bluuebunny/crossref_metadata_2025 huggingface dataset:
Protein Synthesis In Megakaryocytes Stimulated By Thrombo- Poietin In Vitro
Authors: Kellar K L , Evatt B L , McGrath C R , Ramsey R B
Published: July 2018

Studies in our laboratories have been concerned with the responses of megakaryocytes to thrombopoietin in vitro. We have shown that preparations of thrombopoietin stimulate DNA synthesis in guinea pig megakaryocytes. The increase in 3H-thymidihe incorporation correlates with an increase in the labeling index of the megakaryocytes. After 2 and 3 days of incubation an increase in the ploidy levels of the megakaryocytes has been observed in thrombo- poietin-supplemented cultures compared to controls.

Recent studies have examined the incorporation of 3H-leucine in megakaryocyte cultures. Megakaryocytes were prepared on BSA or Percoll gradients to purities of 70-95%. 3H-leucine incorporation was measured after a 15 hr incubation of the megakaryocytes in medium containing 10% thrombopoietin or control preparations of normal plasma or phosphate-buffered saline. Utilization of isotope increased over a 24 hr period and was higher in the thrombopoietin-supplemented cultures. In addition, synthesis of specific proteins was analyzed by using SDS- polyacrylamide gel electrophoresis and quantitation was achieved by employing rocket immunoelectrophoresis. The results indicate that thrombopoietin stimulates endoredu- plication and protein synthesis in megakaryocytes in vitro and that this system may serve as a model for studying the mechanism of action of thrombopoietin in megakaryocytopoiesis.
Interaction Of Atherogenic (VLdL; LdL) And Non Atherogenic Lipoproteins (HDL) With Heparin Fractions Of Various Molecular Size
Authors: Duthilleul P , Fievet P , Bouillet L , Fruchart J C
Published: July 2018

Some anticoagulant properties of various heparin fractions (with molecular weights from 5 000 to 20 300 daltons) have been investigated in purified and plasmatic systems with and without Lipoproteins isolated by ultracentrifugation from hyperlipemic subjects (type IIa). Amidolytic (Tos-Gly-Pro-Arg-pNA) and coagulometric (Yin and Wessler) anti Xa activity of a HMW fraction (231 USP/mg. MW: 20 300) was decreased from 15 %m both systems supplied with LDL or VLDL since HDL have no effect. No influence was detected on anti Ila activity (Phe-pro-arg-AIE fluorimetric method). LMW (20 USP/mg ; MW 6 000 - 8 000) and ULMW (20 USP.MW 4 000-5 000) anti Xa and anti Ila activities was modified neither by VLDL/LDL neither by HDL. These findings point out to differences in the mechanisms of inhibition of thrombin and Xa by various molecular weigh heparin fractions.

Two dimensional crossed Immunoelectrophoresis in agarose 1 % with mixing various quantities of each heparin fraction (0 - 100 μg/ml) in the first phase of electrophoresis of LDL/VLDL, LDL/VLDL and AT III, LDL/VLDL and FXa reveal the presence of LDL or VLDL/heparin fractions complexes and LDL or VLDL/AT III complexes, which are isolated by Gel filtration on Ultrogel A 6. We conclude that FXa can escape from neutralization by inactive complexes.
Effects Of Heparin On The Rates Of Inhibition Of Thrombin And Factor Xa By Antithrqmbin III
Authors: Griffith M J
Published: July 2018

The rate of inhibition of thrombin and Factor Xa by antithrombin III has been investigated as a function of heparin concentration. Three general observations were made. First, the amount of heparin required to maximally enhance the inhibition of thrombin is much less than the amount of heparin required for Factor Xa inhibition by anti thrombin III. Second, the maximum rate of thrombin inhibition was approximately 5-fold higher than the maximum rate of Factor Xa inhibition with optimal heparin levels. Third, heparin concentrations greater than 10-6M decreased the rates of inhibition of both Factor Xa and thrombin. Mathematical modeling was attempted to describe the mechanism of action of heparin. At low heparin concentrations (< 10-6M) the inhibition of thrombin and Factor Xa could best be described by a model which presumed that binding of heparin to the enzyme accelerates enzyme inhibition by antithrombin III. The effect of heparin at concentrations < 10-6 on the rates of enzyme inhibition could not be explained by direct binding of heparin to either antithrcmbin III or thrombin (Factor Xa). This observation has led to the hypothesis that the interaction of thrombin and Factor Xa with heparin is altered at relatively high heparin concentration which results in a decrease in the effectiveness of heparin in accelerating enzyme inhibition by anti thrombin III.
Physico-Chemical Studies On Oligosaccharides From Procine Mucosal Heparin
Authors: Choay J , Lormeau J C , Petitou M , Torri G , Sinay P
Published: July 2018

Our recent studies have revealed that low molecular weight antithrombotic (in vivo) and anti Xa fractions and fragments can be obtained from porcine mucosal heparin (PMH) using three different processes: 1) Direct alcoholic fractionations, 2) depolymerization with nitrous acid and 3) by bacterial heparinase digestion. Ihe crude products are further fractionated by anti thrombin-III affinity chromatography and gel filtration. Physiocochemical studies were carried on each fragment employing nitrous acid degradation, colorimetric and NMR analysis. A potent antithrombotic octasaccharide (h-ULMF) is isolated from heparinase depolymerized fractions and in the NMR analysis gave some unexpected signals one of which was consistant to a 3-0-sulfated glucosamine unit (unsaturated sulfated uronic acid/N-Sulfo-Glucosamine/Iduronic acid/N- Acetyl-G1ucosamine/Glucouronic acid/N-Sulfoglucosami ne/Sulfated Idouronic acid/N-Sulfo-Glucosamine). ULMFs obtained by extraction and clemical degradation were also found to exhibit similar signals. Further degradation of h-ULMF-8 resulted in formation of smaller fragments with biological activity. Our results suggest that a smaller oligosaccharide fragment contained in the biologically active octasaccharide molecule is primarily responsible for the antithrombotic actions. Furthermore these studies provide additional data on the structure activity relationship for the antithrombotic actions of heparin, its fraction and fragments.
Heparin Therapy In Dispholidus Typus Envenomation: An Experimental Study
Authors: Bradlow B A , Atkinson P M , Rebello M , Gaillard M C
Published: July 2018

The coagulant action of Dispholidus typus venom was relatively resistant to inhibition by heparin in vitro. Heparin concentrations that inhibited coagulation due to either intrinsic pathway or Russell’s viper venom activation had little effect on coagulation due to D. Typus venom. At very high heparin to venom ratios, similar to ratios attainable in vivo this resistance could be overcome. The resistance could not be attributed to an abnormal thrombin produced by the venom since the thrombin produced from purified prothrombin by venom action reacted similarly to the thrombin produced by Factor Xa activation with purified antithrombin III. Thrombin produced from whole plasma by venom action also reacted similarly to physiological thrombin with antithrombin III in a crossed immunoelectrophoresis system. Incubation of venom with heparin and with antithrombin III did not alter the activities of these inhibitors. The heparin resistance may therefore be due to the fact that the venom is a direct activator of prothrombin. In vivo studies in rabbits indicated that heparin administered simultaneously with venom delayed the onset and reduced the severity of disseminated intravascular coagulation. Heparin administered later was much less effective. Early heparin therapy may be of value in human victims when specific antivenom is not available.
The Effect Of The Natural Proteinase Inhibitor Aprotinin On The Activation Of Platelet Function During Total Hip Replacement
Authors: Ketterl R , Haas S , Lechner F , Kienzle H , Blüme G
Published: July 2018

Postoperative thromboembolism is a well known problem after total hip replacement. Besides the intravasation of bone marrow and thromboplastic material it is discussed that it is caused by activated platelets. Therefore, we focussed our interest on platelet function during total hip replacement.

In 12 male and female patients (age:52-84 years) with total hip replacement blood samples were taken at the following time intervals: 1.induction of anesthesia, 2.arthrotomy, 3.preparation of the acetabulum, 4. polymerization of the bone cement in the acetabulum, 5. preparation of the bone marrow in the femur shaft, 6. implantation of the prosthesis shaft, 7.2 hours postoperatively. A second group of 14 patients (age:55-81 years) was treated with 20 000 KIU/ kg BW aprotinin (TrasylolR) immediately after induction of anesthesia. Following tests were carried out: 1. platelet morphology according to KRZYWANEK, 2. platelet adhesiveness according to MORRIS, 3. collagen-induced platelet aggregation according to BORN, 4. spontaneous platelet aggregation according to BREDDIN.

The number of shape-changed platelets is increasing with the duration of the operation and the maximum is to be seen when the femur shaft is prepared for the prosthesis. In contrast to these findings only a slight increase of shape- changed platelets occurs after application of aprotinin. Concerning the adhesiveness of platelets corresponding results were found. Platelet aggregation induced by collagen was also increased during the operation time. The increase of the aggregability characterized by the maximal amplitude was not seen in the aprotinin-group. The spontaneous platelet aggregation was enhanced in the non-treated group (angle α 2↑ maximal amplitude↑) whereas this phenomenon was not observed after application of aprotinin. The total hip replacement is closely related with a stimulation of platelet function. This could be the origin of postoperative thromboembolism.
Homologues Of Fibrin X Oligomers
Authors: Hafter R , Graeff H , Hugo R v
Published: July 2018

Crosslinked fibrin derivatives signalize intravascular coagulation. D-dimer, Y-D and X oligomers are observed in plasma from obstetric patients with severe coagulation disorder. They are also found in ascitic fluid from patients with advanced ovarian cancer and can be produced in vitro by simultaneous action of thrombin, plasmin and factor XIII with fibrinogen. The study was aimed to evaluate the subunit structure of separated molecular entities. The derivatives were separated by 4% SDS-PAGE preceded in case of the in vivo products by gel filtration and/or by immunoabsorption technique. The gels were sliced at the respective migration positions and derivatives therein reelectrophoresed on 7,5% gels after reduction. Subunit characterisation revealed that D-dimer is composed of the chain remnants γ1-γ1, β2, α2, while Y-D is composed of γ-γ1, β2, α3, α2, besides αE, βE and γE Crosslinked X oligomers are composed of γ-γ, γ-γ1, β, β1, γ2, α1 and α2 besides αE, βE and γE Three possible combinations of plasmin degraded and undegraded dimeric γ-chains were observed in vivo and in vitro: γ-γ γ-γ1 and γ1-γ1. The ratio of degraded (γ1) to undegraded γ-chains in dimeric γ-chain patterns indicates the mol. structure of the respective derivative. Two X oligomers could be demonstrated in which the ratio of γ-γ to γ-γ1 in terms of stain intensity was either 1:1 or 2:1. Their subunit compositions are in accordance with structures describ- able as D-X-Y and D-X-X-Y. Their molecular weights, calculated from the subunit compositions are 476,000 and 716,000 respectively. - It is proposed that crosslinked X oligomers exist as a homologous family with increasing X fragment content.
Characterization Of A Crosslink-Containing Fragment Derived From The A Polymer Of Human Fibrin And Its Application In Immunologic Studies Using Monoclonal
Antibodies
Authors: Sobel J H , Birken S , Ehrlich P , Friedman R , Moustafa Z , Canfield R E
Published: July 2018

A high molecular weight crosslink-containing fragment derived from cyanogen bromide (CNBr) digests of the a polymer component of human fibrin has been isolated and characterized. The material has been used to generate monoclonal antibodies toward the goals of (1) producing fibrin-specific probes for use in the early detection of thrombosis and (2) generating monoclonal lines to single determinants in the COOH-terminal region of the Aα chain for use in structural studies of fibrinogen and fibrin.

Biochemical and immunologic characterization data indicate the fragment is comprised, predominantly, of equimolar quantities of the CNBr peptides spanning residues #241-476 (CNBr 8) and #518-584 (CNBr 10) in the original Aα chain. The acceptor and donor units are crosslinked via an average of 2.8-3.2 ε-(γ-glutamyl) lysine bonds per mole of CNBr 8 + CNBr 10 producing heterogeneously sized fragments in the range of 80,000-200,000 daltons.

Two types of monoclonal lines have been obtained. The first react with regions of primary structure and in one instance immunoreactivity could be localized to the Aa tryptic peptide #253-268. The second type appear to recognize conformational determinants as shown by one antibody that reacts well with the CNBr crosslinked fragment but poorly with its constituent peptides.
Platelet And Smooth Muscle Cell Responses After Endothelial Desquamation
Authors: Stemerman M B
Published: July 2018

Although compromise of endothelial integrity occurs through many mechanisms, mechanical removal by balloon catheter is an excellent experimental method to study vascular responsiveness after injury. The interaction of platelets with the vessel wall, as well as proliferation of vascular smooth muscle cells can be assessed in this model. Following platelet attachment to the subendothelium, platelets release materials from their alpha granules. Using an antibody raised against platelet factor 4, a protein stored in alpha granules, we have demonstrated that material released from platelets do enter the vessel wall. A large amount of PF 4 antigen enters the wall shortly after endothelial removal, permeating the wall completely by 30 minutes, but little trace of the antigen can be found four hours after injury. Using infusions of PGI2 to a level of 850 ng/kg/min in rabbits, in vivo platelet adhesion to the exposed subendothelium can be greatly reduced and release of PF4 antigen into the vessel wall markedly diminished. Growth of smooth muscle cells (SMC) after endothelial removal has also been measured by 3H-Thymidine labeling of SMC DNA. As measured by this method as well as direct cell counts, SMC proliferation in the abdominal aorta is significantly greater than the thoracic. Reinjury of only the abdominal aorta by balloon catheter 4 days after the initial total aortic injury causes a proliferative spurt in the thoracic aortic SMC, thus demonstrating that a humoral signal can initiate SMC proliferation. In addition, the response of SMC from 21 month old rats when compared with 3 month old rats is much greater. These studies demonstrate in vivo methods for examining the response of platelets and SMC following endothelial injury. Further, these studies indicate that the response to injury hypothesis of atherosclerosis progression should now be broadened to the concept of a response to signal view of atherogenesis.
The Molecular Defect Of Factor IX Chapel Hill
Authors: Braunstein K M , Griffith M J , Briët E , Roberts H R
Published: July 2018

We had previously described Factor IX Chapel Hill as an abnormal molecule with 100% Factor IX antigenic level but only 5% functional activity as measured by routine one-stage clotting assay. Clinically the patient has a mild bleeding tendency with hemorrhage only after trauma. Initially, we were unable to demonstrate significant cleavage of Factor IX Chapel Hill by a crude human XIa preparation from celite activated plasma and could not determine the specific activity of the molecule. In the present study we have further investigated the cleavage of Factor IX Chapel Hill using a more purified preparation of human Factor XIa. Under similar incubation conditions Factor IX Chapel Hill was cleaved to Factor IXaβ at approximately one half the rate of normal Factor IX. Clotting activity was generated concomitantly with the cleavage of Factor IX Chapel Hill, reaching a maximum level at a time corresponding to complete cleavage of the molecule. The specific activity of Factor IXaβ Chapel Hill was estimated from a standard curve relating clotting time to normal Factor IXa8 concentration. Four determinations were made on separate days and found to correspond to a specific activity for Factor IXaB Chapel Hill of approximately 30% of normal Factor IXaβ. From these results we conclude that the structure of Factor IX Chapel Hill is sufficiently different from normal Factor IX that the interaction with Factor XIa and/or Ca++ is altered resulting in a slower rate of cleavage to Factor IXaβ. In addition, the structure of Factor IXaβ Chapel Hill is also sufficiently different from normal Factor IXaβ that the apparent activity of the molecule in plasma is reduced. Further these results suggest that the molecular defect in Factor IX Chapel Hill is not within the activation peptide region of the molecule as a defect in this region would not have affected the specific activity of the molecule.
Activation of Bovine Factor IX By Bovine Factor XIa . Active Site Titration Of Factor IXa and Development of A spectro-Photometric assay for Factor IX
Authors: Tans G , Janssen-Claessen T , Dieijen G v , Rosing J , Hemker H C
Published: July 2018

Activation of factor IX by factor XIa occurs via an intermediate which has no esterase activity towards synthetic arginine esters or coagulant activity as determined with a clotting assay. Factor IXa can be active site titrated using p-nitrophenyl-p1-guanidinobenzoate (p-NPGB) as a titrant. The rate and equilibrium constants describing the active site titration will be presented. To determine whether the intermediate occurring during factor IX activation by factor XIa has its active site exposed for p-NPGB the time course of activation of factor IX by factor XIa was followed l) by active site titration of the active sites generated, 2) by gel- electrophoretic analysis in the presence of sodium dodecyl sulfate, 3) by a clotting assay for factor IXa and 4) by measurement of factor IXa using a spectrophotomefric assay. It will be shown that the intermediate occurring during activation of factor IX by factor XIa does not interact with p-NPGB indicating that the active site is not available in the intermediate.

Factor IXa can be determined spectrophotometrically by measurement of the rate of factor X activation by factor IXa in the presence of phospholipid and CaCl2. The factor Xa generated is measured using the chromogenic substrate S222. Since human factor IX can be activated with bovine factor XIa and since human factor IXa can activate bovine factorX the bovine clotting factors factor XIa and factor X can be used to construct an assay for factor fx in plasma samples. Experiments will be presented in which it is shown that the spec- trophotometric assay for human factor IXa can be used to determine levels of factor IX in plasma samples of healthy individuals, in plasma samples deficient in various clotting factors and in plasma samples from patients under anti-coagulant therapy. The results are in agreement with a factor IX clotting test.
Bradykinin Mediated Hypotension After Infusion Of Prekalli - Krein Activator Containing Plasma Protein Fraction
Authors: van Rosevelt R F , Bleeker W K , Bakker J C , Ufkes J G R , Sinclair D , van Mourik J A , van Aken W G
Published: July 2018

Rapid infusion of plasma protein fraction(PPF) containing prekal1ikrein activator (PKA) may cause severe hypotension. It has been suggested, that such changes in blood pressure are due to PKA-mediated activation of the kallikrein system. We studied both in patients and in animals, whether a relationship exists between PKA activity in PPF, the plasma bradykinin (BK) level and changes in mean arterial pressure (MAP),during rapid infusion of PPF and albumin. Plasma Bk was measured by radioimmunoassay, using blood collected in a mixture of kallikrein and kininase-inhibitors. PKA was determined spectrophotometrically. PPF and albumin were infused (250 ml, 30 sec.) in the bypass circuit of patients undergoing open heart surgery. After infusion of PPF (29 U PKA/L), MAP decreased within 1.5 min. from 76±11 (SD) to 49 ± 9 mm Hg; concurrently the venous Bk concentration increased from 0.7 ± 0.1 to 1.7 ± 0.2 ng/ml (n=6). After albumin infusion (3 U PKA/L) MAP dropped from 71 ± 14 to 66 ± 13 mm Hg; the Bk concentration did not increase significantly (n=6). In rats, pretreated with the kininase II inhibitor BPP9A the effect of rapid infusion of different lots of PPF on arterial Bk concentration and MAP were compared. Both the decrease of MAP and the increase of arterial Bk concentration were proportional to the amount of PKA infused. Infusion of synthetic Bk provoked a similar fall in MAP at corresponding arterial Bk concentrations. After complete inhibition of PKAin PPF by CT-inhibitor, no effects on MAP or Bk level were observed. The ability of several lots of PPF to generate Bk (ng/ml/10 min.) in Hage- man factor deficient plasma closely correlated with the PKA content of PPF (n=25, r 0.95, p 0.005). From these observations we conclude that the hypotensive reactions after PPF infusion are mainly caused by the PKA mediated generation of Bk in the recipient.
Prostacyclin Prevents Endothelial & Platelet Damage During Haemodialysis
Authors: Turney J H , Dodd N , Weston M J
Published: July 2018

We have previously demonstrated that prostacyclin (PGI) enhances the biocompatibility of extracorporeal circuits. Dialysis with & without PGI were studied in 17 patients. We measured platelet count, (β-thromboglobulin, factor VIII related antigen, & Antithrombin III (Bick method). Results are presented as percentage change + SEM of initial values.

The change in all values during dialysis with heparin alone was significant (p<0.005). Additional PGI prevented any change (p<0.0001 compared with heparin at 300 minutes). We conclude that platelet activation & consumption persists throughout dialysis with heparin alone. The rise in factor VIII-RA & AT III reflects vascular endothelial damage induced by the reinfusion of activated blood components. Thus PGI not only protects platelets but also prevents dialysis-induced vascular endothelial damage. Longterm use of PGI should tend to reverse the prothrombotic state in dialysed uraemic patients and may therefore reduce their risk of atherosclerotic cardiovascular disease.
Low Affinity Platelet Factor 4/β-Thromboglobulin (LA-PF4/βTG) Antigen And Circulating Platelet Aggregates During Prostacyclin (PGI2) Therapy
Authors: Musiat J , Szczeklik A , Nizankowski R
Published: July 2018

We have found that continuous intraarterial infusion of PGI2 alleviates rest pain and promotes healing of ischemic ulcers in patients with advanced arteriosclerosis obliterans. Since these effects could be related to the anti-platelet actions of prostacyclin, we studied serum levels of the platelet specific protein, a possible marker of platelet activation in vivo, named LA-PF4/βTG, and compared it with those of serum circulating platelet aggregates. The former was assessed by radioimmunoassay, the latter by method of Wu and Hoak. Determinations were carried out in 16 patients with arteriosclerosis obliterans, initially and during the second day of intraarterial infusion of PGI2 in a mean dose of 6 n9/kg/min. This therapy led to an almost uniform fall in circulating platelet aggregates. In contrast, levels of LA-PF4/β-TG antigen at the beginning of second day or the infusion /mean = 19.19 ng/ml, ± S.D. a 8.97/ did not differ significantly from the initial values /mean = 23.59 ng/ml, ± S.D. = 13.12/.

In conclusion: 1/ In our patients vascular disease did not produce any apparent increase in serum LA-PF4/β-TG antigen levels; 2/ Infusions of PGI2 did not affect basal levels of this platelet specific protein, while it markedly depressed the percent of circulating platelet aggregates.
Assessment Of Energy Costs Of Secretory Responses In Platelets By Abrupt Arrest Of ATP Regeneration
Authors: Akkerman J W N , Gorter G , Holmsen H
Published: July 2018

A new method has been developed for the quantitative assessment of energy consuming processes in platelets. Under carefully controlled metabolic conditions ATP resynthesis is abruptly blocked by a cocktail of metabolic inhibitors. This leads to a fall in metabolic ATP, which is linear with time between 0 and 30 sec after addition of the inhibitors. Evidence is presented that this fall reflects the velocity by which the platelets consume metabolic energy prior to addition of the inhibitors. Resting platelets consume 4 μmol ATP equivalents/min/1011 cells at 37° and 0.5 μmol (same units) at 15°C. When thrombin (5 U/ml) is included in the inhibitor-mixture, aggregation and secretion of dense granules (3H-serotonin), α-granules (β-thromboglobulin) and lysosomal granules (N acetyl β glucosaminidase) follow despite the arrest in ATP resynthesis. The fall in metabolic ATP is now much steeper, reflecting an increase in energy consumption during these functions. Using changes in temperature as a means to affect secretion and energy metabolism, secretion velocity (measured between 0 and 10 sec after thrombin addition) can be compared with simultaneous energy consumption (measured between 0 and 30 sec after thrombin addition). At a consumption of 12 ymol ATP/min/1011 cells secretion velocity of dense-, α- and lysosomal granules is 100, 95 and 50% of uninhibited suspensions, respectively. At 6 μmol (same units) these percentages are 70, 35 and 25%.If thrombin is added after addition of the inhibitors thereby initiating secretion at lowered metabolic ATP levels, secretion is slower as metabolic ATP is lower. Again lysosomal granule secretion is more inhibited than α-granule secretioiy. which is slower than dense granule secretion. These data reflect an increasing need for metabolic energy in the order: dense-, α- and lysosomal granule secretion.
Inconstancy Of PGI2 Administration In Preventing Attacks Of Prinzmetal Angina
Authors: Chierchia S , De Caterina R , Crea F , Bernini W , Distante A , Maseri A , L’Abbate A
Published: July 2018

It has been proposed that vasospastic angina, eventually due to local defects of PGI2 production, might benefit from PGI2 administration. We therefore investigated the effects of PGI2 in healthy volunteers and, then, in patients with frequent ischemic episodes (IE) of Prinzmetal angina, to detennine 1. hemodynamic, antiplatelet and possible side effects of the drug and 2. its possible therapeutic usefulness in the management of IE. In 6 healthy volunteers PGI2 was infused i.v. at doses of 2.5,5,10 and 20 ng/kg/min during consecutive periods of 30 min each. Heart rate (HR) and right atrial pressure were monitored continuously; cardiac output (thermodilution in 2 subjects, indirectly by a Doppler technique in all), arterial blood pressure (BP) and in-vitro platelet aggre- gability (PA) by ADP (Born), intermittently. In 2 subjects we also measured pulmonary arterial pressure and, in one, left ventricular pressure, during the infusion and in control conditions. PGI2 was then infused in 6 pts with frequent IE at maximal well tolerable rate (6-26 ng/kg/min) for periods of 3 hours alternated with equal periods of placebo (P), continuosly recording 2 ECG leads to detect ST-T changes, and sampling blood for PA as before. In all healthy volunteers PGI2, at the highest rates of infusion, decreased significantly (p < .001) both systolic BP (-10 ± 3%, mean ± SD) and diastolic BP (-19 ± 5%) increasing HR (+ 21 ± 5%); no significant changes were observed in the other hemodynamic parameters. The maximal decrease in PA was 58 ± 30%(p <. 001). Skin flushing, restlessness and headache, sometimes observed at the highest doses, rapidly disappeared decreasing the infusion rate. In the 6 pts the same trend in BP, HR and PA was evident. 106 IE were observed. PGI2 did not affect severity, duration and number of IE (44 during P, 62 during PGI2 infusion). One of the pts, however,not clinically different from the others, showed a reduction at 10 ng/ kg/min (6 IE during P, 2 during PGI2) and a complete abolition in the 3 following periods at 20 ng/kg/min (4,3,5 IE during P vs. none duringPGI2). We conclude that 1. PGI2 can be safely administered to humans and 2. it may prevent IE is some vasospastic pts, but not in others. Different pathogenetic mechanisms are perhaps involved in apparently similar Prinzmetal anginas.
Interference Of Sulphynpyrazone With The Platelet And Bronchial Effects Of “Platelet-Activating Factor”
Authors: Vargaftig B B , Lefort J , Wal F , Chignard M
Published: July 2018

Platelet-activating factor (PAF-acether, l-O-alkyl-2-acetyl-sn- glyceryl-phosphorylcholine) stimulates platelets independently from ADP release and formation of arachidonate (A) metabolites, is released from platelets by aggregating amounts of thrombin or collagen, and may account for physiopathologically relevant aggregations. PAF-acether induces reversible aspirin-resistant thrombocytopenia and bronchoconstriction (BC) when given iv at 60120 ng/kg to propranolol-treated guinea-pigs. BC is platelet- dependent, and is suppressed by prostacyclin or by platelet depletion. BC was reduced for 30-60 min by iv sulphynpyrazone (S, 100 mg/kg). Inhibition by S of A-induced BC and thrombocytopenia lasted 6 hrs. This agrees with the fact that S is metabolized to a more effective cyclooxygenase inhibitor. Surprisingly, S was inactive against thrombocytopenia by PAF-acether, suggesting that BC was not inhibited because of a primary platelet site of action of S. Nevertheless, when platelets were collected from S-treated animals, aggregation and the release reaction by PAF-acether were inhibited. Inhibition of aggregation was surmounted by increasing the amounts of PAF-acether, whereas the release reaction was still inhibited by 50 % when 35 times more PAF-acether were added. Aggregation by ADP was unaffected by S, whereas the time course of inhibition of A was as against BC. In vitro, S (0.1-1 mM) inhibited the release reaction by AA and by PAF-acether ; this was surmounted with high amounts of the latter, but not of the former : the mechanism of inhibition by S of PAF-acether and of A thus differs in vivo and in vitro and this may be relevant to its therapeutic effectiveness.
Selenium Deficiency Impairs The Biosynthesis Of Prostacyclin In Rat Aorta
Authors: Bult H , Van den Bosch P , Von den Bossche R , Van Hoydonck A , Herman A G
Published: July 2018

Prostacyclin(PGI2)synthase is inactivated by lipidhydroperoxides and it has been suggested that this occurs in atheromatous plaques (S.Moncada & J.R.Vane, New Engl. J.Med.,300,ll42,1979). Glutathione peroxidase is a selenium(Se) containing enzyme which converts hydroperoxides to less toxic alcohols. Therefore, we investigated whether Se deficiency reduced the biosynthesis of PGI2. Rats were fed a diet with a reduced Se content(25 ppb) or a standard diet (l42 ppb Se). After 7months 4 rats from each group were killed. Urinary Se-excretion and Se content of the liver were significantly (P<0.05, Student-t test) reduced. Aorta's were removed and rings (about 2 mg) were stirred in l ml Krebs'solution. After 5 and 15 min, the release of endogenous PGI2 into the medium was assessed by its anti-aggregating capacity in rabbit platelet rich plasma and after 16 min the 6-oxo-PGF1α content of the medium was measured with RIA. Se deficient aorta’s released less PGI2 like activity (at 5 min, 46 % reduction, P<0.05; at 15 min, 25 % reduction) and less 6-oxo- PGFia (43% reduction, P<0.05). Similar experiments were performed with rings that were exhausted by 6 h preincubation in 100ml 50 mMTris, pH 7.5 in the absence of glucose. This procedure reduced the endogenous PGI2 release in control aortic rings with respectively 81 %(bioassay) or 89 % (RIA of 6-oxo-PGF]a). In Se deficient, exhausted rings the endogenous biosynthesis of PGI2 was strongly impaired as indicated by bioassay and measurements of 6-oxo- PGF1α (82 % reduction, P<0.05). Cyclo-oxygenase and PGI2# synthase activities were measured by incubation of 20 mg aorta with respectively 10/ug l-14C-arachidonic acid or 10μg l4C-PGH2. After 20 min the reactions were stopped and ihe products formed were extracted, radiochromatographed and quantified by liquid scintillation counting. In Se deficient aortas, cyclo oxygenase activity was not affected whereas PGl2-synthase activity showed 37 % reduction (P<0.05).These results support the hypothesis that a low peroxide tone is a prerequisite for an optimum biosynthesis of PGI2.
The Plasmin Inhibitor From Human Platelets Inhibits The Binding Of Modified Plasminogen To Fibrin
Authors: Sandbjerg Hansen M
Published: July 2018

Modified plasminogen (Lys-plg) is known to bind to fibrin and this binding is competitively inhibited by omega-aminocarboxylic acids. An inhibitor of the plasma proteinase plasmin (EC 3.4.21.7) was partially purified from washed and lysed human platelets. This material contained none of the known plasma proteinase-inhibitors when studied by crossed immunoelectrophoresis and electroimmuno assay.

Human fibrinogen was clotted by thrombin in the presence of radiolabelled Lys-plg and the fraction of radioactivity associated with the washed fibrin clot was determined. The presence of the inhibitor preparation in the clotting mixture caused a significant decrease in the fraction of radiolabelled Lys-plg adsorbed onto fibrin. The fibrin-adsorbed fraction decreased as a function of an increasing inhibitor concentration. The incorporation of purified cold insoluble globulin (fibro- nectin) into the fibrin clot did not influence the adsorption of Lys-plg neither in the presence nor the absence of the inhibitor. The presence of calciumions or albumin in various concentrations did not influence the reactions. The addition of 6-aminohexanoic acid to the clotting mixture strongly enhanced the effect of the inhibitor preparation. It has previously been demonstrated that the inhibitor binds to immobilized plasminogen. These results may indicate that lysine-binding sites are important for the inhibitor-enzyme interaction.
A Kinetic Approach To The Definition Of Pathophysiology Of Low Antithrombin III (AT) Levels In Patients
Authors: Reeve E B , Leonard B D , Bies R D , Ambruso D R , Hathaway W E
Published: July 2018

Patients with 50 percent of normal levels of AT are at serious risk of deep venous thrombosis. Possible causes of 50 percent levels are (1) a halving of synthetic rate, or (2) a doubling of fractional catabolic rate. In health fractional catabolic rate is 50 percent of the plasma AT per day. Doubling of fractional catabolic rate-might be due to (a) the presence of a structurally defective AT molecule or (b) might be directly related to a great excess of AT consumption by complex formation with released clotting proteases. These possible causes of low AT levels can be distinguished by turnover studies with 131I-iabelled AT from the patient and from a healthy donor.

We have developed simple, rapid methods of preparing highly purified AT from 30 to 60 ml of citrated blood and of labelling it with 131I without denaturation at about 0.5 iodine atom per AT molecule. A turnover study requires intravenous injection of 4-6 µC of 131I.

Studies in a female patient with hereditary AT deficiency (40-50 percent AT level) compared to normal subjects indicates our approach. Fractional breakdown rate of her own 131l-AT was 50 percent per day indicating no obvious structural abnormality. Fractional breakdown rate of 131I_labelled donor AT was also 50 percent per day further indicating no difference in the behavior of her own and of normal AT and no excess consumption by clotting proteases. The patient's ability to compensate for increased AT-III consumption during an episode of thrombosis was impaired. Thus the patient suffered from a synthetic defect resulting in the ability to synthesize only 50 percent of her AT requirements per day.

AT turnover studies provide an approach to the definition of the etiology of depressed AT levels in various disease states.
Comparison Of Mean Platelet Survival Time In A Non-Radioisotope Method Calculatend With New Model With Mean Survival Time In CR-51 Methods
Authors: Tsukada T , Tango T
Published: July 2018

Platelet survival time using non-radioisotope method measuring malondialdehyde(MDA) generation stimulated by arachidonate prior to and after intake of aspirin(ASA) and Cr-51 method was measured simultaneously in 18 cases with ITP. In cases with normal platelet survival time MDA generation curve showed linear or the letter-S pattern. On the other hand, all cases with shorter survival time had MDA generation curve with letter-S pattern. To describe the S pattern of MDA generation curve following model was devised on the assumption : 1) ASA inhibits MDA generation in megakaryocytes so that newly produced platelets show impaired MDA generation, 2) ability of MDA generation in platelets exposed to ASA recovers during circulating.

where a is the disappearance time of inhibiting effect of ASA on megakaryocyte MDA generation and b is the time necessary for recovery of MDA generation in platelets exposed to ASA. Mean survival time(MST) is calculated by l/λ.

The difference of MST between non-radioisotope method and Cr-51 method( gamma model) in 11 cases with MST of less than 4 days in Cr-51 method were 2.9 ± 0.4 days in platelet survival time (PST) at which MDA generation attained to pre-ASA levels, 2.7 ± 0.4 days in MST calculated with linear regression of MDA generation curve and 1.8 ± 0.4(SEM) days in MST calculated by the above model. On the other hand, in cases with MST of more than 4 days in Cr-51 method the difference of MST in both methods were 1.4 ± 0.3(SEM) days in PST, 0.6 ± 0.2 days in linear regression and 0.6 ± 0.2 days when MST was calculated with the above formula.

These results suggest that the model reported is so far the better fitting model for the MDA generation curve in cases with normal and shortened platelet survival.
Circulating Platelet Factors (Beta Thromboglobulin, Platelet Factor 4) In Patients With Prosthetic Heart Valves
Authors: Andrassy K , Zebe H , Koderisch J , Duczek A , Ritz E
Published: July 2018

Despite anticoagulation, thromboembolic complications are frequent in patients with heart valve prosthesis (HVP). The present study was performed to investigate whether activation of platelets could be demonstrated in such patients. Platelet proteins in the circulation were measured as an index of platelet activation. Pat.: 33 pat. with mitral valve prosthesis (MVP) (19 with Bjφrk- Shiley (BS) and 14 pat. with Starr Edwards type (SE)) and 54 pat. with aortic valve prosthesis (35 BS; 10 SE; 9 bioprosthesis (BP). All pat. were anticoagulated (Dicumarol). Results : In MVP, a significant difference between BS and SE was observed with respect to LDH (247±48 vers. 474± 246) (p<0.05) but not with regard to Beta-TG and PF 4. There was no difference of platelet protein levels in presence/absence of atrial fibrillation. In 35% Beta TG (> 53 ng/ml) and PF 4 ( > 11 ng/ml) were above X±2 SD of CO. ADP and collagen induced aggregation (MA) was unchanged in all pat. (collagen 401 12; ADP 32±11; CO: collagen 39±7; ADP 32±6). In pat. with demonstrable hemolysis (LDH >250 IU), a significant correlation was observed between LDH and Beta TG/PF 4 both in MVP and AVP. MVP and AVP differed with respect to LDH (higher in AVP, p<0.05) and PF 4 (higher in MVP (p<0.01) but not with respect to Beta TG (differences of elimination of TG and PF 4?). Comment : In a high proportion of patients with HVP platelets are activated. The observation of elevated platelet indicator proteins is in agreement with previous findings of decreased platelet survival in HVP (Weily, H., New Engl. J. Med. 290, 534, 1974). Elevated platelet prot.may identify risk of thromboembolism.
Decreased Platelet Survival And Aggregation In Mice Exposed To Hypobaric Hypoxia
Authors: Jackson C W , Whidden M A , Smith P J , Edwards C C , Lyles S A
Published: July 2018

Decreased platelet survival and function have been observed in patients with chronic hypoxemia. To develop a model for investigating these problems, we have studied the mouse exposed to severe hypobaric hypoxia. Mice were exposed to 0.3 or 0.4 atmospheres (ATM) of hypobaric hypoxia 16-19 hours daily for 18 days. Platelet counts first increased with a peak of 1.5 times baseline on day 3 followed by a decline below baseline during the second week of exposure. The greatest effect occurred at 0.3 ATM with a decline of platelets to 15% of baseline as compared to a nadir of 50-80% of baseline at 0.4 ATM. Survival of 51Cr- labeled platelets from untreated donors infused on day 13 of hypoxic exposure was decreased to 1/2 of control in the 0.3 ATM group but normal in the 0.4 ATM group. Megakaryocyte concentration showed the same trend with a decrease to 1/2 of control at 0.3 ATM with only a slight decrease at 0.4 ATM. Platelet aggregation with ADP was markedly reduced after two weeks exposure to 0.3 ATM. Aggregation of control platelets suspended in plasma from the 0.3 ATM group was depressed. Aggregation of control platelets resuspended in equal volumes of hypoxic and control plasmas was also decreased. Resuspension of control platelets in control plasma after a 1 hour incubation with hypoxic plasma partially restored the platelet aggregating activity. These studies indicate that exposure of mice to 0.3 ATM of hypobaric hypoxia for two weeks produces thrombocytopenia and decreased platelet aggregation. The decrease in platelet count is associated with decreased platelet survival and megakaryocytes. Decreased platelet survival is associated with an extrinsic rather than an intrinsic platelet defect since normal platelets have a shortened survival in hypoxic mice at 0.3 ATM. The decrease in platelet aggregation is likewise due to a plasma inhibitor since plasma from hypoxic mice inhibits aggregation of normal platelets. The changes in platelet kinetics and aggregation of the mouse exposed to 0.3 ATM for two weeks mimic in many ways those seen in patients with chronic hypoxemia and provide a laboratory model to study these alterations.
Release Of Slow-Reacting Substance (SRS) From Rabbit Platelets
Authors: Mencia-Huerta J M , Hadji L , Benveniste J
Published: July 2018

In the guinea-pig, platelet aggregation by several agonists results in a bronchoconstriction. Thus we speculated that SRS could be the mediator for this phenomenon, eventhough it has not been yet described as originating from glatelets.

Washed rabbit platelets (2.5 × 108) stimulated by plateletactivating factor (PAF-acether) (0.72 nM), thrombin (0.15 unit/ml) or arachidonic acid (AA) (10 uM) released 2.7 ± 0.9, 2.5 ± 0.2 and 1.4 ± 0.8 units of SRS, respectively (5 exp., 1 unit = guinea-pig ileum contraction induced by 5 ng histamine). By contrast, ADP (10-100 uM) aggregated platelets but did not induce the release of SRS. Preincubation of platelets with aspirin (0.1 mM : 10 min), or the presence of indomethacin (5 uM) in the incubating media, did not modify the yield of SRS, thus excluding prostaglandins as the contracting substance. SRS release was not correlated with the aggregation since aspirin-treated platelets failed to aggregate in the presence of AA but still released SRS, whereas untreated platelets ADP-stimulated, did aggregate without releasing SRS.

L-cysteine (20 mM) increased 10 fold the yield of SRS and eicosatetraynoic acid (ETYA) (10-100 uM) caused a dose-dependent inhibition of SRS release.

Platelet SRS was resistant to base but not to acid hydrolysis and its action on guinea-pig ileum was reversed by FPL 55712 (1 ug/ml). It was further purified on reverse phase high pressure liquid chromatography (RP-HPLC) using a discontinuous gradient of methanol in water. Platelet-SRS was eluted as 3 peaks of activity (retention times : 39 min, 46 min, 54 min) as did SRS from ionophore-stimulated rat peritoneal cells. Synthetic leukotrienes C and D exhibited retention times of 46 min and 54. min respectively. SRS obtained from platelets preincubated with 14C- AA was also analyzed on RP-HPLC. Only one peak of activity (54 min) coincided with radioactivity suggesting the incorporation of AA in this SRS molecule.

These data bring evidence for the release from stimulated rabbit platelets of a SRS closely related to leukotrienes.
Kinetics Of Antithrombin III During Severe Consumption Coagulopathy In An Infant Measured Without Radioactive Tracer Proteins
Authors: Schmidt B , Wais U , Witt I , Pringsheim W , Künz W
Published: July 2018

The precise diagnosis of disseminated intravascular coagulation remains difficult to achieve: Decreased production of coagulation factors and/or thrombocytes can mimic the laboratory pattern of disseminated intravascular coagulation. The measurement of increased turnover rates for coagulation proteins would provide more convincing criteria.

During the course of severe coagulopathy in an infant suffering from septicaemia and shock, antithrombin levels were determined repeatedly before and during treatment with Antithrombin concentrate: Activities and concentrations were measured, using chromogenic substrates and immunodiffusion plates, respectively. By mathematical analysis of these data, using a biexponential function, the plasma elimination half-life of the antithrombin III was estimated to be 7.5 to 10.5 hours. Compared with known plasma half-lives of radioactively labelled antithrombin III in adults, the increase was five- to ten-fold. This indicates an accelerated consumption of antithrombin III in this case of severe coagulopathy.
Interaction Between Plasma Fibronectin And Complement Clq
Authors: Ingham Kenneth C , Brew Shelesa A , Miekka Shirley I
Published: July 2018

Human plasma fibronectin interacts with collagen and gelatin and has been implicated in the clearance of collagenous debris from circulation. The existence of collagenlike regions in Clq suggests a possible affinity between . this subunit of the first component of the complement system and fibronectin. Using the technique of polyethylene glycol precipitation (Miekka and Ingham, Arch. Biochem. Biophys. 203:630-641, 1980) we have obtained evidence for such an interaction. [3H]-fibronectin (0.3 mg/ml, in .05 M phosphate, 0.15 M NaCl, pH 7.35) was half precipitated at a concentration of 10.7% PEG-4000. In the presence of gelatin (0.6 mg/ml) the midpoint of the precipitation curve was shifted to 4.9% PEG. A similar but smaller shift to 7.1% PEG was observed in the presence of Clq (0.2 mg/ml). These effects can be attributed to the formation of complexes of lower solubility than either component alone. Additional evidence for such complexes came from competition experiments in which Clq was shown to inhibit the binding of [3H]-fibronectin (0.17 mg/ml) to gelatin sepharose (70 ul of packed gel/ml). In the absence of Clq, 50% of [3H]- fibronectin sedimented with the gel whereas addition of Clq caused a dose-dependent retention of cpm in the supernate with half-maximal inhibition at approx. 0.5 mg/ml Clq. Immunoelectrophoresis of fibronectin ± Clq at pH 9.0 was also consitent with an interaction in that the latter inhibited the anodal migration otherwise seen with the former, an effect which was enhanced if the Clq was first heated (60°, 1 h). The heat treatment of Clq caused the appearance of turbidity which diminished upon addition of fibronectin. Experiments are in progress to determine the specificity of this interaction which, if confirmed, could signify a role for fibronectin in the clearance of immune complexes.