bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet Membrane Components Involved in Adhesion to Collagen Fibers
Authors: Lahav J.
Published: May 2019

In an attempt to identify platelet membrane components involved in collagen recognition, rabbit platelets suspended in medium containing ImM EGTA were passed through sepharose columns containing immobilized collagen fihers. The percent of platelets retained was proportional to the amount of collagen on the column but independent of the platelet concentration applied. Platelets that did not adhere in a single passage were still capable of adhering. Saturation of the column was not achieved. The adhering platelets were lysed with 0.5% triton x-100 and the proteins retained on the collagen were solubilized in 0.3% SDS. Polyacrylaminde gel electrophoresis revealed at least 4 bands in the SDS fraction (and in whole platelets solubilized in SDS) that were absent from the triton lysate. None of these bands was of the major glycoproteins. Iodination of the platelet membranes did not interfere with their adhesion to the column, when iodinated platelets were subjected to the treatment described above, the SDS fraction contained one iodinated band not present in the triton lysate and another markedly enriched relative to its amount in the lysate. It is suggested that more than one platelet membrane component, or a complex consisting of several different components, is involved in platelet adhesion to collagen fibers.
The Activation of Bovine Factor X by Factors IXa and VIII.
Authors: Neal G.G. , Chavin S.I.
Published: May 2019

The esterase activity of bovine factor xa on the synthetic substrate α-N-benzoyl-L-arginineethyl ester can be used to follow the activation of factor X by the intrinsic pathway of coagulation. Bovine factor IXa activates factor X in a reaction having an absolute requirement for calcium ions, but little affected by phospholipid. At a molar ratio of factor IXa to factor X of 1:10, 3% of the factor X is activated after five minutes incubation at 37°C; the inclusion of bovine factor VIII in the incubation mixture results in 35% activation in the same period. Factor VIII cannot activate factor X in the absence of factor IXa. There is not an absolute requirement for phospholipid in the interaction of factors X, IXa and VIII, but the addition of crude cephalin further accelerates the rate of factor X activation. Hirudin does not affect the interaction of factors IXa and X in the absence of factor VIII, but does reduce the stimulatory effect of factor VIII on the rate of formation of factor Xa by a factor of between two and four. As hirudin specifically inhibits thrombin, this effect on factor X activation is likely to reflect the presence of trace amounts of thrombin in the incubation mixtures. The incomplete inhibition by hirudin of the effect of factor VIII on factor X activation suggests that ‘activation’ of factor VIII by thrombin cannot be a prerequisite for its interaction with factors IXa and X.
Immunchemical Investigation on Antithrombin III/AT-III/in Presence of Heparin
Authors: Bánuergyi D. , Sas G.
Published: May 2019

Various components of AT-III car, be differentiated by crossed ioununo-eleetrophoresis in the presence of heparin as we described previously / Sas et al. 1975 /. The migration of heparin is discontinuous in this original method, i.e. at the end of the electrophoresis the plate does not contain heparin at all. This fact rises the possibility pf the artefactual nature of these AT-III components. To exclude this effect we introduced a modification using double plates incorporated with heparin. The second plate was used as a heparin “reservoir”. By this modification we obtained constant heparin flow in the course of electrophoresis. We found four different AT-III components in normal plasma comparing with three AT-III eomportints of the original method.

Investigating heparins originated from different sources we found a higher mobility of AT-III in the pnsenee of mucous heparin than that of lung heparin. This observation corresponds well with the results obtamed by heparin-agarose affinity chromatography.
Drug Treatment in the Prevention of Coronary Artery Disease
Authors: Mitchell J.R.A.
Published: May 2019

The disappointing performance of anticoagulants in the prophylaxis of coronary heart disease led to the realisation that components other than fibrin play a major part in the structure of arterial thrombi. Attention has therefore been focussed on the possible role of agents which modify platelet behaviour. Novel agents which alter thromboxane synthesis will not be available for large-scale clinical trials for some years, so the present trials are assessing the value of platelet-modifying agents which are already in use for other purposes. The implications of the Antura-Reinfarction study and the role of aspirin and persantin will be discussed.

Attention will also be drawn to the importance of using valid end-points to assess potential anti-thrombotic regimes in coronary disease. The differential implications of using infarction, sudden death, pump failure, dysrhythmias and re-infarction as end-points in trials will be described.
Prostacyclin: Production by Vascular Endothelium and Effects on Platelet Aggregation
Authors: Moncada S. , Bunting S.
Published: May 2019

The inhibitory effect of vascular endothelial cells on platelet aggregation is due to their ability to release prostacyclin. The existence of an ADPase has been confirmed in endothelial cells but this enzymes does not seem to be related to the anti-aggregating properties of vascular endothelium. In vitro, the release of prostacyclin by humand and rabbit endothelial cells persists after several subcultures. The production of PGI2 can be demonstrated by its inhibition by aspirin-like drugs or 15-hydroperoxy arachidonic acid (a specific inhibitor of PGI2 synthesis). Moreover, the antiaggregating activity is antagonised by an antibody to 5,6 dihydro prostacyclin which cross reacts and neutralises prostacyclin.
Structure of the Antithrombin III-Binding site in Heparin
Authors: Lindahl U. , Bäckström G. , Höök N. , Riesenfeld J. , Thunberg L. , Fransson L.-A. , Linker A.
Published: May 2019

Fragments with high affinity for antithrombin III (AT), composed of 12 to 16 monosaccharide units, were isolated from heparin after partial chemical or enzymatic depolymerization of the polysaccharide. Analysis of such fragments based on identification ot deamination products suggested that nonsulfated L-iduronic acid (a minor constituent) is essential for the anticoagulant activity of heparin. The location of this unit in the AT-binding sequence was determined by periodate oxidation. Furthermore, an N-sulfate group essential for activity was located by structural analysis of partially N-desulfated fragments retaining high affinity for AT. It is proposed that the AT-hinding sequence in heparin has a variable structure containing certain nonvanable regions. A tentative structure for this sequence is presented, with indication of identified constant and variable regions.

(Supported by grant No. 2309 from the Swedish Medical Research Council).
Aggregation of Platelet Membrane Vesicles by ADP
Authors: Watanabe K. , Yamamoto M. , Ando Y. , Ikeda Y. , Toyama K. , Deguchi N.
Published: May 2019

The study was performed to examine biochemical and biophysical changes in platelet membrane vesicles(PMV) when aggregated by ADP. PMV was isolated from normal donors by using glycerol lysis technique, and was suspended in 10mM Tris, 0.15M NaCl buffer, pH 7,4. Aggregation of PMV was readily observed under phasecontrastmlcroscopy within 3 min after addition of ADP(5mM), while AMP, UDF or GDP was without effect. Light scattering Intensity of PMV suspension determined by Lasernepherometer increased with addition of ADPCJmM), reaching plateau within 2 to 3 min, also indicating that vesicles were rapidly aggregating in suspending medium. In electronmicroscopic observation, amorphous floccuent materials were seen between aggregated PMV by ADP. This PMV aggregation by ADP was inhibited by the treatment of PMV with either neuraminidase(0.1u/ml) or trypsin(20μg/ml) and was hardly observed in PMV from a patient with thrombasthenia.

The addition of ADF(10mM) to PMV suspension resulted in the reduction in electronhoretic mobility of PW(0.301±0.057μ·cm/sec·V, control ;1.194±0.037) and increase in membrane microviscositv (p value; 0.277, control; 0.255).

Changes in protein profiles in aggregated PMV were examined by SDS Polyacrylamide gel disc electrophoresis. One protein band(M.W. 200,000) disappeared and instead, there observed increased coomassie blue staining intensity at the top of the gel.

The results suggest that these biochemical and biophysical changes in PMV may in part explain the mechanisms of platelet aggregation By ADP.
Effects of L-Enantiomers of Analogues of Adenosine, AMP and ADF on Human Platelets
Authors: Cusack N.J. , Hickman M.E. , Bom G.V.R.
Published: May 2019

The aggregation of human platelets by ADP and the inhibiting effect of adenosine are apparently mediated by different receptors on the external platelet membrane (R.J. Haslan and G.M. Rosson, 1975, Molec. Pharmacol., 11, 528). One of the criteria for receptors is that they show stereospecificity for their ligarais. We therefore synthesised the unnatural L-enantiomers of adenosine and ADP, 2-chloroadenosine and 2-chloro-ADP, and photolysable 2-azidoadenosine and 2-azido-ADP to test platelet receptor specificity. None of the L-enantiomers of ADP aggregated platelets or prevented ADP itself from aggregated platelets. L-enantiomers of adenosine, 2-chloroadenosine and 2-azidoadenosine did not inhibit ADP induced platelet aggregation, nor did they cause increases in levels of platelet cAMP. L-enantiamers of adenosine and ADP and analogues are proposed as suitab: controls for adenosine and ADP binding studies, and for the estimation of nonspecific labelling; in affinity labelling experiments.
Formation of Thrombin-Antithrombin Complexes in Congenital at III Deficiency Study of 3 Cases.
Authors: Boyer C. , Wolf M. , Larrieu M.J.
Published: May 2019

Congenital AT III deficiency was observed in three members from the same family. One of the patients (28 y, female) demonstrated recurrent venous thrombosis and pulmonary embolism. Comparative values of AT III (35 to 40%) were observed in the three cases by enzymatic assay using a chromogenic substrate (S-2238) and by rocket immunoelectrophoresis with a monospecific anti-AT III antiserum. By crossed immunoe1ectrophoresis the migration of AT III was normal in presence or absence of heparin. The formation of thrombin-AT III (T-AT) complexes was studied during coagulation of whole blood or recalcified plasma sampled at various intervals. Using crossed immunoe1ectrophores is in presence of heparin, patients’ AT III was entirely complexed in the 20 minutes sample from whole blood whereas the major part of AT III remained free in the controls. The appearance of T-AT was already demonstrated in patients’ recalcified plasma after 4 minutes whereas no complexes were formed in the controls’ 6 minutes samples. The early formation of T-AT was confirmed by latex agglutination using anti-T-AT neoan mean antiserum prepared by D. Co11en. In the 20 minutes sample, a significant higher titer was found in patients than in controls. The fast T-AT formation and disap peara nce of AT III in congenital AT III deficiency may be of relevance for the occurrence of thrombosis in such cases.
Heparin-Affinity of Antithrombin III in a Family With Congenital Antithrombin III Deficiency
Authors: Sas G. , Bánhegyi D. , Petö I.
Published: May 2019

We found a new thrombophilic family with antithrombin III/ AT-III / deficiency. In the members of this family both immunologic and functional methods revealed similarly decreased levels of AT-III. Gelfiltration displayed identical size of AT-III molecule of patient and normal alike. On the basis of tnese findings we assumed that in this family normal AT-III is produced Dut only in diminished quantity. Experiments on the heparin-affinity of AT-III did not support this assumption. The AT-III of the proposita migrated slower than that of normal person in the heparinized agarose gel. In the course of the heparin-affinity chromatography the AT-III of the proposita could be eluted at lower salt concentrations than normal AT-III.

Thus we conclude that even in the case of “true” AT-III deficiency the molecule might have some qualitative deviation from normal.
Binding of Myeloma Monoclonal Immunoglobulin with Fibrinogen and Fibrin
Authors: Budzynski Andrei Z. , Olexa Stephanie A. , Pandya Bharat V.
Published: May 2019

Blood and plasma clots are frequently abnormal in patients with multiple myeloma a evidenced by the prolongation of clotting time, formation of bulky and gelatinous cloes and inhibition of clos retraction. It has been demonstrated in several multiple myeloma cases that the isolated immunoglobulin inhibited clot formation in normal plasma and decreased the rate of fibrin polymerization. In this work a hypothesis was tested whether these phenomena originate from binding of multiple myeloma immunoglobulin with fibrinogen and fibrin. Blood was obtained from a patient (J.I.) having IgAH gammopath; with prolonged clotting time and abnormal clot retraction. Washed plasma clots contaii ed largo amount of monoclonal IgAλ demonstrated by precipitation with anti-α or anti-λ antibodies and by the presence of heavy (65,000) and light (25,000) polypeptide chains. This myeloma IgA was incorporated into clots regardless of crosslinking and was extract able. Affinity chromatography of serum or heated plasma on insolubilized fibrin monoment resulted in recovery of large amount of myeloma monoclonal IgA, but not IgG, IgH or Igl. The isolated fibrin-specific IgA was taken up by clots obtained from normal plasma or purified fibrinogen, lnso]ubilized fibrinogen bound myeloma monoclonal IgA From plasma. Two-dimensional agarose/polyacrylamide slab gel electrophoresis demonstrated the presen of a fibrinogen-IgA complex in patient’s plasma. The results indicate that a myeloma monoclonal immunoglobulin binds with fibrinogen and fibrin, probably forming an antibot antigen complex, implying the Formation of autoantibodies against these proteins.
Differentiation Between the Plasma Inhibitor of Plasminogen Activation and an Inhibitor of the Amidolytic Activity of Urokinas
Authors: Gallimore M.J. , Fareid E. , Hedner U. , Martinsson G.
Published: May 2019

A plasma component which inhibits the amidolytic activity of urokinase (UKI)has been found in human plasma and partially purified. An inhibitoin of plasminogen activation(PAI) has also been identified and partially purified. Both proteins have similar physical properties(migrate as α2-globulins on agar gel electrophoresis and have molecular weights of approximately 75.000). As the partially purified UKI preparation cross-reacted immunochemically with PAI antiserumit was initially thought that the two inhibitors were the same. However when PAI was removed from parti ally purified UKI no loss of urokinase inhibition in an amidolytic assay as observed and a preparation of PAI failed to produce inhibition in thiassay. PAI has been found to inhibit plasminogen activation in clot l”sis and other assays and binds to active Hageman factor. Initial results with UKI indicate that it does not inhibit plasminogen activation and appears to act as a competitive substrate in the amidolytic assay. Both PAI and UKI failed to bind I125-labelled urokinase. It is therefore concluded tha UKI and PAI are different.
High Resolution Two Dimensional Gel Electrophoresis of Surface Labelled Normal and Thrombasthenic Platelets
Authors: McGregor J.L. , Clemetson K.J. , James E. , Dechavanne M.
Published: May 2019

Glycoproteins and proteins on the surface of human platelets were labelled by techniques specific for sugar or protein moieties. Such labelled samples were separated by a high resolution two dimensional gel technique (O’Farell’s technique). Platelets were surface-labelled with sodium periodate + NaB3H4, galactose oxidase + NaB3H, neuraminidase + galactose oxidase + NaB3H4 and laetoperoxidase 125I. Comparison with one dimensional separation (Laemmli system) allowed the identification of the major glycoproteins (Ia, Ib, IIb, IIIa, IIIb). In general the labelling patterns were very similar to those obtained with stainning techniques. Following neuraminidase treatment glycoproteins lb and lib moved to more basic pH regions while the others were largely unaffected. Analysis of platelet membranes from patients with Glanzmann’s thrombasthenia by these techniques showed the absence of glycoproteins lib and Ilia or their presence in greatly reduced concentrations. In addition both glycoproteins lb and lllb were much more basic than in normals and appeared to have a reduced amount of sialic acid. The use of surface labelling and two dimensional electrophoresis provides additional evidence on membrane defects of Glanzmann’s thrombasthenic platelets.
Platelet Functional Defect Causing Glazmann’s Thrombasthenia
Authors: Gartner T. K. , Phillips D. R. , Gerrard J. M. , White J. G.
Published: May 2019

Clanzmann’s thrombasthenia is an inherited bleeding disorder characterized by an absence of two membrane glycoproteins and by defective platelet aggregation. This defect appears to be restricted to an inability of the platelets to interact since their other functions are normal. We previously showed that platelets generate lectin activity in response to thrombin and that this lectin appears to mediate at least the initial phases of platelet aggregation by specifically binding to receptors on adjacent platelets. The lectin and lectin receptor activities of thrombasthenic platelets were characterized to determine it either function was missing. Control and thrombasthenic platelets were washed and the lectin activity determined by their ability to agglutinate either bovine erythrocytes or formaldehyde fixed human platelets. The lectin activities of normal and thrombasthenic platelets were Indistinguishable. Normal and thrombasthenic platelets were fixed with formaldehyde to facilitate the independent assay of lectin receptor activity. The fixed platelets lacked platelet lectin activity. The control fixed platelets had normal lectin receptor activity. Fixed thrombasthenic platelets, however, lacked platelet lectin receptor function in our aasay. Therefore thrombasthenic platelets fail to aggregate because they lack the platelet lectin receptor function.
Thrombasthenia: A Model for Studying Platelet Activation in the Absence of Aggregation
Authors: Levy-Toledano S. , Maclouf J. , Hardisty R.M. , Caen J.P.
Published: May 2019

In response to the ionophore A 23 187, thrombasthenic platelets undergo normal shape charge, serotonin release and metabolism of 14C-arachidonic acid (including liberation from prelabelled platelet phospholipids and conversion to thromboxane B2 and HETE). These responses, using either PRP or washed platelets, are accompanied by a characteristic optical density (OD) change in the absence of aggregation. This change was associated with the formation of a central contractile gel mass and central apposition of organelles, but lactic dehydrogenase determinations showed no evidence of platelet lysis. These reisuits can be mimicked by stirring A 23 187 with normal platelets in the presence of EOTA or EGTA. The OD change in this system was inhibited by chlorpromazine, propranolol and other drugs which interfere with membrane transport of Ca2+ but not by colchicine, cyto-, chalasin B, prostaglandin Ej, creatine phosphate/creatine Phosphokinase or antimycin and deoxyglucose.

These findings show that the action of ionophore on thrombasthenic platelets provides a model for dissociating platelet thromboxane formation, contraction and secretion, all of which are dependent on intracellular calcium shifts, from aggregation, for which extracellular calcium is required; they suggest that the underlying membrane glycoprotein abnormality in thrombasthenia may result in a failure to react with extracellular calcium.
Effect of Sulphinpyrazone (SP) Aspirin (ASA) and Dipyridamole (DP) on Plateletvessel Wall Interaction after Oral Administration to Rabbits
Authors: Davies J.A. , Menys V.C.
Published: May 2019

Clinical trials of anti-platelet drugs have suggested that they may be useful in the prevention of thrombotic disease. While such drugs inhibit platelet function, those which act on cyclooxygenase also reduce PGI2 synthesis and may interfere with tne natural antithrombotic properties of the vessel wall. We studied the effects of SP, ASA and OP ex vivo on the platelet-vessel wall interaction. Rabbits were dosed by mouth with drug (at about twice the weight-adjusted human dose) or placebo for 5 days, then exsanguinated and aortas removed. Washed platelets prepared from the blood were labelled with 51Cr. and their adhesion to everted aortapr epared from treated or control rabbits was measured in a perfusion device. PGI2-like activity in aortic rings was assayed by its inhibitory effect on platelet aggregation to ADP. Adhesion of platelets to aort as from SP- treated rabbits was i ncreased (p < 0.025), PGI2 - like activity was partially inhibited, but over all adhesion of SP-treated platelets to aor tas f rom SP-treated animals reduced by 30% (p < 0.02). Adhesion to aortas of ASA- treated rabbits was sliahtly inc r ea=-.ed (p > 0 . 1) , PGI 2 - l ike act ivi ty abolished , and no overall reduc tion in platelet adhesion seen. DP had no effecton adhesion or PGI-like activity. These results support the evidence that cyclo-oxygenase inhibitors reduce the inherent resistance of the vessel wall to platelet adhesion. However with SP, inhibitory effects on platelets appear to be more important.
Removal of Potentially Thrombogenic Material from a Concentrate of Factors II, IX & X by Polyethylene Glycol Precipitation
Authors: Foster P. R. , Patterson M. R. , Johnson A. J. , Middleton S. M.
Published: May 2019

Polyethylene glycol(PEG) precipitation has beer used for the removal of hepatitis B surface antigen (HBsAg) from a concentrate of factors II, IX & X of intermediate purity. HBsAg is precipitated into a fraction PI at 20% (w/v) PEG and pH 6.5 while factors II, IX & X remaining in the supernatant are subsequently precipitated at 30% (w/v) PEG and pH. 5.2

These various fractions have been assessed for the presence of potentially thrombogenic materials by in vitro tests. In comparison with the starting material the precipitate rich In factors II, IX & X has a significantly increased non-activated partial thromboplastin time (NAPTT) and a decreased rate of factor Xa generation. By contrast the waste fraction (PI) exhibits a significantly decreased NAPTT and an increased rate of factor Xa generation suggesting that potentially thrombogenic material has been selectively precipitated. This procedure has been used to prepare a factor II, IX & X concentrate containing 100 U FIX/ml with reduced contamination by both HBsAg and thrombogenic materials.
A Platelet-Derived Factor Chemotactic for Rabbit Arterial Smooth Muscle Cells
Authors: Ihnatowycz I.O. , Winocour P.D. , Moore S.
Published: May 2019

Following endothelial injury intimal proliferation of smooth muscle cells (SMC) is preceeded by migration of cells from the media. Factors in serum and/or released from plat lets were examined for their ability to induce directed migration of SMC across a gelatin coated polycarbonate filter in a modified Boyden chamber. A suspension of SMC in serumf ree Eagle is medium was placed in the upper chamber and the test material in the bottom chamber. After a 6 hour incubation at 37°C the cells on the lower side of the filter were counted. Washed platelets were freeze thawed or exposed to thrombin or collagen, centrifuged, and the supernatant heat inactivated and dialysed. There was slight migration in response to platelet poor plasma serum. Whole blood serum, platelet rich plasma serum and the platelet sup ernatant (PSN) caused significantly more migration. Migration did not occur when PSN was placed in the top chamber. The amount of chemotaxis caused by PSN from thrombin or collagen treated platelets was dose dependent and independent of plasma factors. Indomethacin (20μM) or sulfinpyrazone (lmM) significantly reduced the release of he chemotactic factor from collagen treated, but not from thrombin treated platelets. PSN of a denosine diphosphate treated platelets, Tyrode or medium alone caused no migration. Thus platelets release a nondialysable, heat stable chemotacti c factor. Release does not dept end on primary aggregation but is associated with the release reaction and is independent of the endoperoxide pathway. Supported by Medical Research Council, Canada MT2168.
Polymorphism of Platelet Secretory Proteins and Their Mitogenic Activity
Authors: Niewiarowski S. , Paul D. , Rucinski B. , Varma K.G. , James P. , Walz D.A.
Published: May 2019

Three low molecular weight proteins: β-thromboglobulin (βTG), low affinity plalelet factor 4 (LA-FF4) and platelet basic protein (PBP) have been purified from the material secreted by human platelets stimulated by lonophore A231S7. The preparative steps included heating at 100°C for 5 min, isoelectric focusing (pH gradient 3.5-10.9 in sucrose) and affinity chromatography on hepar in-agarose (elution with 0.4-0.5M NaCl). BTG, LA-PF4 and PBP showed complete immunological identity using anti-LA-PFi, antibody. Their respective isoelectric points were at pH 7.0, 8.0 and 10.9. Purity was confirmed by NH2 terminal amino acid analysis (βTG; gly; U1-PF4 asp; PBP: gly) and electrophoresis. At the NH2 terminal end LA-PF4, had four additional amino acids (Asn-Leu-Ala-Lys) while residues 5-21 of LA-PF4 and 1-16 of βTG were identical. As determined by Isoelectric focusing and specific radioimmunoassay the proportions of βTG, LA-PF4 and PBP released from platelets were 70%, 14% and 16%, respectively. Evidence is presented that LA-PF4 is a nrotein originally secreted by human platelets and converted to STG by a heat labile protease associated with the platelets. The three proteins Initiated DMA-synthesis in serum-arrested Swiss 3T3 cells in 0.4% serum. Activity of PBP was observed below 1 ng/ml and saturated at 50 ng/ml. LA-PP4 and βTG were 10 or 40 fold less active, respectively.
Human Albumin Radio-Immumo Assay
Authors: Forestier F. , Guennec J.Y. , Solé Y. , Pelage A. , Tallobre P.
Published: May 2019

Knowing the importance of carrier proteins in platelet function and especially the role of albumin in prostaglandin metabolism we have established a radio-immuno assay (RIA) for quantifying the platelet albumin content. We used a double antibody RIA. The first one is raised in rabbit againat human albumin (Behring Ref.ORCB0405 and used at a dilution of 1/80.000. The second antibody is against rabbit IgG and is filed on to cellulose. Highly purified human albumin (Kabl Vitrum Ref. 58801) is. labelled with I125/chloramine T. Under our experimental conditions the specific activity is T. μCi/μg. The labelled protein is purified by Polyacrylamide gel electrophoresis (8,4%). The incubation medium contains 0.5% bovine albuain in a 0,05 M phosphate buffer (pH 7.5). By using outcherlouy plates, the anti-human albumin antibody did not interact with bovine serum albumin. After an incubation of 24-36 hours at room temperature the iamiuno-adsorbent was added. The tubes were then shaksd on rotary mirer for one hour, centrifugea and the precipitate was washed twice. This method allowed us to detect amounts of albumin in nanogranma quanti-ties. Platelets were pretreated with inhibitors of release (PGE1, Theophylline and EDTA), washed with Tris/EDTA buffer and solubilised with Triton X 100. Platelets prepared in this way contained approxinativel 1 mg of albumin/109 platelets. Zonal ul tracen trifuga-tion showed that the albumin was localised to the non lysosomal α granules and studies showed that the release of albumin occurred at the same times as the release of fThrombo-Globulin and Platelet Fee tor 4.
Inhibitory Activity of Foy on Thrombin and Activated Factor X.
Authors: Kōsakl G. , Ohno H. , Imaoka S. , Hirata F.
Published: May 2019

When hypercoagulable state prolongs, DIC will develop regardless of the presence of naturally circulating anticoagulants. And heparin has been used In order to reinforce them in the treatment and prevention of DIC. The mechanism of anticoagulant action of FOY [Ethyl-p-(6-guanldlnohexanoyloxy)benzoate methanesulfonate] was Investigated, since It was reported that it has anticoagulant activity and that it is very effective in the treatment of DIC.

POY was found to possess inhibitory effect on hydrolytic activity of thrombin on synthetic substrate as well as that of activated factor X, plasmin and plasma kallikrein. The mode of action was competitive and the strongest inhibition was against plasma kallikrein (Ki:0.2μM), secondly to thrombin(0.97μM), thirdly to plasmln(1.6μM) and then to activated factor X(8.5μM), judging from their Ki values. The inhibitory effect of FOY in coagulant activity of thrombin on fibrinogen was stronger than that against fibrlnolyi » activity of plasmin. Synergetic effect of FOY with AT III was then studied. FOY was found to inhibit residual activity of thrombin or activated factor X which was not neutralized by AT III and heparin. It was postulated that the inhibitory action of FOY was not affected by the presence of AT III and heparin, since Km values of thrombin and activated factor X and Ki value of FOY were not altered in the oresence of AT III and heoarin.
Template Bleeding Time (TBT): Influence of Venostasis and Direction of Incision Upon the Influence of Aspirin (ASA)
Authors: Mielke C.H. , Ramos J. , Rodvien R.
Published: May 2019

It is commonly assumed that because ASA prolongs the TBT, that ASA produces hemostatic impairment. To test this assumption, we assessed the effect of two variables (incision direction and controlled venostasis) on ASA’S prolongation of the TBT in 15 normal subjects. Following the single ingestion of 10 grains of ASA, the TBT was significantly prolonged (p <. 05) up to 72 hrs. only if venostasis at 40mm Hg was maintained and the incision made parallel to the skin creases (SC). If venostasis was maintained and the incision was perpendicular to SC, significant TBT prolongation induced by ASA occurred only up to 24 hrs. TBT’s done without venostasis and with an incision perpendicular to SC never significantly differed from baseline. Collagen-induced aggregation and release of serotonin after ASA ingestion returned to normal first (13 of 15 individuals). Normalization for collagen occurred between 48 and 120 hrs. Epinephrine and then ADP recovery occurred later (up to 216 hrs.). These data suggest that ASA’s effect on TBT, aggregation and release can be exaggerated by laboratory techniques and that ASA’s effect on hemostasis may be minimal in the normal subject.
Platelets Release a New Mediator, Platelet-Activating Factor, Which Accounts for ADP and Thromboxane-Independent Aggregation
Authors: Chignard M. , Le Couedic J.P. , Tencé M. , Benveniste J. , Vargaftig B.B.
Published: May 2019

Platelet aggregation induced by low concentrations of ionophore A23187(I) or thrombin (T) is due to ADP and to metabolites of arachidonic acid(AA) as shown by its inhibition by aspirin and by ADP scavangers. High concentrations of I or T surmount inhibition, thus involving other mediator(s) Platelet-activating factor (PAF)is a 1-lysophospholipid released from macrophages among other cells, in the presence of I. We now show that PAF is released from rabbit platelets during aggregation by I, T and collagen but not by AA nor by PAF itself. Formation and release of PAF by platelets is unaffected by cyclo-oxygenase blockers or by ADP scavengers, but is suppressed by inhibitors of phospholipase A2 activity (dibutyrylcyclic AMP and bromophenacylbromide). Platelet PAF exhibits similar absorption characteristics on silicic acid thin layer and hight pressure chromatography, and sensitivity to N. naja phospholipase A2 as compared to PAF from leukocytes. PAF may be like ADP and thromboxane A2, a Final effector for platelet aggregation and be responsible for the aspirin-resistant third pathway of platelet aggregation.
Prophylaxis for Postoperative Deep Vein Thrombosis. Synergistic Effect of Heparil and Dihydroergotamine
Authors: Kakkar V.V. , Bentley P.G. , Lawrence D. , de Haas H.A. , Ward V.P.
Published: May 2019

Changes in blood coagulation and stasis in the deep vein of the lower limbs are both considered to be important factors in the pathogenesis of deep vein thrombosis (DVT). It is logical to propose, therefore, that prophylaxis against DVT might be better achieved by methods that minimize or eliminate both of these factors Dihydroergotamine (DHE) has been shown to prevent stasis due to its potent venoconstrictor effect on the capacitance vessels of the limb. Randomised clinical trials in 300 patients undergoing major abdominal surgery or hip replacement arthroplasty were performed. The diagnosis of DVT was established by the 125I-fibrinogen uptake test and/or phlebography. Group 1-100 patients, DVT developed in 10 (20%) of 50 p tients receiving DHE compared with 2 (4%) of 50 receivering heparin (p < 0.05). Group 11-100 patients; 3 patients were excluded for various reasons. DVT was detected in 2 (4%) of 48 patients receiving 5000IU of heparin 8 hourly compared to 3 (6%) of 49 patients receiving 2.5000 IU of heparin + 0.5mg of DHE every 8 hourly. Group III-100 patients undergoing total hip replacement, DVT developed in 26 (52%) of 50 patient who received 5000IU of heparin 8 hourly compared to 10 (20%) of 50 patients who received 5000IU of heparin ± 0.5mg of DHE, p < 0.01. A total of 315 blood samples were withdrawn before, during and after total hip replacement. The mean plasma heparin level (±SEM) was 0.019±0.004 IU/ml in the heparin group compared to 0.028±0.003 IU/ml, p < 0.02. The clinical significance of these results will be discussed.
Contact-Activated Factors: Contaminants of Immunoglobulin Preparations with Vasoactive and Coagulant Properties
Authors: Alving B.M. , Tankersley D.L. , Mason B. L. , Rossi F. , Aronson D.L. , Finlayson J. S.
Published: May 2019

Immune globulins (IG) may cause adverse vasoactive reactions. Twenty-four IG solutions (16.5% protein) made by 5 manufacturers were analyzed for components of the contact-activation system, which could mediate such reactions. Kallikrein (K) was measured with S-2302 as the substrate and prekallikrein activator (PKA), by its activation of prekallikrein to K with [3H]TAME, BAEE, or S-2302 as the substrate. Capillary permeability (CP) was determined by intradermal injection of IG into guinea pigs pretreated with Evans Blue dye. The K activity in the 24 lots ranged from <0.01 to 0.67 TAME U/ml. PKA activity varied from 5 to 5000% that of a reference preparation (Plasma Protein Fraction) that had caused PKA-associated hypotension in recipients. Lots exhibiting medium to high K and/or PKA activity caused increased CP. All lots tested shortened the non-activated partial thromboplastin time (PTT) of normal plasma. Two lots that corrected the activated PIT of XI-deficient plasma and reacted with anti-XI antibody by immunodiffusion were gel chromatographed. Coagulant activity was co-eluted with IgG (i.e., M
r ~ that of XIa); only those fractions containing K or PKA caused increased CP. Thus, XIa is responsible for coagulant activity in some IG preparations, whereas PKA and K may be present in sufficient quantities to cause vasoactive reactions.
Complement-Dependent Platelet Agglutination and Lysis of Rabbit Platelets by Endotoxin
Authors: Heinrich D. , Müller-Berghaus G.
Published: May 2019

The effect of bacterial endotoxin on platelets from normal rabbits (NoR) and C6-deficient rabbits (C6defR) was studied. Platelets were washed, labeled with 14C-serotonin and 51chromium and resuspended in Tyrode/albumin (TA), normal rabbit plasma (NoP) or C6-deficient rabbit plasma (C6defR). When incubated with endotoxin (final conc. 100-200 ug/ml) the following results were obtained:

Conclusion: To induce platelet agglutination by endotoxin alternate pathway complement activation up to C3/5 is necessary. Complement activation of C6-9 results in 14C-serotonin release and platelet lysis

Supported by the Deutsche Forschungsgemeinschaft
Platelet Membranes/Drug Combination as Antigenic Stimulus for Lymphocytes of Patients with Autoimmune Quinine- or Quinidine-Dependent Purpura
Authors: Hosseinzadeh P. K. , Firkin B. G. , Pfueller S. L.
Published: May 2019

Quinine- or quinidine-induced thrombocytopaenia appears due to synthesis of an IgG antibody which causes platelet damage. We have studied the nature of the antigenic stimulus in this disorder by measuring the incorporation of 3H-thymidine into DNA of normal or patients’ lymphocytes cultured in the presence of the drugs in comparison to known mitogens (phytohaemagglutin-P or pokeweed mitogen).’ Patients’ lymphocytes responded normally to mitogens but not to the drugs, heterologous or autologous platelets alone. However, significant thymidine uptake occurred when patients’ lymphocytes were cultured with autologous or heterologous platelets in the presence of therapeutic concentrations of the drugs (2.4 x 10-4 . 2.4 x 10-8M). Lymphocyte transformation was maximal after 7 days (up to 93% of phytohaemagglutinin-induced response), suggesting B cell involvement. Supernatants from platelets which had undergone the release reaction could not replace platelets while membranes were more effective than platelets on-a protein basis. Control lymphocytes from 20 normals and 8 patients with non-drug-induced thrombocytopaenia did not demonstrate transformation by the drugs and platelets. Thus the drug in combination with a platelet membrane component act as an antigenic stimulus only for sensitised lymphocytes.
Plasma Betathromboglobulin Measurement to Differentiate Types of Thrombocytopenia
Authors: Han P. , Turpie A.G.G. , Genton E.
Published: May 2019

It is important to differentiate betwteen extravascular (autoimmune thrombocytopenia, ATP) and intravascular (thrombotic thrombocytopenia, TTP) platelet destruction in thrombocytopenia. Betathromboglobulin (BTG), a platelet-specific protein with a plasma half life of 20 minutes is released in-vivo from platelets by various stimuli and may reflect platelet activation or destruction. BTG concentration can be measured in plasma usin a radioimmunoassay to a sensitivity of 1 ng/ml., (nomal 28.0 ± 8.0 ng/ml., n = 70). Plasma BTG was measured in 3 patients with ATP (platelet counts: 17, 20, 16 x 109/L) and 2 patients with TTP (platelet counts: 20, 40 x 109/L). In ATP, BTG was normal (22, 11, 17 ng/ml.) and in TTP, BTG was elevated (80, 72 ng/ml.). Plasma BTG remained normal in ATP after treatment. BTG remained elevated in TTP (120 ng/ml.) even when the platelet count became normal (220 x 109/L) but while fragmented RBC were still present and became normal (21 ng/ml.) on complete recovery. These data suggest that plasma BTG may be useful in differenfiating extravascu1ar from intravascular platelet destruction by detecting increased concentrations of BTG in plasma.
Some Aspects of the Epidemiology of Venous Thromboembolism
Authors: Vessey M.P.
Published: May 2019

A great deal has been discovered about factors predisposing to postoperative venous thromboembolism by means of the radioactive fibrinogen test and venography. These factors will be reviewed by other contributors. Much of the available information about vencus thromboembolism occurring in other circumstances has, however, been obtained by routine clinical and pathological methods. This paper will start with a review of recent vital statistical data about venous thromboembolism for England and wales. In addition to mortality and hospital discharge data, information obtained from the National Morbidity Survey ard from the Confidential Enquiry into Maternal Deaths will be considered. Reference will also be made to vital statistical data from other countries. The epidemiological characteristics of subjects at risk of venous thromboembolism will then be summarised and same of the characteristics will be considered in detail. Special attention will be paid to recent data on the effects of oral contraceptives ard oestrogens.
Hageman Factor (HF, Factor XII) -Dependent Activation of Coagulation, Kinin-Forming, and Fibrinolytic Pathways by Surface Contact
Authors: Griffin J.H.
Published: May 2019

Exposure of plasmo to various negatively charged surfaces (glass, kaolin, connective tissue components, urate crystals, etc.) initiates contact activation of plasma resulting in the activation of the intrinsic coagulation, the kinin-forming, and the fibrinolytic systems. Proteins involved in these reactions include HF, Factor XI, prekallikrein, and high MW kininogen. Recent studies will be summarized that provide new information on the molecular mechanisms responsible for contact activation. Surface-binding of HF does not convert HF from an inactive form to an active enzyme, as previously supposed; rather it makes HF much more susceptible (100 to 500-fold) to proteolytic activation by kallikrein. plasmin, or other proteases. Reciprocal proteolytic activation of prekallikrein and HF occurs, and high MW kininogen is a surface-bound cofactor for each step. The potential influence of different cells on the activation of the contact system will also be discussed. Human platelets have been shown to stimulate the activation of HF and of Factor XI. Endothelial cells contain an enzyme capable of activating Hageman factor. Moreover, human basophils secrete an enzyme capable of activating HF. Thus, it appears that proteins derived from a number of cells can influence the nature and extent ot contact activation reactions.
Role of Bovine HMW Kininogen in Contact-Mediated Activation of Factor XII: Demonstration of a Nicked Form, “Active Kininogen”, With Maximal Cofactor Activity by Limited Proteolysis
Authors: Kato H. , Sugo T. , Ikari N. , Hashimoto N. , Iwanaga S. , Fujii S.
Published: May 2019

Bovine HMW kininogen (HMW-K) has a mol. wt. of 76,000 and the carboxyl-terminal 243 residues consisting of kinin, fragment 1.2 and light chain regions have been sequenced. The purpose of this study is to elucidate the functional regions of the kininogen which are required for the kaolin-mediated activation of Factor XII (XII) in the presence of prekallikrein (Prek). The results were as follows: (1) Kaolin-mediated activation of XII was accelerated 180 fold by adding optimum amounts of HMW-K. (2) The accelerating effect of HMW-K markedly increased by the brief treatment with kallikrein, indicating that a nicked HMW-K, named “Active Kininogen”, is most effective. (3) The same accelerating effect as HMW-K was observed with fragment 1.2-light chain. (4) Prek formed a complex with HMW-K and kinin-fragment 1.2-free protein. Kallikrein also formed a complex with kinin-fragment 1.2-free protein. (5) The adsorption of HMW-K on kaolin was inhibited by fragment 1.2. These results indicate that HMW-K accelerates kaolin-mediated activation or XII by forming a complex with Prek through the light chain region and by interacting with kaolin through the fragment 1.2 region. It was also suggested that plasma kallikrein plays an important role not only In the positive feedback activation of XII but also In the transformation of HMW-K to an “Active Kininogen”.
Inhibition of Haceman Factor by a Fibrinolytic Inhibitor
Authors: Hedner U.
Published: May 2019

Two pathways exist for the activation of the fibrinolytic system: one involves Hageman factor and several additional proteins, and the other involves activators that act directly on the plasminogen molecule. An inhibitor of the plasminogen activation (PA-inhibitor) was first described from our laboratory in Malmo in 1962. This inhibitor was partially purified and found to be distinct from other known fibrinolytic inhibitors, such as antithrombin III, α2-antiplasmin, or Cl inactivator. Furthermore, high levels of PA-inhibitor were found in some patients with thrombotic disease. The PA-inhibitor does not inactivate urokinase or plasmin, but functions as an immediate inhibitor of activated Hageman factor, as measured in a coagulation system as well as a fibrinolytic system. In contrast, the PA-inhibitor has no effect on factor XIIa when TAME esterase activity is measured. Using an autoradiographical technique, 125I-1abeled bovine factor XIIa (two chains) was found in the inhibitor protein peak, in contrast to factor XII (one chain). The relationship between factor XII and the PA-inhibitor was studied in 12 po operative patients. The activities of both proteins were found to decrease. Furthermore, 11 young patients with severe thrombotic disease were found to have IgG antibodies directed against factor XII. These findings suggest that factor XII as well as the PA-inhibitor are of importance for a normal fibrinolytic system and for preventing the development of deep venous thrombosis.
Chromogenic Peptide Substrate Assays for Plasma Inhibitors of Plasma Kallikrein: Identification of the Major Inhibitors
Authors: Gallimore M.J. , Amundsen E. , Larsbraaten M. , Lyngaas K. , Fareid E.
Published: May 2019

Plasma inhibitors of plasma kallikrein(KK) were studied using chromogenic peptide substrate assays. Both “immediate” and “time-dependent” inhibition was detected. Sephadex G-150 gel filtration revealed that fractions containing α2-macroglobulin (α2 M), C1 - esterase inhibitor (CIINH) and a low molecular weight component(KKI3) gave “immediate” inhibition. When fractions were tested for “total” inhibition (incubation of enzyme plus fraction for 300 seconds at 37°C) CIINH was found to be the major inhibitor. Both the α2M and KKI3-containing fractions exhibited more inhibition than in the “immediate” inhibition assay. Studies with purified preparations of CIINH and α2 M indicated that these are the two most important plasma inhibitors of KK. Preparations of α1-antitrypsin (α1AT), antithrombin III (ATIII) and α2-antiplasmin (α2AP) produced insignificant inhibition. When “total” KK inhibition in plasma samples from 20 healthy subjects was compared with plasma concentrations of CIINH, α2M and α1AT (immunochemical assays) a very good correlation (r=0.81) was found between percentage inhibition and CIINH concentration. Correlation values for the other antiproteases were α2M r=0.36 and α1AT r=0.19.
Resistance to Atherosclerosis in Pigs with Cenetic and Therapeutic Inhibition of Platelet Function
Authors: Fuster V. , Fass D.N. , Bowie E.J.W.
Published: May 2019

Platelets appear to play an important role in the development of atherosclerosis. Accordingly, we have investigated whether pigs with genetically impaired platelet function in the form of homozygous von Willebrand’s (vW) disease and normal pigs treated with the platelet inhibitors Dipyridamole (6 mgs/kg/day) plus Aspirin (15 mgs/kg/day) (D+A) are resistant to atherosclerosis when compared with normal control pigs matched by breed and weight. The three groups of pigs received a high (2%) cholesterol diet from age 3 to 9 months and then quantitation of the fibrous atherosclerotic plaques was performed in the distal third of the aortas since in pigs this is the region prone to the development of lesions.

Normal pigs. Of 11 pigs, 8 (73%) had significant plaques involving more than 10% of the aortic surface (mean surface 18%), Homozygous vW pigs. Of 7 pigs, only 1 (14%) had a small plaque (surface 3%) (p<0.01 rank≤test). Normal pigs treated with D+A. Of 7 pigs, only 1 (14%) had significant plaques (mean surface 8%) (p< 0.05 rank≤test).

These findings suggest a resistance to the development of atherosclerosis In pigs with genetic and therapeutic inhibition of platelet function.