bluuebunny/crossref_metadata_2025 huggingface dataset:
Benefit/Risk Profile of Combined Antiplatelet Therapy with Ticlopidine and Aspirin
Authors: De Caterina Raffaele , Sicari Rosa , Bernini Walter , Lazzerini Guido , Strata Giuliana Buti , Giannessi Daniela
Published: July 2018

Ticlopidine (T) and aspirin (ASA) are two antiplatelet drugs both capable of prolonging bleeding time (BT), with a different mechanism of action. A synergism in BT prolongation has been reported and is currently considered an argument for not recommending their combination. However, a profound suppression of platelet function might be a desirable counterpart of a marked prolongation of BT, with a possible use in selected clinical situations. We therefore studied ex vivo platelet function (aggregation by ADP 0.5-1-2.5 μM; adrenaline 0.75-2.5 μM; collagen 1.5-150 μg/ml; arachidonic acid 1 mM; PAF 1 μM; adrenaline 0.17 μM + ADP 0.62 μM; serum thromboxane ([TX]B2 generation) and BT (Mielke) in 6 patients with stable coronary artery disease receiving such combination. Patients underwent sequential laboratory evaluations at baseline, after 7 days of T 250 mg b.i.d., before and after the intravenous administration of ASA 500 mg, respectively, and, finally, after a minimum of 7 days of sole ASA oral administration (50 mg/day). The experimental design, therefore, allowed a comparison of T and ASA effects (2nd and 4th evaluation), and an assessment of the combination effect (3rd evaluation). Platelet aggregation in response to all doses of ADP was depressed more by T than by ASA. Conversely, responses to adrenaline, and arachidonate were affected more by ASA than by T. For all other agents, differences were not significant. T + ASA combination was more effective (p <0.05) than either treatment alone in depressing responses to high-dose collagen (% over control, mean ± SEM: T: 95 ± 3; ASA: 96 ± 5; T + ASA: 89 ± 4). Serum TXB2 (basal, ng/ml: 380 ± 54) did not change with T (372 ± 36), dropped to <1 ng/ml on ASA injection and slightly re-increased to 9.1 ± 3.1 ng/ml on oral low-dose ASA. BT (basal 7.4 ± 0.6 min) was affected similarly by T (9.2 ± 0.8) or ASA (9.7 ± 0.9) alone, but increased to 15.0 ± 0.7 min on combination treatment (106% increase over control). Thus, the strong synergism in BT prolongation by ASA-T combination has a counterpart in the inhibition of platelet function in response to strong stimuli such as high-dose collagen, not otherwise affected significantly by single-drug treatment. This effect is a possible rationale for the clinical evaluation of T + ASA combination.
A Photometric Assay for Blood Coagulation Factor XIII
Authors: Fickenscher Karl , Aab Angela , Stüber Werner
Published: July 2018

An assay for a direct photometric determination of F XIII in untreated and undiluted plasma was developed. In a one-step procedure F XIII is activated by thrombin and Ca2+ and crosslinks glycine-ethylester to a specific glutamine containing peptide substrate. The released ammonia is incorporated into α-keto-glutarate by glutamate dehydrogenase, and the NADH consumption of this reaction is measured photometrically at 340 nm. NADH-consumption is directly proportional to the F XIII activity. Fibrin polymerization and the corresponding turbidity is avoided by the use of a fibrin aggregation inhibitor.

The procedure is rapid and simple and enables to measure within the range of 0 to 150% F XIII. It can be performed with automated analyzers as well as with common photometric equipment.

The normal range of F XIII activity in 167 healthy donors was determined to be 70 to 140%.
The Effect of Agonists and Antagonists of Platelet Aggregation on von Willebrand Factor-Mediated Platelet Agglutination
Authors: McPherson Jean , Zucker Marjorie B , Mauss Evelyn A , Brownlea Sandra
Published: July 2018

Ristocetin-induced platelet agglutination (RIPA) in EDTA-treated citrated platelet-rich plasma was reduced to 49 ± 11% by 1.25 ΜM ADP, 41 ± 14% by 1 ΜM A 23187, and 26 ± 7% by 0.1 Μg/ml platelet activating factor (PAF). The effect of 5-110 ΜM epinephrine was not dose-dependent, but varied between donors, with RIPA from 56-100% of the control. The inhibitory effects of these agonists were not altered by prior treatment of platelets with aspirin. Prior addition of 200 ΜM ATP (an ADP receptor antagonist acting at both high and low affinity ADP receptors) prevented the inhibitory action of ADP but not that of A 23187 or PAF, suggesting that the inhibitory actions of the latter are not mediated by released ADP. As 700 ΜM 8-bromoadenosine 5-diphosphate (an ADP receptor antagonist acting mainly at the high affinity receptor) did not prevent ADP-induced inhibition of RIPA, interaction of ADP with the low affinity receptor is presumably responsible for its inhibitory action. As A 23187, but not phorbol myristate acetate (0.1 ΜM) inhibited RIPA, an increase in intracellular calcium ions rather than direct stimulation of protein kinase C appears to mediate agonist-induced inhibition. Cytochalasin B (10.5-21 ΜM), dibucaine (0.5-1 mM), and prostaglandin E1 (25 nM), added before or after the agonist, prevented or reversed ADP-, A23187-, and PAF-induced inhibition of RIPA, suggesting that the state of the platelet cytoskeleton affects inhibition. N-ethylmaleimide (0.25-0.5 mM), an agent that can penetrate cell membranes and block sulphydryl groups, prevented or reversed ADP, A 23187- and PAF-induced inhibition of RIPA, but 0.5 mM dithionitrobisbenzoic acid, a non-penetrating sulphydryl blocker, had no effect. Diamide (0.1-0.5 mM), an agent that can crosslink cytoskeletal proteins by oxidation of sulphydryl groups, reduced RIPA. Thus an increase in intracellular calcium ions with resultant cytoskeletal changes and reorganisation of intracellular sulphydryl groups may mediate the inhibitory action of agonists on RIPA.
Thromboxane Responsiveness of Dog Platelets Is Inherited as an Autosomal Recessive Trait
Authors: Johnson Gerhard J , Leis Linda A , King Richard A
Published: July 2018

Most mongrel dogs have platelets that form thromboxane A2 (TXA2) from exogenous arachidonate, but they fail to aggregate or secrete in response to it. In contrast to these TXA2 insensitive (TXA2-) platelets, some dogs have TXA2 sensitive (TXA2+) platelets that aggregate and secrete when stirred with arachidonate. To evaluate the possible genetic basis for this difference, we carried out seven matings of mongrel dogs that yielded 48 viable offspring. Four matings of dogs with TXA2- platelets (presumed genotype TT) including 2 back-crosses, produced 32 pups with TXA2- (TT) platelets and 0 pups with TXA2+ platelets. A cross between a male with TXA2+ platelets (presumed genotype tt) and a female with TXA2+ (tt) platelets yielded 9 offspring with TXA2+ (tt) platelets and 0 with TXA2- platelets. Crossing a male presumed homozygous (TT) for TXA2- platelets with a female with TXA2+ (tt) platelets produced 2 pups with TXA2- (Tt) platelets and 0 pups with TXA2+ (tt) platelets. The same female with TXA2+ platelets crossed with a male presumed to be heterozygous (Tt) for TXA2- platelets yielded 2 pups with TXA2+ (tt) platelets and 3 pups with TXA2- (Tt) platelets. Segregation analysis of these data supports the hypothesis that the ability of dog platelets to aggregate and secrete in response to TXA2 is inherited as an autosomal recessive trait.
Upstream Thrombus Growth Impairs Downstream Thrombogenesis in Non-Anticoagulated Blood: Effect of Procoagulant Artery Subendothelium and Non-Procoagulant Collagen
Authors: Sakariassen Kjell S , Weiss Harvey J , Baumgartner Hans R
Published: July 2018

In the present experiments we have investigated the influence of wall shear rate and axial position on platelet and fibrin deposition which results when flowing human non-anticoagulated blood is exposed to either non-procoagulant fibrillar collagen (human type III) or procoagulant subendothelium (rabbit aorta). Platelet adhesion, thrombus volume and fibrin deposition were morphometrically evaluated at axial positions of 1 and 13 mm following perfusions for 5 min at shear rates of 100, 650 and 2,600 s-1.

An axially-dependent decrease of platelet adhesion (34-57%, p <0.01-0.05) and thrombus volume (57-80%, p <0.05) was observed on collagen at all shear rates. On subendothelium, an axially-dependent decrease was observed for platelet adhesion only at 100 s-1 (29% ; p <0.01) and for thrombus volume at shear rates of 650 s-1 and above (49-58%, p <0.01). Deposition of fibrin on subendothelium was axially decreased (16-42%, p <0.05) at all shear rates, while no significant axial differences were seen on collagen. However, substantially more fibrin was deposited on the subendothelium (p <0.05), and the upstream platelet adhesion and thrombus volume were lower than on collagen (p <0.05) at 100 s-1 and 650 s-1. The axially-dependent phenomena on the two surfaces are consistent with the concept of rapid-growing upstream thrombi which deplete the blood layer streaming adjacent to the surface of platelets, leading to decreased platelet deposition farther downstream. The observations suggest that deposition of fibrin is enhanced by subendothelial tissue factor, and that upstream depletion of clotting factors may lower the downstream deposition of fibrin, analogous to the depletion of platelets.
Low Molecular Weight Heparin versus Warfarin in the Prevention of Recurrences after Deep Vein Thrombosis
Authors: Pini M , Aiello S , Manotti C , Pattacini C , Quintavalla R , Poli T , Tagliaferri A , Dettoi A G
Published: July 2018

To evaluate the role of low-molecular weight heparin (LMWH) as an alternative to oral anticoagulants in the prevention of recurrent venous thromboembolism, we compared in a randomized trial conventional warfarin treatment with a three-month course of enoxaparin 4000 anti-Xa units once a day subcutaneously. 187 patients with symptomatic deep-vein thrombosis (DVT), diagnosed by strain-gauge plethysmography plus D-dimer latex assay and confirmed by venography in most cases, were treated with full-dose subcutaneous heparin for ten days and then randomized to secondary prophylaxis. During the 3-month treatment period, 6 of the 93 patients who received LMWH (6%) and 4 of the 94 patients on warfarin (4%) had symptomatic recurrence of venous thromboembolism confirmed by objective testing (p = 0.5; 95% confidence interval [Cl] for the difference, -3% to 7%). Four patients in the LMWH group had bleeding complications as compared with 12 in the warfarin group (p = 0.04; 95% Cl for the difference, 4% to 14%). In the 9-month follow-up period, during which 34 patients on warfarin prolonged treatment for other 3 months and 14 up to one year, 10 patients in the enoxaparin group and 4 patients in the warfarin group suffered a documented recurrence of venous thromboembolism. Of these 14 late recurrences, just one occurred in patients with postoperative DVT. After one year there were 16 recurrences (17%) in the LMWH group and 8 (9%) in the warfarin group (p = 0.07; 95% Cl for the difference, 1% to 16%).

These results confirm the potential of LMWH as an alternative to oral anticoagulants in this setting, and suggest to evaluate in a prospective study a slightly higher dose of enoxaparin in patients with postoperative DVT.
On the Immunogenicity of Recombinant Staphylokinase in Patients and in Animal Models
Authors: Vandaerscheren Steven M F , Marie Stassen Jean , Collen Désiré
Published: July 2018

Streptokinase and staphylokinase, the presently available thrombolytic agents of bacterial origin, are immunogenic in man; their use may cause allergic reactions and/or refractoriness to renewed administration. Infusion of 2 to 10 mg of recombinant staphylokinase (STAR) in 20 patients with acute myocardial infarction or peripheral arterial occlusion induced IgG-related neutralizing activity in plasma with a lag phase of 10 to 12 days, from a baseline of 0.2 ± 0.06 |ig STAR neutralized per ml plasma (mean ± SEM) to a maximum of 30 ± 6.2 µml after 3 to 9 weeks, which persisted at a level of 14 ± 5.8 µml after 18 months (n = 4).

In 4 baboons with a 125I-fibrin labeled clot in an extracorporeal arteriovenous loop, i. v. administration of 63 fig/kg STAR over 1 h, repeated at weekly intervals, induced a progressive increase of STAR-neutralizing activity (from 0.05 ± 0.1 µml at baseline to 4.8 ± 1.5 µml at week 6), which was paralleled by a reduction of in vivo clot lysis (from 60 ± 7% to 8 ± 3%). After temporary discontinuation of STAR-administration, neutralizing activity reverted to baseline within 7 weeks, whereafter the sensitivity of in vivo clot lysis to STAR was restored. In rabbits, i. v. administration of 250 µkg STAR over 1 h, repeated at weekly intervals, also induced a progressive increase of STAR-neutralizing activity (from 0.5 ± 0.2 µml at baseline to 6.4 ± 1.1 [ig/ml at week 6), which was paralleled by a reduction of in vivo clot lysis (from 68 ± 3% to 31 ± 7%). Thus a single STAR-administra-tion elicits an immune response in patients with persistence of neutralizing antibodies for over 18 months. Although antibody induction in baboons and rabbits required repeated administrations and was more transient than in man, these animal models appear to be suitable for the evaluation of the thrombolytic and immunogenic properties of staphylokinase variants.
The Calcium Modulator Nifedipine Exerts Its Antiaggregatory Property via a Nitric Oxide Mediated Process
Authors: Berkels R , Klaus W , Boiler M , Rösen R
Published: July 2018

The in vitro effect of nifedipine, a calcium channel blocker of the dihydropyridine (DHP) type, on platelet aggregation was reinvestigated considering especially the capability of platelets to form endogenous nitric oxide (NO). We studied the dose-dependent antiaggregatory property of nifedipine in porcine platelet rich plasma. Aggregation was stimulated by collagen (7.5 ¼g/ml). Nifedipine inhibited collagen-induced platelet aggregation with an IC50 of 380 nmol/1. The antiaggregatory effect of nifedipine could be significantly diminished by N-nitro-L-arginine (NNA) in a concentration dependent manner, whereas oxy haemoglobin (4 ¼M), a NO scavenger, totally abolished the effect of nifedipine. L-Arginine, the precursor of NO, dose-dependently inhibited the collagen-induced platelet aggregation but did not potentiate the effects of nifedipine. Therefore, we propose that in platelet rich plasma the nifedipine induced inhibition of platelet aggregation is mediated by NO, a potent endogenous inhibitor of aggregation. We could confirm this hypothesis by measuring NO directly with a specific electrode.
The Half-Life of Plasmic Degradation Products of Human Fibrinogen in Rabbits
Authors: Ardaillou N , Dray L , Budzynski A. Z , Marder V. J , Larrieu M. J
Published: July 2018

Human fibrinogen and purified plasmic degradation fragments X (stages 1 and 2), D and E were labelled with 125-1 using the lactoperoxidase method. The chromatographic, elec-trophoretic and immunologic properties of the labelled proteins were found to be similar to those of non-labelled fragments. All the degradation products diffused rapidly (T½ 0.27–0.75 hours) from the intravascular space of rabbits, as compared with fibrinogen (4.26 hours). In addition, the metabolic half-life was found to be 49.3 hours for fibrinogen, as compared with only 5.1, 6.1, 2.3 and 1.4 for fragments X (stage 1), X (stage 2), D and E, respectively. The metabolic half-life roughly reflects the molecular size of the degradation products.
Alterations in the Effects of Warfarin in Dogs by Halofenate
Authors: Weintraub Michael , Griner Paul F
Published: July 2018

The interaction between warfarin and the new lipid lowering agent halofenate (MK 185) [2 - acetamidoethyl (p-chlorophenyl) (m-trifluoromethylphenoxy) acetate] was studied in dogs in both short- and long-term experiments. Our data suggest that halofenate potentiates the anticoagulant effect of warfarin by increasing the degradation of prothrombin (factor II) (Kdeg on placebo = 211 ± 32 × 10−4 × Hr−1 mean ± SEM; on halofenate = 268 ± 39 × 10−4 × Hr−1 mean ± SEM; P < 0.01). However, a concomitant increase in factor II synthesis of 34% results in resistance to warfarin’s effect if halofenate is administered prior to warfarin. The mean prothrombin time of 4 dogs on 2 mg of warfarin following halofenate pretreatment for 8 weeks was 74.8% ± 17.3 (SE) of the anticoagulated control dog. On 2 mg of warfarin alone, it was 133.7% ± 42.0 (P < 0.001). Cessation of halofenate from combined therapy results in a delayed augmentation of warfarin effect. These data suggest that the nature of the interaction between warfarin and drugs such as halofenate which alter the kinetics of prothrombin may depend on the sequence of administration.
Absence of Blocking Antibody in Non-Inhibitor Haemophilic Plasma
Authors: Pool Judith , Kaelin A. C
Published: July 2018

This study examined the hypothesis that non-inhibitor haemophilic plasma contains antibodies which are specific for sites other than the active procoagulant site on factor VIII, and that some of them might be sufficiently close to the active site that pre-incubation of such plasma with factor VIII would block the subsequent binding of inhibitor antibody. Among the 26 non-inhibitor plasmas examined, none was found to contain such blocking antibody. This result does not eliminate the possibility that antibody is present in such non-inhibitor plasmas which is neither specific for the active enzyme site of factor VIII nor capable of blocking the binding of antibody which does have that specificity.
Purification Of A Hemostasis Activating Factor (HAF) From Human And Porcine Subcutaneous Tissue
Authors: Bender N , Kirchmaier C M , Breddin K
Published: July 2018

Platelet shape change up to 90% was induced in freshly drawn citrated blood after incubation with small amounts of porcine or human subcutaneous tissue extract for 2-3 seconds. The normal value of platelets with pseudopodes in freshly drawn blood is about 20 to 25%. Subcutaneous tissue was suspended in 30% propanol in a ratio of 1:4 (v/v) after passing a meat chopper. It sedimented after centrifugation (25 min at 100G) in four zones: an upper and lower lipid layer, a liquid intermediate zone and a pellet containing tissue particles and cell fragments. The highest platelet stimulating activity (= Hemostasis activating Factor) was found in the liquid intermediate zone, which contained total lipid concentrations of 140 mg% to 160 mg%, protein concentrations of 0.15 mg/ml to 0.3 mg/ml and an osmolarity 900 to 1000 mOsm. This intermediate zone was separated into two peaks by gelfiltration on Sephadex G 50. Fractions of the second peak contained the highest platelet stimulating activity. After ultrafiltration of the intermediate zone through Amicon CF 25 cones the activity was found in the ultrafiltrate. The ultrafiltrate has been lyophilised without losing its activity. 2.5μg/ml lyophilised HAF induced shape change in more than 90% of human platelets in freshly drawn whole or citrated blood after incubation for 1 min at 37°C. The lyophilised material contained 11 peptides which were identified by gelchromatography and peptide fingerprinting. However, none of them showed any platelet stimulating activity. The peptide patterns of human HAF and HAF from porcine subcutaneous tissue were identical. As revealed by thinlayer chromatography in different solvents most of the lipid compounds of lyophilised HAS were phospholipids. Besides others cephalin, cardiolipin and phosphatidylcholin have been identified. It seems likely that a mixture of peptides with phospholipids is essential for the platelet stimulating activity.
Rapid Isolation Of Native Urokinase From Normal Human Urine
Authors: Huber K , Kirchheimer J , Binder B R
Published: July 2018

Urokinase (UK) has been isolated to complete homogeneity starting mainly from commercially available prepurified preparations. When native human urine was the starting material, high amounts of urine were necessary because of low yields due to time consuming and complicated procedures. Furthermore, without addition of inhibitors a shift to lower molecular weight (Mr) forms could not be avoided. In order to develop a rapid isolation procedure yielding final UK preparations of complete homogeneity and as unaltered as possible, we avoided concentration procedures at the beginning of the preparation as well as antibody columns because of high activity losses and binding of non active UK antigen, respectively, and employed two affinity chromatography steps.

Two liters of native human urine were dialysed and adsorbed to gelatine-Sepharose; UK was completely bound and could be eluted with 0.1M Tris-HCl buffer, 0.7M CaCl2, pH 7.4. Active material was pooled, dialysed, and adsorbed to agmatine-Sepharose. Again, activities applied were totally bound and could be eluted with 0.1M phosphate buffer, 0.4M KC1, pH=7.4. Active material was freeze dried and gel filtrated on Sephadex G-150 in 0.1M Tris-HCl buffer, 1M NaCl, pH=8.0. During the whole isolation procedure Tween-80 (0.1%) was used in the buffer systems. Gelatine (0.02%) was present during dialysis. The procedure resulted in a UK preparation with a Mr=56.000 (SDS-PAGE) of complete homogeneity, and with a yield of l0-20% calculated from the starting material. Specific activity was about 16 pmoles of active enzyme per μg of protein (3H-DFP incorporation). The final product cleaved Glu-plasminogen to plasmin with an apparent KM≃lμM and a kcat≃0.5 s-1.

The method described is a simple and efficient procedure resulting in a product with a specific activity comparable to those described for completely purified preparations and in a Mr form identic to that predominantly present in native urine.
Isolation And Characterization Of A Plasminogen Activator (PA) From Human Myocardial Tissue
Authors: Binder B R , Reissert G , Beckmann R
Published: July 2018

Pig heart is one of the major sources for isolation of PAs. However, no attempt has been made to isolate PA from human heart. It was the aim of the study to isolate a PA from human myocardial tissue and to compare it with the vascular plasminogen activator (VPA) derived from cadaver vessel eluates.

Human heart was obtained from cadavers and myocardial tissue was prepared, homogenized, and after extraction with a neutral buffer, a pH=4.2 extract was obtained containing about 60-70% of PA activity originally present in the homogenate. The latter extract was made 2 M with (NH4)2SO4 at pH=7.0 precipitating all of the activator activity. PA activity could be purified to apparent homogeneity by reverse ammonium sulfate gradient solubilization followed by hydrophobic interaction chromatography on octly- Sepharose, affinity chromatography on arginin-Sepharose and gel filtration on Sephadex G-150. The material obtained showed a single band in SDS polyacrylamide gel electrophoresis corresponding to Mr=70.000 and the region of the gel where PA activities could be eluted.

Kinetic analysis of the purified activator with synthetic paranitroanilide substrates revealed a KM of 0.8 mM,4 mM, and 0.4 mM for H-D-Ile-Pro-Arg-paranitroanilide, H-D-Val- Gly-Arg-paranitroanilide, and H-D-Phe-Aze-Arg-paranitro- anilide, respectively, which were almost identic to those obtained with the VPA using the same subtrates. Plasminogen activation with the myocardial PA showed in a purified system a strong dependence on the presence of fibrin as it could be observed with VPA.

From the similar isolation characteristics, the molecular weight, the amidolytic activities, and the fibrin effect on plasmin formation it can be concluded that the PA isolated from human myocardial tissue is similar or identic to the VPA.
The Carboxylation Of Bovine Vitamin K-Dependent Proteins By Bovine Carboxylase
Authors: Vermeer C , Soute B A M , Hemker H C , de Metz M
Published: July 2018

Bovine vitamin K-dependent carboxylase was prepared, both from normal cows and from warfarin-treated ones. The two carboxylating enzyme systems were similar except for the fact that carboxylase from warfarin-treated cows contained a 100-fold higher amount of endogenous substrate. About 65% of this substrate consisted of one-chain factor X-precursor (s) and 25% of prothrombin-precursor(s). Solubilized carboxylase could be purified more than 100-fold by immuno- specific adsorption onto immobilized antifactor X antibodies. The purified enzyme contained 40% (w/w) phospholipid (mainly phosphatidylcholine), which was absolutely required for the carboxylating activity.

Carboxylase from non-anticoagulated cows was used for studying exogenous substrates. Bovine descarboxyprothrombin, descarboxyfragment-1 and the pentapeptide Phe-Leu-Glu- Glu-Leu were carboxylated with a low efficiency (Km, between 0.4 and 11 mM). On the other hand a peptide, identical to the amino acid residues 13-29 in descarboxyprothrombin, had a Km of 0.001 mM. It seems probable therefore that the latter peptide contains all information required for a good substrate.
The Extravascular Coagulation System: The Production Of Prothrombin, Factors V, X, IX, VII And Tissue Factor In Macrophages
Authors: Østerud B , Lindahl U , Bøgwald J , Seljelid R
Published: July 2018

The synthesis of coagulation factors and tissue factor (T.F.) has been investigated in macrophage cultures. The macrophages were obtained from mice by peritoneal washing. Serum free medium collected after 24 h macrophage culturing contained 4-12% of prothrombin, Factors VII, IX and X. These factors were not produced when either cycloheximide or warfarin were added to the growth medium. Factor V, secreted from the macrophages, was detected by the use of a clotting assay and a coupled amidolytic assay. Warfarin had no effect whereas cycloheximide blocked the synthesis of Factor V.

T.F. activity in lysed macrophages was determined by incubation of the test sample with purified F.VII, F.X and CaCl2. The production of the T.F. was dependent on lymphocytes. Lymphocytes themselves did not synthesize T.F., but they were required for the production of T.F. in macrophages as the use of anti-theta antibody + complement blocked the generation of T.F.. Endotoxin added to the cultures enhanced the synthesis of T.F. 10-40 fold (i.e. expressed in F.Xa generated in the T.F. assay: macrophages alone 0.5%, macrophages + endotoxin 18.0%, lymphocytes alone + endotoxin < 0.1%).

Macrophages secreted variable amounts of activated F.X due to the tissue factor production. However, endotoxin stimulated macrophages with high concentration of T.F. secreted less F.Xa probably caused by the synthesis of an inhibitor to F.Xa.

This study demonstrates that there exists a complete extravascular coagulation system that can be triggered by the synthesis of T.F..
Response To Infusion Of Polyelectrolyte Fractionated Human Factor VIII Concentrate In Human Haemophilia A
Authors: Kernoff P B A , Lane R S , Middleton S , Tuddenham E G D
Published: July 2018

Plasma fractionation using polyelectrolytes (PEs) has potential advantages which include simplicity of procedure, high product purity, increased yield and removal of hepatitis viruses. The purpose of this study was to assess the in-vivo response to PE-fractionated human factor VIII concentrate (PE VIII) given to 3 volunteers with severe haemophilia A. PE VIII fractionated from bulk cryo- precipitate had a high specific activity (6.74 u/mg protein), no detectable isoagglutinins, a low fibrinogen content, high ratio of VIII coagulant activity (VIII:C)to VIII-related antigen (VIIIR:Ag), and a ratio of VIII:C to VIII coagulant antigen (VIII:CAg) of approximate unity. Single infusions, each of about 2000 i.u.(25 - 40 i.u./kg), were administered i.v. over 10 mins. There were no clinical, haematological or biochemical adverse effects during the 48 hr. post-infusion observation period. Immediate post-infusion recoveries of VIII:C were 119, 102 and 70 per cent with late phase half disappearance times of 17, 19 and 17 hours respectively. These values were similar to those obtained after infusion of intermediate-purity factor VIII concentrate to the same patients, and are in accord with the results of previous studies using conventional concentrates. PE VIII has potential as a therapeutic material for patients with haemophilia A.
Obesity Is Not A Risk Factor In Thrombophlebitis
Authors: Kerstein M D , O’Connell R C , McSwain N E
Published: July 2018

It is alleged that obesity is a risk factor in the occurrence of thrombophlebitis. It is the purpose of this investigation to study that inter-relationship.

One hundred consecutive patients (21 male, 79 female) with an average age of 34 years were the subjects for this study. All patients were candidates for surgical intervention (gastric bypass procedure) because of obesity with minimum of 100 lbs over predicted weight. The mean weight was 279.2 lbs with a range of 191 - 500 lbs. The mean body mass index was 48 (weight kg/ (height cm)2). The incidence of deep vein thrombophlebitis by history was 3/100. The history of documented thromboembolism was zero.

The incidence of post-operative clinical deep vein thrombophlebitis was zero. The incidence of postoperative thromboembolism was 3/100 (3.0%). None of these patients had a history of deep vein thrombophlebitis. The diagnosis of thromboembolism was confirmed by ventilation-perfusion scan. The mortality rate secondary to thromboembolism was 1/100 (1.0%).

Obesity is not a risk factor in the occurrence of postoperative deep vein thrombophlebitis and thromboembolism. The various prophylactic medications and therapies for this group of patients may be an inappropriate risk, an undue cost and un-necessary discomfort.
Studies On Thrombotic Thrombocytopenic Purpura: Purifaction And Properties Of Platelet Aggregation Factor Inhibitor A
Authors: Lian E C Y , Mui P , Chiu L
Published: July 2018

Patients with thrombotic thrombocytopenic purpura (TTP) can be treated with plasma infusions, suggesting that deficiency of certain plasma factor(s) may be responsible for the syndrome. We demonstrated that TTP plasma induced the aggregation of autologous as well as homologous platelets, an activity not affected by hirudin, diisopropylfluorophos- phate, aspirin, or prostacyclin, but inactivated when preincubated with normal plasma. One of the plasma factors designated TTP-PAFI A which inhibits the platelet aggregation caused by TTP plasma has been purified to homogeneity. The preparation procedures include ammonium sulfate fractionation, ion exchange chromatography, ultracentrifugation, gel filtration and preparation gel electrophoresis. The purified protein showed a single line In analytical gel electrophoresis and Immunodiffusion. The amino acid composition of the inhibitor has been characterized, and its molecular weight was estimated to be 180,000 daltons. This inhibitor did not reduce the platelet aggregation induced by ADP, collagen, ristocetin, or thrombin, suggesting a specific interaction with an as yet undefined platelet aggregating factor in TTP plasma. This Inhibitor could be attached to, and then eluted from, protein A. Preliminary studies indicate that this inhibitor as isolated from TTP plasma was partially defective. It appears that functional deficiency of this plasma inhibitor may contribute to the development of TTP.
Structure And Function Of The Human Platelet Von Willebrand Factor Receptor
Authors: Clemetson K J , Naim H Y , Lüscher E F
Published: July 2018

The interactions between platelets and subendothelium involving von Willebrand factor play an important role in haemostasis and thrombosis. The identity of the von Willebrand factor receptor with human platelet membrane glycoprotein Ib has now been fairly conclusively established but the precise nature of the binding site and the changes induced by binding have yet to be demonstrated.

Asialoglycoprotein Ib and asialoglycocalicin have been isolated from neuraminidase treated platelets by affinity-chromatography on peanut agglutinin and compared by tryptic peptide mapping. The results clearly show that glycocalicin is closely related to the α-chain of glycoprotein Ib and is probably a proteolytic fragment of it. Unreduced and reduced glycocalicin showed minor differences in their tryptic peptide maps indicating that glycocalicin and by extension the α-chain of glycoprotein Ib probably contain at least one intramolecular disulphide bond.

Treatment of platelets with reducing agents such as dithiothreitol results in loss of response to bovine von Willebrand factor. Similar treatment of surface-labelled platelets did not release any glycoprotein subunits into the supernatant, implying that disulphide bridge-linked subunits are tightly associated with the membrane or with other subunits. Hydrophobic chromatography of reduced, purified glycoprotein Ib indicates that both α and β subunits are hydrophobic and thus probably integral membrane glycoproteins. Intact disulphide bridges seem to be a prerequisite for von Willebrand factor receptor activity.
The Receptor For Factor VIII/VWF On Human Platelets: Its Appearance And Disappearance Upon Action Of Thrombin And Prostacyclin
Authors: Hawiger J , Fujimoto T , Ohara S
Published: July 2018

Under normal physiologic conditions the receptor for F. VIII/VWF on human platelets is not readily available unless the antibiotic, ristocetin, is added. Searching for a physiologic mechanism inducing the receptor for F. VIII/VWF on human platelets, we found that low doses of thrombin (0.01-0.05 U/ml) induce steady state binding of 125I- F. VIII/VWF to human platelets. The binding is saturable and specific. Specificity was established by demonstrating that 100-fold molar excess of unlabeled F. VIII/VWF inhibited the binding while an excess of fibrinogen or fibro- nectin, two common contaminants of F. VIII/VWF, did not inhibit binding of 125I-F. VIII/VWF. Furthermore, rabbit antibody against F. VIII inhibited the binding. Thrombin- induced binding to human platelets can be observed only with fresh, metabolically active platelets. Glutaralde- hyde-treated platelets did not bind 125I-F. VIII/VWF following their treatment with thrombin, although they exhibited good binding in the presence of ristocetin. Thrombin appears to induce binding by acting on platelets rather than on F. VIII/VWF itself. Prostacyclin (PGI2 10-9M) inhibited thrombin-induced binding of 125I-F. VIII/VWF to human platelets, while ritcocetin-induced binding remained unaffected. Strikingly, PGI2-induced inhibition of binding was observed not orly when PGI2 was added before thrombin but also when PGI2 (10-9 M) was used 30 minutes after thrombin treatment and addition of 125I-F. VIII/VWF. Thus, binding of F. VIII/VWF to platelet receptor is induced by low concentrations of thrombin and is inhibited by PGI2. Since both agents are generated in the vicinity of injured vessel wall they may contribute to platelet-F. VIII/VWF- vessel wall interaction in vivo by regulating “up and down” platelet receptor for F. VIII/VWF.
Salicylate, Sulphinpyrazone, Indomeihacin And Aspirin May Interact With Platelet Cyclooxygenase At A Site Different From The Substrate Binding Site
Authors: Cerletti C , Livio M , Rajtar G , de Gaetano G
Published: July 2018

In vitro studies have shown that salicylate(SA), inactive by itself, prevents the inhibitory effect of aspirin (ASA)on platelet prostaglandin production. The purpose of this study was to verify this interaction in vivo and the ability of other non-steroidal anti-inflammatory drugs to counter ASA effect on platelet cyclooxygenase. Groups of male CD-COBS rats received one of the following drugs : SA (25-200 mg/kg i.p.),sulphinpyrazone(200 mg/kg p.o.), indomethacin (0.5 mg/kg i.p.) or the corresponding vehicles, followed after 30 min by ASA(1-50 mg/kg i.p.)as its soluble lysine salt. One or 24 hours later platelet-rich plasma was prepared and MDA (spec- trophotanetric assay) or TxB2 (RIA) generated on stimulation with 0.4-1 mM Na arachidonate were measured. All 3 compounds blunted ASA inhibitory action. In particular: 1. The preverting effect of SA was dose-dependent and could be overcome by increasing the dose of ASA. At a dose ratio (SA:ASA) of 20 the effect of ASA was reduced by about 50%, but no interaction was apparent at a ratio below 5. 2. Sulphinpyrazone had a greater preventing effect when it was administered 6 h (100%) than 3 h or 30 min (60%) before ASA (5 mg). 3. Indo-methacin reduced by 50% the effect of 5 mg of ASA but not of 10 mg. Tests were made 24 h after ASA administration, when the inhibitory effect of indcmethacin was no longer detectable. 4. 200 mg/kg SA reduced by 50% the inhibitory effect of indcmethacin.

This data suggests that the 4 drugs interact with the same enzyme and that competition does not necessarily involve the substrate arachidonic acid. This supports the existence of a supplementary binding site which regulates cyclooxygenase activity, but is not directly involved with the substrate site. The clinical implications of these results include the interference of ASA with its hydrolysis product SA and a reappraisal of the pharmacological basis for the association of sulphinpyrazone and ASA in thrombosis prevention trials.
Induction Of Circulating Platelet Aggregates By Release Of Endogenous 5-Hydroxytryptamine In The Rat
Authors: De Clerck F , Gorp L Van
Published: July 2018

The intravenous injection of the mast cell degranulator compound 48/80 in rats resulted in the formation of platelet aggregates (index 0.36 ± S.E.M. 0.03) and in a reduced blood coagulability as measured thromboelastographically. E.M. examinations revealed aggregates of shape changed platelets without degranulation. Radioinxnunoassay showed absence of thromboxane B2 formation. In combination with anti- histaminics, the serotonin-antagonist R 41 468 at the low dose of 0.16 mg/kg completely blocked platelet activation. Cyproheptadine, dipyridamole, VK 774 were also inhibitory. Pyrilamine, acenocoumarine, aspirin, suprofen, propranolol, lidoflazine, flunarizine, Trasylol and phentolamine were ineffective. These data support the concept that 5-hydroxy- tryptamine contributes to platelet activation in vivo.
Tissue Factor-Like Activity Of The Human Monocytic Tumor Cell Line U937
Authors: Hudig D , Rapaport S I , Bajaj S P
Published: July 2018

Cells of the human monocytic cell line U937, derived from a patient with histiocytic lymphoma (Sundstrom and Nilsson, Int. J. Cancer 17:565, 1976) have procoagulant activity similar to that of activated peripheral blood monocytes, although about 10-fold more U937 cells than monocytes are required for equivalent activity. Procoagulant activity of the cells is Ca2+ dependent and is not demonstrable in factor VII deficient or factor X deficient plasma. Culture with E. coli 0127:B8 1ipopolysaccharide increases the procoagulant activity of washed U937 cells two-fold. Exposure of U937 cells to lymphokines from normal lymphocytes does not induce further coagulant activity. The slope of the log/log plot of cells vs. clotting time parallels that of human brain thromboplastin. Other cell lines of myeloid or lymphoid origin, e.g., K562 cells, WI-L2 cells, do not have procoagulant activity. Thus, U937 cells have constitutive factor VII-dependent coagulant activity similar to the tissue factor activity induced by activation of normal monocytes.

In further experiments, U937 cells were incubated with purified human factor VII in the presence or absence of Ca2+ and then repeatedly washed. When subsamples of the cells were then added to recalcified factor VII deficient plasma in the absence of added tissue factor, the following clotting times were obtained: for cells incubated with factor VII in the presence of Ca2+,45"; for cells incubated with factor VII in the absence of Ca2+, 150". These data suggest that U937 cells can bind factor VII in a reaction requiring Ca2+, which then enables the cells to express their tissue factor-like activity in factor VII deficient plasma.
Subcellular Localisation Of ADP-ASE In Cultured, Porcine, Endothelial Cells
Authors: Lieberman G E , Leake D S , Peters T J
Published: July 2018

A role for vessel wall ADP-ase in the maintenance of haemostasis has been suggested by a number of studies. For such a role to be likely the enzyme ideally should be an ecto-enzyme. The purpose of this study was to localise the ADP-ase using the technique of subcellular fractionation on a sucrose-density gradient in conjunction with marker enzyme assays for the different organelles.

Firstly, homogenates of cultured, pig aortic endothelial cells which had been harvested following the 5th-7th subculture were prepared. These were used to determine the kinetics of ADP-ase activity using the recently described rapid assay for ADP-ase which employs[β-s2P]ADP. The activity was found to be linear with respect to incubation time and protein concentration. The pH optimum was found to be 7.4 and the Km to be 45μM.

Post-nuclear supernatants derived from these homogenates (controls) and those from cells which had first been incubated with digitonin which binds to the membrane cholesterol thus making membranes heavier, were fractionated. In the controls, the profile of ADP-ase activity throughout the gradient was dissimilar to lactate dehydrogenase (cytosol), malate dehydrogenase (mitochondria), neutral α glucosidase (endoplasmic reticulum) and N-acetyl β glucosaminidase (lysosomes). It most closely corresponded to the 5’ nucleotidase profile (plasma membrane). Following digitonin treat ment, both 5’NT and ADP-ase shifted to a denser zone in the gradient confirming their coincident localisation.

Lastly, intact cells and sonicated cells were incubated with the enzyme inhibitor: diazotised sulphanalic acid which is a poorly permeant reagent. With intact cells, ADP-ase and 5’NT were inhibited whereas LDH was not, but with sonicated cells all three enzymes were inactivated.

Thus ADP-ase is an ecto-enzyme on the plasma membrane of pig aortic endothelial cells.
The Effects Of Heparin On The Activation Of Factor X And Prothrombin In Antithrombin III-Depleted Plasma
Authors: Ofosu F , Cerskus A , Hirsh J , Blajchman M A
Published: July 2018

The predominant mode of the anticoagulant action of heparin is considered to be the enhancement of the rate of inactivation, by antithrombin-III, of several activated clotting factors. Recent evidence, however, suggests that heparin can reversibly inhibit the activation of prothrombin and factor X even in the absence of anti- thrombin-III. This antithrombin-III-independent action of heparin has been demonstrated only in purified clotting factor systems. In order to determine the significance of the antithrombin-III-independent effects of heparin in plasma, the effects of heparin on the activation of factor X and prothrombin were studied in antithrombin-III- depleted plasma produced by affinity chromatography of normal plasma on heparin-Sepharose. Heparin partially inhibited the activation of factor X and prothrombin on the addition of either factor IXa or factor Xa in anti- thrombin-III-depleted plasma. This inhibition was demonstrable only when high concentrations (1 or 10 units/ ml) of heparin were used. In contrast, when as little as 1% antithrombin-III was added to antithrombin-III-depleted plasma containing 1.0 u of heparin per ml of plasma, no factor X or prothrombin activation could be demonstrated. Thus, it appears that in comparison with the magnitude of the antithrombin-III-dependent effect, the contribution of the antithrombin-III-independent anticoagulant effect of heparin on the activation of factor X and prothrombin in normal plasma is limited.
BIS-ANS Binding To Human Thrombins
Authors: Metz G , Berliner L J
Published: July 2018

Bis-anilinonaphthalenesulfonate(bis-ANS) binds extremely strongly to the human thrombins accompanied by a much enhanced fluorescence for the dye. One unique feature of bis-ANS binding is an enhancement of TAME hydrolysis by α-thrombin to approximately 175% times the rate for native α-thrombin at pH 8.1 (0 M NaCl). Non-coagulant γ-thrombin is distinguished from highly coagulant α-thrombin by enhancing bis-ANS fluorescence ∼2 times more than that by the latter form. The very strong binding of this dye Kdiss<10-8 M, pH 6.5, 0.05 M Phosphate, 0.75 M NaCl) as welras its apparent binding loci situated near, but not obstructing, the catalytic center displays the powerfully useful properties of this probe, extremely sensitive to thrombin environment while not inhibiting its action. This is to be compared with the active site specific fluorophore inhibitor DAPA which prevents binding of other ligands to the thrombin active site.
The Primary Structure Of Crotalase, A Thrombin-Like Venom Enzyme, Exhibits Strong Homology With Kallikrein
Authors: Pirkle H , Markland F S , Bajwa S S , Theodor I , Baumgartner R , Kirakossian H
Published: July 2018

Edman degradation of crotalase and its tryptic and CNBr fragments yielded an N-terminal amino acid sequence, Val- Ile-Gly-Gly-Asp-Glu-Cys-AsN-Ile-AsN-Glu-His-Arg-Phe-Leu-Val- Ala-Leu-Tyr-Asp-Tyr-Trp-Xxx-GlN-Xxx-Phe-rLeu-, and internal sequences, -Leu-Ile-Arg-Leu-AsN-Lys-Pro-Val-Ser-Tyr-Ser-Glu- His-Ile-Ala-Pro-Leu-Ser-Leu-Pro-Ser-Ser-Pro-Pro-Ile-Val-Gly- Ser-Val-Cys-Arg-Ala-Met-Gly-Trp-Gly-GIN-Thr-Thr-Ser-Pro-GIN- Glu-Thr-Leu-Pro-Asp-Val-Pro-His-Cys-Ala-AsN-Ile-AsN-Leu-Leu- Asp-Tyr-Glu-Val-Cys- and -Ser-Val-GIN-Phe-Asp-Lys-Glu-GIN- GIN-Arg-. The longer internal sequence aligns homologously with positions 105-168 of chymotrypsinogen with gaps, each 5 residues long, following Pro 128 and GIN 143. No homologous alignment has been found for the shorter internal sequence. These findings led to experiments which demonstrated that crotalase has specific enzymatic properties resembling kallikrein.
Sex Difference In Platelet Response To Aspirin Treatment
Authors: Nicholson N S , Smith S L , Saunders R N
Published: July 2018

This study was done to determine if a sex difference in response to aspirin similar to that seen in the clinic could be demonstrated in an animal model with hyperactive platelets. The platelet hyperactivity which results in the spontaneous formation of platelet aggregates in retired breeder rats was reduced in both male and female rats by sulfinpyrazone, dipyridamole and indomethacin administered at 20 mg/kg. Aspirin blocked spontaneous aggregation in the male, but had no effect in the female even at doses of 100 mg/kg. Because aspirin is known to be an inhibitor of cyclooxygenase, the metabolism of arachidonic acid was studied in these rats. Arachidonic acid at 20 mg/kg was active in reducing spontaneous aggregation in the male, but had no effect in the female. However, in an in vitro study of the metabolism of arachidonic acid, no significant differences were seen between males and females in the conversion of arachidonic acid to PGF2α, PGE2, TXB2 or HHT. Aspirin was equally effective in both males and females in blocking the in vitro conversion of arachidonic acid via the cyclooxygenase pathway. The retired breeder rat provides a system for meaningful investigations toward understanding the human sex-related differences in platelet sensitivity with aspirin, although the mechanisms of the in vivo male/female platelet sensitivity have not been explained by in vitro studies thus far.