bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purine Metabolism In Megakaryocytes And Platelets
Authors: Levine R F , Webster H K 
Published: July 2018

Levels of purine nucleotides (PU NTD),incorporation of labelled adenosine (Ar) and hypoxanthine (Hyp), and the distribution of these labels in the purine pathways were studied in intact human, monkey, and guinea pig (gp) platelets (pits) and in megakaryocytes (megas) from monkeys and gp. Following incubations of 5-180 min., perchloric acid extracts were made of 109 pits or of 0.6-1.0×l06 megas per experiment. PU NTD in pits were separated by gradient anionic exchange HPLC; PU nucleosides and bases were separated by reversed-phase HPLC. Simultaneous quantitation was accomplished by on-line UV and radioactivity detectors. Concentrations of PU NTD in pits were comparable in all 3 species and to those in the literature for human pits. No major differences were found in the profiles of PU NTD of megas from monkeys and gp. The concentrations of adenylates in 106 megas were the same as in 109 pits. Differences were observed in purine interconversion pathways between pits and megas for Hyp, the only purine base available in a measurable quantity for salvage. Megas synthesized both adenylates and guanylates via IMP from HYP.No significant incorporation of Hyp was found in pits studied under the same conditions. In contrast, Ar was incorporated in both pits and megas, and then deaminated or directly converted to AMP. These studies found that megas have retained the capacity to synthesize PU NTD by salvage of Hyp. They are thus closer to other kinds of rapidly proliferating cells than to end stage cells such as pits, in which this pathway did not appear to be significant.
Morphological Study Of 5-Ht Organelles In Human Megakaryocytes
Authors: Hourdille P , Fialon P , Belloc F , Bernard P , Boisseau M R 
Published: July 2018

Human blood platelets possess dense bodies which have been clearly demonstrated to be the storage site for 5-hydroxytryptamine and the non-metabolic pool of ADP. Previous electron microscopy investigations performed on human megakaryocytes have revealed electron-opaque granules, but only in platelet-forming megakaryocytes. In this study different methods of visualizing platelets 5-HT storage organelles were applied to human megakaryocytes. The observations were facilited by the high enrichment of megakaryocytes obtained using the Percoll density centrifugation. By conventional electron microscopy we rarely observed dense bodies similar in size and opacity to those observed in platelets. Using uranaffin cytochemical method we observed typical and atypical uranaffin-positive organelles. Distribution of these organelles was heterogeneous with numerous megakaryocytes containing only few granules. Using mepacrine labelling test, human megakaryocytes could bedetected easily since they are large and have multilobulated nucleus. Their nucleus was intensely fluorescent and was surrounded by a slightly-fluorescent cytoplasm. For each sample, 50 megakaryocytes were examined 96 ± 1.4 % of the megakaryocytes exhibited greenish yellow, very fluorescent granules with a flashing phenomenon. These granules were randomly distributed in the whole cytoplasm. The other 4 ± 1.4 % detected by their morphological characteristics no granules were observed. We found no correlation between megakaryocytes size and the presence of granules.These results indicate that mepacrine labelling test and uranaffin cytochemical electron microscopy are very useful tests for megakaryocytes 5-HT organelles study, and allow us to correlate the results obtained by these different methods.
Role Of Hmw Kininogen In Polysaccharide Sulfate-Mediated Activation Of Factor XII
Authors: Kato H , Shimada T , Sugo T , Iwanaga S 
Published: July 2018

We have shown that bovine HMW kininogen (HMW KGN) accelerates the activation of Factor XII (XII) and prekallikrein (Prek) in the presence of kaolin, adsorbing on kaolin through fragment 1.2 region and forming a complex with Prek through light chain region. The present study was undertaken to examine the functional role of HMW KGN in the activation of XII and Prek with other negatively-charged surfaces than kaolin. XII was incubated with a variety of substances, HMW KGN and Prek, and plasma kallikrein generated was estimated using a fluorogenic peptide substrate, Z- Phe-Arg-MCA.Among 8 kinds of polysaccharide sulfates (dextran sulfate, xylan sulfate, amylopectin sulfate, chondroitin polysulfate, heparan sulfate, amylose sulfate, heparin and carrageenin), amylose sulfate had the most potent ability to activate XII. The experiments using fourteen amylose sulfates with different sulfur content and different viscosity revealed that some amylose sulfates with a higher sulfur content (18%) activated strikingly XII and Prek in the presence of HMW KGN. In the reaction, fragment 1.2-light chain region in HMW KGN was shown to be essential, using the various derivatives of HMW KGN. The rate of XII activation was quite dependent on the concentration of HMW KGN and amylose sulfate. At higher concentrations, both of them inhibited the activation reaction. On the other hand, sulfatide has been shown to accelerate the activation of XII with plasma kallikrein by Fujikawa et al. and the reaction was inhibited by the addition of HMW KGN. In the present study, the activation of XII and Prek with sulfatides as estimated by the generation of kallikrein, was accelerated by HMW KGN. This reaction was also dependent on the concentrations of sulfatides and HMW KGN, and fragment 1.2-light chain region in HMW KGN was shown to be essential.These results indicate that HMW KGN is required to accelerate the activation of XII and Prek with polysaccharide sulfate and sulfatide as well as kaolin.
Molecular Weight Determinations Of Low Molecular Weight (LMW) Heparins By Ultracentrifugation And High Pressure Liquid Chromatography
Authors: Barlow Grant , Sugisaka N , Petracek F J 
Published: July 2018

Molecular weights were independently determined on nitrous acid depolymerized LMW heparin fractions ranging from 2-15 daltons using the analytical ultracentrifuge and high pressure liquid chromatography (HPLC).Sedimentation-diffusion equilibria were obtained in the analytical ultracentrifuge using speeds ranging from 20,000 to 56,000 rpm. Near theta conditions were obtained using 0.5M NaCl as the solvent. Calculations of molecular weight distributions and, from those figures, weight average molecular weights were made using the method described by Scholte (N.Y. Acad Sci. 164, 156, 1969). The results show that weight average values as low as 2,000 daltons can be determined.Sedimentation-diffusion equilibria were obtained in the analytical ultracentrifuge using speeds ranging from 20,000 to 56,000 rpm. Near theta conditions were obtained using 0.5M NaCl as the solvent. Calculations of molecular weight distributions and, from those figures, weight average molecular weights were made using the method described by Scholte (N.Y. Acad Sci. 164, 156, 1969). The results show that weight average values as low as 2,000 daltons can be determined.The HPLC results were obtained using previously described methods (Fed Proc. 36, 89, 1977) and a new highly efficient gel column (TSK gels). Fractionated dextrans were used as reference standards.
Significance Of Tryptophan Residues In Fibrinogen In Fibrin Polymer Formation
Authors: Matsushima A , Saito Y , Okano K , Inada Y 
Published: July 2018

Human fibrinogen and its fragment were modified with 2-hydroxy-5-nitrobenzyl bromide(HNBB) or with H2O2-dioxane to see the functional significance of tryptophan residues in the fibrinogen molecule. Tryptophan residues were modified with HNBB in a stepwise manner. Modification of two out of the total 64 tryptophan residues in the molecule did not reduce the polymerization activity with thrombin but further modification of the next two residues led to a complete loss of the polymerization activity. The latter two tryptophan residues modified were in Fragment E and the position may be either Trp 33 or Trp 41 in the a-chain in the fibrinogen molecule.Fragment D inhibited the association activity of fibrin monomer. This inhibitory effect was lost completely by the modification of approximately six out of the total 21 tryptophan residues in the fragment with H2O2-dioxane. These resul ts indicate the significance of tryptophan residues in Fragments D and E of fibrinogen molecule in fibrin polymer formation.
Treatment Of Venous Thromboembolism - Case For Thrombolytic Therapy
Authors: Kakkar V V 
Published: July 2018

Thrombolytic therapy has a unique advantage in the treatment of patients suffering from thrombotic disease, since it is capable of inducing the dissolution of intravascular fibrin and thus causing the reduction or elimination of thrombi. The rapidity of thrombus removal distinguishes this form of treatment from anticoagulant therapy, in which normal physiological processes are allowed to restore the obstructed circulation. By quickly removing the obstruction, it should be possible to reduce the mortality arising from acute thromboembolic episodes.The results of therapy for deep-vein thrombosis have been fairly uniform. The published studies can be broadly classified into two main groups; in uncontrolled trials, partial or complete lysis of thrombi was obtained in approximately 65-80% of the patients who received streptokinase, while only 10-25% of the patients receiving heparin showed this change.In patients suffering from acute major or massive pulmonary embolism, a number of trials have demonstrated a more rapid resolution of the embolus than would be expected by treatment with heparin alone.The role of lytic therapy in preventing the late sequelae of deep vein thrombosis at present remains uncertian. Studies involving large numbers of patients and longer periods of follow-up are required to determine the extent to which post phlebitic venous insufficiency is reduced by early thrombolytic therapy.
Inhibition Of Contact Activation By Platelet Factor 4
Authors: Weerasinghe K , Scully M F , Kakkar V V 
Published: July 2018

The contact activation of FXII by dextran sulphate (DS, MW 500,000) has been shown to be inhibited by platelet factor 4 (PF4). Plasma was prepared from blood taken with EDTA, theophylline and PGE1. Contact activation was initiated by the addition of an equal volume of DS (10μg/ml) and incubating at 0° (8 min) or 37° (2 min). The kallikrein generated was measured using S2302. Addition of purified PF4 to plasma caused a concentration dependent inhibition with complete inhibition at greater than 2.0μg/PF4/ml plasma (molar ratio of 20:1). PF4 was also observed in a concentration dependent manner to inhibit clotting of plasma as initiated by dextran sulphate.Repeated freezing of human PRP (Av. platelet count=3.55 × 108/ml) released PF4 (as measured by radioimmunoassay) and caused inhibition of contact activation. By dilution with PPP, the degree of inhibition was found to be related to the concentration of PF4. The amount needed for 50% inhibition varied from 0.2-1.5μg/ml in different donors. Platelet aggregation with epinephrine (2μg/ml) released 4.8μg/ml (±1.7) of PF4 and showed 55% (±22) inhibition of contact activation (n=ll). Collagen (5.7μg/ml) caused 4.9μg/ml (±1.3) of PF4 to be released, but the degree of inhibition varied (n=13). The discrepancy between this and the freezing experiment may be due to the “contact-product activator” properties of collagen and the intact platelet membrane. Control experiments performed using indomethacin or EDTA-PRP showed no release of PF4 or inhibition of contact activation.The effect observed appears to be related directly to the ability of PF4 to bind to negatively charged polysaccharides since PF4 was not found to inhibit activated factor XII or kallikrein. These results indicate another role for PF4 as an inhibitor of contact activation. Together with its known antiheparin properties it indicates that PF4 may be closely involved in the control of haemostasis.
Altered Regulation Of Prostacyclin (PGI2) Activity In Severe Pre-Eclampsia
Authors: de Gaetano G , Marchesi D , Zoja C O , Schieppati A , Misiani R , Mecca G , Rossi E , Donati M B , Remuzzi G 
Published: July 2018

It has been suggested that prostaglandins of the E series play a crucial role in regulation of fetal vascular tone. The discovery of PGI2 has opened new perspectives for understanding the circulatory physiology of normal pregnancy and the vascular abnormalities of pre-eclampsia. We have bioassayed PGl2 platelet antiaggregatory activity and confirmed it by RIA of 6-Keto-PGF1α and thin-layer radiochranatograpty in human umbilical and piacental vessels. Fetal umbilical arteries obtained at the end of 9 uncomplicated pregnancies generated significantly more PGI2 (191 ± 29 ng/mg tissue) than vessels from control non pregnant adults (43 ± 12 ng/ mg) (p < 0.01). Placental veins generated PGI2 similarly to control veins (33 ± 7 and 37 ± 8 ng/mg respectively) . This could contribute to maintaining the lew peripheral vascular resistance of the fetal circulation with very high cardiac output. Umbilical arteries from 5 vramen with severe preeclampsia generated significantly less PGI2 (65 ± 12 ng/mg tissue) than the corresponding vessels from women with normal pregnancy (p < O.Ol). Similarly placental veins frem preeclampsia patients generated less PGI2 (11 ± 2) than normal pregnancy veins (p<0.01).We propose that in severe pre-eclampsia,placental ischemia and fetal distress could be triggered by deficiency of the adaptive mechanism which maintains high vascular levels of PGI2 in normal pregnancy. The abnormally high PGI2 stirnulating activity in plasma frem patients with severe pre-eclampsia in comparison with plasma from women with normal pregnancy supports the contention that regulation of PGI2 synthesis is deranged in pre-eclampsia.
The Effect Of Prostacyclin On The Participation Of Platelets In X-Activation And Thrombin Formation
Authors: Bevers E , Dieijen G V , Rosing J , Hornstra G , Zwaal R F A 
Published: July 2018

Damaged vascular tissue triggers the intrinsic and extrinsic clotting system, resulting in the clotting of plasma. Platelets, especially after their activation, promote this clotting response, which is inhibited by prostacyclin (PGI2).The present experiments were devised to characterize this anticoagulant effect of PGI2.Using specific chromogenic substrates, the intrinsic activation of factor X and the conversion of prothrombin into thrombin (IIa) was measured in reaction mixtures containing highly purified clotting factors. Phospholipids were added as vesicles, platelet lysates, or as whole platelets, either or not activated with a mixture of collagen and thrombin. Phospholipid vesicles and lysed platelets greatly promoted Xa and IIa formation, which was not affected byPGI2.The very low formation of Xa and IIa occurring in the presence of non-activated platelets was not inhibited byPGI2 either. Platelets activated with a mixture of collagen and thrombin stimulated Xa and IIa formation considerably. PGI2 inhibited this effect in a dose-dependent way.These results demonstrate that prostacyclin does not interfere with Xa and IIa formation as such, but specifically inhibits the process by which a mixture of collagen and thrombin stimulates the participation of platelets in these reactions.The inhibiting effect of PGI2 is only partial. This is most probably explained by the fact that prostacyclin can only inhibit but not block platelet activation by a mixture of collagen and thrombin.
The Effect Of Prostacyclin (PGI2) On The Procoagulant Activity Of Human Platelets
Authors: Brox J , Østerud B 
Published: July 2018

Platelets from healthy donors were isolated by albumin- gradient centrifugation and gelfiltration. The platelets were exposed to thrombin, collagen, and ADP separately, and thrombin and collagen in combination. The concentrations used were the lowest that gave maximal aggregation. The following parameters were assayed: aggregation, platelet factor 3(PF 3), Factor V-Va(F.V-Va), total procoagulant activity (TPA, which measures the combined activity of PF 3 and F.V-Va), and serotonin release. The effect of various concentrations of PGI2 on these parameters was examined.Thrombin was more potent than collagen, and collagen was more potent than ADP in stimulating the procoagulant activity and serotonin release (i.e. thrombin generated 33%, collagen 14%, ADP 3% TPA as compared to 100% for lysed platelets). Thrombin alone was equally strong as thrombin and collagen in combination in regard to TPA. The platelet aggregation was maximal in all these experiments.PGI2 (1.4x10-8M) inhibited very efficiently aggregation, serotonin release, TPA, PF 3 and F.V-Va activity when the platelets were stimulated with thrombin, collagen or ADP. When platelets were exposed to thrombin and collagen simul- tanously, the inhibitory effect of PGI2 on TPA decreased. PGI2 concentration of 1.4xlO-7M in such platelet mixtures inhibited TPA by 30-40% whereas the same PGI2 dose inhibited TPA by 80-90% when thrombin and collagen were used separately. In these experiments platelet aggregation was less than 20%.This study demonstrates that PGI2 strongly inhibits the availability of the platelet procoagulant activity, and PGI2 may therefore also slow down the generation of thrombin.
Reduced Production Of 6-Ket0-PGF1α By Umbilical Artery In Pre-Eclampsia
Authors: Carreras L O , Van Assche A , Defreyn G , Van Houtte E , Vermylen J 
Published: July 2018

Decreased production of PGI2 activity by fetal and maternal vessels and low values of PGI2 activity in amniotic fluid have recently been observed in patients with severe pre-eclampsia. In this study we measured the production of 6-keto-PGF1α by the umbilical artery and placental veins in 5 cases of severe pre-eclampsia (diastolic blood pressure > 100 mmHg) and 5 normal pregnancies. No mother had ingested aspirin for at least a week before delivery. Vascular specimens were obtained immediately after expulsion of the placenta, immersed in tris buffer 0.05 M, pH 7.4 and cut into fine rings. The production of 6 keto-PGF1α , was studied within 2 hours. Vascular fragments were incubated on a gentle rocker platform at 22°C in 2.5 ml of the following media :
a) tris buffer ; b) tris buffer containing sodium arachidonate (AA) 5 pM. 100 pi subsamples were removed from the incubation dishes at 0, 4, 8, 12, 16 and 24 minutes. The 6-keto-PGF1α level in each of the subsamples was measured by radioimmunoassay. The mean values obtained, expressed as pg.mgcr-1± SEM.A significant reduction in the production of 6-keto-PGF1α by umbilical artery incubated in tris buffer (p<0.01) and tris buffer containing sodium arachidonate 5 pM (p< 0.025) was observed in the cases of severe pre-eclampsia. In contrast, no significant decrease in the production of 6-keto-PGF1α by placental veins was obtained. Considering that fetal umbilical arteries generate much greater PGI2 activity than vessels from normal adults, a central role for this substance in the regulation of fetal circulation has been postulated. The marked reduction in the production of PGI2 could seriously affect fetal outcome in severe pre-eclampsia.
In Vitro And In Vivo Effects Of An Inhibitor Of Thromboxane Synthetase
Authors: Defreyn G , Carreras L O , Machin S J , Vermylen J , Verstraete M 
Published: July 2018

UK-37,248, 4-(2-(lH-imidazol-l-yl)ethoxy)benzoic acid hydrochloride, completely inhibits platelet aggregation in plasma by low concentrations of arachidonic acid at 0.5 uM and thromboxane B2(TXB2) generation in washed platelets at 10 uM. In the latter test system the total amount of cyclooxygenase metabolites is unaltered, the decrease in TXB^ and hydroxyhepatadecatrienoic acid being compensated by an increase in prostaglandins E2 and F2α . Arachidonic acid challenged platelets pretreated with UK-37,248 do not accumulate cyclic AMP ; they however strongly stimulate the production of prostacyclin by aspirin pretreated cultured endothelial cells.In a double blind placebo controlled study ingestion of 200 mg of the compound resulted in a complete inhibition of arachidonic acid induced platelet aggregation, whereas the threshold concentration for irreversible platelet aggregation with ADP was unaltered. Serum TXB2 levels were markedly decreased from the normal pre-values (200-700 pg/ml) to low (60-80 pg/ml). Stimultaneous plasma 6-keto prostaglandin F2α , levels from citrate blood increased from 46 ± 23 pg/ml (mean ± SD) to 409 ± 185 pg/ml).It is concluded that a thromboxane synthetase inhibitor modifies cyclic endoperoxide metabolism in such a way that there is not only a decreased formation of pro-aggregatory thromboxane A2 but also an increased production of antiaggregatory prostacyclin. Thromboxane-synthetase inhibitors may be superior to aspirin as antithrombotic agents. The platelet function defect induced by UK-37,248 is only distinct from that in congenital thromboxane synthetase deficiency in that in the latter condition ADP aggregation always is reversible and arachidonic acid challenged platelets accumulate cyclic AMP (Defreyn et al, 1981). Probably UK-37,248, like imidazole itself, activates phosphodiesterase, thus abolishing part of its anti-aggregating effect.
Aggregating Activity Of Structural Analogues Of Platelet Activating Factor (PAF): High Stereospecificity
Authors: Baumgartner H R , Hirth G , Michael C , Saroka H , Bannwarth W , Barner R 
Published: July 2018

12 structural analogues of PAF, a 1-0-alkyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine, were synthesized in order (1) to study their structure-activity relationship and (2) to find competitive inhibitors of PAF.The compounds were dissolved in ethanol and diluted with 0.1 % albumin in isotonic saline. They were added to stirred platelet-rich plasma (PRP) obtained from citrated blood. For each PRP the proaggregatory EC50 for ADP and 1-0-octadecyl-2-0-acetyl-sn-glyceryl-3-phosphorylcholine (Ro l4-8l6l) was determined using a Born MK3 or an elvi 840 aggregometer.In human PRP Ro l4-8l6l had an EC50 of 43 ± 5 nM (mean ± SE, n = 6) and was 33 × more potent than ADP. Ro l4-8l6l was 160 x (n = 20) more potent than ADP and 1300 x (n = 6) more potent than its S-enantiomer in aggregating rabbit platelets (EC50 for Ro l4-8l6l = 3.8 ± 0.3 nM). Rabbit PRP was used to compare additional analogues. Introduction of a double bond (cis-9-octadecenyl) in the alkyl chain of racemic 1-0-alkyl-2-0-acetyl-glyceryl-3-phosphorylcholine caused a threefold increase of the aggregating activity. With the replacement of the acetyl group by H or CH3 in Ro l4-8l6l platelet aggregating activity virtually disappeared. Replacement of choline by ethanolamine caused considerable loss of activity. Permutation of the substituents attached to the glycerol moiety was also associated with partial loss of activity. Plasmalogen was inactive. None of the analogues inhibited Ro l4-8l6l induced aggregation.Our findings demonstrate that the platelet aggregating effect of the PAF molecule is highly stereospecific: the natural configuration at C (2) of l-0-alkyl-2-0-acetyl-glyceryl-3-phosphorylcholine is of particular importance for activity.