bluuebunny/crossref_metadata_2025 huggingface dataset:
The in Vivo Evaluation of the Hemostatic Function of Stored Human Platelets
Authors: Blajchman M. , Senyi A. , Hirsh J.
Published: May 2019

The assessment of the hemostatic function of stored human platelets is difficult to assess in human subjects. The use of thrombocytopenic rabbits treated with ethyl palmitate to produce reticuloendothelial blockade, has made it possible to study the hemostatic function of human platelets in vivo. The assessment of hemostatic function has been made using both a jugular bleeding time technique and an ear bleeding time technique, and in both, a close correlation between bleeding time and platelet count has been established. Using both methods, both fresh and human platelets stored for 72 hours at 22°C correct the bleeding time of thrombocytopenic animals to levels appropriate to the platelet count achieved. Platelets stored at 4°C using standard methods of preparation and storage were ineffective hemostatically after 24 hours storage. Platelets prepared and stored at 4°C at a pH of 6.4 were hemostatically effective in thrombocytopenic rabbits for as long as 10 days of storage. No correlation, however, was noted between the hemostatic effect of stored platelets and in vitro tests of platelet function. Similarly, the intravenous infusion of ADP and collagen produced similar falls in platelet count for both hemostatically effective and non-effective platelets. These studies provide further evidence for the limitations of in vitro tests of platelet function for the assessment of the potential in vivo function of stored human platelets. Furthermore, these findings raise the possibility for the prolongued liquid storage of human platelets at conditions which minimize bacterial contamination, yet maintain hemostatic efficacy.
A 3-D View of Splenic Pathophysiology in Idiopathic Thrombocytopenic Purpura
Authors: Barnhart Marion I. , Lusher Jeanne M.
Published: May 2019

The spleen plays a dual role in the pathogenesis of chronic idiopathic thrombocytopenic purpura (I.T.P.) It is generally the major site of phagocytosis of antibody-coated platelets and is also an important site of production of autoantibody against platelets. As splenectomy is the treatment of choice in chronic I.T.P. in children, as well as adults, we have had the opportunity to study the freshly excised spleens from children with chronic I.T.P. We have previously described splenic ultrastructure in I.T.P. as viewed by SEM in two publications (Excerpta Medica ,#357:462-471, 1974 and Am. J. Hematol. 1: 243-264, 1976). Our most recent studies, to be presented here, employ an additional tool - that of latex particle probes. We have used these probes to identify platelets and macrophages containing phagocytized platelets, and to locate the sites of platelet autoantibody production. 15 spleens were immediately perfused (via the splenic artery) with heparinized Tyrode’s solution under physiologic pressure to partially remove blood cells from major vessels. This was followed by perfusion with 0.025% buffered glutaraldehyde and then 0.2 μm latex spheres coated with either specific antiplatelet antibody or normal rabbit immunoglobulin. In I.T.P. spleens perfused with latex spheres coated with normal rabbit immunoglobulin, none of the spheres adhered to splenic cells. In those spleens perfused with latex spheres coated with antibody against platelets, the spheres adhered to splenic macrophages and to morphologically obvious platelets (thus identifying platelet loci in the spleen). In addition, latex coated with platelet membrane protein adhered to lymphocytes in lymphatic nodules, indicating sites of autoantibody production.
Molecular Properties of Bovine Hageman Factor
Authors: Claeys H. , Van Hoef B. , Collen D.
Published: May 2019

Hageman factor was isolated from bovine plasma by CM-Sephadex, Ultrogel AcA-44 and DEAE-Sephadex chromatography, with a yield of 1.1 + 0.2 mg per liter plasma and a specific activity of 51 + 12 units per mg protein. The amino acid composition was similar to that reported for Human Hageman factor [Revak et al. 1974). The preparation previously characterized in our laboratory (Vth Cong. Paris 1975, Abstract n° 118) now appears to have been contraminated with a histidine rich protein. The present material could be adsorbed to and eluted from celite without loss of activity and without apparent generation of spontaneous activity.

By SDS-polyacrylamide gel electrophoresis, two molecular forms of bovine Hageman factor could be distinguished : a single chain molecule with an estimated molecular weight of 80,000 and a two chain disulfide bonded molecule with mol. wts of 50,000 and 32,000. The amount of the two chain protein in different preparations varied between 0.25 and 1.00.

Incubation with DF32P revealed that the two chain protein molecule was completely inhibited with the incorporation of 1.2 mol of DFP per mol of protein, whereas the single chain molecule did not incorporate DFP. These data suggest that the single chain molecule is a precursor form with inaccesible active site serine and the molecule in which the two chains are connected by disulfide bridge[s] is an activated or degraded form with accessible active serine. Similar structural properties have recently been described for human Hageman factor, in which the two chains of the activated form are however not disulfide linked (Griffin et al. 1976).
Bacterial Heparinase Permits Quantitative in Vitro Assays of Clotting Factors, and Neutralizes Heparin in Vivo
Authors: Kingdon Henry S.
Published: May 2019

We have reported previously that preparations of heparinase from the bacterium F. heparinum eliminate heparin prolongation of partial thromboplastin times and prothrombin times (J. Lab. Clin. Med. 79, 1027, 1972). Therefore, when the PTT is prolonged, this deviation from normal can be shown to be due to heparin (or otherwise) before embarking on expensive specific assays to define a possible hemorrhagic disorder. Similarly, the use of heparinase in prothrombin times renders them insensitive to heparin effect and makes them a more accurate measure of the effect of oral anticoagulants. In the current studies, it was demonstrated that quantitative assays of factor VIII in normal human plasma, while uninterpretable after the plasma was heparinized, returned to normal baseline values after the heparinized plasma was treated with heparinase. The quantitative values for factor VIII were identical when heparinized plasma treated with heparinase were compared with the same plasma before heparinization. In other experiments, heparinized rabbits were rendered hemostatically normal within 15 minutes of heparinase infusion, whereas control rabbits exhibited significant heparin inhibition of blood coagulation for 3–4 hours after heparin infusion. Finally, mutants of F. heparinum which produce large quantities of heparinase when grown in the absence of heparin have been isolated and appear to provide an inexpensive and readily available source of heparinase, so that the enzyme might conceivably become commercially available for use in laboratory evaluation of coagulation disorders, and also for definitive studies of its possible therapeutic applications.
Arachidonic Acid Metabolites not Always Crucial for Thromboregulation?
Authors: Hornstra G. , Haddeman E.
Published: May 2019

When blood platelets are triggered for aggregation, phospholipase A2 is activated resulting in the release of arachidonic acid (AA) from cell membrane phospholipids. AA is converted into prostaglandin endoperoxides (EP) by the cyclo-oxygenase enzyme system (CO). From EP’s, Thromboxane A2 (TxA2) is formed enzymatically. There is now considerable evidence that the AA-EP-TxA2 axis is of major importance for thromboregulation.

However, recent findings indicate that under certain conditions an alternative regulatory mechanism may exist.

In essential fatty acid (EFA-) deficient rats, platelets contain hardly any AA, the efficiency of which as a CO-substrate is severely depressed because of the competition of a fatty acid, specific of EFA-deficiency (C 20:3 n-9). The collagen- and thrombin-stimulated production of thrombogenic AA-metabolites (measured as malondialdehyde) is almost absent in EFA-deficient platelets. Nevertheless, platelet release is normal to increased.

This finding points to a thromboregulatory mechanism different from the AA-EP-TxA2 axis. This, probably secondary, mechanism may be of significance for the prevention and therapy of thrombosis.
Oscillatory Response of Ristocetin Induced-Platelet Aggregation in Thrombasthenia
Authors: Chediak J. , Telfer M. , Vander Laan B. , Cohen I.
Published: May 2019

Since the introduction of ristocetin as an aggregating agent for the diagnosis of von Willebrand’s disease, variable results have been reported on its effect on thrombasthenic platelets. The platelets from 10 thrombasthenic patients were subjected to aggregation with serially increasing concentrations of ristocetin. Unlike normal control platelets which showed normal irreversible aggregation using final concentrations of 1.25 mg/ml or higher, these platelets required increased concentrations of 1.8 and 2 mg/ml for maximal aggregation and intermediate concentrations (1.25 to 1.75 mg/ml) showed a cyclic oscillatory pattern of aggregation-disaggregation for 12–15 minutes, ending in partial disaggregation. If at this point creatine phosphate/creatine Phosphokinase (CP/CPK) or ATP were added the oscillatory pattern was reinstituted. Measurements of pH of the stirred platelet rich plasma (PRP) at the height and depth of oscillations changed only from 7.6 to 7.8 without correlation with the pattern. Incubation of the PRP with ADP prevented the ristocetin induced platelet aggregation; it was restored with the occurrence of oscillatory pattern upon addition of CP/CPK or ATP.

The oscillatory phenomenon of thrombasthenic platelets in response to ristocetin is a unique finding. The constancy of the pH at different stages of the oscillatory waves ruled out the possibility of a gross electrostatic effect. The membrane defect reported in thrombasthenic platelets may play a role in these findings.
Platelet Aggregation in Patients with Ischemic Heart Disease, Transient Cerebral Ischemia and Peripheral Thrombo-Atherosclerosis
Authors: Gormsen J. , Nielsen J. D. , Andersen L. A.
Published: May 2019

The threshold concentrations of ADP and adrenaline inducing platelet aggregation in vitro were determined in a Payton aggregometer in 90 normal controls, 30 patients with ischemic heart disease (IHS), in 34 with transient cerebral ischemia (TCI) and 22 with peripheral thrombo-atherosclerosis (PTA). The threshold concentrations were significantly lower in normal women ≥ 50 years old than in normal women < 50 years old.

Compared with the corresponding control groups significantly lower threshold concentrations were found in following groups of patients: men and women ≥ 50 years with IHS (p < 0.005 and p < 0.001 respectively), men + women < 50 years with IHS (p < 0.05), men + women with TCI (p < 0.01), men + women ≥ 50 years with PTA (p < O.002 and p < 0.001 respectively), men + women < 50 years with PTA (p < 0.005).
3’5’-Adenosine Diphosphate (3′5′ADP):A Physiological Inhibitor of Platelet Aggregation and Platelet Release Reaction
Authors: Subbarao K. , Jaya K.
Published: May 2019

Certain analogues of adenosine have been shown to inhibit ADP-induced platelet aggregation. We therefore studied the in vitro effect of 3′5′ADP and coenzyme A on human platelet aggregation and [14C]-serotonin release reaction induced by the addition of ADP, thrombin, collagen and epinephrine to human platelet rich plasma (PRP). It was found that coenzyme A Li3·2H2O at a concentration of 0.12 mM strongly inhibited ADP-induced platelet aggregation of PRP but did not show similar effect on the aggregation of platelets induced by other aggregating agents. The 3′5′ADP which is a part of coenzyme A structure, on the other hand, inhibited both ADP and thrombin induced platelet aggregation. The extent of inhibition of platelet aggregation by coenzyme A and 3′5′ADP was found to depend upon the concentration of the inhibitor and the incubation time. Whereas 3′5′ADP Li2·3H2O at a concentration of 10 μM produced about 70% inhibition of ADP-induced platelet aggregation of human PRP, total inhibition of thrombin induced platelet aggregation was observed when platelets were incubated with 60 μM of 3′5′ADP. The 3′5′ADP also inhibited the [14C]-adeonsine uptake by platelets in a concentration dependent manner. The inhibitory potency of 3′5′ADP on platelet aggregation was found to be 10-fold higher than that of N6-2′-0-dibutyryl-cyclic 3′5′-adenosine monophosphate. The inhibition of platelet aggregation by coenzyme A and 3′5′ADP was always accompanied by the inhibition of [14C]-serotonin release reaction. If coenzyme A and 3′5′ADP are indeed physiological inhibitors of platelet aggregation, then aggregation of platelets should depend on metabolic events that regulate the concentration of these agents in blood.
Measurement of Nucleotide Pools in Platelets and Red Blood Cells Using High Pressure Liquid Chromatography
Authors: D’Souza L. , Glueck H. I.
Published: May 2019

Release of adenine nucleotides during platelet aggregation is well established. The released adenosine diphosphate (ADP) is thought to cause secondary platelet aggregation. A high pressure liquid chromatographic technique has been used to measure accurately the various nucleotide pools in intact platelets, as well as their levels during various stages of aggregation. Accurate and highly reproducible measurements of 10-12 moles of all nucleotides are possible using the bonded microparticulate, Partisil −10 SAX column, a strong anion exchanger (Whatman Inc., Clifton, N. J.) and an electronic integrator. The method is a modification of an earlier method of P. Brown (1973), and G. Rao et al (1975). Using this technique we are able to obtain within 75 minutes a single step gradient separation of cyclic nucleotides (cAMP, cCMP, cGMP) as well as mono-, di-, and tri-phosphonucleosides. Sample sizes of 0.5 ml of platelet rich plasma are adequate for at least 3 estimations. Normal donor platelets show the following adenine nucleotide levels;* AMP 0.13 (0.14); ADP 1.64 (0.81); and ATP 4.90 (0.42) nanomoles per 1011 platelets. The method has certain advantages over the enzymatic or luciferase assays, since all the nucleotides can be simultaneously measured in a single step from a very small sample and there is no cross reactivity with any of the nucleotides. Similar studies of plasma and red blood cell nucleotides are possible with this technique.

*Mean (standard deviation)
Physicochemical Characteristics of the Human Factor VIII-Von Willebrand Factor Oligomers
Authors: Bolhuis P. A. , Seinen A. J. , Mochtar I. A.
Published: May 2019

Analytical ultracentrifugation of the factor VIII-Von Willebrand factor showed an apparently homogenous protein with an S° of 29. With the diffusion coefficient D of 1.24 x 10-7 cm2/ sec.(obtained by 20,w double diffusion of the protein and the immunochemically equivalent amount of rabbit anti-factor VIII) a mean mol. wt. of 2.2 x 106 and a friction ratio of 1.9 are derived. Turbidity measurements confirmed the high mol. wt. and resulted in an estimation of the co-volume of the protein. The free electric mobility wasfound to be 3.6 x 10-5 cm2/Vsec at pd 7.00 and 8.5x 10-5cm2/V sec at pH 8.80, while iso-electric focussing patterns of the native protein and the polypeptide (mol.wt. 260 000) obtained by reduction in the presence of urea exhibited a maximum at about pH 4.5. The apparent D found upon electrophoresis is an order higher than given above and is dependent on the field strength. The heterogeneity indicated by this phenomenon was confirmed by reversal electrophoresis (yielding a decrease in the apparent D). Since continuous immuno precipitation lines are found upon cross-electrophoresis, we interprete the results in terms of a series of closely related oligomers with a mean number of 8 polypeptidechains and a variation from about 6 to 10.
Isolation and Properties of the Abnormal Factor IX Molecule of Hemophilia BM
Authors: Østerud B. , Lavine K. , Kasper C. K. , Rapaport S. I.
Published: May 2019

Abnormal factor IX from a hemophilia Bm patient (F. IX-Bm) has been isolated to homogeneity on SDS Polyacrylamide gel electrophoresis by the same technique utilized for purifying normal F. IX. F. IX-Bm generated no measurable procoagulant activity when incubated with F. XIa in a two-stage F. IXa assay (normal F. IX, 55 sec; F. IX-Bm, >30 min).F. IX-Bm inhibited the activation of F. X by F. VII and ox brain thromboplastin as measured in an amidolytic assay for factor Xa. Normal F. IX also inhibited this reaction but to a five times lesser degree. F. IX-Bm has the same molecular weight on SDS gel electrophoresis as normal F. IX (55,000) and does not differ from normal F. IX in its amino acid composition. F. XIa cleaves F. IX-Bm in the presence of Ca ions at the same rate as it cleaves normal F. IX, yielding a heavy chain of 27,000 molecular weight and a light chain of 16,000 molecular weight. However, the cleavage does not give rise to procoagulant activity. Like normal F. IXa, the cleaved forms of F. IX-Bm appear to bind phospholipid since F. IX-Bm protein was precipitated with phospholipid in the presence of Ca ions. These data support an hypothesis that the abnormality in the F. IX-Bm molecule stems from a defect at the active site.
Post-Transfusion Purpura
Authors: Aster Richard H.
Published: May 2019

Post-transfusion purpura is characterized by severe, destructive thrombocytopenia with bleeding manifestations occurring 7–8 days after blood transfusion. In nearly all cases, patients have been women with a past history of pregnancy who received their first blood transfusion. A potent platelet antibody detectable by a variety of methods is invariably present in serum at the onset. In all but two cases studied, this antibody has been directed toward the platelet-specific antigen, PlAl, present in 98% of the general population. Patients untreated or given prednisone recover spontaneously in 10–48 days unless fatal hemorrhage supervenes. Exchange transfusion with whole blood or by plasmapheresis appears to have shortened the duration of thrombocytopenia significantly in numerous reported and non-reported instances. Following recovery, patients whose antibody had P1A1 specificity are found to have platelets that lack this antigen. It is not yet clear how an isoantibody, apparently provoked by blood transfusion, causes fulminant destruction of autologous platelets. Possible explanations include a) cross-reactivity between the isoantibody and an autoantigen, b) transfusion of soluble P1A1 antigen in the plasma of certain unusual donors with subsequent formation of immune complexes having anti-platelet activity, and c) coating of recipient platelets with transfused P1A1, causing transient conversion of platelet type to Prepositive and permitting destruction of such platelets by anti-P1A1 antibody. Evidence for these pathogenetic mechanisms will be summarized.
The Apparent Separation of Factor VIII Related Antigen and Coagulant Activity from von Willebrand Factor Activity by an Immunoadsorbent
Authors: Cooper H. A. , Lee D. , Lamb M. A. , Wagner R. H.
Published: May 2019

An antibody was raised in rabbits to the small active fragment of human factor VIII. The antigen was obtained by Ca2+ dissociation of a human factor VIII preparation made from a multidonor pool of plasma. After two adsorptions with 0.1 volume of normal human plasma, the antibody neutralized the F. VIII coagulant activity of normal human plasma, but did not precipitate with any plasma or plasma fractions nor did it neutralize vWF activity as measured by ristocetin aggregation of fixed washed platelets. A solid phase immunochemical reagent was prepared by CNBr binding of the partially purified rabbit antibody to 1% agarose beads. Non-immune beads were similarly prepared with IgG fractions from a normal non-immunized rabbit. Using a batch technique the beads were studied for their ability to remove F. VIII coagulant, F. VIII Ag, and vWF activity from normal human plasma. Assay of the supernatant plasma after 2 hrs, 22°, from 10 replicate experiments gave the following results for residual activity, as per cent of non-immune bead control:F. VIII (37.5±4), F. VIII-Ag (30.8±9.7), and vWF (72.1±16). The experiment was repeated with 6 replicate samples with higher ratio of beads to plasma with essentially similar results. This unexpected separation of F. VIII-Ag from vWF activity prompted further investigation into how these activities are related to the molecular structure of F. VIII and vWF.
Lipases Produce Functionally Intact Subunits of Factor VIII (Antihaemophilic Factor)
Authors: Furlan M. , Jakab T. , Beck E. A.
Published: May 2019

Cryoprecipitates of fresh human plasma were fractionated by gel filtration on Sepharose CL-2B. Factor VIII was eluted in the void volume together with a non-proteolytic enzyme which was capable of degrading factor VIII into smaller subunits. Similar subunits were observed following treatment of factor VIII with a triglyceride lipase from Rhizopus arrhizus. They retained full functional activity (procoagulant and ristocetin cofactor). The subunits reaggregated spontaneously at 37° into a reconstituted complex which remained functionally fully active. The resulting aggregate could be repeatedly dissociated by addition of fresh lipase. The reaggregation process was enhanced by phenylmethylsulfonyl fluoride or ethylenediamine tetraacetate, but was inhibited at 0°. The treatment with lipase renders factor VIII more susceptible towards plasmin which destroys its functional properties.
Kinetics of Thrombin Inhibition by Alpha2 Macroglobulin: Changes Induced by Prior Proteolysis of the Inhibitor by Trypsin
Authors: Tapon-Bretaudiere J. , Josso F.
Published: May 2019

Plasma alpha2 macroglobulin (α2M) is able to inhibit serine esterases by a two-step reaction involving a first cleavage of the inhibitor molecule by the proteinase, followed by the formation of a molecular complex between the enzyme and the proteolyzed inhibitor. The rate of the inhibition reaction varies greatly from one proteinase to one another. Thus, α2M appears to be a ‘progressive” inhibitor of thrombin.

Kinetics of thrombin inhibition by α2M was evaluated after prior treatment of the inhibitor by trypsin, either immobilized to avoid the secondary fixation of the enzyme to the proteolyzed inhibitor, or soluble, the enzyme excess being neutralized by soybean trypsin inhibitor (STI). In the second case, trypsin bound to α2M was assayed using a low molecular weight synthetic substrate (BApNA).

When α2M was pretreated by immobilized trypsin, the reaction rate of the inhibition was increased without modification of the over-all neutralizing activity of α2M.

Whenα2M was pretreated by soluble trypsin, it was found that STI in excess was able to displace trypsin bound to α2M. In these conditions, the treated α2M inhibited thrombin at a very rapid rate, the over-all neutralizing activity of α2M depending on the ratio STI/Trypsin.

These findings demonstrate that (i) the binding of α2M to proteinases is reversible-(ii) the limiting factor of the inhibition rate of thrombin by «2M is the first proteolytic step of the inhibition reation.
An Acquired Platelet Storage Pool Deficiency in Patients with Severe Valvular Heart Disease
Authors: Harbury C. B. , Galvan C. A.
Published: May 2019

The purpose of this study was to assess whether patients with severe valvular disease damage their platelets in vivo and acquire a platelet storage pool deficiency. Forty-three preoperative valve patients, 43 coronary artery bypass patients (CABG) and 22 concurrent normal controls were studied. Platelets were counted by phase and ZBI Coulter Counter. Total and releasable platelet ADP and ATP was measured by the luciferase assay (Holmsen). Nucleotides are expressed in nanomoles per 108 platelets. Analysis is by a non paired t test. Re-leasable ADP: control = 2.35 ± 0.6, valve 1.86 ± 0.5 t = 3.35, p<0.01, a significant difference. Releasable ATP: control= 1.7 ± 0.4, valve 1.2 ± 0.4 t = 4.05 p < 0.01 a significant difference. Total ADP control = 3.3 ± 0.5, valve 2.6±0.6, t = 4.12, p <0.01 a significant difference. Total ATP control = 5.7 ± 0.8 valve = 5.3 ± 1.1. This is not a significantly lower value t = 1.07. CABG patients were not significantly different from controls, but were significantly different from valve patients. Twenty-six valve patients were studied postoperatively, 10 had received platelet transfusions and were excluded from quantitative assessments. Estimated blood loss (OREBL) valve =1556 cc, CABG = 734 cc, p<0.001. Transfused red cells (RBCTx) valve =8.5u, CABG 6.5 u, p<0.01. Postoperative chest tube drainage (CTD) above and below 400 cc, CABG vs valve χ2 = 5.12, p< 0.02. Ten valves and 2 CABG received platelet transfusions. If these are included and considered bleeders χ2 = 11.01 p<0.001. These patients with severe valvular heart disease appear to have a mild acquired platelet storage pool deficiency and a significantly greater bleeding tendency at surgery.
Effect of Photooxidation on Human Fibrinogen
Authors: Inada Y. , Hessel B. , Blombäck B.
Published: May 2019

Photooxidation of human fibrinogen and of the amino terminal fragment N–DSK, obtained by cyanogenbromide treatment of fibrinogen, has been studied. Fibrinogen and N–DSK were illuminated in the presence of methylene blue. Polymerization of fibrinogen after addition of thrombin was abolished by illumination as measured spectrophotometrically. Fibrinopeptides were released at a normal rate from the photooxidized fibrinogen by the action of thrombin. During the course of the polymerization of fibrinmonomers, a short period of illumination gave rise to the inhibition of the subsequent polymerization reaction. Histidine residues in photooxidized fibrinogen and photooxidized N–DSK were shown to be modified by illumination. Illumination of thrombin activated fibrinogen-Sepharose (fibrinmonomer-Sepharose) and thrombin activated N–DSK-Sepharose in the presence of methylene blue resulted in loss of affinity to fibrinogen. On the other hand, photooxidized fibrinogen which had no polymerization activity showed the same affinity for fibrinmonomer-Sepharose as did nonoxidized fibrinogen. The latter result suggests that histidine residues in the N–DSK domain of fibrinogen play an important role in the polymerization process.
Fibrinogen-Collagen Interactions
Authors: Stemberger A. , Jilek F. , Hörmann H. , Blümel G.
Published: May 2019

Recently fibrinogen-collagen sponge preparations have been used surgically for tissue adhesion (Austria, BRD, Swiss). Therefore fibrinogen-collagen interactions were studied by affinity-chromatography and by investigation of fibrin crosslink formation in presence of collagen.

Affinity chromatography of plasma on collagen-Sepharose yielded retention of fibrinogen on conjugates of native collagen type3 and denatured collagen typel. Vice versa these collagen types were adsorbed by fibrinogen-Sepharose.

Fibrinogen-collagen mixtures were clotted by adding thrombin, Ca2+ and factor 13. In contrast to dissolved collagen, collagen fibers interfered with fibrin crosslinking, i.e. – dimer formation was restricted as shown by acrylamid gel elctrophoresis of the reduced coagulum. Covalent coupling of fibrin to collagen was not observed by DEAE and CM-cellulose chromatography.

The affinity of collagen to fibrin was demonstrated by adhesion of collagen sponges (containing thrombin) with 10% fibrinogen. Breaking strength (p/cm2) is dependent on sponge. Studies on collagen-fibrinogen tissue adhesions are in progress. Scanning electron microscopic pictures showed inclusion of fibrin into collagen sponges.

According to our results, collagen fibrinogen adhesion refer mainly to mechanic sticking. The affinity of plasma fibrinogen to collagen type3 will stimulate further investigations, since Gay et al.(Klin. Wschr. 53, 1975) have shown that collagen type3 is the main connective tissue component under endothelial cell-layers of arterial walls.
Dose-Dependent Inhibition of Experimental Arterial Thrombosis by Carbenicillin and Ticarcillin
Authors: Lyman B.T. , Johnson G.J. , White J.G.
Published: May 2019

Drugs that inhibit platelet (PL) function may be useful in the prophylaxis of arterial thrombosis. We have shown that carbenicillin (CARB) and ticarcillin (TIC) cause dose-dependent inhibition of PL aggregation (AGG) in dogs, and that high doses of either of these penicillins significantly decrease the frequency of thrombosis in pronase (PN) injected arteries of dogs. Using this model we have compared the thrombosis prophylaxis effectiveness of these penicillins administered in low dose (250mg/Kg/24 hr, s.c.) (LD-P) versus high dose (750mg/Kg/24 hr, s.c.) (HD-P). Treatment for 3 to 7 days with LD-P resulted in marked inhibition of ADP-induced AGG, but had little or no effect on collagen-induced AGG, while HD-P routinely inhibited collagen as well as ADP-induced AGG. Control dogs were untreated, and their PL function remained normal. Thrombosis with total occlusion occurred in 13 of 15 arteries in control dogs, while only 3 of 29 segments totally occluded in HD-P treated dogs (χ2 = 21.7; p<0.001). However, total occlusion developed in 13 of 20 arteries in dogs receiving LD-P (χ2 = 1.1; p<0.3). The frequency of occlusion was significantly lower (p<0.001) following treatment with HD-P than LD-P. Aspirin (ASA) administration in low dose (650mg/24 hr p.o.) (LD-A) or high dose (2600mg/24 hr p.o.) (HD-A) uniformly inhibited collagen-induced PL AGG, but total occlusion developed in 11 of 16 PN injected arteries in dogs given LD-A, and in 10 of 12 arteries in dogs given HD-A. We conclude that CARB and TIC are effective experimental antithrombotic agents. Their effect is dose-dependent and closely correlated with inhibition of collagen-induced PL AGG. In high doses CARB and TIC are more effective than ASA in preventing PN induced arterial thrombosis. CARB and TIC warrant further study as potentially useful antithrombotic agents in humans.
Changes in Thermodynamic Parameters in the Inhibition of Thrombin by Bovine Antithrombin III
Authors: Yue Robert H. , Starr Toby , Gertler Menard M.
Published: May 2019

The inhibition of thrombin by isolated bovine antithrombin III (1, 200 units/mg of protein) was studied under a variety of experimental conditions. This inactivation reaction follows a second-order reaction and the rate constant depends on a number of parameters. The second-order rate constant decreases with an increase of thrombin concentration. However, at a constant initial concentration of thrombin, the measured amount of antithrombin III present in a sample is directly proportional to the aliquot of the sample. The rate of inactivation was investigated with changes of temperature. For example, at an initial thrombin concentration of 6. 8 units/ml and antithrombin III concentration of 4. 2 units/ml, the reaction proceeds with Ea of 25 Kcal/mole and Δ S‡ of 42 e. u. /mole. In the presence of 0. 0015 unit of heparin/ml and with similar concentrations of thrombin and antithrombin III, the reaction proceeds with Ea of 16 Kcal/mole and Δ S‡ of 14 e.u. /mole. Therefore, the presence of heparin causes a lowering of the activation energy and a concomitant decrease in the entropy of activation. This change in the thermodynamic parameters allows the inactivation reaction to proceed much faster in the presence of heparin. The effect of heparin may be the result of solvent macromolecular interaction in this inhibition reaction.
Studies on the Mechanism of the Rate Enhancing Effect of Heparin on the Thrombin – Anti-Thrombin III Reaction
Authors: Andersson L-O. , Holmer E.
Published: May 2019

The rate of the reaction between thrombin and antithrombin III is greatly increased in the presence of heparin. Several different mechanisms has been suggested to explain this effect. One possible mechanism is that the binding of heparin to antithrombin III transforms it to a much more rapidly acting inhibitor. Another possible mechanism is that the heparin is bound to thrombin and that this complex then reacts with antithrombin III. This study was performed to clarify how heparin actually works. A commercial heparin preparation was fractionated by affinity chromatography on matrixbound antithrombin III into a high and a low affinity fraction. The high affinity fraction had strong anti-coagulant activity both when assayed by activated partial thromboplastin time and in purified thrombin-antithrombin III system. The low affinity heparin fraction had very low activity in both test systems. The strength of the binding of the two heparin fractions to antithrombin III and thrombin, respectively, was determined by a crossed Immunoelectrophoresis technique. The results obtained showed that the high affinity heparin was strongly bound to antithrombin III as expected. The low affinity heparin was also bound but with a much lower strength. In contrast thrombin had essentially the same affinity for both heparin fractions indicating that the mechanism suggested first is the correct one. Competion experiments using both heparin fractions and the thrombin-antithrombin III system also yielded results in good agreement with that. Thus heparin acts as anti-coagulant by forming complex with antithrombin III making it a rapidly acting inhibitor.
The Use of 99m Tc-Urokinase and 99m Tc-Macroaggregates of Albumin in the Diagnosis of Deep Venous Thrombosis
Authors: Reitsma D.J. , Cox P.H. , Pompen W.B.v.d. , den Ottolander G.J.H.
Published: May 2019

In 32 patients suspected of DVT 3 methods were carried out: 1. 99m Tc-urokinase (Tc-UK) labelled according to Millar was injected i.v. Both legs were scanned with a gammacamera in search for hot spots. 2.99m Tc-macroaggregates of albumin (Tc-MAA) was injected in veins on both feet simultaneously. With a gammacamera the isotope flow pattern was followed. Hot spots were registrated and a lungscan carried out. 3. In l4 of these patients afterwards a röntgen phlebography was carried out. Results: With MAA the presence of hot spots could be used as proof for DVT. Isotope rheography gave only contributary evidence. In 9 cases out of 32 there was a discrepancy between the Tc-UK and Tc-MAA scan. There was a better positive correlation between röntgen phlebography with the Tc-UK (11 out of 14) than with the Tc-MAA scan (7 out of 14). In 6 out of 7 patients röntgenphlebography correlated in localisation with isotope phlebography. In 6 out of 7 patients there was an abnormal lungscan. In 6 of these patients there was also a positive Tc-UK legscan but only twice a positive Tc-MAA legscan. Conclusion : Tc-UK is an easy test in the diagnosis of DVT. Tc-MAA can give additional evidence and provides at the same time a useful lungscan.
Cyclic Nucleotide Phosphodiesterase in Platelets and Platelet Aggregability
Authors: Yamazaki H. , Motomiya T. , Mashimo N. , Asano T. , Hidaka H.
Published: May 2019

Though roles of cAMP and cGMP in platelets are thought to be important on platelet aggregation, little information on their phosphodiesterase (PDE) is available. Platelet interaction with various vessel walls might be reflected on cyclic nucleotides and their PDEs. cAMPPDE and cGMPPDE in platelets and platelet aggregability were measured in 22 healthy volunteers (41.0±14.2 yrs, mean±SD), 26 arteriosclerotic patients (58.4±8.7 yrs) and other miscellaneous patients excluding vascular diseases (44.9±16.4 yrs). PDE activities were measured by the Hidaka and Shibuya method (Biochem. Med., 10:301, 1974). Platelet aggregation induced by ADP and epinephrine was recorded by Sienco aggregometer. Activities of cAMPPDE and cGMPPDE of platelets were 2.37±0.52, 7.23±1.87 in the healthy, 2.50±0.85, 7.53±2.60 in the arteriosclerotics and 2.38±1.02, 6.98±2.59 pmol/min/107 platelets in the miscellaneouus patients respectively. No significant difference was observed among the three groups. Platelet aggregabilities also showed no significant difference. However there was a significant inverse correlation between the aggregability by 1 ug/ml of epinephrine and the PDE activities only in the arteriosclerotic patients. The correlation coefficients were -0.61 between the primary aggregation and cAMPPDE, -0.65 between the primary aggregation and cGMPPDE, -0.58 between the 5 min aggregation and cAMPPDE and -0.76 between the 5 min aggregation and cGMPPDE. The slopes in regression lines of cGMPPDE were far steeper than those of cAMPPDE. Aggregability and cyclic nucleotide metabolism in circulating platelets may be affected by arteriosclerotic vessel walls.
Effect of paraChloromercuribenzoate (PCMB) on Platelet Function and Metabolism AT pH 5.3
Authors: Davenport K. , Mürer E.H. , Stewart G.J.
Published: May 2019

The effect of different SH blockers on blood platelets at low pH has been studied. Washed human platelets secrete 70% of preabsorbed serotonin at pH 5.3 when exposed to 1 mM NaF for 5 min at 37°C. The secretion is 60% inhibited by 0.1 mM PCMB. With higher concentrations of PCMB the extracellular concentration of preabsorbed serotonin rises to 75%. In the absence of fluoride 0.1 mM PCMB causes liberation of 43% of preabsorbed serotonin, while the extracellular [serotonin] found with higher [PCMB] alone does not vary from the values found in the presence of NaF and PCMB. At the same time cytoplasmic nucleotides are found extracellularly, but amount to only 1/2 to 1/3 of the liberated serotonin. This indicates that PCMB inhibits secretion induced by fluoride, while at the same time causing selective liberation of stored material from platelets by a process which may or may not be a secretory process. In contrast to NaF, 3 min exposure to 0.2 mM PCMB produces no distinct ultrastructural changes and no acceleration of the effect of exposure to pH 5.3. In the presence of PCMB the breakdown of metabolic ATP is impaired, and there is no production of inosine and hypoxanthine. PCMB (0.1–0.3 mM) does not induce any of the described changes when added at pH 7.4. When, however, the platelets are first incubated with PCMB at pH 5.3, then the changes induced by the compound continue at pH 7.4. Neither N-ethylmaleimide nor parachloromercuriphenylsulfonic acid induce similar changes at the low pH. The platelet membrane seems therefore to be specifically receptive to the effect of PCMB at this pH, or PCMB itself has been converted to a form to which the platelet membrane is sensitive.
Proteases in Blood Platelets: Collagen Induced Activation of Proelastase by a Trypsin-Like Enzymes
Authors: Legrand Y.J. , Caen J.P.
Published: May 2019

Arterial elastolysis is an important feature of atherosclerosis and the comprehension of the activation mechanism of platelet elastase (proelastase) is essential to the knowledge of the role of platelet proteases as an agent of vascular alterations. Trypsin, chymotrypsin and elastase-like proteases were purified from a platelet protein fraction by a three step affinity chromatography procedure. Three electrophoretically homogenous proteins of molecular weights equal to 40 × 103 (trypsin), 32 × 103 (chymotrypsin) and 26 × 103 (elastase) daltons were obtained. Proelastase is strongly activated by the purified trypsin-like enzyme, whereas the chymotrypsin-1ike enzyme is ineffective. After the separation of platelet organelles on a discontinuous sucrose gradient proelastase, elastase and trypsin were found associated with the light granules and membranes. Fibrillar collagen incubated with platelets produces a rapid activation of proelastase ; elastase is then released in its active form. This effect of collagen is likely indirect and may involve an intermediary step during which proelastase becomes available to the trypsin-like enzyme.
Heparin and Venous Thrombo-Embolism
Authors: Kakkar V.V.
Published: May 2019

Venous thromboembolism represents a serious hazard in patients confined to bed in hospital. Though the search for an effective method of prophylaxis has been going on for nearly the last 90 years, a method which is effective in the total elimination of this condition has yet to be developed. One promising approach is the use of low-dose heparin given subcutaneously.

In this review: (a) the rationale for low-dose heparin will be considered in the context of present concepts of pathogenesis of venous thrombosis; (b) factors which govern the kinetics of clearance of heparin from the circulation will be compared following its intravenous and subcutaneous administration; (c) the recently published studies, where the efficacy of this form of prophylaxis against deep vein thrombosis and pulmonary embolism has been assessed, will be analysed, and (d) further possible developments in heparin prophylaxis includingthe use of semi-synthetic heparin analogue will be discussed.
Evaluation of Three Methods Used to Identify Accelerated Coagulability
Authors: Cohen E. , Caprini J.A. , Zuckerman L. , Vagher J.P. , Robinson B.
Published: May 2019

Recently, the thrombelastograph (TEG) has proved useful in the identification of accelerated coagulability. However, several laboratories use different measurements and standards by which they judge the severity of the accelerated coagulation from the output of the TEG. This paper describes a statistical comparison of three of the most widely used TEG measurements. We used actual TEG tracings obtained from two well-defined clinical groups from Evanston Hospital (A), composed of normals and patients with recently diagnosed malignancies. The latter comprise the hypercoagulable class. Our own measurements of the TEG profile include the r, r+k, angle, and MA. Pieptea’s lab (B) used much more sophisticated measurements involving parameters of the equations which mathematically describe the TEG output. Raby’s lab (C) utilized the r+k and ITP for their determination of kinetic hypercoagulability. At A, an additional set of TEG parameters is obtained from a simultaneous analysis of a celite-activated TEG. When the celite-activated measurements are added to the parameters of the three laboratories, all three methods have significant discriminating power. The discriminant function equation was derived using 90 normals and 90 patients with cancer. The cases correctly classified according to their degree of coagulability in laboratories A, B, and C were 98.3%, 92.2%, and 93.9%, respectively. When the equations were verified on an additional 82 cases, the percentages were 98.7%, 92.7%, and 85.4% for laboratories A, B, and C, respectively. (The sample included 51 normals and 31 with cancer). The general clinical agreement of all three laboratories demonstrates the biologic significance of the TEG as a valuable analytical tool.
Molecular Binding Ratio of Anti-Factor VIII to Purified Factor VIII.
Authors: Cockburn C.G. , Reay Diana M.L. , Hardisty R.M.
Published: May 2019

A simple method has been developed for cutting washed FVIII:anti-FVIII immunoprecipitates from agarose gels and applying the SDS-denatured, reduced protein to SDS-polyacrylamide gels. The relative amounts of IgG heavy and light chains and FVIII have been quantitated by recording the areas under the densitometer traces of the Coomassie brilliant blue-stained gels. The results show that approximately 20 molecules of anti-FVIII bind per 200,000 daltons of native FVIII. We conclude from this that the factor VIII subunits hava a high surface-area-to-volume ratio.
Preliminary Results on the Discontinuous Long-Term Administration of Subcutaneous Calciumheparin to Arteritic Patients. A Pharmacological Action Hypothesis
Authors: Raby C. , Bertrand H.
Published: May 2019

Thirty five patients with severe evolutive arteriopathy were selected since following the advent of their first accident and although they received classical treatment they experienced at least one or sometimes several relapses of thrombosis. These patients were subjected to discontinuous subcutaneous ambulatory heparin treatment. This treatment consisted of two daily injections of 5,000–10,000 IU calcium-heparin (Calciparine) depending on each patient’s weight, during 30 days every three months. A backward survey of nine years for the patients first treated and of over two years for those most recently examined indicated that such patients never underwent recurrence of the disease and were considerably functionally improved. Experimentally in the animal and after subcutaneous injection of low doses of using 35s labeled heparin we have shown that heparin remains bound both on arterial and venous endothelium at least 15 hours after all anticoagulant activity has disappeared from circulating blood. As a rule and depending on individuals the relationship between the dose and the persistence of heparin bound on the wall is much more constant than that existing between the dose and the duration of the anticoagulant effect. Low doses (100-150 IU/kg in man) repeated twice a day will suffice to ensure the persistence of an endothelial coating. This coating could account for the excellent clinical results obtained.
Anti-Heparin and Platelet Aggregation Activities of Polyamino Acids
Authors: Fareed Jawed , Messmore Harry L. , Balis John U. , Moncada Rogelio
Published: May 2019

An earlier report from this laboratory has described the antagonism of the anticoagulant effect of heparin by certain basic polyamino acids. Of the numerous polyamino acids tested, only basic polyarnino acids such as poly-L-lysine (MW 85,000) and poly-L-omithine (MW 120,000) effectively neutralized heparinized plasma (1 u/ml) in concentrations less than 10 μg/ml. Addition of these two polyamino acids in quantities up to 50 μg/ml citrated plasma, significantly shortened the thrombin time. Poly-L-proline (MW 19,000), poly-L-histidine (MW 16,000) and poly-L-lysine (MW 85,000) possessed weak anti-heparin action. These polyamino acids also neutralized the anticoagulant activity of hirudin and polyanetholsulfonic acid in varying degrees. The effects of polyamino acids on platelet aggregation was also tested. Of the 15 basic polyamino acids tested, only poly-L-α-omithine was found to induce aggregation of platelets. Polyornithine in the amounts of 50.0, 25.0 and 12.5 μg/ml to platelet rich plasma caused a 65, 45 and 30% change in transmittance, respectively. The polyornithine induced aggregation (PIA) of platelets was only partially blocked by acetylsalicylic acid. Contrast media (6.0 mg/ml) used in diagnostic radiology and meglumine (5 mg/ml) totally blocked the PIA. The PIA of platelets was found to be a biphasic process, an initial lag time of 30 seconds, after which irreversible aggregation was observed. These studies suggest that basic polyamino acids may be used clinically to antagonize overheparinization; in addition, polyornithine may prove useful in the diagnosis of platelet function defects.
Hypercoagulable State Expressed as the Molecular Increase in Theminute Population of Plasma Fibrinogen
Authors: Kazama M. , Maruyama Y. , Kinoshita T. , Matsuda J. , Nakamura I. , Abe T.
Published: May 2019

It is expected that the hypercoagulable state would induce the qualitative alteration in the minute population of plasma fibrinogen.

The plasmas of 120 cases were chromatographed on Biogel A-15m column and the elution profiles of fibrinogen complexes eluted at the initial part were analysed using staphylococcal clumping test on microtitration plate. The arbitrary scores for the hypercoagulability were given to each result according to the profile.

All of the normal plasma gave the profiles shown at the top of figures and the scores ranged 1-4. The plasmas of DIC used to give the profile shown at the middle with scores larger than 10, of which finding was highly coincident with the clinical diagnosis and sometimes more specific than any other test. The chromatography in cases of various thromboses including cerebral, myocardial and diabetes mellitus with vascular changes revealed the specific profile shown at the bottom with the scores 5-10. The thrombolytic therapy of these cases with urokinase resulted into the temporal disappearance of the specific profiles, but they re-appeared soon after the discontinuation of the therapy.