulin to a fibrillar derivative which exhibited improved binding properties for the rod-like native collagen molecules. – Supported by Deutsche Forschungsgemeinschaft, Project Ho 740/1.
What can Crystallographic Analysis tell us About Fibrinogen?
Authors: Cohen Carolyn , Weisel John W.
Published: May 2019

A major project in our laboratory has been the determination of the molecular structure of fibrinogen and its Interactions to form fibrin. We have approached this problem by attempting to obtain ordered forms of fibrinogen and, In fact, to crystallize the molecule. X-ray crystallography is the only objective method that can yield the detailed structure of the native hydrated protein molecule. In order to obtain crystals of fibrinogen, we found that limited proteolytic digestion is required. We have now produced three macroscopic crystal forms and a variety of microcrystals using several different enzymes and fibrinogens from different species. These modified molecules are largely intact and retain their biological function to form clots with thrombin similar in appearance to native fibrin by electron microscopy. The degree of order in all these aggregates is, moreover, far superior to that of fibrin. Some of the microcrystalline forms have been shown to be closely related to fibrin. Since the fibrinogen molecule is several times larger than any protein yet solved by X-ray methods, the technical problems in the crystallographic analysis are formidable. Because of the large size of the molecule, however, electron microscopy provides Information essential for the solution. We have completed a preliminary X-ray characterization of the ctystals and, using coordinated X-ray diffraction and electron microscopy, deduced plausible packing models. The results from this first stage in the X-ray analysis of fibrinogen give insight also into the packing of the molecules to form fibrin. Supported by USPHS grant #AM17346.
The Inhibition of the Vitamin K Dependent Carboxylation by Free Radical Scavengers
Authors: Walter S. J. , Burgess A. I. , Esnouf M. P. , Green M. R. , Hill H. A. O. , Okolow-Zubkowska M. J.
Published: May 2019

Prothrombin contains γ-carboxyglutamic acid residues, synthesised at a post-translational step requiring vitamin K quinol, carbon dioxide, molecular oxygen, and a carboxylating system solubilised from liver microsomal fractions of rats (normal and vit.K deficient) and calves by non-ïonic detergents. The reaction is assayed by the incorporation of 14C into prothrombin precursor or the pentapeptide, Phe-Leu-Glu-Glu-Val. Superoxide dis-mutase reduces the incorporation (50% inhibition at 300μM) and so too do copper chelates with known dismutase activity: 50% inhibition at 60μM copper (II) aspirinate, 127μM copper (II) penicillamine and 152μM copper (II) tyrosine. The superoxide anion is postulated as a primary product of reduced Vitamin K and dioxygen. Catalase also inhibits at high concentrations (> .5mM), possibly by shifting the equilibrium to favour dismutation or b; preventing production of an ‘active carbon” species via hydrogen peroxide. So far it has not been possible to replace Vitamin K with active intermediates, such as t-butyl hydroperoxide, which also inhibits the action of the vitamin, possibly due to the formation of Vitamin K 2,3-epoxide. These results point to the involvement of radical species in the Vitamin K dependent carboxylation of prothrombin.
Measurement of Des-Arginine-Fibrinopeptide B (DES-ARG-FPB) in Plasma by Radioimmunoassay
Authors: Eckhardt T. , La Gamma K.S. , Nossel H.L.
Published: May 2019

During thrombin induced clotting of fibrinogen, fibrin I(des-fibrinopeptide A(FPA)-fibrin) and fibrin II (des-FPA-FPB-fibrin) are formed sequentially. As fibrin II forms thicker strands and is more rapidly crosslinked by factor XIIIa than fibrin I, measurement of FPB, reflecting fibrin II-formation, is of special interest. The radioimmunoassay for FPB cannot he applied to plasma, because 1. cross-reacting fragments cleaved from the N-terminal portion of the Bß chain of fibrinogen by plasmin and fibrinogen are present and 2. cleavage of arginine 14 of FPB in blood by carboxypeptidase B results in a 20-fold reduction in inmunoreactivity. These difficulties have been overcome as follows: fibrinogen is precipitated with ethanol; N-terminal fragments such as Bβ 1-42 are selectively adsorbed to bentonite; FPB is converted by plasma carboxypeptidase B to des-arg-FPB which is measured using antiserum R29 (sensitive to des-arg-FPB), 125I-tyrosinated synthetic des-arg-FPB analogue as tracer and native des-arg-FPB standard. When added to blood, FPB is completely recovered as des-arg-FPB immunoreactivity. The lowest detectable level of des-arg-FPB in samples processed and assayed as described is 0.4pmol/ml. Preliminary data suggest that the des-arg-FPB level in normals is less than 0.7pmol/ml.
Conformational Alterations of α2-Antiplasmin Induced by Complex Formation with Plasmin
Authors: Plow E.F. , Wiman B. , Collen D.
Published: May 2019

The conformational and structural changes induced in the α2-antiplasmin (AP) molecule by complex formation with plasmin have been analyzed utilizing quantitative radioimmuno-chemical analyses. Complexes prepared in plasmin excess -(PAP-P) and therefore subjected to limited proteolysis and complexes prepared in AP excess (PAP-A) have been compared with free AP. With AP antiserum, PAP-A, PAP-P and AP yielded reactions of complete identity by immunodiffusion analysis. In radioimmunoassay, however, these were clearly distinguished, and four distinct sets of antigenic determinants were delineated. Set I determinants were expressed equivalently by PAP-P, PAP-A and AP and were, therefore, not altered by complex formation. This set was recognized by 90% of the antibodies, and the determinants were all included within a large fragment of Mr 60,000 derived from the NH2-terminal region of AP. The other three sets of determinants were modulated by complex formation. Set II was expressed by PAP-A and AP but not by PAP-P, and these were sensitive to proteolysis by plasmin. Set III determinants were expressed only by AP and were localized to a peptide of Mr 8,000 derived from the COOH-terminal region of AP. Set IV determinants were also present only on AP but were not present in the peptide and required an intact reactive site in AP for expression. Thus, there is evidence for multiple conformational modulations in AP induced by complex formation, and these modulations can be pinpointed to specific loci within the AP molecule.
Pathways of Stimulation of Tissue Factor Activity Generation by Leukocytes
Authors: Niemetz J. , Muhlfelder T.
Published: May 2019

Leukocytes nay generate a potent procoagulant thromboplastic (tissue factor) activity (TFa) if appropriately stimulated. Among the stimulants endowing leukocytes with TFa are endotoxins (lipopolysaccharides: LPS), antigen-antibody, complexes and a complement component: the chemotactic fragment of C5 (C5fr) vhieh is a particularly potent activator. As LPS and antigen-antibody complexes can activate complement with ensuing generation of C5fr, the question arose whether C5fr is the sole terminal pathway for activation of leukocyte TFa. Studies of the amount of activity generated with the LPS and C5fr showed that normal leukocytes stimulated with C5fr produce significantly more TFa than leukocytes stimulated with LPS. In contrast, leukocytes from patients with paroxysmal nocturnal hemoglobinuria which respond normally to LPS, generate considerably less TFa when stimulated with C5fr. Furthermore, the-TFa in normal leukocytes - appears sooner following LPS stimulation than following C5fr stimulation. These results suggest that at least in vitro C5fr need not be the sole external stimulant for the generation of TFa.
Electrophoretic Distribution of FVIII:C Measured by Chromogenic and Clotting Methods and Comparison with other FVIII-Associated Activities
Authors: Seghatchian M.J.
Published: May 2019

The new technique of meesuring FVIII-associated activities in agrose gel after electrophoresis has made it possible to investigate the molecular nature of FVIII components and their electrophoretic distribution. The method has been successfully used for the characterization of the neterogeneous nature of FVIIIR. Ag in commercially available FVIII concentrate and cryoprecipitate obtained from normal, haemophilia and WD patients and their response to transfusion of FVIII concentrates. The mobility of FVIIIR:Ag measured by X-immunoelectrophoresis in these samples was compared with the VIII:C distribution by chromogenic, one stage end two stage clotting, and FVIIIR. HCF measured by both the slope of ristocetin induced platelet aggregation (RIPA) and by the measurement of non aggregated platelets. Different methods measured various forms of FVIII to differing degrees. FVIII:C methods had the highest sensitivity for the identification of FVIII, particularly the smaller forms, while RIPA and antigen methods preferentially measured the larger aggregates. The presence of a FVIII subunit with FXa generation activity and o faster mobility than the main FVIIIR:Ag became apparent in all the preparation analysed. The high sensitivity, and the staining properties of chromogenic methods, particularly after the colour intensifying reaction made this method also a good visual marker of FVIII distribution in agrose: Thus in certain types of immunochemical investigation the FVIIIR:Ag assay may be replaced by quantitative chromogenic FVIII:C. The parallel measurement of FVIII related activities provided a new approach to the diagnosis and the control of m:. najo mese of FVIII functional abnormalities.
Separation of factor VIII by Ion Exchange Chromatography
Authors: Austen D.E.G.
Published: May 2019

Using a chemically treated agarose, it has been possible to separate factor VIII clotting activity from factor VIII related antigen and ristocetin co-factor in human factor concentrates, or alternatively from antigen and platelet aggregating factor in porcine materials. It appears that components are separated by an ion-exchange mechanism although hydrophobic interactions and steric influences cannot be ruled out. A column technique is employed and products appear to be stable. The latter is an important feature since factor VIII which has been exposed to charged surfaces is often unstable. However, part of this good stability may be attributable to the lysine-containing buffer which is employed. Factor VIII clotting activity has a normal antibody reaction after separation and the antigen is of normal mobility. The factor is recovered from the column at relatively low ionic strength (0.5 molar sodium chloride) and this might suggest that the components are separated without factor VIII being disaggregated into sub-units. Unlike some ion-exchange separations of factor VIIIitis not necessary to purify the starting materials to a higher quality than that of a washed and adsorbed cryoprecipitate and one passage of suet] a material through the column results in high purification
Generation of Factor VIII:C by Isolated Pig and Rat Livers
Authors: Owen C. A. , Bowie E. J. Walter , Fass D. N.
Published: May 2019

When isolated neonatal pig livers were perfused with blood from pigs with von Wille-brand’s disease, no factor VIII coagulant activity (VIII.C) emerged in the perfusate. When purified porcine Willebrand factor, lacking VIII:C, was added to the perfusate it rapidly diminished, presumably being taken up by the liver. Concurrently VIII:C rose steadily in the blood during the 5 hours of perfusion. Similarly, isolated adult rat livers released no VIII:C when they were perfused with rat erythrocytes suspended in Tyrode’s solution containing 6% bovine serum albumin. VIII:C did emcrRe if cryoprecipitate from aged rat plasma were added. The neo-VIII:C was rapidly inactivated by plasma from a patient with classic hemophilia who had developed an antibody to VIII:C. Addition of cycloheximide to the rat before extirpation of the liver, as well as to the perfusate, prevented the emergence of VIII:C. We suspect that VIII:C is derived from the liver but only when Willebrand factor is present.
Blood Viscosity and Platelet Aggregation in Male and Female Patients with Transient Ischaemic Attacks (TIA)
Authors: Palareti G. , Poggi M. , Fortunato G. , Coccheri S.
Published: May 2019

A series of 40 patients with TIA (25 males and 15 females) was thoroughly investigated by means of angiography and computerized tomography, and divided into a group (A)of 15 “sine materia”, and a group (B) of 25 with direct or indirect evidence of vascular occlusive or stenotic changes. Blood viscosity at 230 sec-1 37°, was cp 4.2 ± 0.3 in the controls, cp 4.7 ± 0.7 in all patients (p < 0.05) cp 4,98 ± 0.7 in all malepatients (p < 0,01 versus male controls), and cp 4.75 ± 0.8 in group B (p < 0.02). Hacmatocrit and Fibrinogen were also significantly increased in all male patients and in group B. Circulating platelet aggregates (CPA) were increased in 40% of thepatients. Almost all patients with elevated CPA were males, with a slight prevalence in group B. Changes in blood viscosity parameters and in platelet aggregation in TIA patients were therefore related both to evidence of vascular lesions, and to sex, since they were found to prevail in male patients of both groups.
Detection of Circulating Platelet Aggregates in Thromboembolic Disease
Authors: Marie Lorène Scrobohaci , Petrilǎ Teodora , Constantinescu M. , Stadler Magdolna , Mihǎilǎ Doina , Pop I. , Popa D.
Published: May 2019

Summary: The method of Wu and Hoak in det rmining circulating platelet aggregates in vivo was used.

In different cardiac and vascular states, a low aggregation index is found: aorto-iliac occlusion (Leriche syndrome)- 38 csses(index X ± SD 0.52 ± 0.05 in comparison with normal subjects 0.91 ± 0.05); valvular diseasses (0.40±0.006); during extracorporeeal circulation - 14 cases (0.5±0.02l); thrombophlebitia-25 casea(0.84±0.065).

After an antiaggaagation treatment (Dipiridamol) in 12 cases of aortoiliac occlusion the value of the aggregation index normalised(0.84±0.065) Experimenntlllly, 10 rabbits were perfused wi th thrombin soution (2 u./body woight k.); the lower values of the aggreeation index (0.91±0.085 brfore Emd 0.43±0.035 after the thromhin perfuaion) prove the fidelity of the method.

The simplicity of the technique, the reproducti bili ty in experimental conditions and the normalisation of the valuwa after treatment prove the practical value in determining the circulating platelet aggregates.
Platelet Aggregation and Circulating Fibrinogen Complexes in Patients with TIA
Authors: Abbate R. , Gensini G.F. , Serneri G.G. Neri , Inzitari D. , Taiuti R. , Amaducci L.
Published: May 2019

In the Atherosclerosis Project of the National Research Council we investigated 31 controls and 25 patients with transient ischaemic attacks (TIA) in whom an angiographic study of the carotid arteries and of the aortic arc has been performed. Circulating platelets aggregates (Wu and Hoak 1974) and circulating high molecular weight fibrinogen complexes (HMWFC) by gel-filtration of plasma beta-alanine precipitate (Graeff and von Hugo 1974), fibrinolytic activity (EI.T) and antithrombin III (Howie et al. 1973) were determined. The patients who had suffered from TIA showed an increased pis s-let aggregation (17/25, P<0.001) and an increased concentration of HMWFC (15/25, P<0.001). Fibrinolytic activity and AT III activity showed no significant differences between the two groups. The angiographic study showed in 11 patients identifiable atherosclerotic lesions (IAL), but no correlation between artériographie lesions and increase of platelet aggregation and HMWFC concentrations was found.
Severe Acute Disseminated Intravascular Coagulation (DIC): Clinical Importance; Heparin Therapy; and Etiology
Authors: Mant M.J. , King E.G.
Published: May 2019

We have reviewed 47 patients (pts.) with severe acute DIG (mean fibrirogen 100.5 mg/dl, platelets 45,700/μl, prothrombin time 19.4 sec, partial thrcmboplastin tiire 86.5 sec). Most pts. were critically ill. Predisposing conditions included infection (32), shock (32), and surgery or trauma (21). Routine treatment included aggressive therapy for underlying diseases and metabolic disturbances, vitamin K, folate, and blood components. Twelve pts. were treated with heparin and 10 (83%) died. Ifeparin apparently benefited only 1 pt, and in 7 bleeding increased. Thirty (86%) of 35 pts. who did not receive heparin died. Analysis suggested that heparin would not have affected the outcome in any of them. In all pts. who died multiple potential causes were present. Thrombosis, ischemia, or hemorrhage were the apparent immediate cause of death in only 2 pts. but were contributory causes in others. In most pts. with significant complications potentially attributable to Die other causative pathology was present. Twenty-five pts. had autopsies. Microvascular thrombi were not found but large vessel thrombi were relatively common. We conclude that 1) Severe DIG is usually a preterminal event in criticallv ill pts. 2) Other pathology may be more important than DIC when death or significant thrombosis or bleeding occur. 3) Heparin is rarely beneficial and often increases bleeding. 4) Microvascular thrombi may not be the only mechanism responsible for DIC.
Reversible Factor VIII-Abnormalities in Reye’s Syndrome.
Authors: Joist J. H. , Vargo J. , Haymond M. W. , Keating J. P. , DeVivo D. C.
Published: May 2019

Reye’s syndrome, an acute and frequently fatal viral infection with severe metabolic disturbances is frequently associated with marked hemostatic alterations. Hypoprothrombinemia and deficiencies of other clotting factor activities as well as findings suggestive of disseminated intravascular coagulation have been reported. This report deals with detailed sequential qualitative and quantitative studies of blood coagulation factors in four children with this disorder. Depression of factors IX, X, VII, V and II and marked disproportionate elevation of VIIIAHF, VIIIAGN VIIIVWF associated with a loss of the slow moving component of VIIIAGN on crossed Immunoelectrophoresis were consistently observed during the initial phase of the illness. In three of the four children who survived, the changes were fully reversible. Since laboratory evidence for DIC was lacking (normal fibrinogen, consistently negative protamine sulfate precipitation test) the findings seem to indicate that Reye's syndrome is associated with an acute, transient stimulation of synthesis of normal and abnormal factor VIII as well as decreased synthesis or synthesis of functionally abnormal clotting factors by the liver.
Factor VIII and Ristocetin-Induced Aggregation in sickle Cell Disease
Authors: Mackie I.J. , Bull H. , walford D. , Brozovic M.
Published: May 2019

Haemostatic variables were studied in 23 patients with sickle cell disease (SCD) who were followed over a year. Investigations included measurements of factor VIIIC, VIIIAg, antithrombin III biological and antigen levels, fibrinogen and platelet aggregation to ADP, collagen, porcine AHG and ristocetin. All eight SS patients in the steady state had normal levels of F VIIC and high levels of F VIIIAg with marked discordance between the two values (mean VIIIc: VIIIAg ratio 0.3). By contrast, 15 patients with 15 disease or S thalassaemia tended to show less Barked discordance between levels of F VIIIC and F VIIIAg (mean VIIIc VIIIAg 0.7). Patients in the steady state showed normal platelet aggregation to ADP, collagen and porcine AHG, but ristocetin induced aggregation was absent or œarkedly reduced. Platelet aggregation to ristocetin is absent in most individuals of Afro-Carribean origin and the ristocetin co-factor activity may be difficult to assess in this ethnic group.

During crisis both factor VIIIc and factor VIIIAg were invariably high. Factor VIIIAg was studied using crossed Immunoelectrophoresis in 5 patients. In one patient two peaks were found and in the others the F VIIIAg had more anodal mobility than normal. These findings are suggestive of the presence of lower molecular weight fragments of factor VIIIAg and may account for the apparent very high levels as measured by Laurell rocket technique.
Antithrombin III Deficiency in a Japanese Family
Authors: Nakagawa M. , Kawamura T. , Tsuji H. , Okajima Y. , Urano S. , Kitani T. , Ijichi H.
Published: May 2019

Antithrombin III has been reported to be decreased in the cases of several thrombotic disorders and the decreased Antithrombin III is known to induce the hypercoagulable state. This study was started from the 28 year-old male patient who developed the superior sagital sinus thrombosis after appendectomy and it was followed by deep vein thrombosis of extremities. Antithrombin III level was 19 mg/dl and activity was 68% by the progressive antithrombin assay and other laboratory examinations were within normal range excepts for the elevated serum lipids. Antithrombin III was assayed for his family members in three consecutive generations by single radial immunodifusion method, coagulation assay, and chromogenic assay. Four out of eight members were confirmed to have low Antithrombin III level and activity ranging from 59%-68% of normal values, although the two of this four members had no history of thrombosis. Mother of this propositus is deceased, but it was suspected of having the defect of Antithrombin III. History of peptic ulcers were found in all members of this family. The inheritance pattern of Antithrombin III deficiency was characteristic of an autosomal dominant disorder.
Prostacyclin can Replace Heparin for Haemodialysis in Dogs
Authors: Woods H.F. , Weston M.J. , Bunting S. , Moncada S. , Vane J.R.
Published: May 2019

Prostacyclin, produced by vascular endothelium, is the most potent inhibitor of platelet aggregation known to man. To assess its effects on platelets during dialysis ten greyhounds were dialysed for 90 minutes with cuprophan coils. At the end of dialysis arterial platelet counts (% of initial value) were significantly higher in the five dogs in which prostacyclin had been infused (115.7 ± 8.6) than in the animals in which heparin alone had been used (77.8 ± 16.8, < 0.05). Prostacyclin reduced the extraction of platelets by the dialyser. The screen filtration pressure, a measurement of platelet aggregates in blood leaving the dialyser, remained unelevated in the prostacyclin treated animals (80 ± 11.3 mm Hg) but rose significantly in those infused with heparin alone (249 ± 57 mm Hg, < 0.02). In another five dogs infused with prostacyclin but no heparin, dialysis did not reduce platelet count, elevate the screen filtration pressure or alter the overall clotting tests.

Thus prostacyclin enables haemodialysis to be carried out without the undesired effects on platelets and haemostasis that are associated with the use of heparin.
The Use of Prostacyclin [PGI2) During Charcoal Haemoperfusion
Authors: Woods H.F. , Weston M.J. , Bunting S. , Moncada S. , Vane J.R.
Published: May 2019

Although charcoal haemoperfusion has been used in the treatment of hepatic and renal failure and of drug and other intoxications, its potential has been limited by thrombocytopenia and other effects on platelet function. To try to improve blood-compatibility of charcoal haemoperfusion we infused PGI2, the most potent platelet inhibitor known to man, in addition to heparin during charcoal haemoperfusion carried out for 120 minutes in healthy dogs. With PGI2; platelet counts fell toonly 86 ί 2.3% of initial compared to 17 ± 3.7% when heparin alone was used. PGI2 prevented any rise in screen filtration pressuring during charcoal haemoperfusion (a measure of platelet aggregate formation) whereas with heparin alone the screen filtration pressure quadrupled. During charcoal haemoperfusion with heparin only the plasma fibrinogen levels fell to 54 ± 4% of initial but with heparin plus PGI2 the plasma fibrinogen levels were maintained at 92 ± 3.8%. Furthermore the use of PGI2 prolonged the activity of heparin as measured by thrombin clotting time during charcoal haemoperfusion.

This dramatic improvement in biocompatibility during charcoal haemoperfusion when PGI2 is used may allow more satisfactory assessment of its use for detoxification.
The Influence of Pregnancy upon Blood Coagulation and Plasma Fibrinolytic Enzyme Function
Authors: Fletcher A.P. , Alkjaersig N.K. , Burstein R.
Published: May 2019

Fibrinogen catabolism has been studied in 27 patients from early pregnancy through delivery by plasma fibrinogen chromatography. This technique quantifies in plasma, the concentration of high molecular weight fibrino(ogen) complexes (HMWFC), reflecting the rate of fibrin formation in vivo, native fibrinogen and derivatives of fibrinogen smaller than the native molecule (Fragment X) resulting from the actions of the enzyme plasmin on fibrinogen; this reflects the rate of fibrinogenolysis. During the second gestational month plasma HMWFC concentration was elevated 2-3 fold over control values, increased with gestational age being 5 fold elevated at delivery. Fragment X was mildly elevated throughout pregnancy.

These results demonstrate that intravascular fibrin deposition, presumably involving the uteroplacental circulation is the earliest and most persistent alteration in blood coagulation function noted during pregnancy. Further, the significant increase in fibrin deposition occurring during pregnancy is accompanied by a significant increase in compensatory fibrinolysis.
Decreased α-Adrenergic Receptors in Newborn Platelets: Cause of Abnormal Response to Epinephrine?
Authors: Corby Donald G. , O’Barr T.P.
Published: May 2019

Platelets of newborn infants (NBP) fail to aggregate or release adenosine diphosphate in response to epinephrine (E). Because E-induced aggregation is an α-adrenergic event, we considered the possibility that NBP possess fewer E receptors than do those of adults. Therefore we compared the specific binding of the α-adrenergic antagonist, 3H-Dihydroergocryptine (DHE), in intact washed platelets prepared from paired samples of maternal and cord platelet rich plasma. NBP demonstrated normal kinetics of 3H -DHE binding and normal affinity for 3H-DHE. Scatchard analysis of 3H-DHE binding indicated a single class of binding sites that exhibited a high affinity for the radioligand (Kd = 7.5nM). Maternal platelets (MP) were found to bind approximately 2-fold more DHE than NBP (3.70 + 0.28 vs. 1.7 fmol/107 platelets) at saturation. This corresponds to 223 ± 17 vs. 105 ± II binding sites per platelet (p < 0.001). Repeat washing of NBP did not yield increased DHE binding suggesting the binding sites had not previously been masked by elevated circulating levels of E and/or nor-E in venous cord blood. When control platelets were incubated with concentrations of 3H-DHE that half-saturated the α-adrenergic receptors, diminution of platelet function comparable to that seen in HBP was observed. Since NBP and MP are similar size, it appears that a deficiency of α-adrenergic receptors may account for the diminished response of NBP to epinephrine.
Fibrinogen Seattle: A Heritable Disfibrinogen with an Isolated Impairment in Fibrinopeptide B Release
Authors: Branson H.E. , Theodor I. , Baumgartner R. , Schmer G. , Pirkle H.
Published: May 2019

The slow clotting, asymptomatic, fibrinogen Seattle first described in a patient with tetralogy of Fallot has been traced through four generations as an autosomal dominant with 70% penetrance. The dysfibrinogen was indistinguishable from normal fibrinogen by radial immunodiffusion and Immunoelectrophoresis. Transamidation and plasmin digestion studies gave no evidence of abnormality. Thrombin and botroxobin monomer reprolymerization followed turbidometrically (A350) disclosed a shorter lag phase, a more acute maximal slope, and a lower maximal turbidity. Electron microscopy revealed that thrombin incubated fibrinogen Seattle more readily formed clots with aperiodic fibers than comparable controls. By amino acid analysis the final yield of fibrinopeptide B was slightly more than half normal (54% at 2 hours with 2.5 NIH units/ml). The dysfibrin manifested evidence of retent ion of f ibrinopept ide B as equal quantities of chains with Bβ and β charge mobility were demonstrated on low pH (2.7) Polyacrylamide electrophoresis in 2M urea. A distinction is made between fibrinogen Seattle and fibrinogen Detroit both with disordered fibrinopeptide B release on the basis of the patterns of fibrinopeptide release, immunoelectrophoretic mobility, botroxobin monomer aggregation, Chronometric fibrinogen concentration, and clinical symptomatology. No other dysfibrinogens have been shown to manifest disordered release of fibrinopeptide B alone.
The Pathogenetic Role of Disseminated Intravascular Microcoagulation (DIMC) in Progress of Ischemic Heart Disease (IHD)
Authors: Furkalo N.K. , Bolshakova R.M. , Dukhina M.A.
Published: May 2019

Hemostatic parameters (including the contact activated phase, platelet functional state, anticoagulational activity, detection of soluble fibrin and fibrinogen (fibrin degradation products) as well as blood viscosity and the character of microcirculation disturbances evaluated by biomicroscopy, microphotographing and filming of bulbar conjunctival vessels, were studied in 394 patients with chronic IHD. Microthrombosis parallel with generalized sludge were discovered within the microcirculatory bed. Analysis of hemostatic rheologic and microcirculatory data has revealed features of chronic, moderately pronounced DIMC, which did not result in marked consumption of coagulational factors. At equal severity grades of IHD, myocardial infarction occured more often in those patients, who bore microthrombosis, microhemorrhage and perivascular edema in the microcirculation system. Hence the detection of DIMC features may become an early diagnostic criterion of IHD severity and its possible complications.
Platelet Hypersensitivity in Acute Myocardial Infarction (M.I.) Patients (PTS)
Authors: Corteliarof M. , Fassio G. , Boschetti C. , Polli E.E.
Published: May 2019

A national multicentre controlled trial (ARIS) to determine the effect of sulfinpyrazone (S) (400 mg twice a day) on m.i. recurrence and cardiac death prevention is now in its 3rd year. Admission of new pts terminates on June 30, 1979. Various platelet (pi) and coagulatory parameters are measured in a randomised series of 175 pts at trial entry (10-20 days after m.i. episode) and at various times during treatment with S or placebo. This has provided the opportunity of comparing (factorial ANOVA analysis for unbalanced groups) basal results in m.i. pts and in 45 normal matching controls: no difference in pl count, pl retention and PTT; m.i. pts had significantly shorter bleeding time (146. 99±80,47 vs 264.11±112.98 sec), heparin thrombin clotting time (HTCT) (29. 97 + 16,47 vs 36,31±11.31), and significant increase of fibrinogenemia (339.85±179.94 vs 237. 85 + 35.77 mg%) and PF4 release (37.30±15.13 is 28.00 ± 12.3.4 U/ml). ADP (0.2, 0.4 and 0.8 μM) produced pi aggregation curves in m. i. pts showing significantly increased 1 st wave slope with 0.2 μM and maximal extent at 0. 2 and 0.4 μM: significantly higher frequency of 2nd wave at 0.4 and 0.8 μM, Collagen (1.25, 2.5 and 5 μg) produced pl aggregation curves with significantly reduced lag period at 2.5 μg, steeper slope and higher maximal aggregation at 1.25 and 5 μg. The only important correlation (inverse) was between HTCT and PF4 release in the m.i. group.
Abnormal Platelet Function in Raynaud’s Phenomenon.
Authors: Zahavi J. , Hamilton W.A.P. , O’Reilly M.J.G. , Clark S.E. , Cotton L.T. , Kakkar V.V.
Published: May 2019

Platelet aggregation (PA) induced by adenosine diphosphate (ADP) 0.6 and 4μM, 1-ephinephrine 6μM, collagen lu/ml and thrombin 0.2u/ml has been measured in citrated platelet rich plasma of 38 patients (mean age 42.2) suffering from Raynaud’s Phenomenon. In 29 of them, plasma β-thromboglobulin (βTG), measured by radioimmunoassay was determined and the results were compared to age and sex matched healthy subjects and correlated to serum immunoglobulin (Ig) and plasma fibrinogen levels. Plasma βTG (mean 72.2ng/ml, ranged 20-209) was significantly elevated in the patients compared to the controls (p ≤ 0.005). The rate and extent of platelet aggregation however, though higher in the patients than controls were significant only to ADP 0.6μM (p ≤ 0.045) rate and p ≤ 0.002 extent) and 4μM (p ≤ 0.005 rate and p ≤ 0.004 extent). Serum IgM levels were also abnormally increased in 12, IGA in one and IgG in 4 patients. There was no correlation between the platelet studies are plasma fibrinogen or serum immunoglobulin suggesting that these tests are measuring various aspects of the disease state. These results indicate that in-vivo platelet activation and “release reaction” are enhanced in patients suffering from Raynaud’s Phenomenon presumably not only by abnormal plasma factors. Furthermore, they suggest that platelets may be involved in the pathogenesis of the disease and its complications.
Defective Active Glucose Transport in Platelets of Atherosclerotic Patients
Authors: Yardimci T.U.
Published: May 2019

The glucose transport system in normal human platelets is temperature dependent and is a saturable process obeying Michaelis Menten Kinetics. It is inhibited by DNP, KCN, NaN3 and by Oubain. Therefore this system uses cells’ metabolic energy and/or is coupled to Na+ gradient. The Km of this system is 3.44±0.91x10-5 M and V max is 2.03±0.13 nmole/109 pietelets/30". 3-0-methylglucose does not use the same transport system whereas 2-deoxyglucose inhibits the glucose transport in platelets. In the platelets of nondiabetic atherosclerotic patients showing hypercoagulability, the active transport of glucose is decreased. In most of the tases this system is temperature insensitive, and is insensitive to metabolic inhibitors. The importance ot this alteration in the pathogenesis of atherosclerosis is discussed.
The Assay of Factor VII in its Native and Activated Forms
Authors: Snape T.J. , Griffiths D.
Published: May 2019

The preparation and use for factor VII assay of an artificial human factor VII-deficient substrate plasma is reported. Results of factor VII assays carried out using this plasma as substrate were closely comparable with values obtained using congenitally deficient plasma.

Neither of these assay systems distinguishes between factor VII in its native and activated forms. The activation state of factor VII in plasma and in a therapeutic factor VII concentrate has been investigated using an amidolytic assay (Seligsohn et al, 1978, Blood, 52, 978) and also by a method involving conventional assay before and after incubation with heparin (Godal et al, 1974, Thromb. Res., 5, 773). The results suggest that factor VII in the concentrate is unactivated and is stable with respect to attempts to activate it by cold activation in the presence of contact activators.
The Interaction of Human Factor VII and Tissue Factor, and the Effect of DFP.
Authors: Østerud B. , Bjørklid E. , Brown S.F.
Published: May 2019

When partially purified F.VII (absolutely free of F.X) was incubated with tissue factor (T.F.) and Ca++, a product was formed that over time increased its potency to clot F.VIII d.p. (58 sec at 0 min and 32 sec C.T. at 5 min incubation time). The effects of monospecific antibodies to F.VII and the apo-protein of T.F. were tested on these products. Thus, when anti-F.VII was added to a 10 min incubation mixture of F.VII, T.F. and Ca++, the clotting time increased from 32 sec to 125 sec. However, adding anti-T.F. to an identical 10 min incubation mixture had no effect on this mixture’s ability to clot F.VIII d.p.. Anti-T.F. allowed to react with T.F. prior to incubation with F.VII and Ca++, abolished the formation of a F.X. activator.

DFP neutralized purified human F.VII and F.VII in human plasma. The inhibition was relatively slow. However, when F.VII had been preincubatcd with T.F. and Ca++, the neutralization of F.VII activity by 2 mM DFP occurred rapidly. We conclude that there is a time consuming reaction between F.VII and T.F. resulting in a product of F.VII that apparently no longer needs T.F for its activation of F.Y.
Some Effects of Factor VII on Rates of Thrombin Production and one Stage Pi’s
Authors: Baughman D. J. , Lytwyn A.
Published: May 2019

Attempts to automate a chromogenic one stage PT assay required hastening the onset of thrombin production without altering the rate of production. Assays were performed by combining 0.25ml chromogenic substrate S-2238 (2mM), 0.40ml tissue thromboplastin,0.050ml plasma, and 0.050ml diluted serum (40%v/v) in 1.35ml TRIS buffer (pH=8-5,I=0.15). Sera were prepared by using the supernatant of a) whole blood, b) recalcified plasma, c) recalc fied plasma and tissue thromboplastin, d)recalcified plasma and partial thromboplastin (cephalin and ellagic acid) and e) thrombin clotted plasma. The results indicated that S-2238, a potent thrombin inhibitor, delayed the onset and altered the rate of thrombin; production. Small amounts of thrombin shortened the initial generation of thrombin without altering its rate. Sera from a), c) and d) but not b) and e) resulted in immediate thrombin production at most plasma concentrations without affecting the rate, had no residual prothrombin, and had similar residual S-2238 activity not reduced by hirudin or At III-heparin. In particular, scrum b) had ~80% plasma Factor X which could be adsorbed without affecting thrombin production, had ~40% Factor V and ~300% Factor VII. These results suggest that when Factor VII activity is greater than ~7% there is an effect on the time but not on the rate of thrombin production via positive thrombin feedback scheme. Thus, the standard PT can measure the time of thrombin appearance, and/or the rate of thrombin production, and/or fibrin polymerization.
Characterization of Antithrombotic Effects of Antiplatelet Drugs and Anticoagulants in Experimental Thrombosis
Authors: Zimmermann R. , Zeltsch C. , Zieboll L. , Lange D. , Petri A.
Published: May 2019

Drugs with possible antithrombotic properties have to be investigated in models for inducing experimentalthrombosis. Some of the well known antiplatelet drugs and anticoagulants were studied in the venous (4 models, 370 experiments) and arterial system (2 models,200 experiments). Thrombi were produced by different endothelial lesions, by stasis and hypercoagulability or a combination of these thrombogenic factors. In the venous system heparin reduced the thrombus size in comparison with controls by 98% (significantly, p<0.05 = s), phenprocoumon 91% (s), acetylsalicylic acid (ASA) 30%, ASA and dipyridamole (DIPY) 69%, carbenicillin 54%. In the arterial system a minor effect could be seen: Carbenicillin 72 %(s), ASA and DIPY 36%, ASA 8%, heparin 52%, phenprocoumon 17 %. Dependent on the experimental conditions ASA may demonstrate venous thrombus size reductions by 0, 6 or 30% and heparin by 33,98,81,68 or 88%, respectively. Comment: A drug may demonstrate an effect in one model but not necessarily in another one. The antithrombotic efficacy of new substances has to be investigated in models characterized by their thrombogenic conditions and thrombus structure and compared with that of well known antithrombotic substances.
Platelet Function and Vascular Responsiveness in Spontaneously Hypertensive Rats (SHR)
Authors: Motomiya T. , Mashimo N. , Yamazaki H.
Published: May 2019

SHR has been known to develop arterial and venous thrombosis at advanced age. However cerebrovascular and cardiorespiratory disturbances induced by injection of ADP or arachidonic acid have been reported as being milder in SHR than in normal Wistar rats (NWR). In this study, utilizing 14 mature SHR (24-week of age) and 6 young SHR (6-week) and same number of age-matched NWR, platelet aggregation, 14C-serotonin release and adenine nucleotides content were measured. In addition ADP and ATP contents were determined in experimentally induced pulmonary thromboembolism in SHR and NWR. Vascular responsiveness to rabbit aorta contracting substance (RCS) and serotonin was also studied. ADP-induced platelet aggregation was significantly decreased in mature SHR as compared with NWR (P<0.) and showed a tendency to decrease in SHR at 6-week. Serotonin release was significantly decreased in SHR as compared with NWR at 24 and 6-week (P<0.01). Decrease in ADP content after induction of thrombosis was smaller in SHR than NWR (P<0.05). Aorta smooth muscle strip contraction Induced by RCS and serotonin was not different between SHR and NWR at 24 and 6 weeks of age. Decrease in platelet function in SHR even at 6-week of age when hypertension has not been established seems to represent an inherited defense mechanism against thrombus formation.