bluuebunny/crossref_metadata_2025 huggingface dataset:
The Role Of Superoxide Dismutase(SOD) In The Platelet Functions
Authors: Kimura A , Fujimura K , Kuramoto A
Published: May 2019

We previously reported that SOD and catalase(CAT) as well as glutathione peroxidase(GPx) exist in human platelets, which are known to protect membrane or enzyme from oxidative damage and might regulate the prostaglandin synthesis by scavenging active oxygen(O2
- H2O2 1O2, HO.),and that the compensatory high SOD activity might be important for the maintenance of normal platelet function in the cases of glutathione reductase deficiency and the cases of myeloproliferative disorders with decreased platelet CAT and/or GPx. In order to make clear the roles of SOD, further study was done by utilizing diethyldithiocarbamate(DDC); specific inhibitor of SOD.

DDC inhibited the activity of platelet SOD after two hours incubation in a dose dependent manner. Platelet treated with 10-4 M of DDC inhibited the SOD activity to 86 % of control and showed the increase of platelet aggregability induced by ADP, epinephrine and collagen. The higher concentration of DDC (10-3 M) treated platelet returned to show nearly the same maximal aggregability as that of control, and platelets with l6 % residual activity (10-2 M DDC) rather showed the inhibition of aggregation. The basal malondi- aldehyde (MDA) was not changed at any concentration of DDC treated. However, N-ethylmaleimide(NEM)or thrombin-induced MDA production of DDC treated platelet showed an almost similar manner to platelet aggregability; increased at low concentration of DDC and decreased at higher concentration.

These results suggested that SOD plays a regulatory role on platelet function and metabolism, possibly by affecting the pathway of prostaglandin biosynthesis.
Role Of Albumin In The Regulation Of Platelets Production
Authors: Cherbit G , Forestier F , Solé Y , Tersarkissian M H , Gaschard J C
Published: May 2019

We measured Serum Albumin (S.A.) and Platelet Albumin (p. A.) on 144 normal individuals, 70 pregnant women (by using a Radio-Iramuno-Assay method) and platelet counts.

All the data were studied according to the method of multivariate analysis on a UNIVAC 1110 computer.

The aim of our study was to quantify the influence of unknown parameters involved in S.A. synthesis and P.A. content.

Two distinct parameters were put forward by these experiments : First called S.A. parameter (decreased during pregnancy) second, P.A. parameter (increased during pregnancy).

On the other hand we established the existence of a third parameter controlling the regulation of platelet albumin content by altering the counts of circulating platelets.

The less we got platelets, the more the P.A. content was important, showing a feed back mecanisra between S.A., P.A. and platelet counts.

Albumin is involved in the regulation of platelets production.
Localisation Of Thrombotic Processes With In-111 Labeled Thrombocytes
Authors: Hardeman M R , Vreeken J , van der Schoot J B
Published: May 2019

Human platelets were labeled with In-111 oxinate in order to establish their in vivo kinetics as well as their possible use for the detection of the early events in various thrombotic, i.e. platelet consuming, processes. Autologous platelets were incubated at roomtemperature with 250-500 uCi In-111 oxinate; the labeling-efficiency was 75-90 %.

In vivo survival of In-111 labeled platelets was found to be equal to that of Cr-51 labeled platelets: 8-10 days,while the in vivo recovery was 60-70 %(in the presence of a spleen) indicating that nearly all of the injected platelets were viable. Injected, not platelet-bound radio-activity disappeared rapidly from the plasma during the first day after injection; on the following days, plasma as well as urine activities were negligable.

Thrombotic processes could be visualised in renal artery thrombosis, DVT, atherosclerotic and dissecting aneurysms, rejecting kidney-transplants and shunt-thrombosis. Most results correlated well with those of other techniques, i.e. angiography and clinical, histological and biochemical parameters. A false negative result, however, was found in a patient with an a.carotis thrombus, existing for longer time. Pathological accumulation of radio-activity seen at scintigraphy could always be correlated with an increased rate of disappearance of circulating radio-active platelets.

Thus, using In-111 oxinate, platelets can be labeled with a high efficiency, a high in vivo recovery and maintenance of normal in vivo kinetics. These platelets are suitable for scintigraphic detection of thrombotic processes. The method seems to be limited to new, growing thrombi.
Giant (Bernard-Soulier) Platelets are Normal Sized in Circulation
Authors: Frojmovic M. M. , Milton John G. , Caen J. P.
Published: May 2019

Bernard-Soulier Syndrome (BSS) is a platelet disorder for which one of the characteristics is the appearance of “giant” platelets on peripheral blood smears. A comparison is made between the shape distributions of platelets obtained from normal (9) and BSS (3) donors. Geometric parameters are evaluated from a cinematographic analysis of freely rotating glutaraldehyde-hardened and unfixed platelets and of platelets on blood smear. On blood smear, normal platelets have a mean diameter of 1.8 ± 0.2 μm, where 10% have a diameter greater than 2.5μm (Weiss, et al (1974). Am. J. Med. 57: 920). On the other hand, BSS platelets are significantly larger, having a mean diameter of 3.3 + 0.7/μm, with 80% having a diameter greater than 2.5μm. These observations are consistent with all published reports concerning BSS platelets and suggest donors homozygous for this disorder (Bithell, et al (1972) N. Y. Acad. Sei. 201: 145). In contrast, the freely circulating disc-form of the BSS platelet is essentially indistinguishable from a normal platelet: mean diameter and thickness of 3.2 ± 0.3μm, and 1.4 ± 0.4 μm respectively, as compared with 3.2 ± 0.3 μm and 1.1 ± 0.4 μm for normal platelets.

It is concluded that the giant size of BSS platelets results from abnormal behaviour of these platelets during the preparation of the blood smear.
Biochemical Properties of Human Platelet Factor 4
Authors: Walz D. A. , McCoy L. E. , Wu V. Y.
Published: May 2019

Human platelet factor 4 (PF4) was isolated from outdated platelet concentrates to evaluate its biochemical properties. Platelets were washed in saline, lysed by freeze-thawing, and centrifuged to remove particulates from the lysate. PF4 was adsorbed on heparin immobilized on Sepharose. The yield of PF4, eluted in molar saline, was 9.3 ug/ml platelets. Polyacrylamide electrophoresis indicated a single-chain protein with an apparent molecular weight of 9,600. Amino acid composition indicated a 92 residue chain having 2 disulfide bridges and free of MET, PHE, and TRP. Electrophoresis of equal molar ratios of heparin and PF4 in the presence of 0.1% SDS resulted in the appearance of a slow migrating band having an apparent molecular weight of 20,000. The isolated PF4 was poorly soluble in the absence of heparin or at low salt concentrations. Elution profiles of gel and membrane filtration studies were indicative of molecular aggregation. PF4 appeared to be totally resistant to tryptic and chymotryptic hydrolysis but not to pepsin. However, PF4 bound to heparin was resistant to pepsin. Reduced-alkylated PF4 retained its ability to bind heparin but lost its resistance to tryptic or chymotryptic hydrolysis. The presence of heparin did not alter the peptide pattern following tryptic or pepsin hydrolysis of reduced-alkylated PF4 while the peptide pattern after chymotrypsin was modified. Peptides released after chymotryptic hydrolysis of reduced-alkylated PF4 had no apparent affinity for heparin unless heparin was present originally. These peptides after chymotryptic hydrolysis of heparin-bound-reduced-alkylated PF4 may serve as models for the design of small, synthetic heparin antagonists. (Supported by USPHS NIH grant #HL-20317-01 and the Charles DeVlieg Foundation.)
DDAVP : A New Pharmacologic Approach to the Management of Hemophilia and Von Willebrand’s Disease
Authors: Mannucci P. M.
Published: May 2019

I-deamino – 8 D – arginine (DDAVP) infusion causes a marked increase of F VIII – related properties in patients with moderate and mild hemophilia (H) and von Willebrand’s disease (VWD) The possibility was evaluated that such an autologous F VIII response might be hemostatically effective, allowing patients (pts) to undergo surgery without blood products. 0.3 μ/Kg of DDAVP given before dental surgery and repeated in the early post–operative period were followed by a 2 – to 3 fold rise of F VIII coagulant activity (VIII : C) in 4 pts with moderate and mild H. In two, there was no abnormal bleeding after dental extraction, whereas plasma concentrates became necessary to control oozing in the remaining 2 pts. The adoption of higher dosage (0.4–0.5 μ/Kg) in pts with higher starting VIII : C (9% or more) was followed by a more marked response (4 to 6 fold). Hence, VIII : C levels up to 100% of average normal could be achieved and dental extractions as well as major surgical procedures (such as cholecystectomy, thoracotomy and two tonsillectomies) were carried out successfully in 6 pts with mild H and 2 with VWD. The mean halfdisappearance time of autologous VIII : C was 9.4 hours (range 7.5–11.6). Plasma and urine osmolality showed no consistent variation. DDAVP appears a promising pharmacologic alternative to plasma concentrates in the management of a number of pts with H and VWD.
Mechanism of Activation of Human Factor IX by Activated Human Factor XI
Authors: Østerud Bjarne , Bouma Bonno N. , Griffin John H.
Published: May 2019

Human Factor IX (F. IX) has been highly purified using: BaSO4 adsorption and elution, DEAE-cellulose chromatography, preparative polyacrylamide gel electrophoresis, and chromatography on heparin-agarose. The purified F. IX appeared homogeneous on SDS-gels and revealed a single polypeptide chain of 55,000 MW in the Weber-Osborn system. Human Factor XI was purified by ion exchange chromatography to greater than 95% homogeneity as judged on polyacrylamide gels. Following activation of factor XI by purified human factor XIIa, the factor XIa was a potent activator of F. IX in a Ca++-dependent reaction. As judged on SDS gels, activated F. IX at 43,000 mw contains two disulfide-linked polypeptide chains of 27,000 and 16,000 mw. These mw values for the zymogen and activated form of F. IX are identical to those reported for bovine F. IX. And the amino acid compositions of human F. IX and of the isolated heavy and light chains of F. IXa are quite similar to those reported for bovine F. IX and F. IXa. Based on these observations, it is suggested that activated human F. IX contains its active site in a C-terminal 27,000 mw polypeptide which is linked by disulfide bonds to a 16,000 mw N-termina| polypeptide containing gamma carboxyglutamic acid.
Anti-Inflammatory Agents which do not Inhibit Platelet Prostaglandin Synthesis
Authors: Cerskus I. , Philp R. B.
Published: May 2019

There is overwhelming evidence to support the theory that non-steroidal anti-inflammatory drugs (NSAID) act by interfering with production of prostaglandins (PG). Since PG intermediates are intimately involved in platelet aggregation, NSAID such as aspirin (ASA) also have adverse effects on platelet function. In the present study three compounds bearing structural resemblance to ASA were investigated for effects on platelet aggregation, PG synthesis and carrageenin-induced rat paw oedema.

ASA was more potent than 3-methylphthalide (3-MP) in inhibiting platelet aggregation in response to ADP and collagen. 2-Acetylbenzoic acid (ABA) inhibited aggregation only at very high doses (I50 5mM), whilst 3-propionyloxybenzoic acid (PBA) significantly potentiated aggregation. Neither ABA nor PBA, up to 5mM final concentration, affected PG synthesis from labelled precursor in platelet lysates, and 3-MP inhibited PG synthesis by only 20% at ImM, at which concentration aggregation was virtually abolished. However, 3-MP was slightly more potent than ASA in suppressing carrageenin-induced rat paw oedema, whilst ABA and ASA were of approximately equal potency. PBA had no effect.

These results indicate that anti-inflammatory activity may be separated from effects on platelet function, and that the PG synthetase of platelets may differ from that involved in PG elaboration in an acute inflammatory response. The possibility that ABA and 3-MP may be selective inhibitors of the latter enzyme, cannot be excluded.
Studies on the Structure and Subunit Composition of Human Antihaemophilic Factor
Authors: Gorman J. J.
Published: May 2019

Human antihaemophilic factor has been purified by hydroxylapatite chromatography following precipitation from plasma and gel filtration on Sepharose 6B.

Application to hydroxyiapatite was in 0.02 M tris HCl (pH 7.35) – 0.14 M NaCl and after washing with 5mM phosphate (pH 6.8) – 0.1 M NaCI the antihaemophilic factor was eluted with 0. 1M phosphate (pH 6.8) – 0.1M NaCl. Factor VIII coagulant activity, factor VIII related antigen and von Willebrand factor activity eluted simultaneously.

The protein(s) had a molecular weight in excess of 500,000 and multiple subunits as shown by electrophoresis in 5% acrylamide gels containing sodium dodecyl sulphate;without reduction the protein failed to enter these gels but following reduction multiple bands were observed, the major band had a molecular weight around 200,000.

Thin layer peptide mapping demonstrated structural inter-relationship between the 200,000 dalton protein and three of the smaller species, however, two other unrelated smaller species were evident.

It is apparent from these findings that human factor VIII may exist as multiple molecular forms due to heterogeneity of one subunit (MW around 200,000) and the molecular structure may include other smaller non-identical subunits. The structure-function relationships of these subunits remains to be elucidated.
Synthesis of Coagulation Factors and Factor VIII Related Antigen by the Isolated Perfused Rat Liver
Authors: Bloom A. L. , Shaw Eryl , Giddings J. C. , Peake I. R.
Published: May 2019

The synthesis of coagulation factors, including procoagulant factor VIII (FVIIIC) and factor VIII related antigen (FVIIIRAG) was studied using the isolated perfused rat liver. The perfusion fluid consisted of rat blood cells suspended in Tyrodes solution containing bovine albumin. Synthetic function was confirmed by the addition of 35S l-methionine to the perfusion medium. Using two dimensional crossed Immunoelectrophoresis and autoradiography progressive incorporation of radioactivity into plasma protein was demonstrated. This was inhibited by cyclohexamide. Coagulation factor assays demonstrated the synthesis of factors II, IX and X and of factors V and VIIIC. Demonstration of synthesis of FVIIIRAG was attempted using an immunoradiometric assay and a cross-reacting rabbit anti-human antiserum. FV and FVIIIRAG synthesis was more clearly demonstrated when the perfusion fluid was depleted of leucocytes and platelets. Addition of Warfarin inhibited the synthesis of factors II, IX and X but not of factors V and VIII. Cyclohexamide completely inhibited synthesis of all coagulation factors but actinomycin acted only after a latent period. No coagulation factor synthesis was demonstrated in the perfusion system without a liver. The results confirmed the functional capacity of the isolated perfused liver for synthesising proteins and the vitamin K dependent factors and demonstrated similar kinetic features for the synthesis of factors V, VIIIC and, possibly, VIIIRAG.
Plasminogen Activator Release by Oral Contraceptives and Exercise
Authors: Hedlin A. M.
Published: May 2019

Oral contraceptives are capable of inducing an increase in blood fibrinolytic activity and our study was undertaken in an attempt to determine whether this is accomplished by stimulating the release of plasminogen activator.

For this study 25 women, twelve non-users and thirteen users of the oral contraceptive, Demulen, provided blood and urine samples. Samples were obtained following a one-half hour of rest (pre-exercise), and following a 5 minute interval of moderate exercise on a treadmill. Subjects were tested early in the menstrual cycle (when the oral contraceptive was withdrawn) and late in the cycle when Demulen had been used for about two weeks.

Plasma plasminogen activator levels were essentially the same for the two groups, early in the cycle, with exercise causing a doubling of the pre-exercise activator level. However, late in the cycle the contraceptive users had a much higher pre-exercise activator level than the non-users, and exercise caused a three-fold increase in this level.

Urine plasminogen activator levels were measured in samples obtained before and after exercise. The urine plasminogen activator levels, unlike the plasma plasminogen activator levels, showed no significant difference between non-users and users. An even more remarkable difference was that at both intervals in the cycle and in both groups the urine plasminogen activator level decreased, rather than increased, following exercise.

We conclude that the oral contraceptive Demulen does stimulate the release of plasma plasminogen activator but this does not interfere with release of this activator by exercise.
Shear Rate Dependence of Platelet Adhesion to Collagenous Surfaces in Willebrand Factor-Depleted Blood
Authors: Baumgartner H. R. , Tschopp Th. B. , Meyer D.
Published: May 2019

A platelet function defect similar to that in von Willebrand’s disease, was produced by adding to human blood an antibody raised in rabbits against human factor Vlll/Willebrand factor. The effect of this antibody (F VIII Ab) on platelet adhesion was tested in an annular perfusion chamber. Only a small adhesion defect was observed at a shear rate corresponding to that in large arteries (830 s-1) . Since bleeding usually occurs from small vessels, platelet adhesion to collagenous surfaces was investigated at higher shear rates corresponding to those in small vessels. The surfaces were exposed to human blood (l5mM citrate) at 1, 2 and 4x103 s-1 shear rate for 6,

and 3 min, respectively. Different exposure times were chosen in order to obtain in control perfusions 50 – 70 % coverage of subendothelium with platelets at each shear rate. Platelet adhesion and adhesion-induced aggregation were measured morphometrically. As compared to control, F VIII Ab inhibited adhesion to subendothelium by 15±6, 32±21, 40±10 and 90±2 % (mean ± SE) at 0.83, 1, 2 and 4x103 s-1 shear rate, respectively. The corresponding inhibition by F VIII Ab observed on the fibrillar collagen of α-chymotrypsin-digested subendothelium was 62±11, 75±20, 99±1 and 100 %. In platelet rich plasma, F VIII Ab abolished Ristocetin-, inhibited collagen- and had no effect on ADP-induced aggregation.

Thus the defect in platelet adhesion to collagenous surfaces observed in Willebrand factor-depleted blood is minimal at low (venous) and maximal at high (small vessel) shear rates.
Structure-Function Studies of Factor VIII/Von Willebrand Protein(s)
Authors: McKee Patrick A.
Published: May 2019

Factor VIII (FVIII) procoagulant activity was initially thought to be a glycoprotein with a molecular weight (MW) >1 million and composed of disulfide-1inked ~200,000 MW subunits. A protein with similar properties, except lacking procoagulant activity, is in hemophilic plasma; it was identical to normal FVIII by SDS-gel analyses, isoelectric focusing, and PAS staining. Subsequently it was shown that the FVIII glycoprotein also has von Willebrand factor (vWF) activity, suggesting that both FVIII and vWF activities might be properties of the same molecule. When the FVIII/vWF protein(s) is rechromatographed on 4% agarose and 0.25 M CaCl2, virtually all the protein and vWF activity elute in the void volume, but most of the FVIII procoagulant activity elutes much later. The extent of separation of the two activities depends on the amount of protein applied to the column. Also, exposure of the FVIII/vWF to thrombin before gel filtration strikingly accentuates separation of the two activities. The reduced SDS-gel pattern of the void volume protein peak showed the 200,000 MW subunit while that of the procoagulant peak contained several subunit bands which ranged from ~30,000–100,000 MW. Removal of sialic acid from FVIII/vWF is associated with reduced ristocetin induced platelet aggregation and causes a 50-fold increase in the rate of clearance of protein from the circulation by the hepatocyte. Currently, our data suggest that FVIII procoagulant and vWF activities are properties of a single molecule composed of disulfide-bound identical subunits. Cleavage by thrombin then results in FVIII procoagulant activity. Additional cleavages, to which the molecule appears very sensitive, results in FVIII inactivation. The vWF activity is very stable—even to proteolysis—and it appears to be a function of the carbohydrate side chains of the molecule.
Mechanism of Action of Halofenate, A Platelet Inhibitor
Authors: Favis G. R. , Colman R. W.
Published: May 2019

Halofenate (Hal) has previously been shown to inhibit epinephrine (Epi) and ADP induced platelet aggregation and C14-serotonin release. We further investigated the site of action of Hal by examining platelet shape change as a membrane event and malondialdehyde (MDA) formation as a measure of prostaglandin synthesis. Platelet-rich-plasma (PRP) with and without Hal wasdiluted in an EDTA buffer and examined in a spectrophotometer modified for stirring and maintained at 37°. ADP induced increase in absorbance was recorded and the velocity of the shape change curve was plotted against ADP concentration. MDA production was measured by the thiobarbituric acid assay and utilized a DEAE-52 cellulose column to concentrate the chromogen. Hal in pharmacologic concentrations (.96mM) had no effect on Epi induced primary aggregation or on ADP induced shape change. However, at higher than pharmacologic amounts (3.36mM), Hal did inhibit ADP induced shape change. Epi-induced MDA formation (.18μM-.33μM) normally occurs concomitant with the second phase of aggregation and serotonin release but was markedly decreased by Hal (.06μM-.085μM). This inhibition was not due to a direct effect on prostaglandin synthesis since sodium arachi-donate (1mM) caused secondary aggregation in PRP treated with Hal but not PRP treated with aspirin (4mM). Hal (.96mM) does not seem to inhibit platelet aggregation through an inhibition of ADP induced shape change or of Epi induced primary aggregation. Since Hal treated platelets respond to arachidonate, Hal must work at some earlier step than arachidonate induced prostaglandin synthesis. We suggest that this may be an alteration of the platelet membrane structure which makes ADP and Epi binding sites less accessible or which impairs arachidonic acid release by phospholipase. Decreased MDA formation and inhibition of aggregation would then be secondary to this membrane change.
Reduction of Surface Negative Charge in Human Platelets by Ristocetin
Authors: Muraki H. , Watanabe K. , Ando Y. , Toyama K. , Hasegawa M.
Published: May 2019

The effect of ristocetin on the platelet surface charge was examined. One part of citrated PRP or platelet suspensions was mixed with nine parts of lOμM Tris-buf f ered saline (pH 7.4) and the mixtures were incubated at 25°C with or without ristocetin. These mixtures were applied for a cell electrophoresis microscope to determine the electrophoretic mobility(EM), from which the platelet surface charge was estimated. A significant reduction in surface negative charge of platelets was observed when ristocetin was added to the mixtures. EM of non-treated and risto-cetin(lmg/ml)-treated platelets was −1.269±0.045 and −0.870±0.040 μ/sec/V/cm, respectively. A dose-response relationship was observed between platelet EM change and ristocetin concentra-tion(0.1~1.2mg/ml). The change in EM reached to the maximum within seconds. Similar reduction of EM was observed when formalin-fixed platelets were incubated with ristocetin. Heparin at concentrations(10u~100u/ml) inhibited, but protamine sulfate(0.05mg/ml) enhanced the change of EM and platelet aggregation by ristocetin. Both the ristocetin-induced aggregation and the reduction of EM were optimally observed at pH 7.3 and were inhibited at pH above 8.0. The presence of plasma component was found to be essential in the reaction mixture for this reduction of EM. EM was decreased in the presence of ristocetin proportionally with the increase in per cent of plasma in the mixture up to 10(v/v)%. The ristocetin-induced reduction in EM of platelets was not observed when normal platelets were suspended in plasma from a patient with Willebrands’ disease(EM= −1.278±0.070 μ/sec/V/cm, with ristocetin, 1mg/ml).

These results strongly suggest the involvement of decreased surface negative charge in the mechanism of platelet aggregation induced by ristocetin.
Effect of Heparin on Circulating Antithrombin III Level
Authors: Marciniak E. , Gockerman J. P.
Published: May 2019

Both the natural defense against thrombosis and the anticoagulant effect of heparin rely essentially on the presence of antithrombin III (AT III) in blood. The rationale for heparin therapy is to strengthen the existing inhibitory potential of AT III by increasing the velocity of binding. It has not been established whether there is a modifying effect of heparin on the circulating levels of AT III. Twenty-two patients with documented or suspected thromboembolism, including one with hereditary AT III deficiency, were studied before, during and after continuous intravenous infusion of 750–2000 heparin u/hr. AT III was evaluated in plasma 1) as a total factor Xa binding capacity in a method independent of the velocity of inhibition, 2) by immunodiffusion, 3) by rocket Immunoelectrophoresis.In all patients, regardless of their initial AT III level, there was a marked progressive decrease in AT III during heparin infusion, with an abrupt return to original levels in 2–3 days after cessation of therapy. The average maximum decrease in the 5th-7th day of therapy was of 0.38+0.09 u/ml (normal AT III = 1.0 u/ml) or 11.2 + 2.5 mg/dl and was not affected by concurrent therapy with Coumadin.Heparin given in repeated intravenous bolus injections also gradually lowered the quantity of circulating AT III but no changes were seen after a single intravenous injection in normal volunteers or subjects with congenital AT III deficiency. These results give evidence for a possible mechanism of a paradoxical, thrombosis inducing effect of heparin and indicatethat low AT III levels in heparinized patients should not be mistaken for a cause or result of the underlying thromboembolic episode.
Production by Human Lung Cells of Trypsin and Plasmin Inhibitor(s) Differing from a1-Antitrypsin, a2-Macroglobulin, C1-Esterase Inhibitor and Inter-a-Trypsin Inhibitor
Authors: Bernik Maria B.
Published: May 2019

Human lung cells in primary culture and serial subculture were used to study the production of inhibitory activity and to isolate and identify inhibitor(s) of trypsin and plasmin produced and released by the cells into the supernatant medium. Assays of inhibitory activity were performed on fibrin, and casein substrate and results expressed in BAEE units of trypsin inhibited on the former substrate. Inhibitory activity against plasmin and trypsin accumulated progressively in serum free supernates of cultures to concentrations of 80–150 BAEE units/ml and was isolated from the supernates by concentration with Amicon PM 30 membranes, gel filtration on Sephadex G—100 or G-200 columns and polyacrilamide gel electrophoresis. On calibrated columns inhibitory activity eluted in the range of 75,000 mol wt substances. On immunodiffusion, performed using a wide range of concentrations of chromatographed inhibitor preparations with specific activity of about 1,300 BAEE units/mg protein there was no cross-reaction with antiserum to a1-antitrypsin, a2-macroglobulin, C1-esterase inhibitor or inter-a-trypsin inhibitor. There was also no cross-reaction with antiserum to antithrombin III or antichymotrypsin. Immunoelectrophoresis showed no immunoreactive material with the antisera. These observation indicate the production in lung of inhibitor(s) differing from the major protease inhibitors and derivatives or subunits of these inhibitors described to date.
Binding of Adenosine Diphosphate (ADP) to Isolated Human Platelet Membranes
Authors: Lips J. , Sixma J. J.
Published: May 2019

Human platelet plasma membranes were isolated according to the glycerol loading technique of Barber and Jamieson. The binding of 14C ADP was studied with Millipore filtration in a Ca2+ and Mg2+ containing buffer at pH 7,4. At least two types of binding sites were found: A high affinity system with a maximum binding of 160 pMoles/mg protein and an association constant of 1,1 χ 106 M-1; and alow affinity system with a maximum binding of about 4500 pMoles/mg protein and an association constant of 0,6 χ 1θ4 M-1. The binding according to the high affinity system showed little temperature dependency (Q10 = 1,10). The pH optimum was at 7,3. Ca2+ ions were an absolute requirement for binding.

Nucleoside diphosphokinase (NDPK) was found in the membrane vesicles. Evidence that this enzyme was not responsible for ADP binding was obtained. The enzyme is Mg2+ dependent and is inhibited by AMP, in contrast to ADP binding. The Q10 was 1,44.

ADP binding was inhibited by ATP, IDP and β/γ-imidoadenosine triphosphate.
Nonradioactive Semi-Automated Determination of Platelet Survival Time in Man
Authors: Roncucci R. , Deperon R. , Lansen J. , Destailleur J. , Verhaeghe R. , Doumont J. , Lamhelin G. , Stalle F. Van
Published: May 2019

Several methods have been proposed for the determination of platelet life-span. Among them the technique described by Stuart and al (l) based upon the ex-vivo assay of platelet malondialdehyde after one single oral intake of acetylsalicylic acid is receiving an increasing interest. We adapted this method for semi-automated determinations using an AutoAnalyzer system.

Platelet survival time was determined with this technique in healthy volunteers as well as in patients with abnormal platelet life-span.

In some instances chromium-51-determinations were also performed in order to assess the reliability of the method proposed. The results obtained are comparable for both methods and compatible with those described in the literature.
The Effects of Two New Antithrombotic Agents (IM Idazoquinazoli Nones) on Platelet Shape and Aggregation
Authors: Tang S. S. , Frojmovic M. M.
Published: May 2019

Recently, one member of a new series of compounds was reported as a potent, nontoxic and long lasting antithrombotic agent, based on in vitro and in vivo animal tests (Bristol Labs; Fleming et al (1975), J. Pharmacol. Exp. Ther. 194, 435).We here report on the effects of this compound, 6-methyl-l,2,3,5-tetrahydroimidazo [2,1-b]quinazolin-2-one hydrochloride monohydrate (BL-3459), and its metabol ical ly more stable 6,7-dichloro analogue (BL-4162) , on rabbit and human platelet shape change and aggregation, and compare them with other agents known to affect platelet adenosine 3′:5′-cyclic monophosphate (cAMP). In aggregometer studies with citrated (0.29%) platelet-rich plasma (PRP), both BL-compounds were found to inhibit platelet shape change and aggregation induced by ADP, thrombin, serotonin and adrenaline-serotonin. Typically for human PRP, aggregation induced by 10 μM ADP was inhibited by 90% with 10 μM BL-4162 or 0.1 μM prostaglandin E1 (PGE1), and by 60% with 10 μM BL-3459. In corresponding rabbit PRP tests, 60% inhibition was caused equally by 1 μM BL-3459 or BL-4162, and by 0.05 μM PGE1,Both BL-compounds, like methylxanthines, were found to potentiate the inhibitory effect of PGE1 on platelet aggregation but did not potentiate the action of methylxanthines. Moreover, they both slightly increased the basal level of rabbit cAMP and potentiated the elevation of cAMP by PGE1, These BL-compounds are potent inhibitors of human and rabbit shape change and aggregation and appear to act by a mechanism distinct from that of PGE1.
Non-Specific Binding of Thiocholine Ester of Cinnamic Acid to Fibrin
Authors: Seelich T. , Perret B. A. , Furlan M. , Beck E. A.
Published: May 2019

Thiocholine esters inhibit the enzymatic crosslinking of fibrin (1). Thiocholine ester of 14C-labelled cinnamic acid (2-diethylbenzylaminoethyl thioltranscinnamate bromide) was prepared and incubated with human fibrinogen in the presence of factor XIII and thrombin. Polyacrylamide gel electrophoresis with sodium dodecyl sulphate, followed by liquid scintillation counting of the separated protein bands, showed that all three chains of fibrinogen had bound the crosslinking inhibitor(2 moles/mol α chain, 1.5 moles/mol β chain, 2.2 moles/mol γ chain). Cyanogen bromide cleavage of the isolated α chain resulted in 6 major fragments all of whichwere radioactive. These results indicate that the binding of the thiocholine ester of cinnamic acid was not restricted to the specific donor crosslinking sites of fibrin(ogen). This thiocholine ester differs in this respect from dansyl-cadaverine which binds exclusively to the acceptor sites involved in fibrin(ogen) crosslinking.
Fibrinogens of Common Laboratory Mammals - A Comparative Study
Authors: Hawker R. J. , Hawker Linda M.
Published: May 2019

Many laboratory mammals are used as in vivo or in vitro models of haemostatic mechanisms and require readily available purified fibrinogen suitable for trace labelling. A comparative study was made on the fibrinogens extracted by a standard 45 minute procedure from man and the following common laboratory mammals: baboon, rhesus monkey, dog, cat, rabbit, guinea pig, rat and the mini-pig. The method* utilising 5.5 mis of plasma is applicable for autologous preparations and gives 60–70% yields of pure fibrinogen from all species being dependent upon the fibrinogen concentration of the starting plasma. Clottability was consistently in excess of 90% and the final solution was suitable for direct labelling with 125-lodine using Chloramine T.

The fibrinogens were examined for purity on sodium dodecyl sulphate Polyacrylamide gels from more than 60 different individuals, using reduction with mercapto-ethanol to compare the subunît molecular sizes (Aα B β γ). No other proteins were detectable nor were enzymatically derived modifications. We demonstrate molecular size differences as high as 9,000 daltons between the same designated subunit within a phylogenetic order, for example, the Aasubunit of the non-human primates compared with that of man.

* Hawker, R. J. and Hawker, Linda M., 1976. J. Clin. Path. 29, 495–501 .
Circulating Factor IX Antigen-Inhibitor Complexes in Hemophilia B- Following Infusion of a Factor IX Concentrate
Authors: Goodnight S.H. , Britell C.W. , Wuepper K.D. , Østerud B.
Published: May 2019

A persistent low titer IgG factor IX inhibitor (0.8–2.3u/ml) has been previously described in an 11 year old boy with severe hemophilia B-. Since the disappearance of factor IX antigen (IXAGN) following Konyne(R) infusion was markedly delayed (

60 hr) compared to factor IX clotting activity (T

8 hr) (Blood 48:977, 1976), a search for circulating factor IX antigen-inhibitor complexes was undertaken. The inhibitor binds firmly to staphlococcal protein A – Sepharose 4B (SPA-Sepharose) but may be eluted in the IgG 1, 2, and 4 fraction with 3M NaSCN. SPA-Sepharose chromatography of post-infusion plasma (IXAGN 2.7u) from the patient with the inhibitor showed IXAGN (0.5u) in the IgG 1, 2, and 4 fraction, whereas IXAGN was not found in that fraction of chromatographed samples of post infusion plasma from non-inhibitor patients with hemophilia B- or B+. Using a highly specific antibody to factor IX, two dimensional Immunoelectrophoresis of purified factor IX or normal plasma showed a single symmetrical fast moving peak. When an equal volume of inhibitor plasma was added to either factor IX or normal plasma a second, slower moving component was also seen. Two dimensional Immunoelectrophoresis of plasma obtained from the patient 45 minutes after infusion showed both the slow and fast components. The presence of IXAGN in the IgG 1, 2, and 4 peak after SPA-Sepharose chromatography and the demonstration of 2 components on two dimensional Immunoelectrophoresis indicates the presence of circulating factor IX antigen-inhibitor complexes in the plasma of this patient following the infusion of a factor IX concentrate.
Localization of Crosslink Sites in α-Chain of Human Fibrin
Authors: Fretto L.J. , Ferguson E.W. , McKee P.A.
Published: May 2019

To determine the position of potential crosslink acceptor sites in the α-chain, fibrin was specifically labeled with the fluorescent pseudo-donor monodansylcadaverine (MDC) and then digested to obtain labeled α-chain peptides. These were isolated and characterized by sodium dodecyl sulfate (SDS) gel electrophoresis, amino acid analysis, amino-terminal sequence analysis and ultracentrifugation. The α-chain of MDC-labeled fibrin was selectively cleaved to a series of fluorescent peptides of molecular weights 45,000 to 21,000 daltons using plasmin and 34,000 to 12,000 daltons using ancrod. A labeled plasmic peptide of molecular weight 23,000 displayed all the fluorescence of the MDC-labeled α-chain and when digested to the plasmin-resistant peptide of 21,000 daltons (P21), an amino-terminal segment having a fraction of the fluorescence was released. SDS-gel analysis of the residual, particulate plasmin-treated fibrin showed that the disulfide-bound amino-terminal 25,000-dalton portion of the α-chain was not MDC-labeled. However, the residual fibrin from ancrod digestion had an amino-terminal 38,000-dalton α-chain derivative which contained two MDC-labeled sites that were separable by CNBr cleavage. Taken together with the sequence data, the above findings established the existence of three acceptor sites located approximately at residues 260, 315 and 390 of the Aα-chains. That the physiological crosslink acceptor sites also reside in the middle 1/3 of the α-chain was demonstrated by plasmin digestion of highly crosslinked fibrin; a peptide having electrophoretic properties and an amino acid composition very similar to P21 was isolated which also contained at least one crosslink as determined by cyanoethylation. These findings combined with pre-existent information about fibrin structure permitted construction of a model for α-chain polymerization.
Combined Administration of Low Dose Heparin and Aspirin Versus low Dose Heparin Alone as Prophylaxis of Deep Vein Thrombosis (D.V.T.) After Total Hip Replacement. A Preliminary Report
Authors: Kher A. , Flicoteaux H. , Jean N. , Blery M. , Judet T. , Honnart F. , Pasteyer J.
Published: May 2019

In a prospective randomized trial, the efficacy of a prophylaxis of D.V.T. by low dose heparin alone (t.i.d.-group I-18 patients) and by low dose heparin (t.i.d.) combined with aspirin (500 mg b.i.d.-group II-15 patients) was investigated using both radioactive fibrinogen uptake test and venography. The treatment groups were comparable for all pretreatment characteristics. The incidence of isotopic D.V.T. between group I (22.2%) and group II (26.6%) was not statistically significant. There was also no significant difference in the frequency of popliteal vein thrombosis between the two groups. One non fatal pulmonary embolism occurred in group I. Correlation of phlebography with radioactive fibrinogen uptake test is 87.5%. The combination of low dose heparin and aspirin does not improve the results obtained with low dose heparin alone in the prevention of post operative D.V.T. and a tendency in increased bleeding and wound haematomas were observed with such a combination.
Postoperative Increase of Heparin in Prevention of Deep Vein Thrombosis (DVT) After Total hip Replacement
Authors: Schöndorf T.H. , Hey D.
Published: May 2019

Acetylsalicylic-lysine (ASL) and a modified low dose heparin prophylaxis were examined to prevent postoperative DVT. 71 patients over 40 years undergoing hip replacement were randomised according to odd or even year of birth. Group I: 1.8g ASL were injected i.v. after the operation and then 24 hourly for 9 days. Group II: 5 000 IE heparin were given s.c. 10 and 2 hours before operation and continued in 8 hour-intervals until the evening of the first postoperative day. Heparin was subsequently increased to 3 × 7 500 IE/24 hours. DVT was diagnosed by the 125-I-fibrinogen test; in all legs with a positive scan a venography was performed. Partial-thromboplastin time and platelet aggregation was regularily controlled.

The total incidence of DVT under the heparin prophylaxis (4/58) was significantly less than under the ASL-treatment (19/33); the rate of popliteal-femoral DVT revealed a similar significant difference. DVT opposite to the operated hip and bilateral DVT developed only under the ASL-treatment. The postoperative haematocrit decrease and the amount of blood from the wound drainage of the two groups did not differ statistically. Major wound bleeding occurred under ASL in one and in two patients under heparin.

ASL did not prevent DVT sufficiently, but DVT was effectively reduced by the postoperative increase of heparin in these high risk patients.
The Carbohydrate Components in the Action of Antithrombin III.
Authors: Danishefsky I. , Zweben A. , Slomiany B.
Published: May 2019

The carbohydrate components of human antithrombin III were identified and the contribution of the sialic acid component to the interaction of the inhibitor with thrombin and heparin was studied. Gas chromatographic analyses showed the following percent composition:galactose, 0.77; mannose, 1.49; N-acetylglucosamine, 1.68; sialic acid, 1.88, corresponding to a molar ratio of approximately 2:4:4:3, respectively. Colorimetric analysis by the thiobarbituric acid method, showed an average value of 3.3% neuraminic acid, indicating the presence of three residues of the latter per galactose unit. The neuraminic acid was removed by treatment with neuraminidase from CI. perfringens, and the product was purified by affinity chromatography on heparin-aminohexylsepharose. Except for sialic acid, the modified antithrombin III had the same carbohydrate composition and circular dichroism spectrum (300-190 nm.) as the original inhibitor. The desialylated product retained its effect in the progressive inhibition of thrombin and in the rapid deactivation of the latter in the presence of heparin.(Thrombin activity was assayed by its effect on fibrinogen.) Gel electrophoresis experiments on the desialylated product also showed that it forms a complex with thrombin similar to that with native antithrombin III. It is concluded, therefore, that the sialic acid component is not involved in the binding of the inhibitor to thrombin or heparin.
Energy Requirement for Maintenance of Adenylate Energy Charge and Metabolic ATP in Platelets During Starvation
Authors: Akkerman J.W.N. , Gorter G. , Sixma J.J.
Published: May 2019

Energy requirements for maintenance of stable adenylate energy charge (AEC) and metabolic ATP(ATP-m)level were studied in gel filtered platelets at various degrees of starvation. Platelets gel filtered and subsequently incubated during 40 min.at 37°C with 1mM CN- and without glucose consumed their glycogen at a rate of 0.79 ± 0.23(± SD, n=6)/μmol glycosyl residues .min-1 10-11 cells. During this period AEC and ATP-m decreased linearly with time at rates of 5-6.10-3 and 0.75-1.05% of total radioactive adenine nucleotides .min-1.10-11 cells respectively. Addition of 25–1000μM glucose increased lactate production and decreased the fall of AEC and ATP-m proportional to the amounts of glucose added. Glycogenolysis remained active below 100μM glucose but ceased at higher glucose concentrations. From these data ATP-m production from glycogenolysis and glycolysis was calculated and compared with the decrease of steady state levels of AEC and ATP-m. A production of 3μmol ATP-m.min-1.10-11 cells was required to maintain initial AEC and ATP-m level. At lower rates of ATP-m production these values fell without reaching stable steady state levels in a lower range. After 40-50 min variations in AEC and ATP-m ceased and lactate formation stopped leaving the cells in a state of hybernation. Subsequent addition of glucoserestored lactate accumulation, AEC and ATP-m. On the basis of formation and steady state levels of ATP-m its consumption was calculated. A lowering production was not completely met by a lowering consumption. Energy consumption in resting platelets is therefore partly independent from energy production.
Ultrastructural Study of Platelet Aggregation by Mouse 15091A Tumor Cells
Authors: Stewart G.J. , Kuprionas R.A. , Gasic G.J. , Catalfamo J. , Gasic G.P.
Published: May 2019

Most tumor cells cause aggregation of platelets in heparinized plasma via material shed into culture medium. In this study we investigated the events by transmission electron microscopy. Freshly washed cells were covered with closely spaced microvilli, many of which pinched off during 1 hour of incubation at 37°C. Both cells and shed microvilli were membrane enclosed. Shed microvilli became spherical vesicles containing cytoplasm. Platelets aggregated when stirred with incubated tumor cells or shed material. The aggregates were composed of platelets that showed pseudopod formation, centralization of granules and increase in the open channel system. Platelets around the periphery of aggregates had bulbous portions free of granules (ballooning) but many granules remained in platelets in the interior of aggregates suggesting that release of lysosomal enzymes may have been somewhat limited. Aggregates resembled those induced by ADP rather than by thrombin. Tumor cells were not incorporated into the aggregates. Vesicles were not selectively associated with platelets prior to or during aggregation. While some vesicles were incorporated into aggregates, it appeared that this was a consequence rather than the cause of aggregation. Therefore, vesicles may have produced soluble material that induced platelet aggregation.
A Chromogenic Semi-Micro Method for the Determination of Factor Xa, Factor Xa Inhibitor and Mini-Dose Heparin Levels
Authors: Scully M.F. , Kakkar V.V.
Published: May 2019

Conditions influencing the measurement of heparin levels in the range 0 to 0.3 units/ml of plasma using the chromogenic substrate, B2-Ile-Glu-Gly-Arg-pNA HCl (S2222, KabitVitrum Ltd) have been analysed to find the most suitable incubation mixture.

Factor Xa chromogenic activity was observed to be stable between pH 5.5 and 9.0 at 37°C. An optimal pH of 7.9 was found for the action of Factor Xa upon the substrate and also for the action of plasma inhibitor upon Factor Xa. A Michaelis constant (Km) of 0.6mM was determined for the hydrolytic cleavage of the substrate by Factor Xa. At a substrate concentration of 3 times Km, Factor Xa was measureable in the range of 0.02 to 0.12 units/incubation mixture with a change in optical density at 405 mμ of 0.06/second/unit corresponding to 5.7 n Kat/unit.

Using 100 μl pooled plasma/unit of factor Xa, 55% of the Xa activity was inhibited at 3 minutes. Taking 3 minutes as a preincubation period, Xa inhibitor was measureable in the range 5 to 25 μl normal plasma per 0.25 units of Xa with a net change in optical density at 405 mμ of 0.8. The greatest difference between the percentage inhibition of Factor Xa by heparinised (0.3 units/ml) and non heparinised plasma, however, was observed to occur after 1 minute preincubation. Using these observations a heparin calibration curve was constructed using 10 μl aliquots of heparinised plasma. Heparin was measured in the range 0.06 to 0.3 units/ml plasma with a net change in optical density at 400 mμ of 0.8.