bluuebunny/crossref_metadata_2025_split huggingface dataset:
MODE OF ACTION OF A NOVEL ANTI-PLATELET AGENT (E-5510)
Authors: Saeki T , HARADA K , Youshimura T , Nakamura Y , Fujimori T , Katayama K E , Yamagish Y , Yamastu I , Ikeda I 
Published: August 2018

A novel anti-platelet agent, 4-cyano-5,5-bis(4-methoxy-phenyl)-4-pentenoic acid(E-5510), has been shown to inhibit platelet aggregation and secretion induced by thrombin as well as by other inducers such as ADP, collagen and PAF. Although E-5510 can act as a cyclooxygenase inhibitor,inhibition of cyclooxygenase may not be the primary mode of action since this compound effectively inhibits thrombin-induced platelet activation. In this paper, effects^of E-5510 on arachidonic acid (AA) metabolism, intracellular Ca++ and cAMP in human platelets are examined.(1) Effect on AA metabolism. Platelets prelabeled with [14 C]-AA were stimulated with thrombin. E-5510 inhibited not only thromboxane A2 and HHT generation but also 12-HETE generation in a dose-dependent fashion. The total AA released was also reduced by E-5510. An almost 50% reduction was obtained by 10 uM of this compound. On the other hand, a cyclooxygenase inhibitor such as U-53059 increased 12-HETE generation in a dose-dependent fashion. In addition to the inhibition of AA metabolism, E-5510 exerted inhibitory effects on phosphatidic acidgeneration, which suggests the possible inhibition of phospholipase C activity by this compound.(2) Effect on intracellular Ca++ and protein phosphorylation. Intracellular Ca++ mobilization was examined using Fura-2 loaded human platelet suspension. The increase in intracellular Ca induced by thrombin was inhibited by E-5510 and the increase in phosphorylation of 40 K protein was also suppressed by this compound after the stimulation of human platelets by thrombin.(3) Effect on cAMP. Platelets were incubated with 10-100 uM of E-5510 and the cAMP content in human platelets was measured. E-5510 increased the cAMP content in a dose-dependent fashion. In platelet homogenate, E-5510 inhibited phosphodiesterase activity with an IC50 of 10 uM.These results suggest that E-5510 may inhibit platelet aggregation and secretion through the multiple modes of action, such as inhibition of phospholipase C, phosphodiesterase and cyclooxygenase, in the process of platelet activation.
CORRELATION BETWEEN THE ANTICOAGULANT AND ANTIPLATELET EFFECT OF D-PHE-PRO-ARG-H (RG-2958).
Authors: Bagdy D , Bara-bás É , Sebestyén L , Diószegi M , Fittler Zs , Bajusz S , Széll E 
Published: August 2018

Anticoagulants usually have no antiplatelet effect and platelet function inhibitors do not interact with the coagulation factors. Since thrombin has a decisive role in thrombus formation (growth and stabilization), inhibitors of the effect of thrombin on platelets may be of special importance in developing a novel type of anticoagulant with antiplatelet properties.D-Phe-Pro-Arg-H /I/ designed and synthetized in our Institute was found to be a highly specific,reversible non-competitive inhibitorgOf thrombin,a specific platelet agonist. (K.= 1x10-8 M). /I/ was administered parenterally and orally to white New Zealand rabbits and to beagle dogs. The kinetics of action was recorded by measuring the WBCT, APTT, PT, TT,platelet count (PC) and platelet aggregation (PA). Optimum degree of anticoagulation was considered by the values proposed by Verstraete and Verwilghen. /I/ was shown to be a highly specific inhibitor of PA induced by thrombin.No direct interaction between the inhibitor and the platelet membrane could be detected. Aggregability of human platelets in citrated PRP and that of the gel-filtrated platelets induced by ADP or collagen did not change after incubation with /I/. The antiplatelet effect of /I/ was studied by ex vivo experiments where the inhibitor was the anticoagulant ( 30 ug/ml whole blood ) instead of citrate. Comparing the aggregability caused by several inducers in citrated human PRP with that of in /I/-PRP a significant difference was observed when epinephrine was the PA-inducer. /I/ acts via formation of an enzyme-inhibitor complex that inhibits the binding of thrombin on their receptor-sites at the platelet membrane. In vivo experiments showed a close correlation between TT and PA induced by thrombin. /I/ proved completely harmless to platelets and red blood cells. No significant change in PC could be detected.
PLATELET FUNCTION IN VASOSPASTIC DISORDERS
Authors: Wilkinson D , Vowden P , Gilka L , Rajah S M , Kester R C 
Published: August 2018

If vasospastic disorders are associated with abnormal eicosanoid metabolism then disturbed platelet function may be observed. We have measured platelet aggregation (PAG) at 37°C to ADP (8 1:1 sequential dilutions from 10μM), collagen (2mg/l) and adrenaline (5μM) in 37 patients with Raynaud's syndrome (21 primary (RD), 9 secondary to scleroderma (RP) and 7 vibration white finger disease (VWF)) and 10 normal subjects (N). In addition we have determined the platelet aggregate ratio (PAR). PAG was expressed as the percentage fall in optical density after the addition of the aggregant. We also recorded the duration of the collagen lag phase and the concentration of ADP at which disaggregation occurred (DAC).Patients with RP showed enhanced PAG compared to normals. This reached significance for collagen lag phase (p<0.001), percentage PAG at 3 minutes to ADP 1.25μM (p<0.05) and the DAC (p<0.01). Similar differences were observed between patients with RP and VWF. Patients with RP also showed significantly enhanced PAG with regard to ADP 1.25μM (p<0.01) and the DAC, (p<0.05) compared to patients with RD. There was no significant difference in platelet function between normal subjects and patients with VWF or between normal subjects and patients with RD. Only with regard to DAC did patients with RD differ from patients with VWF (p<0.05). The differences observed in PAG were not reflected by the PAR. Patients with RP have significantly enhanced PAG when compared to all other groups. This may relate to eicosanoid metabolism and provides a rationale for treatment.
SYNTHESIS OF PLATELET ACTIVATING FACTOR BY HUMAN BLOOD PLATELETS
Authors: Struk A , Prins A , Schaap M C L , ten Cate J W , van den Bosch H 
Published: August 2018

Synthesis of platelet activating factor (PAF) by human blood platelets is a controversial issue. Whereas some groups have reported the synthesis, induced by thrombin, collagen or Ca2--ionophore Azsio?, others were unable to obtain for instance the thrombin-induced PAF synthesis. Also, synthesis of only up to 6 pMoles PAF/10 platelets has been reported, but leucocytes may synthesize up to 6000 pMoles PAF/10 cells. Only an 0.1% leucocyte contamination would thereby explain the “ platelet PAF synthesis” . We therefore optimized the PAF synthesis by human blood platelets and leucocytes, induced by thrombin and A23i07. As platelets have been reported to show an increased PAF synthesis upon treatment with phenylmethylsulfonylfluoride (PMSF), this was also investigated.Leucocytes were optimally stimulated with 10 uM Azale? (mean ± SD 4678 ± 2033, range 1698-7058 pMoles PAF/10 cells, n=6), but could not be stimulated by thrombin. PMSF treatment itself induced PAF synthesis by these cells, but this was not influenced by thrombin or A23ia7.Platelet suspensions, with <0.005% leucocyte contamination as determined by light microscopy after Jenner-Giemsa staining, could synthesize PAF when treated with PMSF and stimulated with 2.5 IU/ml thrombin (mean ± SD 0.6 ± 0.3, range 0.3-1.0 pMoles PAF/10 platelets, n=6), but in these suspensions A23io7-induced synthesis could not be demonstrated.The results indicate that synthesis of PAF by human blood platelets is not due to contaminating leucocytes, if thrombin is used as the stimulus. These results were confirmed by 3H-acetate uptake experiments.
SELECTIVE ANTAGONISTS OF PAF: INTERFERENCE WITH PLATELET FUNCTION AND EFFECT ON THROMBOGENESIS INDUCED BY SUBENDOTHELIUM.
Authors: Hadvary P , Baumgartner H R 
Published: August 2018

Platelet activating factor (PAF) is a very potent excitatory agonist of blood platelets but the physiological importance of this mediator in platelet thrombus formation is not known. We investigated the effect of two chemically unrelated selective inhibitors of PAF-induced platelet aggregation on thrombogenesis induced by rabbit aorta subendothelium (SE) using an ex vivo perfusion system.Ro 19-3704 is a highly potent inhibitor structurally related to PAF. This compound inhibits PAF-induced aggregation of rabbit platelets in platelet rich plasma in vitro competitively. Against 4 nM PAF, a concentration resulting in submaximal platelet aggre-gregation velocity, the IC50 was 70 nM. Inhibition was highly selective for PAF-induced aggregation, since aggregation induced by collagen (HORM, 5 yg/ml), ADP (1 yM) or thrombin (0.4 U/ml) was not inhibited even at a concentration as high as 10 yM. Bro-tizolam, a triazolobenzodiazepine reported to be a selective inhibitor of PAF-induced platelet activation, had in our system an IC50 of 200 nM. The selective benzodiazepine antagonist Ro 151788 was without effect on inhibition of PAF-induced platelet activation by brotizolam.Ro 19-3704 was given intravenously to rabbits as a bolus of 0.2 mg/kg followed by constant infusion of 0.02 mg/kg/min. This dosage provoked ex vivo a constant right shift ratio of the dose response curve for PAF-induced aggregation (RSR[PAF]) by a factor of 25 to 35. Brotizolam was given orally at a dose of 100 mg/ kg together with 300 mg/kg of Ro 15-1788 (to antagonize the central effects) 90 minutes before starting the perfusion experiment, resulting in a RSR[PAF] of 35 to 135. ADP induced platelet aggregation was not impaired by either compound. SE was exposed to the non-anticoagulated blood withdrawn from the carotid artery for 3 min at 2600 s-1 and for 20 min at 200 s-1 shear rate. Quantitative morphometric evaluation showed that SE coverage by platelets and by fibrin, thrombus area and thrombus height were all unchanged by the PAF antagonists at low and at high shear rates despite a very substantial inhibition of PAF-induced platelet aggregation. Therefore a major role of PAF in SE-induced thrombogenesis seems unlikely.
A MONOCLONAL ANTIBODY AGAINST AN ERYTHROID ONTOGENIC ANTIGEN IDENTIFIES GP IV ON HUMAN PLATELETS
Authors: Kieffer N , Edelman L , Edelman P , Legrand C , Breton-Gori us J , Vainchenker W 
Published: August 2018

A murine monoclonal antibody (FA6-152), obtained by immunizing mice with fetal human erythrocytes agglutinated fetal but not adult erythrocytes and bound to both adultand fetal monocytes, platelets andreticulocytes. The antibody did not react with lymphocytes or granulocytes (P. Edelman et al., Blood, 1986, 67, 56). Fluorescent labeling of marrow cells and of in vitro BFU-E, CFU-GM and CFU-MK derivedcolonies revealed that the antigen defined by FA6 was absent from the granulocytic precursors and was detected on the megakaryo-cytic lineage at a later stage of differentiation than major platelet membraneglycoprotein markers. In contrast,the antigen appeared as a very early marker of erythroid differentiation.In the present report, we performed immunoprecipitation experiments on surface labeled platelets toidentify the platelet FA6 antigen.A band of apparent Mr - 85,000 wasimmunoprecipitated from iodinated platelets. This band was also revealed after periodate/Na[3H]BH4 orneuraminidase galactose oxidase/Na[3H]BH4 surface labeling of the platelets, providing evidence that the FA6 antigen corresponds to platelet GP IV. Preliminary studies revealed that FA6-IgG inhibited platelet aggregation induced by low doses of thrombin without affecting the platelet release reaction. Our results therefore suggest that GP IV, which isa multi-lineage hematopoietic differentiation marker, could play a role in cell-cell or cell-substrateinteractions common to different hematopoietic cell types.
A MORPHOLOGICAL APPROACH TO THE MECHANISMS OF RISTOCETIN INDUCED PLATELET AGGLUTINATION(RIPA)
Authors: Escolar G , Monteagudo J , Villamor N , Garrido M , Castillo R 
Published: August 2018

Changes in the morphology of human platelets induced by Ristocetin (RIPA) have been analyzed at ultraestructural level by means of a tannine acid procedure. Studies were completed with aggregation and binding experiments. Modificationsof these tests induced by apyrase,a monoclonal antibody (Mab) to GPIIb/IIIa and EDTA were also investigated.Transmission electron microscopy reveals that ristocetin precipitates adhesive proteins on plateletmembrane. An electron-dense deposit was noticeable within 20 secondsafter ristocetin was added. When experiments were carried out in theaggregometer cuvette under stirring, groups of platelets become activated, change shape, and finally aggregate releasing part of their content. The morphology of aggregates did not differ from those formed in the presence of ADP.Aggregation studies demonstrated that a Mab to GPIIb/IIIa modifies the extent and the rate of the aggregation curve when RIPA was performed in citrated platelet rich plasma (c-PRP). Apyrase modified the extent but not the slope of the curve. Neither the antibody nor apyrase modified RIPA when it was performed in PRP obtained in presence of EDTA. Binding experiments confirmed that I-vWF bound to platelets in presence of ristocetin was not modified by apyrase or anti-GPIIb/IIIa Mab. All these facts together suggest that RIPA,when performed in c-PRP besides reflecting the interaction of GPI with vWF, is also testing other mechanisms of the platelet function including exposure of GPIIb/IIIa complex, interaction of fibrinogen with this glycoprotein, and the contribution of the release reaction.
THE DENSE TUBULAR SYSTEM IS PRESENT IN BOVINE PLATELETS
Authors: White James G , Rao Gundu H R 
Published: August 2018

Human platelets contain two channel systems. The open canalicularsystem (OCS) consists of tortuous channels, continuous with the cellsurface and each other, and spreadthroughout the platelet cytoplasm.The dense tubular system (DTS) derives from rough endoplastic reticulum of the parent megakaryocyte.It is also spread randomly through the platelet and forms specialized membrane complexes with elements of the OCS in one or two areasof the cytoplasm. The OCS serves as the conduit for discharge of granule products during the platelet release reaction. Elements of the DTS contain enzymes involved in prostaglandin synthesis and serve asreservoirs for calcium involved inplatelet physiological activities.Recent studies have shown that bovine platelets lack the OCS and usethe surface membrane as a conduit for the release of granule associated products.The existence of a DTS and its functional role in bovine platelets has not been examined previously. Bovine platelets converted 14C-arachidonic acid to thromboxane B2 as efficiently as human cells. Fura 2 loaded bovine platelets stimulated by thrombin in the presence of EDTA developed cytoplasmic levels of calcium flux comparable to human cells, suggesting that bovine platelets must have a DTS. Ultrastructural procedures for cyto-chemical demonstration of platelet peroxidase and glucose 6 phosphatase resulted in selective deposition ofreaction product in channels of a DTS in bovine cells. Membrane complexes formed by interaction of theOCS and DTS in human platelets were absent. However, some elements of the DTS in cattle platelets are located very close to the cell surface. Results of these studies demonstrate that bovine platelets possess a functional DTS involved in prostaglandin synthesis and calcium flux.
ARACHIDONIC ACID (AA) INDUCED AGGREGATION OF RAT PRO- MEGAKARYOBLASTS (RPM)
Authors: Gladwin A M , Martin J 
Published: August 2018

Platelet aggregation can be induced in vitro by a variety of platelet agonists acting upon membrane receptors. Since platelets have only a limited ability to synthesise proteins, these receptors must be present in megakaryocytes. This was investigated using an eternal line line of RPMs. Cells were suspended in rat platelet free plasma (PFP) at a concentration of 105 cells ml-1 . 200μl aliquots of this were placed in a light aggregometer. For this suspension, and for an aliquot of PFP, light transmission was adjusted to zero and 100% respectively. Addition of ADP (plasma concentrations 1-100μM), thrombin (0.5-5 I.U. ml-1 ), and adrenaline (0.1-1 I.U. ml-1 ) to the suspension caused no change in transmission. However, addition of AA (1.5-6mM) increased light transmission, indicating RPM aggregation. Radioimmunoassay (RIA) on the resultant supernatant showed no thromboxane B2 was produced. Scanning and transmission electron microscopy showed the aggregate to be composed of non-lysed cells. Aggregation of RPMs was not inhibited by preincubation with PGL (1500ng ml-1 ) indomethacin (100μg ml-1 ) or fenoprofen (1002μg ml-1 ). However, preincubation with aspirin (30μM) blocked aggregation.These results indicate that RPMs can aggregate in response to AA. Mechanism of this is unlike that observed for platelets, since PGI2, indomethacin and fenoprofen did not block aggregation. The response was only inhibited by aspirin. Indomethacin, fenoprofen and aspirin are all known inhibitors of cyclo-oxygenase. In addition, aspirin also blocks 12-lipoxygenase. Therefore, this may suggest that the effect of AA on RPMs is mediated via 12-lipoxygenase pathway. Further investigations are in progress
CYCLIC NUCLEOTIDE PHOSPHODIESTERASE INHIBITORS PREVENT AGGREGATION AND SECRETION OF HUMAN PLATELETS BY RAISING CYCLIC AMP AND REDUCING CYTOPLASMIC FREE CALCIUM MOBILIZATION
Authors: Beretz A , Lanza F , Stierlé A , Cazenave J-P 
Published: August 2018

Drugs that raise platelet cyclic AMP (cAMP) are potent inhibitors of platelet activation. We have studied the effects of 5 inhibitors of cyclic nucleotide phosphodiesterase (PDE) of different chemical structures (quercetin, Ro 15-2041, HL-725, cilostamide and MY-5445), which are all potent inhibitors of platelet function. The concentrations that inhibit by 50 % crude cAMP-PDE activity (IC50) from human platelets are: 0.06 μM(HL-725), 0.15 μM(Ro 15-2041 ), 0.23 μM(cilostamide), 6.9 μM(MY-5445) and 44.4 μM(quercetin). We measured on the same preparation of washed human platelets loaded with quin2, the aggregation and the increase in intracellular Ca2+ ([Ca2+]i) induced by 5 μM ADP alone or in the presence of PDE inhibitors.PGE1 (2 nM) potentiates significantly (1.6 to 3.3 fold) the inhibitory effects of PDE inhibitors on [Ca2+]i rises and platelet aggregation. Adrenaline, an inhibitor of adenylate cylase, prevents the effect of PDE inhibitors on ADP-induced [Ca2+]i rise and platelet aggregation. These results suggest that these compounds inhibit [Ca2+]i mobilization and subsequent ADP-induced aggregation through a rise in cAMP, because both effects are potentiated by PGE1 and inhibited by adrenaline. The inhibitor concentrations which potentiate the action of PGE1, on [ Ca2+]i levels also potentiate the rise in platelet cAMP induced by PGE-<. These results suggest that PDE inhibitors inhibit platelet aggregation Ly raising cAMP levels and subsequently inhibiting [Ca2+]i mobilization.
VON WILLEBRAND FACTOR PROTECTS FACTOR VIII FROM INACTIVATION BY ACTIVATED PROTEIN C AND PROTEIN S
Authors: Joost S , Koedam A , Meijers Joost C M , Sixma Jan J , Bouma Bonno N 
Published: August 2018

Activated protein C (APC) inactivated the cofactors factor V (FV) and factor VIII (FVIII). In the case of FV, this reaction and the respective roles of Ca2+ , phospholipids and protein S have been well documented. We investigated the role of protein S and von Willebrand factor (VWF) on the inactivation of FVIII.Purified human factor VIII (3 units/ml) was incubated with protein C (0.2 μg/ml) in the presence of 8 μg/ml phospholipid, 5 mM CaCl, and 1 unit/ml hirudin. Factor VIII coagulant activity decreased with a pseudo first-order rate constant of 0.09 min . The reaction rate increased linearly with the concentration of prot^ig S in the incubation mixture.
12I-FVIII was incubated under the same conditions. SDS-polyacrylamide gel electrophoresis showed cleavage products of Mr 43 and 22 kDa. High Mr bands (FVIII-heavy chain) ranging fromMr 108 to208 kDa disappeared while the Mr 80 kDa FVIII-lightchain remained unchanged. The degradationpattern was not changed by addition of protein S.The FVIII-VWF complex was reconstitutedby mixing the two components (±2 units VWF/units FVIII) and lowering the calcium concentration to 2 mM. The inactivation of the FVIII-VWF complex by APC proceeded at a 15- to 20-fold slower rate as compared to the isolated FVIII, indicating a protection of FVIII by VWF. Protein S exhibited no cofactor activity on the inactivation of FVIII-VWF by APC. The protective effect of VWF was lost completely after activation of the FVIII-VWF complexwith thrombin (0.05 units/ml).When FVIII (0.1 units/ml) was added toplasma of a patient with severe von Willebrand's disease, 96% of its activitywas lost in 20 min after the addition of APC. All of the FVIII activity was retained when haemophilic plasma was used. Mixing experiments showed that one unit ofVWF unit FVIII is needed to fully protec FVIII against APC. These results may explain the observed lability of FVIII in von Willebrand's disease patients.
KINETIC BEHAVIOUR OF VON WILLEBRAND FACTOR AND FIBRONECTIN EXPLAINS DEPENDENCE OF PLATELET ADHESION ON PHYSICAL PARAMETERS
Authors: van Breugel Hans H F I , de Groot Philip G , Sixma Jan J 
Published: August 2018

To study the kinetics of the contribution of von Willebrand Factor (vWF) and fibronectin (FN) in platelet adhesion we developed a method with which we can perform binding studies of platelets to these purified proteins under static and flow conditions. Glass coverslips were incubated for one hour with vWF (50 (jg/ml) or FN (300 pg/ml) in saline and were perfused with washed platelets (resuspended in human albumin solution) in the flat perfusion chamber as developed by Sakariassen (J.Lab.Clin.Med. 102, 522-535, 1983). Static conditions were achieved by incubating the coated coverslips with the platelet suspension.In this system, adhesion of platelets to FN coated coverslips strongly decreased at shear rates above 300 /s. The adhesion to this surface could be inhibited with antibodies against platelet glycoprotein Ilbllla and against lb, under static and under flow conditions.Adhesion to vWF coated surfaces increased with increasing shear rate and ultimately reached a plateau at about 800 /s. Adhesion to a vWF coated surface could be totally inhibited by anti GP-Ib and only partially by GP-IIbllla.When after perfusion of a FN coated surface with platelets, the same surface was perfused with a platelet free perfusate, the coverage of platelets on this surface decreased. No decrease in platelet coverage was found when this experiment was performed with a vWF coated coverslip.From these results we conclude that platelets bind to FN at a high rate and with a low affinity, while they bind slowly but with a high affinity to vWF, probablyvia similar platelet receptors.