bluuebunny/crossref_metadata_2025 huggingface dataset:
Adenosine Diphosphate-Induced Aggregation of Human Platelets in Flow Through Tubes: III. Shear and Extrinsic Fibrinogen-Dependent Effects
Authors: Goldsmith H L , Frojmovic M M , Braovac Susan , McIntosh Fiona , Wong T
Published: July 2018

The effect of shear rate and fibrinogen concentration on adenosine diphosphate-induced aggregation of suspensions of washed human platelets in Poiseuille flow at 23°C was studied using a previously described double infusion technique and resistive particle counter size analysis (1). Using suspensions of multiple-centrifuged and -washed cells in Tyrodes-albumin [3 × 105 μl−1; (17)] with [fibrinogen] from 0 to 1.2μM, the, rate and extent of aggregation with 0.7 μM ADP in Tyrodes-albumin were measured over a range of mean transit times from 0.2 to 43 s, and at mean tube shear rates, Ḡ, = 41.9, 335 and 1,335 s−1. As measured by the decrease in singlet concentration, aggregation at 1.2 μM fibrinogen increased with increasing Ḡ up to 1,335 s1, in contrast to that previously reported in citratcd plasma, in which aggregation reached a maximum at Ḡ = 335 s−1. Without added fibrinogen, there was no aggregation at Ḡ = 41.9 s1; at Ḡ = 335 s1, there was significant aggregation but with an initial lag time, aggregation increasing further at Ḡ = 1,335 s−1. Without added fibrinogen, aggregation was abolished at all Ḡ upon incubation with the hexapeptide GRGDSP, but was almost unaffected by addition of an F(ab’)2 fragment of an antibody to human fibrinogen. Aggregation in the absence of added fibrinogen was also observed at 37°C. The activation of the multiple-washed platelets was tested using flow cytometry with the fluorescently labelled monoclonal antibodies FITC-PAC1 and FITC-9F9. It was shown that 57% of single cells in unactivated PRT expressed maximal GPIIb-IIIa fibrinogen receptors (MoAb PAC1) and 54% expressed pre-bound fibrinogen (MoAb 9F9), with further increases on ADP activation. However, incubation with GRGDSP and the F(ab’)2 fragment did not inhibit the prebound fibrinogen. Moreover, relatively unactivated cells (8% expressing receptor, 14% prebound fibrinogen), prepared from acidified cPRP by single centrifugation with 50 nM of the stable prostacyclin derivative, ZK 36 374, and resuspension in Tyrodes-albumin at 5 × 104 μl−1, aggregated with 2 and 5 μM ADP at Ḡ = 335 and 1,335 s−1 in the absence of added fibrinogen. We therefore postulate that a protein such as von Willebrand factor, secreted during platelet isolation or in flow at sufficiently high shear rates, may yield the observed shear-rate dependent aggregation without fibrinogen.
Antithrombotic Effects and Bleeding Time Prolongation with Synthetic Platelet GPIIb/llla Inhibitors in Animal Models of Platelet-Mediated Thrombosis
Authors: Collen Désiré , Lu Hua Rong , Stassen Jean-Marie , Vreys Ingrid , Yasuda Tsunehiro , Bunting Stuart , Gold Herman K
Published: July 2018

Cyclic Arg-Gly-Asp (RGD) containing synthetic peptides such as L-cysteine, N-(mercaptoacetyl)-D-tyrosyl-L-arginylglycyl-L-a-aspartyl-cyclic (1→5)-sulfide, 5-oxide (G4120) and acetyl-L-cysteinyl-L-asparaginyl-L-prolyl-L-arginyl-glycyl-L-α-aspartyl-[0-methyltyrosyl]-L-arginyl-L-cysteinamide, cyclic 1→9-sulfide (TP9201) bind with high affinity to the platelet GPIIb/IIIa receptor.

The relationship between antithrombotic effect, ex vivo platelet aggregation and bleeding time prolongation with both agents was studied in hamsters with a standardized femoral vein endothelial cell injury predisposing to platelet-rich mural thrombosis, and in dogs with a carotid arterial eversion graft inserted in the femoral artery. Intravenous administration of G4120 in hamsters inhibited in vivo thrombus formation with a 50% inhibitory bolus dose (ID50) of approximately 20 μg/kg, ex vivo ADP-induccd platelet aggregation with ID50 of 10 μg/kg, and bolus injection of 1 mg/kg prolonged the bleeding time from 38 ± 9 to 1,100 ± 330 s. Administration of TP9201 in hamsters inhibited in vivo thrombus formation with ID50 of 30 μg/kg, ex vivo platelet aggregation with an ID50 of 50 μg/kg and bolus injection of 1 mg/kg did not prolong the template bleeding time. In the dog eversion graft model, infusion of 100 μg/kg of G4120 over 60 min did not fully inhibit platelet-mediated thrombotic occlusion but was associated with inhibition of ADP-induccd ex vivo platelet aggregation and with prolongation of the template bleeding time from 1.3 ± 0.4 to 12 ± 2 min. Infusion of 300 μg/kg of TP9201 over 60 min completely prevented thrombotic occlusion, inhibited ex vivo platelet aggregation, but was not associated with prolongation of the template bleeding time.

TP9201, unlike G4120, inhibits in vivo platelet-mediated thrombus formation without associated prolongation of the template bleeding time.
Effect of Dried Ginger on Human Platelet Function
Authors: Lumb Andrew B
Published: July 2018

Eight healthy male volunteers took part in a randomised double blind study of the effects of 2 g dried ginger or placebo capsules on platelet function. Bleeding time, platelet count, thromboelastography and whole blood platelet aggregometry were performed before, 3 h, and 24 h after the capsules. There were no differences between ginger and placebo in any of the variables measured, It is concluded that the effect of ginger on thromboxane synthetase activity is dose dependent, or only occurs with fresh ginger, and that upto 2 g of dried ginger is unlikely to cause platelet dysfunction when used therapeutically.
Studies of Fibrinogen Metabolism in Healthy and Hypertensive Female Subjects with the Use of Autologous I-125-Fibrinogen
Authors: Takeda Y , Takeuchi T
Published: July 2018

10 healthy female subjects and 15 female patients with essential hypertension were studied with respect to fibrinogen metabolism in a steady state. Autologous I-125-labeled fibrinogen (I-125-fibrinogen) was used as a tracer. Comparison of the results showed that plasma volume, t½ of plasma I-125-fibrinogen and fractional catabolic rate (j3p) of plasma fibrinogen were not appreciably different in both groups of subjects, but plasma fibrinogen concentration, and catabolic flux of (synthesis rate) were significantly higher in hypertensive subjects, whereas extravascular fibrinogen and fractional transcapillary transfer rate (j1) of were significantly decreased in the patients. Further analyses of the data in both groups showed that there were significant correlations between diastolic blood pressure (P) and j1 with a regression equation of j1 = e−0.0285P, between 1 and between and j1 with a regression equation of 1. These results indicate that the observed decrease of j1 triggered the transition from normal to pathological steady state and is responsible for the altered metabolism and distribution of fibrinogen in hypertensive subjects.
Platelet Aggregation and Cyclic Nucleotide Phosphodiesterase Activity in Arteriosclerotic Patients
Authors: Yamazak Hiroh , Motomiya Takeshi , Mashimo Nobuaki , Asano Tomiko , Hidaka Hiroyoshi
Published: July 2018

Although roles of cyclic AMP and cyclic GMP in platelets are thought to be important on platelet aggregation, little information on their phosphodiesterase (PDE) is available. Cyclic AMP and cyclic GMP hydrolytic activities of platelets (cAMPPDE and cGMPPDE in platelets) and platelet aggregation by ADP and adrenaline were measured in 22 healthy volunteers, 26 arteriosclerotic patients and other 20 miscellaneous patients excluding vascular diseases. Activities of cAMPPDE and cGMPPDE of platelets were 2.37±0.52, 7.23 ± 1.84 in the healthy, 2.50±0.85, 7.53±2.60 in the arteriosclerotics and 2.38± 1.02, 6.98±2.59 pmol/min/107 platelets in the miscellaneous patients, respectively. No significant difference was observed among these three groups. Platelet aggregabilities also showed no significant difference. However, there was a significant inverse correlation between the aggregability by 1 μg/ml of adrenaline and the PDE activities only in the arteriosclerotic patients. The correlation coefficients were −0.61 between the primary aggregation and cAMPPDE,−0.65 between the primary aggregation and cGMPPDE, −0.58 between the 5 min aggregation and cAMPPDE and −0.76 between the 5 min aggregation and cGMPPDE. The inverse correlation between platelet aggregation and cyclic nucleotide metabolism in circulating platelets of the arteriosclerotic patients may suggest that interaction of platelets with arteriosclerotic vessel walls would produce a certain change in platelets.
The Subcellular Distribution of [3H]-Colchicine-Binding Activity and Tubulin in Pig Blood Platelets
Authors: Castle Alan G , Crawford Neville
Published: July 2018

The subcellular distribution of the [3H]-colchidne-binding protein, believed to be tubulin, the subunit protein of microtubules, has been investigated in mammalian blood platelets. Studies on a soluble extract from pig platelets and two particulate fractions (viz. membrane-rich and granule-rich fractions) have shown that about 98% of the colchicine-binding activity in a platelet homogenate is located in the soluble phase. This result is in agreement with poly-acrylamide gel electrophoresis experiments which show that the soluble fraction contains a substantial amount of 55,000 MW tubulin, whereas the membrane and granule-rich fractions contain very little of this component. The [3H]-colchicine-binding activity of the platelet soluble phase is largely precipitated by 40-50% ammonium sulphate and also by vinblastine sulphate in millimolar concentrations. Moreover the colchicine-binding protein in the platelet soluble fraction has a sedimentation coefficient of 5.9 S, is eluted in the void volume of a Sephadex G-100 column, and binds to DEAE-Sephadex at low ionic strength and is eluted from this ion-exchanger at an ionic strength of 0.47 M-KC1. In addition, most of the col-chi cine-binding activity of the platelet soluble phase is associated with protein which will undergo temperature-dependent polymerization in vitro and which has a molecular weight on SDS-polyacrylamide gels of 55,000. All these experimental findings suggest that the col-chi cine-binding activity of pig platelet homogenates is due to the presence of the microtubule protein, tubulin, which is largely found in the soluble compartment of the cells.
Specific Inhibition of Platelet Agglutination and Aggregation by Aromatic Amidino Compounds
Authors: Geratz J D , Tidwell R R , Brinkhous K M , Mohammad S F , Dann O , Loewe H
Published: July 2018

A series of aromatic amidino compounds were investigated for their inhibitory effect on platelet agglutination and platelet aggregation. Agglutination of fresh or fixed platelets was produced by bovine plasma or by human plasma in combination with ristocetin, while aggregation of fresh platelets was induced by ADP, thrombin or collagen. Highly effective inhibitors were found for both types of platelet clumping, but there was no parallelism between the inhibitory activities in the two test systems. 5-(5-amidino-2-benzimidazolyl)-2-(4-hydroxyben-zene)benzimidazole suppressed agglutination exclusively. Pentamidine, on the other hand, strongly blocked the aggregation reactions, but did not interfere with agglutination, even at high concentrations. Compounds which inhibited aggregation also prevented the liberation of serotonin from the platelets.
Automation of Two-Stage Factor VIII Assay
Authors: Stirling Yvonne , Howarth D J , Vickers Marguerite , North W R S , Meade T W
Published: July 2018

Two automated methods for two-stage factor VIII assays have been compared with one another, and evaluated in practice. The Depex method records the clotting time when an electric circuit is completed by the formation of a fibrin thread across a hook-type electrode; the Electra method is based on an optical density technique of clot detection. The two methods gave comparable results for measured levels of factor VIII when haemophilic or “normal” plasmas were assayed. Results from the two methods in practice also suggest that both are valid at low and “normal” factor VIII levels. The Electra method is also probably suitable for assays of concentrates; however, the Depex method appears to give falsely high values in these circumstances, and experimental findings suggest that the reason may be that increased viscosity due to the high fibrinogen levels in factor VIII concentrates causes premature closure of the circuit between the two ends of the Depex electrode. The main advantage of the Depex method is that, provided 3 or 4 machines are available, a given number of assays can be completed more quickly than on Electra. The main advantages of Electra are that it is probably subject to less laboratory error than Depex, and that it is suitable for assaying concentrates as well as haemophilic and “normal” plasmas.
Determination of Soluble Fibrin Monomer Complexes by Adsorption on Immobilized Fibrinogen
Authors: Stemberger Axel W , Hӧrmann Helmut
Published: July 2018

Determination of fibrin in plasma, also containing fibrinogen, is possible by selective adsorption on affinity columns prepared from highly purified fibrinogen. Desorption is complete using 1.0 M potassium bromide, pH 5.3. In addition, the cold-insoluble globulin is desorbed. Therefore, the eluate is checked on microtiter plates by the staphylococcal clumping test which selectively detects fibrin or fibrinogen. The procedure works well with simple laboratory equipment. The diagnosis of fibrinogen-fibrin complexes is demonstrated in some pathological plasmas.
Inhibition of Urokinase by Complex Formation with Human Antithrombin III in Absence and Presence of Heparin
Authors: Clemmensen Inge
Published: July 2018

Human antithrombin III was purified from fresh human plasma by affinity chromatography on heparin-Sepharose®, affinity chromatography on concanavalin A Sepharose®, gel filtration on Ultrogel® AcA 34, ion exchange chromatography on DEAE A-50 Sephadex® and preparative agarose gel electrophoresis. The hydrolytic activity of urokinase (plasminogen activator from urine) on acetyl-glycyl-L-lysine methyl ester acetate (Ac-gly-lys-OMe Ac) was inhibited by antithrombin III in a slow time-dependent manner. Heparin accelerated the reaction between activator and inhibitor. Inhibition of catalytic activity was associated with the formation of an 1:1 molar complex between activator and inhibitor as revealed by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The complex was also demonstrated by crossed Immunoelectrophoresis against anti-antithrombin III.
Quantitative Estimation of Coagulation Factors in Liver Disease. The Diagnostic and Prognostic Value of Factor XIII, Factor V and Plasminogen
Authors: Biland L , Duckert F , Prisender S , Nyman D
Published: July 2018

20 coagulation parameters were investigated in 144 patients with different liver diseases. The groups of acute hepatitis, chronic active hepatitis and liver cirrhosis were compared and the prognostic value of the coagulation analyses investigated. It is clear that the determination of the factor V activity is a good and easy test for detection of actual liver function. Repeated controls over several weeks revealed with a statistical significance (p <0.0005) that all patients with a factor XIII below 35& and a plasminogen below 19& will die in liver coma, if they have not died beforehand from acute gastrointestinal haemorrhage, acute infection or cardiac arrest.

Plasminogen is also lower in the group of non-survivors but the values of the two groups are overlapping and of no prognostic help in a single case. The possible causes of the diminution of factor XIII activity are discussed.
The Role of Prostaglandins in the ADP-Induced Aggregation of Rabbit Platelets Shown by the Use of 15-Hydroxyprostaglandin Dehydrogenase
Authors: Wallis Robert B
Published: July 2018

The initial shape change and subsequent aggregation of platelets in citrated rabbit platelet-rich plasma caused by ADP in vitro was inhibited by 15-hydroxyprostaglandin dehydrogenase. This inhibition was NAD-dependent and was also seen when shape change and aggregation were initiated by sodium arachidonate or by collagen. The aggregation of gel-filtered rabbit platelets by thrombin was not, however, affected by removal of 15-hydroxyprostaglandins.

Indomethacin was found to inhibit ADP-induced aggregation but at a concentration (250 μM) much higher than that required to inhibit collagen-induced aggregation. Moreover the platelet release reaction had not taken place 3 min after ADP stimulation. The direct role 15-hydroxyprostaglandin production in ADP-induced aggregation of rabbit platelets is proposed. The involvement of 15-hydroxyprostaglandins in platelet aggregation caused by other inducers is also discussed.
The Inhibitory Effect of Aspirin on Human Endothelial Cells
Authors: Nordoy Arne , Svensson Birgit , Schroeder Connie , Hoak John C
Published: July 2018

Human endothelial cell monolayers prepared from umbilical veins have been incubated with aspirin (1–2 mM) dissolved in Hepes modified solution and in platelet-rich plasma. They have also been incubated with plasma prepared from subjects before and after intake of aspirin giving a mean plasma concentration of 0.5 mM. The effects of the endothelial cells on ADP and collagen-induced platelet aggregation and malondialdehyde production in platelet-rich plasma have been tested. The endothelial cells had a spontaneous inhibitory effect on all three parameters. This effect was abolished when the cells were incubated with aspirin dissolved in MHS for 20 min and the increase in effect observed when platelet-rich plasma was incubated with endothelial cells for a period of 30 min was similarly inhibited when aspirin was dissolved in plasma or when plasma prepared from subjects who had taken aspirin were used. Aspirin had no inhibitory effect on prostacyclin (PGI2) with regard to the effect of PGI2 on platelets. On the contrary, the two compounds had an additive inhibitory effect on platelet aggregation induced by ADP and collagen. These findings should be considered with regard to the use of aspirin as an antithrombotic agent.
The Platelet Dense Tubular System: its Relationship to Prostaglandin Synthesis and Calcium Flux
Authors: Gerrard Jonathan M , White James G , Peterson Douglas A
Published: July 2018

The platelet dense tubular system, an internal smooth endoplasmic reticulum membrane system occupies a pivotal position in the initiation and modulation of platelet activation. The best available evidence suggests platelet prostaglandin and thromboxane synthesis and an internal calcium store critical to platelet activation are both found in the platelet dense tubular system. Studies of the structural, physiologic and chemical properties of thromboxane A2 support the concept that this product of platelet prostaglandin synthesis acts to carry the calcium from the dense tubular system into the cytoplasm where the calcium is released to initiate contraction of the platelet contractile proteins. Evaluation of platelet phospholipase A2 suggests that calcium may be critical to activation of this enzyme. Two of the steps occurring during platelet activation which appear to require calcium 1) phospholipase A2 activation and 2) internal contraction may be linked up in series with the thromboxane A2 acting as the critical carrier of calcium from one location to the other.
Studies on the Effect of Plasminogen Activator on the Interaction between α2-Macroglobulin and Plasmin
Authors: Toki Naotika , Takasugi Sumiyoshi , Sumi Hiroyuki , Yamura Takuso
Published: July 2018

Six different plasmins were prepared by incubating human plasminogen with various amounts of streptokinase or urokinase. It was confirmed that the six different plasmins possessed similar caseinolytic activities, and the inhibitory effects of α 1-antitrypsin on caseinolytic activities of the six different plasmins were all the same. On the other hand, interactions between the six different plasmins and α2-macroglobulin were complicated. Plasmins activated by cleavage of plasminogen were almost immediately or effectively inhibited by α2-macroglobulin. However, plasmin activated by complex formation of plasminogen with streptokinase was not so immediately or effectively inhibited by α2- macroglobulin. It was supposed that the difference between these two results on the interaction between plasmin and α2-macroglobulin might be due to the difference in molecular form of plasmin. In the present study, it was also confirmed that streptokinase or urokinase, in free form in the reaction mixture, interfered with the interaction between plasmin and α2-macroglobulin. The cause for such interference was discussed.
Fibrin Deposition and Removal in Disseminated Intravascular Coagulation Induced by Endotoxin and Saline Loading: Radioautographic Findings in Rats
Authors: Heyes H , Mohr W , Theiss W
Published: July 2018

In rats a single injection of endotoxin followed by an infusion of normal saline induced the generalized Shwartzman reaction. The presence of disseminated intravascular coagulation (DIC) was demonstrated by measuring plasma fibrinogen, platelet counts, schistocytes, plasma haemoglobin, fibrin(ogen) degradation products, and fibrin thrombi in the glomerular capillaries. 125I-fibrinogen was given after triggering DIC in order to examine the fibrinogen turnover in plasma and the kinetics of fibrin deposition and removal in kidney, liver, and spleen. 125I-fibrinogen turnover was found to be highly accelerated. Early deposition and removal were observed in the kidneys, while a later peak with a more delayed fall of radioactivity was noted in liver and spleen. On histological examination fibrin could be seen only in the glomerular capillaries and only in the early phase of DIC. In radioautographs radioactive material was localized in the glomerular capillaries, the Kupffer’ cells of the liver, and in the perifollicular macrophages of the spleen. Comparing the results obtained by scintillation counting to those obtained by light microscopy it can be assumed that radioactivity in kidneys is correlated to fibrin deposition in glomerular capillaries and to an accumulation of fibrin(ogen) degradation products in liver and spleen.
Natural Oestrogen in the Female Climacteric - Influence on Blood Coagulation and Fibrinolysis
Authors: Lagrelius Anders , Lunell Nils-Olov , Blombäck Margareta
Published: July 2018

The aim of the present study was to investigate the effect on blood coagulation and fibrinolysis of a natural oestrogen preparation, piperazine oestrone sulphate, prospectively in menopausal women. Scopolamine was given to the control group.

The women were investigated before and during treatment with regard to factors VIII, VII, X, V, fibrinopeptide A, antithrombin III, plasminogen, rapid antiplasmin and α1-antitrypsin. There was no significant change towards hypercoagulability or decreased fibrinolysis in any group. In the oestrogen group, however, a tendency towards an increased level of plasminogen and a decreased level of antiplasmin was demonstrated. In the scopolamine group there was an unexpected fall in factors X and V and also in plasminogen and α1,-antitrypsin. A low level of some blood coagulation factors in some of the women before treatment is somewhat astonishing; none of them had any history of excessive bleeding.
Shear Stress-Induced Changes in Platelet Reactivity
Authors: Yu Simmon K , Latour Jean-Gilles , Marchandise Baudouin , Bois Marc
Published: July 2018

We have investigated the effects on platelet function of a physiologic shear stress. The aggregation to thrombin and collagen, the release reaction [(14C) serotonin] and the procoagulant activity of washed platelet suspensions were assayed on samples undergoing laminar oscillatory flow for 20 minutes at 37° C in polyethylene tubes, and on paired samples kept at rest. The pulse rate was established at 72 cycles per minute and the shear stress at the wall estimated at 26.2 dynes/cm2. The platelet suspensions were prepared at 37°C from blood samples of 15 healthy volunteers and 15 patients with the diagnosis of coronary artery disease proven by angiography. Our results show enhancement of platelet aggregation in samples undergoing oscillatory flow. Furthermore, platelets from coronary patients gained additional procoagulant activity. However, no change was encountered in the rate and speed of the release reaction. It is concluded that exposure to a high shear stress within an oscillatory flow system enhances platelet reactivity; this reaction may take part in the production of platelet and coagulation changes seen in the atherosclerotic patient and after strenuous physical exercise.
Low Doses of Heparin and Heparin Dihydergot in Postoperative Thromboprophylaxis in Gynaecological Patients
Authors: Urlep-Šalinović Vera , Jelatancev Biserka , Gorišek Borut
Published: July 2018

A prospective study of postoperative thromboembolic prophylaxis involving 500 gynaecological patients was performed to compare the effectiveness and safety of low doses of heparin (LDH) and heparin-dihydergot (HDHE). In the LDH group 255 patients (51%) aged 26-84 were included, in the HDHE group there were 245 patients (49%) aged 34-86. Both groups were well matched with respect to risk factors, duration of surgery, type of operation, volume of blood transfused and duration of prophylaxis. Thromboembolism was detected in 48 patients (9.6%): 26 patients (10.2%) in the LDH group and 22 patients (9%) in the HDHE group. In the LDH group isotopic deep vein thrombosis (DVT) was found in 23 patients (9%), five of them had pulmonary embolism (PE), detected by lung perfusion scanning without clinical signs of PE, three patients developed clinical PE without detected DVT. In the HDHE group, 22 patients (9%) had isotopic DVT and five of them had PE detected by lung perfusion scanning. In the incidence of TE there was no statistically significant difference between the two groups (p >0.1). Within two months after surgery late clinically manifest TE confirmed by isotopic venography developed in five patients (1.9%) in the LDH group and in one patient (0.4%) in the HDHE group. Wound haematomas appeared in 8 patients (3.1%) in the LDH group and in 9 patients (3.7%) in the HDHE group. There were no ischemic complications (ergotism) in the HDHE group. The HDHE prophylaxis proved no more effective and was ten times more expensive than the usual prevention with LDH.
Factor Vila and other Haemostatic Variables following Bone Marrow Transplantation
Authors: Collins P , Roderick A , O’Brien D , Tuddenham E , O’Driscoll A , Chopra R , Goldstone A , Newland A
Published: July 2018

Hepatic venocclusive disease causes considerable morbidity and mortality following bone marrow transplantation. There are two hypotheses regarding the aetiology of this syndrome; firstly that changes in plasma coagulation factors and natural anticoagulants lead to a prothrombotic state and secondly that endothelial cell activation stimulates intravascular deposition of fibrin. We have investigated these mechanisms by measuring the changes in proteins C and S and factors VII and X in the post transplant period and by using the plasma concentration of factor Vila as an in vivo marker of potential endothelial cell tissue factor expression. Protein C fell in both allograft and autograft patients but more so in the allografts. Similar results were found for factors VII and X. These changes were predominantly due to hepatic dysfunction induced by the chemo-radiotherapy. Factor Vila levels were unchanged in both the allograft and autograft patients. We conclude that there is no convincing evidence for a procoagulant state following BMT as there are both anticoagulant and procoagulant changes. The absence of any changes in factor Vila levels suggests that tissue factor was not exposed to the general circulation following BMT but does not exclude focal expression at the sites of thrombosis.
Clinical Factors Associated with Progression to AIDS in the Italian Cohort of HIV-Positive Hemophiliacs
Authors: Schinaia N , Ghirardini A M G , Mazzucconi M G , Tagariello G , Morfini M , Chiarotti F ,   the G.I.C.C
Published: July 2018

This study updates estimates of the cumulative incidence of AIDS among Italian patients with congenital coagulation disorders (mostly hemophiliacs), and elucidates the role of age at seroconversion, type and amount of replacement therapy, and HBV co-infection in progression. Information was collected both retrospectively and prospectively on 767 HIV-1 positive patients enrolled in the on-going national registry of patients with congenital coagulation disorders. The seroconversion date was estimated as the median point of each patient’s seroconversion interval, under a Weibull distribution applied to the overall interval. The independence of factors associated to faster progression was assessed by multivariate analysis. The cumulative incidence of AIDS was estimated using the Kaplan-Meier survival analysis at 17.0% (95% Cl = 14.1-19.9%) over an 8-year period for Italian hemophiliacs. Patients with age greater than or equal to 35 years exhibited the highest cumulative incidence of AIDS over the same time period, 32.5% (95% Cl = 22.2-42.8%). Factor IX recipients (i.e. severe B hemophiliacs) had higher cumulative incidence of AIDS (23.3% vs 14.2%, p = 0.01) than factor VIII recipients (i.e. severe A hemophiliacs), as did severe A hemophiliacs on less-than-20,000 IU/yearly of plasma-derived clotting factor concentrates, as opposed to A hemophiliacs using an average of more than 20,000 IU (18.8% vs 10.9%, p = 0.02). No statistically significant difference in progression was observed between HBsAg-positive vs HBsAg-negative hemophiliacs (10.5% vs 16.4%, p = 0.10). Virological, immunological or both reasons can account for such findings, and should be investigated from the laboratory standpoint.
Severe Homozygous Protein C Deficiency: Identification of a Splice Site Missense Mutation (184, Q → H) in Exon 7 of the Protein C Gene
Authors: Soria J M , Brito D , Barceló J , Fontcuberta J , Botero L , Maldonado J , Estivill X , Sala N
Published: July 2018

Single strand conformation polymorphism (SSCP) analysis of exon 7 of the protein C gene has identified a novel splice site missense mutation (184, Q → H), in a newborn child with purpura fulminans and undetectable protein C levels. The mutation, seen in the homozygous state in the child and in the heterozygous state in her mother, was characterized and found to be a G to C nucleotide substitution at the -1 position of the donor splice site of intron 7 of the protein C gene, which changes histidine 184 for glutamine (184, Q → H). According to analysis of the normal and mutated sequences, this mutation should also abolish the function of the donor splice site of intron 7 of the protein C gene. Since such a mutation is compatible with the absence of gene product in plasma and since DNA sequencing of all protein C gene exons in this patient did not reveal any other mutation, we postulate that mutation 184, Q → H results in the absence of protein C gene product in plasma, which could be the cause of the severe phenotype observed in this patient.
Protein C Is Responsible for the Rapid Inactivation of Factor Va following Blood Clotting In Vitro
Authors: Jackson Denise E , Mitchell Christina A , Salem Hatem H
Published: July 2018

When whole blood is allowed to clot in vitro, factor V is rapidly activated to factor Va which is subsequently inactivated. We developed two monoclonal anti-protein C antibodies, one of which inhibits protein C activation and the other inhibits protein C activity. The addition of either antibody to blood before clotting in vitro significantly inhibited the inactivation of factor Va, confirming the essential role of protein C in mediating the rapid inactivation of factor Va.
Different Regulation of Plasminogen Activator Inhibitor 2 Gene Expression by Phorbol Ester and cAMP in Human Myeloid Leukemia Cell Line PL-21
Authors: Niiya Kenji , Taniguchi Taketoshi , Shinbo Masahiro , Ishikawa Tai-ichi , Tazawa Shigeki , Hayakawa Yumiko , Sakuragawa Nobuo
Published: July 2018

Previous studies have shown that protein kinase C (PKC) activators and dibutyryl cyclic AMP (Bt2cAMP) synergistically increase the antigen level of plasminogen activator inhibitor type-2 (PAI-2) in a human myeloid leukemia cell line PL-21. To clarify the mechanism, PAI-2 gene expression induced by phorbol myristate acetate (PMA), a PKC activator, and Bt2cAMP was investigated by Northern blot hybridization using a PAI-2 cDNA probe cloned from a human placental library. The level of PAI-2 mRNA was markedly increased in response to PMA and reached a maximum 5-9 h after stimulation. Nuclear run-on assay revealed an increase in PAI-2 gene transcription in PMA-treated cells. The induction was inhibited by inhibiting de novo protein synthesis with cycloheximide (CHX). cAMP also increased PAI-2 mRNA level in a dose-dependent manner. The increase began within 2 hours and, contrary to the case of PMA, the mRNA levels were maintained. Moreover, cAMP-induced increase in PAI-2 mRNA was not inhibited by CHX, rather enhanced. PMA and cAMP synergistically induced PAI-2 gene expression, which was completely inhibited by CHX. The cells pretreated with PMA for 24 h did not any more respond to stimulation with PMA but responded to cAMP and PAI-2 mRNA level was increased. The apparent half-life of constitutive level PAI-2 mRNA in PL-21 cells, determined by actinomycin-D-decay experiments, was approximately 2 h. Those induced by PMA and cAMP were approximately 5 h and 2 h, respectively. These data suggest that PAI-2 mRNA induced by PMA is relatively stable and the expression requires de novo protein synthesis, whereas cAMP increases PAI-2 mRNA level without affecting the stability and the induction does not require de novo protein synthesis. Judging from these data, PAI-2 gene expression appears to be differently regulated by the PKC and cAMP signalling pathways.
The Significant Enhancement of Fibrinolysis by Calcium Ion in a Cell Free System: The Shortening of Euglobulin Clot Lysis Time by Calcium Ion
Authors: Kojima Yumi , Urano Tetsumei , Kojima Kazuhisa , Serizawa Kiyohito , Takada Yumiko , Takada Akikazu
Published: July 2018

We investigated the roles of calcium ion (Ca2+) on euglobulin clot lysis time (ECLT) and found that the physiological concentration of Ca2+ significantly (4-5 times) shortened ECLT. The shortening was observed at the concentration of Ca2+ higher than 1.5-2.0 mM. Other divalent cations such as Mg2+, Zn2+ or Mn2+ didn’t change ECLT. Anti-tPA antibody or plasminogen activator inhibitor-1 prolonged ECLT in the absence of Ca2+, whereas they had no effect on ECLT shortened by Ca2+. Cl inactivator also had no effect. When barium absorbed plasma was employed, the shortening of ECLT by Ca2+ wasn’t observed, whereas it was recovered by the readdition of barium absorbed fraction. When factor X deficient plasma was employed, the shortening of ECLT by Ca2+ was also not observed. Thus, Ca2+ enhances fibrinolysis in a cell free system by a novel pathway in which the presence of factor X is prerequisite.
Neutrophil Derived Cathepsin G Induces Potentially Thrombogenic Changes in Human Endothelial Cells: a Scanning Electron Microscopy Study in Static and Dynamic Conditions
Authors: Kolpakov Valeri , D'Adamo Maria Cristina , Salvatore Lorena , Amore Concetta , Mironov Alexander , lacoviello Licia , Donati Maria Benedetta
Published: July 2018

Activated neutrophils may promote thrombus formation by releasing proteases which may activate platelets, impair the fibrinolytic balance and injure the endothelial monolayer.

We have investigated the morphological correlates of damage induced by activated neutrophils on the vascular wall, in particular the vascular injury induced by released cathepsin G in both static and dynamic conditions.

Human umbilical vein endothelial cells were studied both in a cell culture system and in a model of perfused umbilical veins. At scanning electron microscopy, progressive alterations of the cell monolayer resulted in cell contraction, disruption of the intercellular contacts, formation of gaps and cell detachment.

Contraction was associated with shape change of the endothelial cells, that appeared star-like, while the underlying extracellular matrix, a potentially thrombogenic surface, was exposed. Comparable cellular response was observed in an “in vivo” model of perfused rat arterial segment. Interestingly, cathepsin G was active at lower concentrations in perfused vessels than in culture systems. Restoration of blood flow in the arterial segment previously damaged by cathepsin G caused adhesion and spreading of platelets on the surface of the exposed extracellular matrix. The subsequent deposition of a fibrin network among adherent platelets, could be at least partially ascribed to the inhibition by cathepsin G of the vascular fibrinolytic potential.

This study supports the suggestion that the release of cathepsin G by activated neutrophils, f.i. during inflammation, may contribute to thrombus formation by inducing extensive vascular damage.
Bridge Anticoagulant, a New Entity?
Authors: Mulder E
Published: July 2018

After a critical analysis of the work of Nour Eldin and Wilkinson on the so-called Bridge anticoagulant, the author presents his own experimental work on this subject. It is concluded that one part of the evidence for a new inhibitor is not reproduceable, and that the other part depends on the presence of an inhibitor, anticephalin. This inhibitor has been known to exist for many years.
Über die Kontrolle der Wirkung fibrinolytischer Präparate
Authors: Fonio A
Published: July 2018

1. Wird ein fibrinolytisch wirkendes Präparat dem Zitratplasma vor der Rekalzifizierung zugesetzt, dann befindet sich das Präparat bei Eintritt der Gerinnung und Beginn der Retraktion (in statu nascendi) bereits in den lockeren Fibrinmaschen und es wird ihm beim weiteren Eindringen kein Widerstand entgegengesetzt, so daß die fibrinolytische Auflösung des Gerinnsels unbehindert erfolgen kann (Prüfung 1).

Wird im Gegensatz dazu das Präparat erst nach erfolgter Gerinnung und Retraktion zugeführt, dann setzen die fest retrahierten und einander eng anschließenden Fibrillenstränge seinem weiteren Eindringen Widerstand entgegen, so daß die totale fibrinolytische Auflösung des reträhierten Gerinnsels ausbleibt (Prüfung 2).

2. Es werden sechs als fibrinolytisch-wirkend angegebene Präparate — drei Aktivatoren, zwei Plasminpräparate und ein weiteres in vitro nicht fibrinolytisch wirkendes Präparat — nach besonderen Testmethoden am Zitratplasma als Untersuchungssubstrat geprüft. Prüfung 1 : Zusatz des Präparates vor der Rekalzi-fizierung, und Prüfung 2: Zusatz nach Eintritt der Gerinnung und Retraktion.

3. Eintragung der Befunde beider Methoden in je zwei Tabellen pro Präparat nach Prüfung 1 und 2 am Zitratplasma (Tabellen 1—14). Resultate: Prüfung 1: Drei Aktivatoren erwiesen sich als fibrinolytisch wirksam, ein Plasminpräparat desgleichen, zwei weitere Präparate wiesen keine Wirkung auf. Prüfung 2: Alle Präparate vermochten ausnahmslos keine totale Fibrinolysis des retrahierten Gerinnsels zu erzielen.

4. Die gleichen Prüfungen mit gleicher Methodik und Dosierung wurden am Vollblutzitrat bei drei Aktivatoren durchgeführt (Tabellen 15—17). Die Resultate entsprechen den am Zitratplasma erhaltenen. Vorteile der Methode: Geringere Blutmenge, Wegfall der Zentrifugierung. Nachteil: Leicht erschwerte Beurteilung der Befunde durch den Gehalt an Erythrozyten.

5. Übertragung der Befunde der Prüfung 1 und 2 auf die klinische Verwendung fibrinolytischer Präparate: Prüfung 1: Eine beginnende Thrombosierung mit einer Retraktion in statu nascendi, spontan erstmalig oder sekundär im Anschluß an bereits bestehenden Thrombus entstehend, wird durch ein wirksames fibrinolytisches Präparat günstig beeinflußt werden können. Prüfung 2: Ein bereits bestehender fest retrahierter Thrombus wird durch ein fibrinolytisches Präparat nicht zur Auflösung gebracht werden können. Bei wandständiger Haftung des Thrombus wird zudem nur eine frontale Angriffsfläche der fibrinolytischen Einwirkung ausgesetzt sein.
Substitution Therapy in Haemophilia B
Authors: Loeliger E. A , Hensen A , Mattern M. J , Alsbach E. E. J
Published: July 2018

Conclusions

1. Substitution therapy in haemophilia B without daily control of the Factor IX level in vivo by means of a reliable Factor IX assay cannot be adequate.

2. The level of the Factor IX activity which, in cases of haemophilia B, ensures safe haemostasis after major trauma or in major surgery is at least 25% of normal.

3. Assuming a normal t½ for Factor IX of 30 hours and a distribution pool of 7 l (normal adult), in cases of severe haemophilia the minimum amount of Factor IX to be rapidly transfused at the beginning of substitution (loading up of the pool up to 25%) is the amount present in 1.75 l of freshly drawn normal net plasma. The minimum daily dose necessary to maintain the 25% level is contained in 1 l. Due to the influence of body temperature, the required daily maintenance dose increases probably up to 2.4 l.

4. Since rapid infusion of more than 1 l net plasma is not tolerated and since a daily dose of 2.4 l net plasma can hardly be achieved by means of exchange transfusion, partially purified and concentrated human Factor IX must be available. The plasma product PPSB, prepared by the “Centre National de Transfusion Sanguine” in Paris, contains, according to in vivo assays, about 10 times the activity of normal plasma for comparable volumes. The t½ of the Factor IX from PPSB appeared to be the same as from fresh plasma. The disappearance rate of the concentrated Factor IX from serum (CSB), prepared by the same institute, has not been estimated experimentally. According to clinical experience, however, the activity of CSB seems to be of a similar magnitude as that of PPSB. Adequate substitution with PPSB alone would require as initial loading-up dose 175 ml and as daily maintenance dose 100—240 ml of a 10-fold concentrated product, to be given in a continuous drip-infusion.

5. In a case of mild haemophilia B (Factor IX ≅ 20%), transfusion of 0.8 l freshly drawn net plasma daily resulted in Factor IX levels of between 23 and 28% instead of the expected 35% of normal. This observation suggests that, for substitution therapy, mild cases of haemophilia B must be considered as rather severely affected.
Peptides Associated with Bovine Thrombin Preparations
Authors: Landaburu Ricardo H. , Harmison Charles R. , Seegers Walter H.
Published: July 2018

When bovine thrombin was prepared by one method the N-terminal amino acid was glutamic acid while with another method it was threonine. In urea solution these N-terminal amino acids were removed in association with peptides and the N-terminal amino acid of the main protein was leucine. Urea treated thrombin had the same specific activity as the original from which it was prepared, and also had the same carbohydrate content. It was, however, less soluble in water and had a higher viscosity. The sedimentation constant was concentration dependent. Before treatment with urea the rate of change of the sedimentation constant with concentration was positive. After urea treatment it was negative. Extrapolation to zero concentration gave the same value for thrombin as for urea treated thrombin. The peptides removed from thrombin function to alter the properties of the protein. They are attached by bonds that are broken in urea solution. Very likely the original prothrombin molecule is, in part at least, composed of sub-units consisting of polypeptide chains held together by bonds broken by reagents such as sodium citrate. In addition to alanine, bovine prothrombin has arginine as N-terminal amino acid, but the latter was uncovered only in urea solution.
Methods for the Concentration of Bovine Ac-Globulin (Factor V)
Authors: Landaburu Ricardo H. , Seegers Walter H.
Published: July 2018

An attempt was made to obtain Ac-globulin from bovine plasma. The concentrates contain mostly protein, and phosphorus is also present. The stability characteristics vary from one preparation to another, but in general there was no loss before 1 month in a deep freeze or before 1 week in an icebox, or before 5 hours at room temperature. Reducing agents destroy the activity rapidly. S-acetylmercaptosuccinic anhydride is an effective stabilizing agent. Greatest stability was at pH 6.0.

In the purification bovine plasma is adsorbed with barium carbonate and diluted 6-fold with water. Protein is removed at pH 6.0 and the Ac-globulin is precipitated at pH 5.0. Rivanol and alcohol fractionation is followed by chromatography on Amberlite IRC-50 or DEAE-cellulose. The final product is obtained by isoelectric precipitation.
Platelet Distribution and Survival in Plasma Clots and Thrombi
Authors: Wolf Peter
Published: July 2018

Viscous metamorphosis of platelets in native and sequestrene plasma, before and after thrombin and plasmin action, has been studied. A method for the examination of platelet distribution in plasma clots and tissue paraffin sections is described.

It was found that not all platelets undergo viscous metamorphosis in plasma clots or in intravascular thrombi. Platelets before and after viscous metamorphosis are not digested by plasmin. After plasmin action intact platelets can still undergo viscous metamorphosis and the fibrils which are then produced are not made of fibrin.

Thrombin in the absence of calcium ions will not cause platelets to undergo viscous metamorphosis.

Total blockage of cardiac blood vessels by platelet masses in cases of cardiac infarction is demonstrated. The significance of these findings in relation to blood sludging, and future lines of treatment are discussed.
Beitrag zur fibrinolytischen Therapie mit Streptokinase und Fibrinolysin®
Authors: Fischbacher Walter
Published: July 2018

Nach einem Vergleich der direkt wirkenden Fibrinolytika (Streptokinase, Fibrinolysin, Urokinase) mit den indirekt wirkenden (Nikotinsäure und -derivate, Pyrexal u. a.) wird die Wirksamkeit verschiedener Streptokinase- und Fibrinolysinpräparate miteinander verglichen. Es werden Methoden im Thromb-elastographen beschrieben, welche sich gut eignen für die Vorkontrolle (und Dosierung) sowie für die Therapiekontrolle (SK-Toleranz; Plasmintoleranz; ε-ACS-Toleranz; Mischversuche zum Nachweis der SK-Aktivität). Die Resultate nach innerem Fibrinolysetyp und nach äußerem (therapeutischem) werden einander gegenübergestellt. Nach Angaben für die Dosierung und Applikationsart folgen vier ausgewählte klinisch-therapeutische Beispiele mit zum Teil sehr schönen Erfolgen. Die Indikationen, Kontraindikationen sowie die von uns gesehenen Komplikationen werden erläutert. Die Kombination mit Antikoagulantien ist obligat. Auf Grund von In-vitro-Versuchen nach äußerem Fibrinolysetyp scheint es, daß das „Fibrinolysin”, welches als Mischung von Streptokinase und Profibrinolysin aufzufassen ist, der Streptokinase alleine etwas überlegen ist.
A Critical Review of the Evidence for a Continuous Hemostasis in vivo
Authors: Hjort Peter F , Hasselback Richard
Published: July 2018

I. Conclusions

Injury to vessels leads to the formation of platelet plugs and fibrin clots. For nearly a century it has been known that these mechanisms stop bleeding and may produce thrombi. They may be triggered by trivial trauma (180), and hemostasis possibly occurs in one or more vessels at all times in the normal body. The blood is capable of removing at least the fibrin part of such deposits; however, some of them may progress into arteriosclerotic lesions (59). The accumulated evidence, therefore, supports the concept of a hemostatic balance on the local level, triggered by an abnormal local factor.

In this review, however, we are concerned with a general and continuous hemostasis which takes place on the normal, uninjured endothelium. Direct examinations have suggested that hemostatic products are present on normal endothelium, but these investigations cannot be accepted as conclusive. Much of the indirect evidence is weak or controversial. However, the purpura and increased capillary fragility in platelet disorders strongly suggest that platelets are important for vascular integrity, and animal experiments suggest that both platelets and coagulation influence vascular permeability. At present, therefore, the theory of continuous hemostasis can neither be accepted nor rejected; further evidence must be collected.