bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and Some Properties of Tissue Plasminogen Activator
Authors: Wallén P. , Rånby M.
Published: May 2019

A highly purified preparation of plasminogen tissue activator from pig heart has been prepared for studies on its chemical properties and on its interaction with fibrin and fibrinogen from the functional point of view. Essential steps in the purification procedure are two affinity adsorbtions involving two different sites in the activator molecule. 1. Affinity adsorbtion to fibrin followed by elution in two steps with potassium thiocyanate and phenylethylamine. 2. Affinity chromatography on Sepharose-arginine. The final product moves as a single band on SDS-polyacrylamidegel electrophoresis although an additional trace component is sometimes observed. The specific activity is at least 150 000 CTA units//mg protein as measured in a fibrinolytic test system. The molecular weight measured by SDS-polyacrylamidegel electrophoresis is 63 000 dalton before reduction. Reduced samples still appear as one fraction on electrophoresis, but the molecular weight has diminished to about 31 000 dalton, i. e. half the original value. The highly purified activator is labile in neutral solutions of low ionic strength but is stabilized by increasing the ionic strength to 1. The firm and specific adsorbtion of tissue activator to fibrin is presumably of physiological importance. Attempts are therefore made to study sites in fibrin (fibrinogen) with affinity for tissue activator. Preliminary experiments indicate that one site is situated in the part of Aα chain which is released from fibrinogen or fibrin early during digestion with plasmin.
Pharmacological Evaluation of Ticlopidine, A Novel Inhibitor of Platelet Function
Authors: Johnson M. , Walton P. L. , Cotton R. C. , Strachau C. J. L.
Published: May 2019

Ticlopidine (T), 5-(2-chlorobenzyl)-4,5,6,7-tetrahydrothieno(3,2-C)pyridine hydrochloride (a product of Parcor Research) has been evaluated as an antiplatelet agent in various animal species and in human volunteers. T was inactive in vitro, but inhibited platelet aggregation induced by ADP, collagen, thrombin, arachidonic acid and prostaglandin (PG) endoperoxide, when administered orally to mice, rats, rabbits, guinea pigs, pigs, dogs and baboons. Platelet adhesiveness was reduced but platelet survival time was normal in treated animals. Basal PG synthesis and platelet ultra-structure were unaffected by T.T protected against acute thrombocytopenia and death from pulmonary embolism induced by i. v. injection of ADP or collagen. Thrombus formation in experimental models of extra-corporeal circulation and deep venous thrombosis was also impaired.

In man, a single oral dose of 500mg was shown to be a potent inhibitor of ADP, collagen, adrenaline, ristocetin, bovine fibrinogen and 5HT-induced aggregation. A dose-effect relationship was apparent, 250 and 500mg resulting in ~47 and 75% inhibition of ADP-induced aggregation respectively. Inhibition was sustained by chronic daily dosing.

There was a delay in the onset of action of T in vivo, but which then persisted after withdrawal for at least 48 hours, with no evidence of rebound hyperactivity. The duration of action of T correlated with platelet survival time, suggesting an irreversible modification of platelet function. T is a potent platelet inhibitor, exhibiting a novel mode of action and lack of agonist specificity, which may be of value in the treatment of thrombotic conditions.
Antithrombotic Activity of a Potent, New Agent, 6,7-Dichloro-1,2,3,5-Tetrahydroimidazo[2,1-B] Quinazolin-2-One Hydrochloride Monohydrate
Authors: Fleming J. S. , Buyniski J. P.
Published: May 2019

A potent, new anti-thrombotic agent, 6 ,7-dichloro-l,2,3,5-tetrahydroimidazo[2,1-b]quinazolin-2-one hydrochloride monohydrate (BL-4162A) has been evaluated for activity against induced platelet aggregation in a series of in vitro and ex vivo experiments using platelet rich plasma (PRP) from various species including man. In addition, the compound’s potential utility as an antithrombotic agent has been tested in various in vivo animal models including two models of induced thrombosis involving both large and small vessels. Aggregometry was employed to test the ability of BL-4162A to inhibit platelet aggregation induced by aggregating agents such as ADP, collagen, thrombin and antigen-antibody complexes. The compound’s antithrombotic activity was evaluated in small vessels using the biolaser-rabbit ear chamber technique while the effect of BL-4162A on large vessel thrombosis was assessed against electrically-induced carotid artery thrombosis in dogs. Results indicate that BL-4162A inhibits platelet aggregation in the range of 0.08 to 0.68 pg/ml. Platelet antiaggregating activity was also observed in ex vivo aggregometry studies in dogs and rats at oral doses in the range of 1 to 10 mg/kg. Of particular interest, is the fact that BL-4162A effectively inhibited thrombosis in both animal models employed over the same oral dosage range. Results of the above investigations as well as an appreciable duration of action suggest that BL-4162A may be an important candidate for clinical evaluation in thromboembolism.
Effect of Diazepam on Platelet Function
Authors: Carvalho A. C. , Colman R. W. , Vaillancourt R. , Cabrai R. , Anaya R.
Published: May 2019

Diazepam (Valium) is one of the most prescribed medications in the world. Patients on Diazepam may need platelet function evaluation. Therefore, a study of its effect on both in vivo and in vitro platelet function was undertaken in 8 normal volunteers. Diazepam (10–40μg/ml) was incubated in vitro with platelet rich plasma (250,000/μl) at intervals of 15, 30, 60, 120, and 240 minutes followed by determination of platelet aggregation and 14C-serotonin release. Fifty percent inhibition of platelet aggregation and release by Diazepam was obtained at 1 hr with epinephrine (p<0.01) and at 2 hrs with ADP (p<0.01), but no significant effect was noted with collagen. The Diazepam inhibitory effect on platelet aggregation and release was overcome by high concentrations of aggregating agents, suggesting that its primary effect is not mediated by inhibition of prostaglandin synthesis.

Following oral ingestion of 5mg of Diazepam, platelet aggregation and 14C-serotonin release were determined serially (2, 4, 8, 12, 24, and 48 hours) in the 8 normal subjects. After 8 hours, Diazepam inhibited ADP-induced aggregation and release by 39% (p<0.01) and epinephrine by 50% (p<0.01). No significant inhibition of collagen was observed. Forty-eight hours after Diazepam intake, platelet function returned to normal in all subjects.

Our data show that Diazepam impairs both platelet aggregation and release in vitro and in vivo. Although the effect of Diazepam on in vivo hemostasis is still uncertain, our results suggest caution in the interpretation of platelet function testing in patients on this drug.
Studies on the Interaction Between (14C)-Epinephrine and Isolated Membranes from Human Platelets
Authors: Vainer H.
Published: May 2019

Previous studies have reported on the main characteristics of epinephrine’s capacity to stimulate resting platelets. This study was designed to further investigate this problem using (14C)-epinephrine (*E) and isolated membranes from human platelets. Membranes were isolated following the method of Barber & Jamieson (1970): a 6 and 2 fold enrichment in p (nitrophenyl) PDEase and ATPase and negligible levels of cytoplasmic marker enzyme activities have been measured. Incubations contained membranes and *E (33 mCi/μM) in Tyrode’s solution at pH 7.4; membrane bound *E was quantitated by Millipore filtration. Binding was a rapid, reversible and saturable process:It reached equilibrium by 30 min. at 37°C; half-maximum displacement ofE occurred at 5x105M-l “cold” E; about 400 pmoles of E were bound at saturation /mg of membrane protein/hour with a Kaff = 4.5–5.2x107M-1. Acid dissociated E from the labelled membranes showed that E structural alteration or degradation does not occur during binding. Impaired binding follows membrane exposure to impermeant -SH groups reagents or to trypsin, chymotrypsin, pronase and beta galactosidase. A specific binding appeared from its inhibition by alpha adrenoceptor agents, whereas beta active compounds had a much lower effect at higher concentrations; E metabolites did not inhibit binding. Mersalyl, a specific inhibitor of Mg2+ATPase of the myosin-like platelet protein, Thrombosthenin M, strongly inhibited binding. It thus appears that E interaction with isolated platelet membranes has properties to be expected of binding to alpha adrenoceptors and involves thrombosthenin; binding occurred at E concentrations consistent with those reported to stimulate highly responsive platelets to E, suggesting that changes in stereochemical configuration of isolated membranes may cause exposure of new reactive sites for E.
Cross Linking of Human Platelet Membrane Aminophospholipids:Changes Induced by Thrombin
Authors: Schick P. K. , Arosteguy P.
Published: May 2019

The proximity of the aminophospholipids, phosphatidylethanolamine (PE) and phosphatidyl-serine (PS), in platelet membranes was probed with difluorodinitrobenzene (D) which reacts with and cross-links aminophospholipids when they are less than 5 A° apart. D can penetrate intact cells and labels lipids on either side of the membrane bilayer. The incubation of mixtures of pure PE and PS can result in the formation of the following derivatives: PE-D-PE, PE-D-PS, PE-D, PS-D-PS, PS-D. Following incubation of washed platelets with D, lipids and derivatives were extracted, separated by thin-layer chromatography, and quantitated by lipid phosphorus. The incubation of platelets with D (125μM) for 2 hours resulted in derivatization of 49% PE and 34.6%PS. When platelets were treated with thrombin (0.1U/ml) for 5 minutes prior to incubation with D, 71.4% PE and 53.8% PS reacted. Results are shown in Table; %PE(PS)=% total platelet PE(PS).

In controls significant amounts of only PE-D-PE and PE-D-PS were detected and thus are intimately associated within platelet membranes. In thrombin-treated platelets significant amounts of PS-D-PS were formed which did not occur in controls. However, there was no increase in PE-D-PE and PE-D-PS. Also, in thrombin-treated platelets additional PE and PS reacted but were not close enough to be cross-linked. The study indicates that incubation of platelets with thrombin results in the unmasking or rearrangement of specific compartments of PE and PS within platelet membranes which may be critical for platelet hemostatic activities.
Fibrinopeptide A (FPA) Levels in Venous Thrombosis and Pulmonary Embolism Before and During Anticoagulant Therapy
Authors: Yudelman I. , Nossel H. L. , Kaplan K. L. , Hirsh J.
Published: May 2019

FPA levels were measured in 60 patients subjected to venography and to lung scan for symptoms suggestive of venous thromboembolism. In all 23 patients with negative venography and/or lung scan, FPA levels were in the normal range (<1.3 pmol/ml, mean 0.6 pmol/ml). The FPA levels were elevated in 34 of the 37 patients with a positive lung scan and/or venogram. The range was 0.4–112 pmol/ml, median 6.2 pmol/ml. The FPA levels were measured serially in patients with confirmed thromboembolism who were treated with heparin. In 14 of the 15 patients there was a marked drop in FPA levels in the first 15 minutes after the initial dose of heparin. In 1 patient the FPA levels only reached the normal range after 48 hours of heparin therapy. FPA levels were measured daily in 10 patients while on anticoagulant therapy. In 4 patients FPA levels became normal and remained so and no symptoms recurred. In the other 6 patients there were 13 episodes of FPA elevations.10 of these were preceded by a recurrence of the initial symptoms. In one patient FPA elevations occurred in the absence of symptoms while the repeat lung scan showed new lesions. Suboptimal anticoagulation and/or the transition from heparin to Coumadin preceded the recurrence of symptoms in 6 out of 10 episodes. FPA levels were frequently normal in asymptomatic patients with evidence of venous thrombosis as shown byfibrinogen uptake scan. These results suggest that FPA measurements may be useful in the diagnosis and in monitoring therapy of symptomatic thromboembolism.
Adhesion of Platelets to Collagen
Authors: Meyer F. A. , Weisman Z.
Published: May 2019

Platelets adhere to collagen fibers, undergo the release reaction and aggregate, soluble collagen, however, does not cause release or aggregation. Platelets, however, recognize soluble collagen since adhesion (but not aggregation) occurs when a soluble collagen coated surface is exposed to washed rabbit platelets in Tyrode and in competitive experiments preincubation of platelets with soluble collagen reduces the number adhering to a collagen surface. The latter effect is concentration dependent; preincubation with 100 μg/ml of soluble collagen reduces platelet adhesion by 70%.

Similar competitive experiments were performed to examine the features on collagen responsible for platelet adhesion. Preincubation of platelets with a variety of soluble collagens, denatured soluble collagen, pepsin- and periodate-treated soluble collagen and cyanogen bromide peptides all decrease the ability of platelets to bind to a collagen surface. Similar effects arise moreover with the synthetic copolypeptides (Gly-Pro-Ala-Gly-Pro-Pro)n,(Gly-Pro-Pro)n and (Pro2Gly)n. In fact, the homopolymers, polyproline and polyhydroxyproline (but not proline or hydroxyproline) as well give effects similar to those of the collagen materials. The effect is rather specific since preincubation with plasma proteins and synthetic polypeptides based on other major amino acids present in collagen, viz. polyalanine, polyglutamic acid, polyaspartic acid, polyarginine and polylysine do not inhibit the binding of platelets. It would therefore appear that proline and hydroxyproline sequences incorporated in a macromolecular chain are the important, if perhaps not the only determinants involved in platelet recognition of collagen.

Soluble collagen coated surfaces were used in this study, however, similar effects of the test material on platelet adhesion to collagen fibers could be demonstrated.
Deposition of Platelets on Collagen as Evaluated by 51Cr-Label or Morphometry: Effects of Aspirin
Authors: Tschopp Th.B.
Published: May 2019

Whether aspirin inhibits platelet deposition on thrombogenic surfaces by its effect on adhesion-induced aggregation only or whether it also affects adhesion per se, is still controversial. This study compares platelet deposition determined from the deposited radioactivity using 51Cr-labeled platelets with platelet surface interaction as measured’by morphometry. Collagen fibrils of α-chymotrypsin-digested segments of rabbit aorta were exposed to citrated rabbit blood for 10 min in a perfusion chamber at 830 s-1 blood shear rate. The number (L) of deposited platelets as calculated from the 51cr-label amounted to 687±87x103/mm2 (mean ± SE of 35 control perfusions). The surface area covered with platelets (D) as determined by morphometry amounted to 31±2 % and that covered by platelet thrombi (T) (aggregates higher than 5 um) to 19±2 %. The deposited platelet mass (M) as measured by the sum of platelet cross section area multiplied by section thickness amounted to 564±66 μm3/mm2. Linear regression analysis showed platelet mass as determined by L and M to be highly correlated (R = 0.9509, 2p (0. 001). Thus platelet deposition on thrombogenic surfaces may be adequately measured by adding a small proportion (l3±1 %) of 51Cr -labeled homologous platelets to citrated blood.

Aspirin (100 μM) significantly inhibited platelet deposition as shown by decreased values of L, M and T but did not decrease the surface area covered with platelets (D) as evaluated morpho-metrically. Thus the inhibition of platelet deposition on thrombogenic surfaces exerted by aspirin appears to be due to inhibition of a platelet reaction subsequent to adhesion.
Inhibition of Collagen-Induced Platelet Aggregation by Antibodies to Distinct Types of Collagen
Authors: Balleisen L. , Timpl R. , Gay S.
Published: May 2019

The reaction of platelets with fibrillar collagen was measured by recording aggregation according to Borns method and by retraction of Ancrod-fibrin clots. These reactions could be completely inhibited by coating the fibrils with stoichiometric amounts of purified antibodies to type I, II or III collagens. The inhibition was specific, i. e. antibodies to type I collagen prevented aggregation by type I collagen but not by type II or III collagen. Comparable amounts ofantibodies to fibrinogen or to serum albumin had no effect on the reaction. The data indicate that platelet aggregation by type I or II collagen fibrils is not due to contamination with type III collagen. The inhibition reaction may be useful for further studies on molecular parameters of the interaction between platelets and collagen fibers.
Interaction of Human Platelets with the 2′, 3′-Dialdehyde Derivatives of ADP (o-ADP) and ATP (o-ATP) as Potential Affinity Labels for the ADP Receptor
Authors: Pearce P. H. , Scrutton M. C.
Published: May 2019

In order to characterise the receptor responsible for mediating platelet aggregation and secretion induced by ADP we have synthesised oADP and oATP by periodate oxidation of ADP and ATP. oADP induces platelet shape change but inhibits aggregation competitively with respect to ADP. The properties of inhibition of aggregation induced by adrenaline, collagen or thrombin are also consistent with the conclusion that the effects of oADP result from interaction with the ADP receptor(s). The effects of oATP generally resemble those of oADP except that oATP inhibits, rather than induces, shape change and causes this effect at a concentration in excess of that required for inhibition of aggregation. ,

Brief exposure of intact platelets to [3H]-oADP or oATP causes stable incorporation of [3H] into the membrane proteins. Analysis of [3H] distribution by SDS gel electrophoresis after prolonged dialysis against SDS/mercaptoethanol shows predominant incorporation of [3H]-OATP into a fraction at 28000 dal tons over a concentration range consistent with that required for inhibition of aggregation. When [3H]-OADP is used, incorporation occurs predominantly into 4 fractions at 28000, 45000 (probably actin), 65000 and 220000 daltons. The [3H] incorporation/ [oADP] relationship and the time course of incorporation suggest that the 28000 and 65000 dalton fractions are likely candidates for identification as ADP receptors. Our data suggest a tentative identification of the 28000 dalton fraction as the receptor responsible for mediating aggregation induced by ADP and also indicate the possibility that different receptors may be involved in induction of shape change and aggregation by this agonist.
Specific Inhibition of ADP-Induced Platelet Responses by 2-n-Amylthio Amp
Authors: Maclntyre D. E. , Gordon J. L. , Drummond A. H. , Steer M. , Salzman E. W.
Published: May 2019

“Primary” aggregation responses to ADP are blocked by 2-n-amylthio AMP (nAmSAMP)*, apparently competitively (Ki ≃ 10 μM). Shape change is inhibited by higher concentrations (> 0.1 mM). nAmSAMP has a modest inhibitory effect on platelet responses to ionophore Lilly A23187 and a greater effect on responses to collagen and blocks secretion and secondary aggregation induced by ADP, adrenaline, arachidonic acid, PGG2, and synthetic analogues of PGE2 and PGH2-nAmSAMP is a much less potent inhibitor than adenosine against all stimulants apart from ADP and is qualitatively unlike adenosine in the following respects:1. primary aggregation responses to the above agents (except ADP) and to serotonin and vasopressin are unaffected;2. inhibition is not increased by preincubation;3. inhibition is not decreased by an inhibitor of adenylate cyclase, SQ22536 (9-[tetrahydro-2-furyl]-adenine);4. basal levels of platelet cyclic AMP are unaffected. We conclude that, unlike adenosine, nAmSAMP does not inhibit platelet responses by stimulating adenylate cyclase. nAmSAMP appears to be a “specific” competitive antagonist of ADP and should therefore be useful in clarifying the role of ADP in platelet reactions.
Heparin Pharmacokinetics
Authors: Swart C. A. M. de , Akkerman J. W. N. , Nijmeijer A. , Sixma J. J.
Published: May 2019

In heparin therapy different schedules for safe and effective treatment have been advocated,but they are not based on firm knowledge of the pharmacokinetics of heparin. We report on the application of two tests (diluted APTT test according to Marder and anti-Xa assay according to Yin) to the study of the pharmacokinetics of the anticoagulant effect of four commercially available brands of heparin. The drug was administered as a single large bolus or by continuous infusion. In contrary to reported pharmacokinetics a non exponential disappearance was observed after single dosis administration. Continuous infusion allowed a mathematical description of elimination of heparin activity,in which two mechanisms could be demonstrated. Pirstly, a mechanism which is saturable and eliminates heparin activity at low levels at a high rate. Secondly, a mechanism which is linearly related to heparin activity. Studies carried out on the elimination of 35S heparin showed that the disappearance of the radioactivity differed from that of the anticoagulant effect. Binding studies with an antibody directed against antithrombin III which was insolubilized on Sepharose 4B showed a close relationship between the disappearance of that part of the radiolabel that bound to the Sepharose-antibody complex and the disappearance of the anticoagulant effect.
Studies with High and Low Molecular Weight Tritium-Labelled Heparin in Vivo and in Vitro
Authors: Shanberge J. N. , Ambegaonkar S. , Kitani T. , Gruhl M. , Kambayashi J. , Nakagawa M. , Sinai Mount
Published: May 2019

When defibrinated platelet-poor plasma 1s chromatographed on Sephadex G-200, fractions with antithrombin-heparin cofactor activity are found 1n only one area. When platelet-poor plasma treated with a tritium-labelled heparin is chromatographed on Sephadex G-200, radioactivity, signifying the presence of heparin, is spread across all of the protein fractions, whereas immediate antithrombin activity is located in two. main areas. Tritiated heparin produced from porcine intestinal mucosa was fractionated on a Sephadex G-200 column with separation of the higher and lower molecular weight fractions. These fractions were added to platelet-poor plasma which was then rechromatographed on Sephadex G-200.With the higher molecular weight, heparin radioactivity appeared in fractions which usually have immediate antithrombin activity, whereas, with the lower molecular weight heparin, it did not. A comparison of the activities of the higher and lower molecular weight material was made after intravenous injection into rats. The higher molecular weight heparin gave higher concentrations of radioactivity in the liver and blood. In addition, the anticoagulant activity as measured by a whole blood recalcification clotting time was maintained for a much longer period. The lower molecular weight heparin was excreted more rapidly in the urine. It is concluded that only the higher molecular weight heparins have anticoagulant activity 1n combining with antithrombin whereas lower molecular weight heparins do not combine with antithrombin but are eliminated in the urine.
The Kinetics of Clearance from the Circulation of Man of Heparin and a Semi-Synthetic Heparin Analogue
Authors: Lane D. A. , Michalski R. , Kakkar V. V.
Published: May 2019

A study has been made of a low molecular weight semi-synthetic heparin analogue, (SSHA) that may be clinically useful as an antithrombotic agent because of itsreported high specificity for potentiating antithrombin III activity. The clearance from the circulation of both heparin and the analogue has been studied in man following intravenous injection. Heparin obeyed almost zero order kinetics when assayed using a specific anti-Xa assay and first order kinetics when measured with KCCT. At high concentrations the heparin analogue was cleared with first order kinetics when assayed both with the anti-Xa assay and with KCCT. At low concentrations the analogue produced between one half and two-thirds of the anti-Xa activity of an equal dose of heparin, producing only a small prolongation of KCCT. With increasing dose, the more specific anti-Xa potentiating effect of SSHA decreased in part because of the difference in kinetic behaviour between heparin and SSHAbut largely because of a flattening of its anti-Xa dose response curve. Because of the initial more rapid clearance of higher doses of heparin from plasma when it is measured by the KCCT, these results suggest that the use of KCCT can cause a small underestimate of circulating heparin anti-thrombotic activity.
Reduced Polymerization of Willebrand Factor in a Family of Pigs with a Variant of von Willebrand’s Disease
Authors: Fass D. N. , Bowie E. J. W.
Published: May 2019

Sodium dodecyl sulfate electrophoresis on 2% agarose allows the separation of porcine Willebrand factor into multiple macromolecular complexes from 1.5 million to 21 million, molecular weight. In non-denaturing buffers these forms are resistant to alteration by 10-4M mercaptoethanol, reduced glutathione, or cysteine. At sulfhydryl concentrations of 10-4M both dithiothreitol (DTT) and dithioerythritol (DTE) seem to catalyze disulfide interchange evidence by the appearance of very high molecular weight polymers. Both DTT and DTE in the absence of detergent or other denaturants lowered the average complex size at sulfhydryl concentration of 10-3M. Concomitant with the reduction in average polymer size there was a loss of ristocetin-Willebrand factor activity. A severe reduction of average molecular weight was found to be a characteristic of the Willebrand factor found in a genetically determined variant of the porcine model of von Willebrand’s disease. These observations provide a molecular basis for a qualitative version of the porcine disease in contrast to all previously described affected pigs which suffer a quantitative defieiency of Willebrand factor activity and antigen.
Characteristics of Factor VIII Related Protein in Different Types of von Willebrand’s Disease
Authors: Bloom A. L. , Peake I. R.
Published: May 2019

The antigenic, biochemical and biological reactions of factor VIII related protein were studied in normal plasma and in the plasma of patients with different types of von Willebrand’s disease (vWd). Antigenic reactivity was compared using the Laurell electroimmunoassay (LA) and an immunoradiometric assay (IRMA). Biochemical characteristics of factor VIII related antigen (FVIIIRAG) were compared by examining its electrophoretic mobility (EM) on two dimensional crossed Immunoelectrophoresis (2DCIE) and by determining its precipitation properties with the glycoprotein precipitant concanavalin A (Con A),Biological reactions were compared using the ristocetin cofactor (RiCoF) assay on fixed platelets and by determining procoagulant factor VIII (FVIIIC). In normal plasma the levels of FVIIIRAG measured by LA correlated with the IRMA and dose response curves were parallel. At a concentration of lmg/ml Con A completely precipitated FVIIIRAG. The biological activities, RiCoF anú FVIIIC, were normal and correlated with those of FVIIIRAG. In patients with vWd in whom levels of FVIIIRAG by LA were normal the EM by 2DCIE was increased. In these patients the dose-response curves of the IRMA were not parallel to normal, FVIIIRAG was not precipitated normally by Con A and the RiCoF activity was reduced. Similar findings were observed in some patients with “typical” intermediate vWd in whom the plasma levels of FVIIIRAG were too low to determine EM, In other patients with vWd the dose response curves of the IRMA were parallel. The results suggest that the non-parallel dose response curves of the IRMA were due to the presence of abnormal FVIIIRAG and were consistent with variations of antigenic reactivity or binding sites in these patients.
Effect of Sulfinpyrazone and Aspirin on Platelet Loss Induced In Vitro by Activated Charcoal Hemoperfusion
Authors: Winchester James F. , Forbes Charles D. , Courtney James M. , Prentice Colin R.M.
Published: May 2019

Activated charcoal hemoperfusion is used for drug overdosage and is potentially useful in acute hepatic failure and chronic uremia. While hemocompatibility of early devices has improved, platelet loss still occurs, and may be associated with hemorrhage.

We have developed an in vitro circuit consisting of hemoperfusion column and lines with a total blood volume of 60 ml for use with human blood. Citrated whole blood hemoperfusion is associated with platelet depletion of similar magnitude for coated or uncoated charcoal (20%) whereas polymer coating of charcoal improved the profound platelet loss (90%) noted with perfusion of heparinized whole blood (3 U/ml) over uncoated charcoal.

At 14 day intervals, in a double blind cross over study, we compared the effects of 2 days treatment with aspirin (ASA)(600 mg/day) or sulfinpyrazone (SP)(800 mg/day) alone or in combination against placebo, on platelet adsorption on 2% hydrogel coated or uncoated charcoal, and platelet retention on cuprophan membranes PT150 and PT250 in a test cell system, in 8 healthy male volunteers. Bleeding time was also measured before and after treatment using a spring loaded device.

In a heparinized system, ASA, SP, and combination reduced mean platelet adsorption on coated charcoal from 53% to 41%, and mean platelet retention to membranes from 20% to 10%, whereas ASA alone reduced platelet adsorption on uncoated charcoal. Bleeding time,was prolonged by ASA but not by SP. These agents are capable of modifying platelet loss in vitro and may do so in vivo.
Decrease in Platelet Aggregability after Total Oophorectomy
Authors: Yamazaki H. , Motomiya T. , Sonoda M. , Miyagawa N.
Published: May 2019

Substantial clinical evidence indicates that large doses of estrogen frequenly result in thromboembolic disorders. Effects of estrogen on platelet aggregability were examined in women with uterine myoma before and after oophorectomy. Bilateral oophorectomy on 15 cases (48.7+0.12 yrs, mean+SE) and unilateral or no oophorectomy on 18 cases (control group : 42.2+0.18 yrs) were performed with myomectomy of the uterus. On one day before and one day, one week and one month after the operation performed, their platelet count by Coulter counter, platelet volume by Coulter channelyzer and platelet aggregability by Sienco aggregometer were measured. 24 hrs total estrogen in urine was also determined. In the control group, platelet counts were 85.1+ 4.9 % of the preoperated value one day after, 127.9+9.0 % one week after and 98.1+7.6 % one month after the operation. In the bilateral oophorectomy group, these were 82.4+5.2 % one day after, 124.0+4.7 % one week after and 96.1+4.8 % one month after. Both the groups showed the same change. Platelet aggregability by 3 μM ADP were 76.9+14.3 % one day after, 203.0+57.1 % one week after and 193.4+59.0 % one month after in the control, while 55.0+13.6 % one day after, 102.5+12.9 % one week after and 60.6+14.7 % one month after the operation in the total oophorectomy group. There was a statistically significant difference in the values obtained one month after the operation between the groups (p<0.05). Characteristic changes in platelet volumes were also observed. A significant correlation was observed between the platelet aggre-gabilities and the daily urinary estrogen excretion levels. The above results suggest that estrogen may enhance platelet aggregability in vivo.
Mild Depression of Platelet Count and Altered Platelet Function in Acute Plasmodium Falciparum Infection
Authors: Essien E. M. , Ebhota M. I.
Published: May 2019

We recently examined a possible role and extent of involvement of Plasmodium falciparum para-sitaemia in our earlier report of significantly lower circulating platelet numbers in healthy adult Nigerians (100 – 300x 103/ul) compared with matched Caucasians (150 – 140 χ 103/ml) ; (P<. 0.001), and in view of recently reported association of Plasmodium parasitaemia with thrombocytopaenia, consumptive coagulopathy (DIC) and other haemorrhagic syndromes. Ninety-eight febrile children aged between 8 months and 10 years whose fever was attributed to malaria parasitaemia only were admitted into the study. Platelet count, platelet aggregation, Factor VIII and FDP were determined on each blood sample which was collected before treatment was started. Tests were repeated 10-Hdays later. It was found that the mean platelet count of 132,000+61, 620/ul (1SD) during illness and immediately after treatment was significantly lower than the count of 234, 400+98, 480/ul, 10–14 days later, (t = 6.496 ; P<c 0.001 ). Significant thrombo cy topa emia (Platelets 70,000/ul) was observed only in Gfo of the subjects and none had any haemorrhagic symptoms. The effect was independent of age or degree of parasitaemia. Pre- and post-treatment leucocyte counts were similarly different (7952+2043 vs. 9221+3071 t = −2.81; P<0.05). Haematocrit values did not change significantly. However, in the response to platelet aggregating agents, it was found that the second wave of ADP-induced aggregation was regularly abolished during the parasitaemia.

It is concluded that in hyperendemic Plasmodium falciparum environment, the malaria may often lead to mild depression of platelet count but that severe thrombocytopaenia is uncommon. The platelets may not be optionally functional.
Plasma ADPase Activity in Myocardial Ischemia
Authors: Heyns A. duP. , Badenhorst C. J. , Retief F. P. , KIeynhans P. H. T. , Potgieter G. M.
Published: May 2019

Adenine nucleotides play a key role in platelet aggregation and in the autoregulation of myocardial perfusion. Low Km ADPase activity in plasma and accumulation of ADP catabolic products were investigated in patients with myocardial ischaemia and necrosis.

ADPase activity was assayed by incubation of 2μM 14C-ADP in plasma at 37°C. Enzymatic reactions were terminated by heat, radio-active nucleotides separated by high voltage electrophoresis and quantitated. ADPase activity was expressed as pmol 14C-ADP catabolized per minute as assayed by two point substrate concentration analysis.

14C-ADP was catabolized in a single exponential manner. The breakdown products AMP, adenosine, inosine and hypoxanthine were detected. Mean ADPase activity, of 25 normal males was 102+ SD 15 and 15 normal females 85+SD 13. In the immediate postinfarction period 25 patients had a significantly (P<0,01) increased plasma ADPase : 127 + 40. ADPase was also significantly increased in the patients with crescendo angina (mean 131 + SD 42) (P<0,01). ADPase levels were higher in the coronary sinus than in peripheral blood of the same subject. In the rat heart >80% of total ADPase activity was located in the cytosol fraction after differential centrifugation of whole heart homogenate.

Low Km ADPase activity is increased in myocardial ischaemia. The enzyme is released in anoxia. It thus may be a measurement of the well known metabolic pathway leading to adenosine accumulation in ischaemic heart muscle.
Selective Inhibition of Platelet Agglutination and Aggregation by Aromatic Amidino Compounds
Authors: Geratz J. D. , Tidwell R. R. , Brinkhous K. M. , Mohammad S. F.
Published: May 2019

A series of aromatic amidino compounds were investigated for their inhibitory effect on platelet agglutination and platelet aggregation. Agglutination of fresh or fixed human 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-hydroxybenzene)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.

This investigation has led to the identification of new specific inhibitors of platelet agglutination and aggregation which can serve an important role in future studies of the two processes. The exact mode of interaction between ami dines and platelets is still being explored.. In the case of agglutination, inhibition most likely occurred at the level of binding of ristocetin cofactor to the platelet membrane. In the case of aggregation, however, interference could have taken place at the membranes or in the cytoplasm and could have been enzymatic or non-enzymatic in nature.
Platelet Coagulant Activities in Metrizamide Gradient Platelets : Application to Bernard-Soulier and Thrombotic Disorders
Authors: Levy-Toledano S. , Dmoszynska A. , Baume H. de La , Dupuy E. , Caen J.
Published: May 2019

A new technique for platelet isolation from five ml of total blood on metrizamide gradients has been applied to the study of platelet coagulant activities.” Assay methods have been described : contact product forming activity (CPFA), collagen induced coagulant activity (CICA) and platelet factor three (PF3) have been measured in the metrizamide gradient platelets (MGP) and found similar to that of platelets in platelet richplasma (PRP).

Platelet coagulant activities of the MGP have been measured in patients with hemorragie tendencies like Bernard-Soulier (B-S) patients or those with thrombotic tendencies including diabetics with or without vascular complications or patients with transient ischemic attacks (TIA). CICA was not found in. all the 3 patients with B-S syndrome. 3 of the 10 diabetics showed an increased CPFA activity while 5 showed an increased CICA activity contrasting with the normal activities found in diabetics without vascular complications ; PF3 activity is increased in the two groups of diabetics. 3 patients of the 7 with TIA had an increased CPFA as well CICA.

The results suggest that these activities may play some role in diabetics with vascular complications and in some cases with TIA.
Inhibition of the Platelet Function by the Effect of Plasmin on Human Factor VIII
Authors: Castillo R. , Maragall S. , Martín E. , Argelagués E. , Qrdinas A.
Published: May 2019

Plasmin-treated human purified factor VIII prepared from normal and afibrinogenemia plasmas inhibits the ADP-induced aggregation and the collagen-induced 14C serotonin release of normal and von Willebrand human platelet rich plasmas (PRP). These results reconfirm the fact that the inhibition of the human platelet function by the effect of plasmin on the plasmatic protein is mainly linked to the digested factor VIII but not to the fibrinogen,fact that we had previously proved when ADP-induced aggregation of normal washed platelets was inhibited by plasmin-treated normal human serum,but not by plasmin-treated von Willebrand plasma. The factor VIII breakdown products were revealed by crossed immunoelectrophoresis,sepharose 4B chromatography and SDS Polyacrylamide electrophoresis.

The defective ADP-induced aggregation that we have observed in PRP from patients between the 8th and the 24th hour of thrombolithic treatment,is only due to the proteolithic effect of plasmin on the plasmatic factor VIII and not to a direct action of the plasmin on the platelet membrane,since washed platelets from these patients aggregate normally by ADP with normal platelet poor plasma(PPP),and the collagen-induced 14C serotonin release is also correct.
Automated Determination of Platelet Aggregation by Continuous Flow System
Authors: Yamanaka M. , Naiki S. , Kaku Y.
Published: May 2019

In general, the determination of aggregation of platelet is performed by aggregometer in which platelet rich plasma (PRP) is agitated mechanically by stirrer in the limited space of tube, and almost all aggregometer has a low ability to perform the tests of many samples as a routine test at the clinical laboratory. To solve the problems of the effect of stirrer and the measurement in the unphysiological condition, we have devised a new method of measurement using the continuous flow system, which is designed finally to determine successively 30 samples per hour. In this system, PRP is passed through only the continuous glass tube partly coiled. One ml. of PRP to be tested is sucked into the tube at a speed of 1 ml. per minute and flowed in the continuous tube line. Solution of adenosine diphosphate (ADP) or physiological saline is simultaneously sucked at a speed of 0.25 ml. per minute and jeted into a flow of PRP at the joint and mixed by coiling tube. The changes in transmittance of PRP during its flow are measured photometrically with the detectors attached to the straight parts of tube at the points arbitrarily settled. An excellent reproducibility of each detector and linearity against the concentrations of platelet in PRP were obtained in this system. Degree of changes in transmittance obtained at the point 20 seconds after an addition of ADP in this system correlated with the maximum aggregation rate of ADP-induced aggregation in aggregometer. Spontaneous aggregation which could not be detected with ordinary aggregometer was obviously observed in this system. These results suggested that this new method might be useful to detect the spontaneous aggregation.
Methylated Type I Collagen as a New Tool for Platelet Function Tests
Authors: Balleisen L. , Kühn K. , Marx R.
Published: May 2019

Methylated type I collagen with blocked carboxylic groups was used for testing platelet aggregation and adhesion on collagen, andas thrombogenic substance in experimental animals. This modified collagen is soluble in buffers at neutral pH, but when added to plasma immediately precipitates. Its platelet aggregation activity as tested by the inhibitory action of ASS and by the depression or absence of collagen induced platelet aggregation in uremia and thrombasthenia was comparable to fibrillar collagen. Preincubation of platelets with Antimycin A and 2-Deoxyglucose also inhibited platelet aggregation by meth. collagen. In a new test for measuring platelet adhesion on collagen, platelets and meth. collagen were rotated in a silastic tube; the decrease of platelets was determined. Furthermore, meth. collagen can be used as a thrombogenic substance in rabbits: The recorded decrease of platelets after injection of meth. collagen can be inhibited by pre-treatment with ASS. Summarizing, meth. collagen, which can be stored over one year in lyophilised or frozen form without a decrease of activity, is a substance with the biochemical characteristics of collagen, which allows handling as a dissolved collagen but behaves like a fibrillar collagen.
Amino Acid Sequence Studies on the γ-Chain of Human Fibrinogen
Authors: Bouma H. , Cottrell B. A. , Friezner S. J. , Takagi T. , Doolittle R. F.
Published: May 2019

The γ-chain of human fibrinogen contains 400±10 residues, eight of which are methionines. As such, we have isolated and characterized nine cyanogen bromide peptides. The alignment of these fragments was attained by the isolation of key overlap peptides derived from the tryptic digestion of citraconylated γ-chains and succinylated and aminoethylated γ-chains. In the latter case we synthesized a radioactive aminoethylating agent which was especially useful in isolating all the cysteine-containing peptides. The amino acid sequence of most of these fragments has been accomplished by a variety of procedures. In general, our results are in harmony with those recently reported by Henschen et al. (Hoppe-Seyler’s Z. Physiol. Chem., 357, 605, 1976), although some differences exist.

The most interesting structural features revealed by the sequence data include significant homologies with the α- and β-chains, especially with regard to the arrangement of certain key cysteine residues. Thus, the sequence at residues γ 135–139 is Cys-Gln-Glu-Pro-Cys, in striking parallel with the sequence at residues γ 19–23, which is Cys-Pro-Thr-Thr-Cys. The occurrence of similar pentapeptide skeins in the α- and β-chains, in each case separated by about the same number of residues, has both structural and evolutionary connotations.
8-Hour Radioimmunoassay for Fibrinopeptide A (FPA)
Authors: Hofmann V. , Straub P. W.
Published: May 2019

To allow emergency measurements of FPA in patients,a RIA method was developed in which both the extraction of FPA from plasma and the incubation time of the antigen-antibody mixture were substantially reduced. Extraction with an equilibrium dialyzer allowed a recovery of 54.75 + 1.26% of added FPA (20 ng) within 2½ hrs. At an incubation of 2½ hrs the limit of detection was 1.12 ng/ ml, as compared to 0.54 ng/ml for 24-hr incubation. Rabbits were immunized with synthetic FPA coupled to thyroglobulin with the carbodiimide method. The tracer was produced acc. to Nossel(1971). To preventlabel damage for 10 weeks, the purified radioiodinated peptide was rechromatographed using a QAE-A25 Sephadex column. In 24 normals FPA was 1.01+0.45 ng/ml using 24-hr incubation. For FPA levels above 2 ng/ml the results obtained with the 2½-hr and the 24-hr incubation showed excellent agreement(r=0.99).3.3–25.2 ng/ml were found in 7 patients with venous thromboembolism,a sharp drop to 1.6–5.1 ng/ml being observed 10 minutes after50 mg of heparin i. v.2.3~32 ng/ml were found in 15 patients with various disorders(4 malignancy, 4 aorticaneurysm,2 nephropathy, 2 anginal attack, 1septicemia, 1 lung abscess, 1 CVI ) . Values of 3.6–80 ng/ml were found after infusion of a prothrombin complex concentrate in 5 patients with hemophilia B. The method is valuable for the emergency coagulation work-up of patients.
Activation Studies on a Human Prothrombin Variant: Prothrombin Barcelona
Authors: Benarous R. , Rabiet M.J. , Labie D. , Josso F.
Published: May 2019

Prothrombin Barcelona tested in the plasma from homozygous patients is characterized by a functional defect (5% activity by a one-stage assay) contrasting with a normal prothrombin concentration found by immunochemical assay.

This low biological activity is the expression of a very slow rate of conversion of prothrombin into thrombin. It may be postulated that the abnormal kinetics of conversion of this variant might involve an inhibition of one of the proteolytic cleavages responsible for the activation process. To test this hypothesis, purified normal and abnormal prothrombins were submitted to proteolysis with factor Xa and thrombin, either with or without protease inhibitors. Prothrombin cleavage was checked at various times by SDS gel electrophoresis.

In the presence of factor Xa, conversion of normal prothrombin into intermediate II started within 30 min and was almost complete within two hours. In the same conditions, conversion into intermediate II of prothrombin Barcelona was only initiated after two hours. Conversely upon treatment of both prothrombins with thrombin, a slight delay in the formation of intermediate I was observed with prothrombin Barcelona, this formation being nevertheless complete after one hr.

In conclusion, the abnormal activation of prothrombin Barcelona seems mainly due to a decreased and delayed formation of intermediate II.