bluuebunny/crossref_metadata_2025 huggingface dataset:
PURIFICATION AND PARTIAL CHARACTERIZATION OF A BIOACTIVE SUBSTANCE FROM RAT'S VESSEL WALL INDEPENDENT OF PROSTACYCLIN PRODUCTION
Authors: Kempfer A C , Maugeri N , Farías C , Bermejo E , Gimeno M , Lazzari M
Published: August 2018

Our previous observations provided evidence that a bioactive substance (BAS) with inhibitory effect on platelet aggregation and with inotropic activity on smooth muscle preparations is present in aortic wall of rats treated previously with indomethacin. The ability to inhibit platelet aggregation was used for monitoring its purification and partial characterization. The original sample was extracted by rinsing aortic rings (1.5 mg dried rings) in buffer Krebs (300 μl) for 40 minutes at room temperature. The substance was purified by gel filtration (Bio-gel P-30, Sephadex G-100, Sephadex G-75) and ion exchange chromagra-phy (DEAE cellulose). In the last method, salt gradient elution was performed. Further purification by Sephadex G-50 resulted in removal of 90% of the contaminating substances without a loss of inhibitory activity. The main peak of both chromatographic procedures was analyzed on SDS-PAGE and PAGE with denaturing solvents. The substance was evident by Coomassie Brilliant Blue and periodic acid Schiff staining.

In order to determine if the BAS was susceptible to proteolysis, an aliquot of the original sample (29 ug total protein) was incubated with trypsin (final concentration 0.3 μg/ml) and with chymotrypsin (final concentration 3 μg/ml). The BAS activity was not detected. An aliquot of the same original sample was incubated with neuraminidase (final concentration 1.2 units). The BAS activity was detected.

The substance appeared to be stable for at least 18 hours at room temperature and 2 hours at 37°C, In addition it was stable over a pH range between 6.8 to 8.6, showing an anionic behaviour.

The protein concentration of this substance determined by the method of Lowry was 1 μg/ml

Partial characterization supports the conclusion that the substance present in aortic wall of rats is a homogeneous protein, which has a molecular size estimated at 55-65 kDa.
INHIBITORY EFFECTS OF ITP SERA ON BINDING OF ANTIPLATELET GLYCOPROTEIN (GP) IIb/IIIa MONOCLONAL ANTIBODIES TO HUMAN UMBILICAL VASCULAR ENDOTHELIAL CELLS (HUVE)
Authors: Nakajima T , Koyama T , Nishida Y , Tanaka H , Kakishita E , Nagai K
Published: August 2018

Some ITP patients have specific autoantibodies to platelet GP IIb/IIIa. On the other hand, HUVE were shown to synthesize platelet GP IIb/IIIa like substances. Therefore, we studied the binding of ITP sera to HUVE by showing the inhibitory effect of ITP sera on the binding of anti-platelet GP IIb/IIIa monoclonal antibodies to HUVE. HUVE were cultured according to the method of Jaffe et al. 125-I-anti-platelet GP IIb/IIIa monoclonal antibody (125-I-Anti-GP) (40.3 mCi/mg), 40 yl, was added to a cell suspension of HUVE (1.5 × 104/500 μl) in a plastic RIA tube. After incubation for 30 min. at 4°C and centrifugation of 10,000 xg for 3 min., the radioactivity of the cell pellet was measured. Specific binding was determined by determining the difference between cell-bound radioactivity in the absence and presence of an excess amount of unlabelled ligand at 100 x concentrations. Scatchard analysis using 125-I-Anti-GP showed that the maximum binding capacity was 8 × 104/cell and Kd was 40.2 nM. The binding rate of 125-I-Anti-GP to HUVE treated with ITP (high PAIgG) sera (n=6) was 15.2±3.3% compared with 24.0±7.5%, observed for HUVE treated with normal sera (n=10). Treatment of ITP sera to HUVE significantly lowered the binding of 125-I-Anti-GP to HUVE (P<0.05). A combined analysis of SDS-PAGE and Western blotting of washed platelet and endothelial cell lysates shows that two proteins from each cells had similar or identical molecular masses to GP IIb/IIIa.

These findings show that there are GP IIb/IIIa on the HUVE, ITP sera from our patients may have antibodies to HUVE GP IIb/IIIa and that anti-platelet GP IIb/IIIa antibodies in the ITP sera may bound not only to some platelets, but also to the HUVE
CAROTID INTIMAL TRAUMA: A MODEL FOR PLATELET INTERACTIONS WITH DAMAGED ENDOTHELIUM
Authors: Galvin D AJ , Meek A C , German K , Harper R A , McCollum C N
Published: August 2018

Radiolabelled platelet deposition on prosthetic arterial grafts may be used as a method to evaluate antithrombotic drugs but requires large animals or patients and involves artificial flow surfaces. A rabbit model has been developed to investigate platelet uptake following carotid intimal damage.

Both carotid arteries of 10 NZW rabbits were exposed by midline incision and intimal damage inflicted by a 3-minute unilateral artery compression using the jaws from non-toothed dissecting forceps mounted on a G-clamp with compression screw. Twenty-four hours later, autologous platelets were labelled with llllndium by the method of Hawker et al [1], The carotid arteries were excised 40 hours later and radiolabelled platelet uptake counted in a well crystal and expressed as a percentage of the activity in 1ml of blood.

Total counts (mean ± sem) in the non-traumatised artery were 2.6±0.35×103 which was similar to background at 2.0±0.07×103. Expressed per gm, radioactivity in the traumatised artery at 4.8±0.38×l05/gm was significantly higher than 2.6±0.43×105 in the undamaged carotid (p<0.01). Platelet uptake on the damaged endothelium, expressed as a percentage of the activity in 1ml of blood, at 55.1±9.8 was also significantly higher than 28.6±6.3 percent on the control artery (p<0.01).

This inexpensive model allows the precise measurement of radiolabelled platelet uptake on damaged arterial intima and may therefore be used to evaluate antithrombotic drugs and investigate the role of platelets in arterial repair, intimal hyperplasia and the pathogenesis of atheroma.

1. Hawker RJ, Hawker LM, Wilkinson AR. Indium labelled human platelets: Optimal method. Clin Sci 1980; 50: 243-248
NIACIN-INDUCED PROSTACYCLIN (PGI2) GENERATION AND THE SEARCH FOR THE IDEAL DOSE OF ASPIRIN
Authors: Jakubowski J A , Deykin D
Published: August 2018

We have previously reported that chronic administration of 80 mg/day of enteric-coated aspirin (ECA) in three divided doses of 27 mg each day for 7 days produced over 90% inhibition of platelet thromboxane production. What we wanted to know was whether that dose of aspirin spared PGI2 production. We developed a sensitive plasma assay for PGI2 (measured as 6-keto-PGF1a). We confirmed the reports of others that normal plasma levels are very low, less than 2 pg/ml. We selected niacin as a provocative challenge to raise plasma levels of PGI2 to test the ability of a given aspirin regimen to spare or suppress PGI2 production in vivo. In 5 normal subjects an oral dose of 3 mg/kg of niacin produced a 3-fold rise in 6-keto-PGF1a from 0.86 to 2.64 pg/ml. A dose of 5 mg/kg produced a rise to 6.6 pg/ml. Administration of 323 mg of regular aspirin/day for 7 days completely abolished niacin-induced elevation of plasma PGI2. The lowest dose of ECA that we have found effective in suppressing platelet thromboxane production in vitro, 80 mg/day in divided doses of 27 mg three times a day for 7 days, also completely suppressed niacin-induced elevation of PGI2. Our data do not support the hyypothesis that a very low dose of ECA selectively suppress platelet thromboxane production but spares generation of PGI2
INFLUENCE OF GLUCOSE AND INSULIN OF PROSTACYCLIN SYNTHESIS IN CULTURED TR0PH0BLAST CELLS
Authors: Rákóczi I , Geró Gy , Demeter J , Gáti I
Published: August 2018

It is known that placental cells can produce prostacyclin /PGI2/. At present, the physiological role of trophoblast PGI2 production can only be speculative. PGI2 produced by trophoblast may help to prevent the platelet aggregation and thrombosis in spiral arteries and it can also explain the maintance of, and blood flow through, the spiral arteries during endovascular trophoblast invasion in early pregnancy. It has been previously shown that increased glucose concentrations in the incubation fluid can inhibit the formation of PGI2 by rat aortic rings. The aim of present investigation was to study the effect of glucose and insulin on the generation of PGI? by trophoblast obtained from early pregnancy. Trophoblast tissue was obtained immediately from surgical termination of first trimester pregnancy /9 specimens/. Trophoblast was cultured using the method of Jogee et al. Cells obtained from trypsinizaticn were cultured at a density of 2×105 cells/ml in medium 199 containing 10% horse,serum. Glucose /5.5, 16.5 and 33mmol/l / and insulin /103 , 104 and 106 mU/1 / were added to culture and the effect on 6-oxo-PGF2 production over a 24-hr incubation was assessed. Control cultures were incubated without glucose and insulin. The concentration of 6-oxo-PGF2 in culture supernatans were measured by specific radioimmunoassay /3H-6-oxo-PGF1, RIA-kit, New England Nuclear, USA /.There was a significant decrease in 6-keto-PGF10c production by trophoblast cells incubated with increased glucose concentrations /16.5 and 33 mmol/1 / compared to controls / p<0.001/. In contrast, insulin in growth medium did not have any effects on the PGI production. These data suggest that high concentrations of 2 glucose inhibit PGIp production by cultured trophoblast cells. This decreased PGIp synthesis may impair the blood supply of trophoblast which could play a role in the development of congenital anomalies in pregnant women with poorly controlled diabetes mellitus in the first trimester of pregnancy.
INCREASED RESPONSE TO ARACHIDONIC ACID AND U-46619 AND RESISTANCE TO INHIBITORY PR0STAGLANDING IN PATIENTS WITH CHRONIC MYELOPROLIFE RATIVE DISORDERS
Authors: Cortelazzo S , Castagna D , Galli M , Barbui T , de Gaetano G
Published: August 2018

The potency of prostaglandins (PGs) D2, I2 and as inhibitors of platelet aggregation induced by threshold aggregating concentration (TAC) of arachidonic acid (AA) and U-46619 was determined in platelet rich plasma from 20 normal subjects and 20 patients with thrombocytosis (≥500×l09 platelets/L) secondary to myeloproliferative disorders. Patients had a significantly increased response to both AA and U-46619 (p< 0.02) than the control group (i.e. TAC for AA, mean+SD, was 0.41±0.10 mM vs 0.48±0.12 mM ; TAC for U-46619 was 220±155 nM vs 375±102 nM). In contrast, platelet sensitivity to all three inhibitoty PGs was significantly lower in patients than in normal subjects. Indeed the threshold inhibiting concentrations (nM) of PGs against AA were the following: PGD2 20.33±4.16 vs 7.00±2.62 (p< 0.001), PGI2 0.76±0.46 vs 0.34±0.22 (p< 0.01) and PGE1 11.83±3.97 vs 6.50±2.22 (p<0.001). The corresponding inhibitory concentrations (nM) against U-46619 were the following: PGD2 4.67±4.24 vs 0.76±0.30 (p< 0.02), PGI2 1.15±0.96 vs 0.03±0.01 (p< 0.0001) and PGE1 21.12±15.27 vs 0.68± 0.30 (p< 0.0001). Selective pharmacologic inhibition of TxA2 sinthase by 40 μM dazoxiben resulted in 6 out of 11 “responders” in patients and 7 out of 10 in normal subjects, a difference not statistically significant. Serum TxB2 was slightly, but not significantly lower in patients than in controls (360±143 ng vs 390±155 ng/3×109 platelets/mL). It is suggested that in patients with myeloproliferative disorders platelet arachidonate metabolism is normal, but the functional response to aggregating and antiag-gregating prostanoids is altered towards a potential hyperaggrega bility. The relevanbe of this “in vitro” finding to thrombotic or haemorragic complications in these patients remains to be establi shed.
ADRENALINE (ADR) INTERACTIONS WITH THROMBIN AND COLLAGEN -EFFECTS ON PLATELET ARACHIDONATE RELEASE AND 5HT SECRETION AND ROLE OF ENDOGENOUSLY FORMED THROMBOXANE A2 (TxA2)
Authors: Patel Y , Krishnamurthi S , Kakkar V V
Published: August 2018

We have examined the effect of combinations of ADR + thrombin (T) and ADR + collagen (C) on platelet arachidonate release and 5HT secretion, and assessed the role of endogenously formed TxA2 on these responses using indomethacin (I). Washed, human platelets prelabelled with [3H]-arachidonic acid (AA) or [14C]-5HT were used, ADR was added 10 sec before T or C and the reaction was terminated 3 min later. In the range 1-100μM, ADR induced no detectable aggregation or 5HT secretion but potentiated platelet aggregation when added with sub-threshold concentrations of T or C, which on their own induced no aggregation. At 2-4 fold higher concentrations of T and C (threshold for 5HT secretion), 5HT secretion and AA/TXB2 release were also potentiated by ADR (1-10μM) by 30-50%. Pre-treatment of platelets with I (10μM) abolished threshold T and C-induced 5HT secretion, as well as its potentiation by ADR. However, approximately 2-fold and 5-fold higher concentrations of T and C respectively were able to induce 'I-insensitive'secretion, which was further potentiated by ADR. In I-treated platelets, C-induced AA release and its potentiation by ADR were also abolished suggesting a role for endogenously formed TxA2 This was confirmed by addition of the TxA2 mimetic, U46619 (0.3μM), which potentiated C-induced AA release in the presence and absence of ADR, even though it induced no AA release on its own or, in combination with ADR alone in the absence of collagen. The latter suggests agonist specificity regarding the ability of TxA2 to synergistically stimulate AA release. Finally, unstirred platelets in PRP pre-incubated with ADR (10μM) for 120 min lost their responsiveness to ADR, when eventually stirred; however, these 'ADR-desensitised' platelets when washed and resuspended, were able to demonstrate synergistic effects on secretion when stimulated with ADR+T or ADR+C. This is analogous to the previously demonstrated ability of ADR to inhibit adenylate cyclase even in 'ADR-desensitised' platelets and re-inforces the separation regarding the mechanisms underlying the various effects of ADR on platelets.
ARACHIDONATE METABOLISM IN RAT PLATELETS IN THE PRESENCE OF Ca2+-SUBSTITUTES (Sr2+, BA2+)
Authors: BLACHE D , CIAVATTI M
Published: August 2018

To further document the involvement of external Ca2+ in the platelet induced-activation process, we have studied the arachidonate (AA) metabolism of intact washed rat platelets in the presence of different concentrations of Ca2+, Sr2+ or Ba2+. The thrombin-induced mobilization of [3H]- or [14C]-AA preincorporated into platelet phospholipids was followed as well as the subsequent formation of labeled cyclooxygenase and lipoxygenase products (CLP). Results indicate that upon thrombin (<0.1 U/ml) stimulation, AA release and CLP formation are reduced by 50 to 90% only by omission of Ca2+ as compared to 1 mM Ca2+ in the suspending medium. In higher Ca2+ concentrations (5 mM), the AA mobilization and CLP formation are inhibited and the data are close to those obtained in the absence of Ca2+. In the presence of Sr2+ or Ba2+, results indicate that these cations can substitute to Ca2+. Similarly to Ca2+, an optimum concentration is found in Sr2+ or Ba2+ (3-5 mM) and higher concentrations inhibit AA release and CLP formation.

We have also studied the activity of a semi-purified preparation of phospholipase A2 (PLA2) from rat platelets. This activity was assayed (pH 9) using heat-denaturated (3H)-AA prelabeled phospholipids as substrate. The results show that this PLA2 activity was strongly Ca2+-dependent. We found that unlike Mg2+, Sr2+ and Ba2+ are able to greatly enhance this activity. Relative effiency (Vmax) are in the order Ca2+>Sr2+>Ba2+. Taken together, these findings as well as our previous results (Blache et al, BBA 1987, in press) suggest that external Ca2+ may play a major role in the regulation of rat platelet activity. Our interpretation is in line with the view that Sr2+ or Ba2+ could enter the platelet through a mechanism common to Ca2+ (a kind of Ca2+ channel). Although direct evidence await the results of further studies which are in progress, it can be considered that Sr2+ or Ba2+ might cause platelet induced-activation mimickigci a rise in the cytosolic Ca2+ and subsequent activation of Ca2+-dependent enzymes.
ABNORMAL PLATELET FUNCTION MEASURED IN RELATIVES OF STROKE PATIENTS
Authors: Grotemeyer K-H , Pilawa I , Schlake H P
Published: August 2018

Platelets are assumed to play a crucical role in stroke. Abnormal reactivity of platelets has been observed in patients suffering from stroke but it seems unclear whether or not this abnormal reactivity represents a basic factor of stroke. If abnormal platelet reactivity is more but a consequence of stroke it should be present before stroke happens.

In families with stroke patients stroke is more frequent as usual. In order to obtain more information we determined platelet reactivity in healthy persons with a family history without vascular events and healthy persons with a history showing stroke in the family.

Platelet reactivity was measured using a modification ofa platelet test system described by WU and HOAK(1)

Platelet-index-value for the 110 healthy persons without history was 0.98 + /- 0.09 and for the 72 healthy relatives of stroke patients 1.18 + /- 0.28 The difference was significant (p<0.01).

The results support the hypothesis that increased platelet reactivity may not only be regarded as a consequence of stroke. It may be possible that this increased platelet function may represent a pathogenetic co-factor in ischemic events and in atherosclerosis also.

1) Grotemeyer K.-H. et al., DMW (1985) 110, 256
PLATELET AGGREGATION INDUCED BY TUMORIGENIC ARTERIAL SMOOTH MUSCLE CELLS
Authors: BLAES N , COVACHO C
Published: August 2018

A focal proliferation of intimal smooth muscle cells is assumed to be an early event in atherogenesis. In addition, platelets have been suggested to play a role at different steps of the disease process. Blood platelets can be aggregated by a number of tumor cells of various tissue origin. Rat arterial smooth muscle cells presenting a tumorigenic and metastatic phenotype (NBC1 and NBC2 cell lines) have been obtained in the laboratory (BLAES N. et al, Arch. Mai. Coeur Vais., 1986, 79, 55a). The aim of the present work was to assay the proaggregant abilities of these tumor cells of vascular origin. Smooth muscle cells derived from normal rat aortic media. NBC1 and NBC2 cells were spontaneously transformed in cell culture. Interactions between cells and platelets were studied in an homologous in vitro system containing 0.5 ml of PRP (300 000 platelets per ul) from heparinized rat blood and 50 ul of rat smooth muscle cells suspension. Proaggregant activity was tested for control non tumorigenic smooth muscle cells and tumorigenic cells (after 10 or 240 passages). Experiments showed that NBC2 cells elicited a proaggregant ability, weak after 10 passages but very significant after 240 passages. This effect depended on the number of cells added to the platelet suspension (it increased from 105 to 2.106 cells per 0.5 ml suspension). Aggregation profiles appeared biphasic and could be abolished by aspirin. The first reversible phase occured immediately and was reduced dose-dependently by apyrase. Hirudin was shown to affect the aggregation profile suggesting an activation of the clotting system in the NBC2-induced platelet activation. By comparison, non tumorigenic arterial smooth muscle cells showed no effect at comparable cell numbers and NBC1 cells (less metastatic than NBC2 cells) exhibited only a monophasic profile. These results suggest that NBC2 cells are able to induce platelet aggregation by mechanisms involving ADP and thrombin generation. This ability could be related to their tumorigenic and metastatic phenotype.
PLATELETS FROM TYPE IIA HYPERCHOLESTEROLEMIC PATIENTS GENERATE MORE INOSITOLPHOSPHATES AFTER THROMBIN STIMULATION IN COMPARISON WITH THOSE OF NORMAL SUBJECTS
Authors: Medini L , Maderna P , Tremoli E , Galli C
Published: August 2018

Functional and biochemical responses of platelets to stimulating agents have been reported to be amplified in several pathological conditions, including hyperlipidemia. Enhanced aggregation and thromboxane formation are, e.g. frequently observed in the presence of high plasma cholesterol levels (type Ila hypercholesterolemia). Stimulation of phosphoinositides breakdown through specific phosphohydrolases (phospholipase C) resulting in the formation of inositolphosphates (IPs), is one of the early events in platelet activation. A study was thus designed in order to investigate IPs generation in thrombin stimulated platelets from type Ila hypercholesterolemic patients in comparison with a group of normocholesterolemic subjects. Preparation of washed platelets, prelabelling with 2[3H] myo inositol and separation of lipid and water soluble inositides was carried out according to Watson et al (1). Product generation (inositoltrisphosphate, IP3; inositolbisphosphate, IP2; inositol mono phosphate, IP) in relation to the 2[3H) inositol incorporated in phosphoinositides was evaluated at 10 and 90 s after platelet stimulation with 1 U/ml NIH in the presence of 10 mM lithium chloride. The major differences found in platelets from type Ila patients in comparison with those of controls were the following:1) in non stimulated platelets a greater incorporation of myoinositol in phosphoinositides and lower levels of labelled IP2; 2) after stimulation, the levels of all labelled IPs were significantly greater, and those of IP2 were double than in controls. In particular the percent increment of IP2 over basal values in platelets of type Ila patients was more than two fold greater. It is concluded that the enhanced generation of IPs in platelets from type IIa patients, following thrombin stimulation, may contribute to the greater sensitivity to agonists of the aggregatory process in this pathological condition.

1) Watson P.S., McConnell R.T. and Lapetina E.G., J. Biol. Chem.21:13199, 1984
TICLOPIDINE AND INDOBUFEN: EFFECTS ON HAEMOSTATIC FUNCTIONS
Authors: Montecchio G P , Custodi P , Carbone S , Bendotti C , Piovella F
Published: August 2018

Many different mechanisms are involved in thrombus formation. We compared the effects on haemostatic function of two drugs having different mechanism of action, the one interfering with arachidonic acid metabolic cascade (Indobufen) and the other (Ticlopidine) independent from it. 18 adult patients of both sexes suffering from cerebral Transient Ischaemic Attack (T.I.A.) or Reversible Ischaemic Neurologic Disability (R.I.N.D.) have been treated with Indobufen (400 mg daily) or Ticlopidine (500 mg daily) for three weeks. The effects on various haemostatic parameters including bleeding time, platelet adhesion to glass beads, platelet aggregation induced by ADP, collagen, platelet activating factor (PAF )f have been evaluated at the beginning and at the end of treatment. Both drugs prolonged the bleeding time, Ticlopidine being more effective than Indobufen. ADP-induced platelet aggregation was more effectively inhibited by Ticlopidine, while Indobufen was more effective on collagen-induced aggregation. PAF-induced platelet aggregation was inhibited by Ticlopidine, while Indobufen was ineffective. Platelet adhesion to glass beads was not influenced by treatment with either drugs. In conclusion, both drugs confirmed to be effective in inhibiting haemostatic function although with different mechanisms. Ticlopidine seems to be involved in more mechanisms, interfering with platelet aggregation induced by ADP, collagen, PAF and prolonging the bleeding time. Indobufen interferes with platelet aggregation induced by ADP and collagen, is less effective in prolonging the bleeding time, and does not affect PAF-induced platelet aggregation.
DIPYRIDAMOLE INDUCES CHANGES IN THE THROMBOGENIC PROPIERTIES OF ENDOTHELIAL CELL EXTRACELLULAR MATRICES
Authors: Aznar-Salatti J , Escolar G , Almirall L , Ordinas A , Bastida E
Published: August 2018

Dipyridamole (DIP) is a pirydo-pirymidine drug widely used as an antiplatelet agent.DIP has also been demonstrated to have effects on various cultured cells. We studied platelet reactivity of extracellular matrices (ECMs) produced by untreated (ECM-c) and DIP-treated (ECM-DIP) endothelial cells (ECs).Confluent monolayers of EC were incubated in buffer containing 0 or 10 pM. DIP for 48 h. The ECMs were prepared by removing the EC with 2% EGTA (1 h at 37°C). Platelet deposition onto the ECMs was measured under flow conditions using a flat chamber perfusion model ECM-c and ECM-DIP were exposed for 5 min to whole blood at shear rates of 300 or 1300 sec~1.Then, the perfused ECMs were processed for cross sectional morphometric evaluation using a computer system. The platelet, deposition is expressed as total surface covered with platelets (mean±SEM ) and shown in the table.

Platelet aggregate formation on ECM-DIP at 1300 sec-1 was also reduced(by 25%)as measured morphometrically. We conclude that DIP-treatment of EC results in ECM modifications,which in turn decrease ECM-platelet interactions.These data also suggest that pharmacological treatment of the endothelium influences basement membrane reactivity.
THE NEWLY SYNTHESIZED COMPOUND E-5510 IS A HIGHLY POTENT ANTIPLATELET AGENT
Authors: Harada K , Fujimori T , Kogushi M , Kogushi M , Yamatsu I , Ikeda Y
Published: August 2018

Our newly synthesized compound, 4-cyano-5,5-bis(methoxy-phenyl)-4-pentenoic acid (E-5510) has highly potent antiplatelet activity. In this paper, the effects of E-5510 on platelet functions in vitro and ex vivo in human and in various experimental animals are examined.

E-5510 inhibited human platelet aggregation induced by collagen, arachidonate, ADP, PAF and epinephrine (IC50: 1.5, 0.7, 2.0, 1.6 and 1.1 uM, respectively). Thrombin-induced platelet aggregation, which was not inhibited by aspirin and U-53059 (lC50s: 100 uM), was also inhibited by this compound (IC50: 21uM). The IC50 of E-5510 in thrombin-induced ATP secretion fromhuman platelets was only 2 uM. Platelet adhesion to a collagen coated disk, whichwas measured by the method of Buchanan et al (Prost. Leuko. Med., 21, 157, 1986) was inhibited by E-5510 (IC50: 19.3 uM) butnot by aspirin and U-53059. In the PRP ofthe guinea pig, the beagle and the monkey, E-5510 inhibited collagen-induced platelet aggregation in vitro to the same degree as in human PRP(IC50: 1.2, 0.6 and 1.5 uM, respectively). After being administered orally to guinea pigs, E-5510 exhibited extremely potent ex vivo inhibitory effect in collagen-induced platelet aggregation with a very low ED50 of 0.05 mg/kg. In contrast, the ED50’s of ticlopidine, aspirin and U-53059 were 300 , 27.2 and 1.0 mg/kg, respectively. In beagles and monkeys E-5510 also showed ex vivo antiplatelet effects at 0.01 and 0.003 mg/kg, respectively. This effect continued for more than 8 hrs. and disappeared within 24 hrs. The antiplatelet effect in human PRP was highly correlated with that in PRP of experimental animals in which the ex vivo effects were confirmed at a very low dose. Thus, E-5510 will ensure to exert the antiplatelet effect after oral administration to human subjects.

In summary, E-5510 is unique among the known antiplatelet agents since it has potent inhibitory effects on thrombin-induced platelet activation and platelet adhesion to collagen. It was also shown that this compound had an ex vivo antiplatelet effect at an extremely low ED50. Our results suggest that E-5510 will be a beneficial agent for antiplatelet therapy in humans.
ANTI-THROMBOTIC EFFECTS OF E-5510 IN EXPERIMENTAL THROMBOSIS MODELS
Authors: Fujimori T , Saeki T , Harada K , Sato M , Ohshima N
Published: August 2018

A new agent developed in our laboratory, 4-cyano-5,5-bis(4-methoxyphenyl)-4-pentenoic acid (E-5510), suppressed various human platelet functions in vitro. The compound also showed quite potent ex vivo anti-platelet effects in many experimentalanimals. It is well known that anti-platelet effects are not always parallel to anti-thrombotic effects. Thus, in order to predict the efficacy of E-5510 in thrombotic disorders, the anti-thrombotic effects were examined in 3 different animal models of thrombosis.

(1) Anti-thrombotic effect in an extracorporeal shunt model Two hrs after oral administration of the drug to guinea pigs,an extracorporeal shunt between the right carotid artery and the left jugular vein was performed. The thrombus formation on a silk thread inserted in the shunt tube was quantitated by measuring the time from the onset of circulation to the stenosis of blood flow. E-5510 dose-dependently inhibited thrombus formation, the minimum effective dose being 0.03 mg/kg.

(2) Effect on microthrombus formation in mesenteric arteriole In order to evaluate the effect on intravascular platelet thrombus formation, thrombosis was induced in vivo in mesenteric arteriole in guinea pigs with filtered light from a mercury lamp and an intravenous fluorescent dye in an intravital microscope system (M. Sato and N. Ohshima, Thromb. Res.,35, 319, 1984). The thrombus formation was quantitated by measuring the time taken for circulating platelets to begin to adhere to vessel wall and the time taken for blood flow to stop completely due to fully developed thrombus. Both the time required for platelet adhesion to vessel wall and for platelet thrombus formation were significantly prolonged after oral administration of E-5510.

(3) Effect on pulmonary thromboembolism Acute pulmonary thromboembolism was induced in mice by a bolus intravenous injection of arachidonic acid, and mortality was determined 3 min later. E-5510 dose-dependently reduced pulmonary thromboembolic mortality, and its ED50 was 0.11 mg/kg.
The results described above indicate thatE-5510 may have beneficial effects in clinical treatments of thrombotic disease.
EFFECT OF THE CALCIUM CHANNEL BLOCKER, NIFEDIPINE, ON HAEMOSTASIS, CLOTTING, AND THROMBOLYSIS EX VIVO.
Authors: Rademaker M , Meyrick Thomas R H , Kirby J D , Kovaos I B
Published: August 2018

Nifedipine, as other calcium channel blockers, has been shown to inhibit platelet aggregation induced by various agonists in vitro. The therapeutic relevance of these findings are, however, questionable, as in vitro inhibition of platelet function occurs at plasma concentrations of nifedipine in excess of 50 uM, while the peak plasma concentration following a single 10 mg oral dose of nifedipine is only 0.2 uM. We have used the Haemostatometer, to assess the effect of nifedipine on haemostasis ex vivo. This instrument allows quantitative assessment of haemostasis by monitoring the pattern of haemostatic plug formation (HPF) in holes punched through polyethylene tubing through which non-anticoagulated blood flows under standard conditions (1,2). The pattern and speed of blood coagulation subsequent to HPF was measured as was the spontaneous thrombolysis time (STT) (taken as the time until expulsion of the haemostatic plug from heparinised blood). All values are for mean + SEM.

Blood samples were taken from 10 healthy volunteers before and ninety minutes after a single 10 mg oral dose of nifedipine. Following nifedipine, the initial phase of the haemostatic reaction (HPF) was prolonged from 0.95 ± 0.12 min to 1.57 ± 0.14 min, (p< 0.01); clotting time was also increased from 13.1 ± 1.1 min to 20.7 ± 2.3 min, (p=0.013), and the STT fell from 48.0 ± 9.9 min to 27.5 ± 4.6 min (p=0.014).

The increased bleeding time (HPF) provides evidence that a therapeutic dose of nifedipine evokes an anti-platelet effect. The prolongation of the clotting time seen in this study could be explained by,an effect of nifedipine on platelets and hence clotting. The mechanism of the enhanced spontaneous thrombolysis following nifedipine is not known.

We suggest that the effect of nifedipine on haemostasis and thrombolysis may contribute to its therapeutic effect.

(1) Gorog P and Kovacs I B. Haemostasis 16: 337-345, 1986.

(2) Gorog P. Angiology 37: 99-105, 1986.
THE INFLUENCE OF SOME HYPOTENSIVE DRUGS ON THE SEROTONERGIC MECHANISMS IN RATS PLATELETS
Authors: Buczko W , Pietraszek M , Chabielska E , Malinowska B
Published: August 2018

Serotonin (5HT) is a vasoactive amine that has been reported to be involved in a number of forms of circulatory failure. The 5HT content and metabolism in platelets is changed in hypertension and in peripheral arteriolar diseases. The present study concerns the effect of verapamil (VER), propranolol (PRO) and cap-topril (CAP) - drugs having different hypotensive mechanisms of action, on serotonergic mechanisms in rat blood platelets. In vitro, VER produced noncompetitive inhibition of 14C-5HT uptake (IC50=8.2μM), PRO inhibited the uptake in a competitive fashion (Km =0.9μM), whereas CAP was ineffective. Only PRO released (24%) radioactive 5HT from incubated platelets. Inhibition of amine uptake was also obtained when plateletswere prepared from-irats pretreated with VER (10 mg.kg−1 ), PRO (5 mg.kg−1) or CAP (10 mg.kg−1 ). Moreover, the concentration of endogenous 5HT in blood platelets was reduced after VER and PRO administration. Platelets aggregation induced by ADP was inhibited by VER and CAP. They also diminished the potentiating effect of 5HT on ADP-induced platelet aggregation. It can be concluded that these effects may be a secondary mechanism of action in vivo. Thus “serotonergic component” of studied drugs should be taken under consideration at least in therapy of hypertension.

Supported by CPBR, no 11.6
EFFECTS OF FEVERFEW EXTRACT AND PARTHENOLIDE ON PLATELET SECRETION.
Authors: Gronewegen W A , Heptinstall S , Loesche W , Spangenberg P
Published: August 2018

Feverfew (Tanacetum parthenium) is used for prophylaxis of migraine and it had been suggested that the plant may have antithrombotic potential. We have prepared extracts from the leaves of feverfew and have demonstrated inhibition of 14C-5HT secretion in platelet-rich plasma induced by the phorbol ester PMA, l-oleoyl-2-acetyl-sn-glycerol (OAG), arachidonic acid, the thromboxane analogue U46619, adrenaline, collagen and ADP. The effects of a solution of parthenolide (an∝-methylenebutyro-lactone isolated from feverfew) were determined in parallel. Both feverfew extract (FE) and parthenolide inhibited 14C-5HT release in a concentration-dependent manner and the effectiveness depended on the nature of the aggregating agent used. Both FE and parthenolide were most effective as inhibitors of the secretion induced by PMA and OAG. When we compared the concentrations of FE and parthenolide which gave 50% inhibition of secretion for all the agents tested, a good correlation was found (r= 0.936). Further studies showed that feverfew extract and parthenolide inhiMt release of β-thromboglobulin from platelets as well as 14C-5HT. FE did not cause liberation of LDH. Inhibition of secretion by FE appears to be irreversible since washing platelets after treatment did not restore secretory activity.

The structure of parthenolide suggests that it can alkylate sulphydryl (SH) groups. When agents containing SH groups (e.g. cysteine) were added to FE, anti-secretory activity was reduced. We also obtained a considerable decrease in the number of acid-soluble SH groups in platelets treated with feverfew extract or parthenolide at concentrations which inhibit secretion. However there was a less marked decrease in the number of acid-insoluble SH groups. FE itself does not induce formation of disulphide-linked proteins but such proteins were formed when platelets were activated in the presence of FE, probably as a result of decreased glutathione levels.

We conclude that parthenolide or parthenolide-like compounds are responsible for the anti-secretory effects of FE, and that alkylation of sulphydryl groups in platelets may be involved.
EFFECTS OF INHIBITORS ON COLLAGEN INDUCED PLATELET AGGREGATION IN SIX DIFFERENT SPECIES.
Authors: Jansen J W C M
Published: August 2018

One approach to the development of antithrombotics is inhibition of platelet aggregation. The pharmacological approach often used is to test compounds on collagen induced platelet aggregation measured in platelet rich plasma. Therefore we have compared inhibitors with different mechanism of action on aggregation of platelets from six different species commonly used in pharmacological studies. Aggregation was induced with submaximal amounts of collagen (Hormone Chemie).

Inhibitors of the cyclooxygenase system, aspirin and indomethacin, were very potent in inhibiting aggregation of platelets from humans guinea pig and dog (IC50 20-60 and 1-3 ¼M resp.). Aggregation of pig and rat platelets was poorly inhibited by both of these compounds (IC5: 700-900 ¼M), whereas platelets from mice showed intermediate sensitivety (IC50 ca.100 ¼M).

The combined lipoxygenase/cyclooxygenase inhibitor BW755C, was extremely active on platelets of guinea pig (IC50 1 ¼M) and was poorly active in mice platelets (IC50 300 ¼M). In the other species the inhibitory activity ranged from 20-80 ¼M.

The phosphodiesterase inhibitors, papaverine and BL3459 inhibited aggregation in all species (IC50 50-100 and 1-5 ¼M resp.). Dipyridamole inhibited aggregation also in all species but with lower activity (IC50 > 100 ¼M).

Conclusion: remarkable species differences are present with respect to inhibition of collagen induced platelet aggregation by the various compounds e.g. rat and porcine platelet aggregation was hardly inhibited by cyclooxygenase inhibitors. The effects of the compounds on human platelets are comparable to the effects on canine plateletes.
INHIBITION OF THE THROMBOPENIC EFFECT OF ELLAGIC ACID BY PCR 4099, AN ANTIAGGRGATING AGENT, IN RATS.
Authors: DAMAS J , GREGG V , REMACLE-VOLON G
Published: August 2018

In the rat, intravenous injection of large doses (30 mg/Kg) of ellagic acid (EA) induced a decrease in the plasma level of fibrinogen and in the blood platelet content and an increase of the activated partial thromboplastin time. EA induced the accumulation of Crs1-labelled platelets into the lung and the liver accompanied by a 64% fall in Cr51 blood radioactivity.

The long-lasting thrombocytopenia induced by EA was inhibited by heparin (4 mg/Kg), defibrase (20 U/Kg), and clocoumarol (4 mg/Kg). It was not inhibited by aspirin (90 mg/Kg), indomethacin (8 mg/Kg) and ketoprofen (4-10 mg/Kg). The thrombocytopenia would depend on an intravascular coagulation.

Using this model of platelet stimulation, we studied the protective effect of PCR 4099. This drug was first given as a single dose, orally four hours before the injection of EA. It reduced the thrombocytopenia for 10 mg/Kg and suppressed it for 40 mg/Kg. The thrombocytopenia was also inhibited by 3 day oral administration of PCR 4099 4 mg/Kg. PCR 4099 is thus a potent inhibitor of this kind of platelet stimulation.
MICROEMBOLISATION DURING SURGICAL SHOCK: EFFECT OF PROSTAGLANDIN E1
Authors: Poskitt K R , Irwin J T C , Backhouse C M , McCollum C N
Published: August 2018

Embolisation of microaggregates following major surgery may be a cause of pulmonary arterio-venous shunt and postoperative respiratory failure (1). Prostaglandin E1 may prevent intravascular aggregation and we studied this possibility in a pig model of surgical shock.

Following autologous platelet labelling with Indium, 16 pigs (20-30kg) were randomised to receive a perioperative infusion of PGE1 (100ng/kg/min) or placebo. Arterial and Swann Ganz catheters were inserted under anaesthesia prior to surgery consisting of midline laparotomy, exteriorisation of small bowel 1.5 hours of aortic clamping and 1 hour of hypotension. On induction, during shock and at 3 days in survivors platelet and leucocyte count, blood radioactivity, venous aggregates (SFP), lung platelet uptake (LPU), pulmonary vascular resistance (PVR) and alveolar-arterial p02 difference (A-ad02) were measured.

All results mean ± sem *p <0.05 Mann Whitney U-test

During surgical shock, the formation of venous aggregates, the fall in circulating radiolabelled platelets and their accumulation in lungs were reduced by PGE1 (p< 0.05). BP, CVP and PWP were all lower on PGE1 and at 3 days the improvement in A-ad02 in PGE1 pigs failed to reach significance.

PGE1 reduced platelet aggregate formation and their subsequent pulmonary microembolisation despite worsening shock due to vasodilation.

1. McCollum CN, Campbell IT. The value of measuring intravascular platelet aggregation in the prediction of postoperative pulmonary dysfunction. Br J Surg 1979: 66; 703-707
THE PLATELET “REBOUND-PHENOMENON” DURING PROLONGED, CONTINUOUS PGI2 INFUSION OCCURS AT THE RECEPTOR LEVEL
Authors: Steurer G , Sinzinger H , Fitscha P
Published: August 2018

During earlier attempts in optimizing the therapeutic regimen with PGI2 we were able to discover an “ intra- and post-infusion platelet rebound” being characterized by an activated platelet function and a diminished responsiveness of platelets to the action of PGI2 in-vitro.

In order to verify this phenomenon at the receptor level we infused continuously 6 patients suffering from peripheral vascular disease (PVD) with PGI2 at a rate of 5 ng/kg/min for 5 days. Anticoagulated venous blood has been drawn at different intervals. Saturation binding experiments on platelet membrane fraction have been performed using [3H]iloprost, a stable PGI2 analoque. Analysis of the binding data according to Scatchard demonstrated a decrease of receptor affinity with an increased number of binding sites.

It is concluded, that intrainfusion rebound occurs at the receptor level, whereas the postinfusion rebound does not. This is a further piece of evidence that an intermittent infusion regimen is preferable.
THE PLATELET ANTIAGGREGATORY EFFECT OF ILOPROST IS ENHANCED BY ASPIRIN:IN VITRO AND EX VIVO STUDIES IN HUMAN SUBJECTS.
Authors: Tremoli E , Maderna P , Colli S , Mannucci L , Sirtori C R , Di Minno G
Published: August 2018

To evaluate whether the activity of Iloprost, a chemically stable prostacyclin analog, on platelet aggregation could be potentiated by aspirin (ASA), in vitro and ex vivo studies in human volunteers were performed. In vitro studies were carried out in human platelet rich plasma (PRP) incubated with different concentrations of ASA (25-150 μM). For ex vivo studies Iloprost (0.5 ng.Kg−1.min−1 for 30 min) was given intravenously to healthy volunteers. After 20 hour wash out a single 50 mg ASA dose was given to the same subjects. Two hours after ASA intake, a second infusion of Iloprost was carried out. Blood was collected at appropriate time intervals thereafter. Platelet aggregation and thromboxane B2 (TXB2) formation were determined in collagen stimulated PRP. ASA, in vitro , dose dependently reduced the concentrations oF Iloprost required to achieve 50% inhibition of platelet aggregation (IC50) in PRP stimulated by 1 g/ml collagen. Also, the IC50S for Iloprost were significantly reduced (p<0.01) in PRP of subjects who ingested ASA two hours before blood collection. Iloprost infusion (0.5 ng.Kg−1.min−1 for 30 min) only minimally affected the concentrations of collagen eliciting 50% aggregation (AC50) and was ineffective on TXB2 synthesis. ASA, administered after a 20 hour wash out period did not significantly affect the AC s for collagen, whereas it inhibited TXB2 synthesis by more than 50%. The mean AC50 for collagen, evaluated at the end of Iloprost infusion in PRP of subjects who previously ingested ASA, was signicantly greater than that evaluated after the two single treatments. No significant changes in hemodynamic and ECG parameters were detected during the study. These findings, indicating an in vivo potentiating effect of ASA on the antiaggregatory activity of Iloprost, observed at doses of Iloprost with no effect on hemodynamic parameters, may be of relevance for the design of treatment schedules aimed to the selective inhibition of platelet aggregation.
REVERSIBLE REDUCTION OF PLATELET PROSTACYCLIN BINDING SITES AFTER ILOPROST INFUSION IN MAN.
Authors: Modesti P A , Fortini A , Boddi M , Poggesi L , Abbate R , Gensini G F
Published: August 2018

The reduction of platelet sensitivity to prostacyclin (PGI2), observed in man after the infusion of PGI2 analogues, could suggest the presence of a down regulation mechanism for PGI2 platelet receptors. Robertson (1980) have shown a dcwn-regulaticn of PGE2 binding after PGE2 infusion in rat and Leigfrt (1985) have provided evidence of an homologous desensitizaticn to PGI2 in cultured cells, but direct evidences of PGI2 receptors dcwn-regulaticn in man are lacking.

This work was performed to study the behaviour of PGI2 platelet receptors after a PGE2 analogue infusion in man.

Eight subjects with peripheral artery disease (stage IV according to Fontaine) treated for 14 consecutive days with six hours iv infusion of Iloprost (Sobering, FRG) at 2 ng/Kg/min were studied. Platelet studies were performed an the 1st, 2nd, 7th and 14th day of therapy, blood sanples being collected immediately before the beginning (between 8.00 and 9.00 a.m.), at the end and 6 and 18 hours (the following morning) after the end of the infusion. PGI2 platelet receptors were investigated by a direct radioligand binding assay. PGI2 inhibitory dose 50 (I.D.50) was evaluated in platelet aggregation induced by ADP 5 ¼M.

After six hours of Iloprost infusion a significant reduction of high affinity PGI2 platelet receptor (HAR) nunber was observed (p<0.005) without any change of affinity for the ligand. After 6 hours from the end of the infusion the reduction of the HAR was still statistically significant (p<0.05). The following morning the receptor nunber was restored (n.s.). After one and two weeks from the beginning of the treatment the basal values of PGI2 HAR were not significantly changed from the values recorded on the first day of therapy.

PGI2 I.D.50 after the infusion was significantly increased when compared to the basal values (p<0.01). Six hours later the basal sensitivity was restored (n.s.). Eighteen hours later, the following morning PGI2 I.D.50 were still not significantly changed in comparison to the basal values.

These data are suggestive for the presence of a reversible down regulation mechanism for the PGI2 receptors.
STRUCTURE-RELATED DIFFERENCES IN ACTION BETWEEN PROSTACYCLIN (PGI2.J, VARIOUS STABLE PGI2 ANALOGUES AND PROSTAGLANDIN E. (PGE1).
Authors: Maurin N
Published: August 2018

PGI2 and PGE. have various effects in common: they inhibit pi at let aggregation (PA), improve erythrocyte fluidity, and produce vasodilation. A problem with PGI2 is the combination of PA inhibition with vasodilation and the resultant, usually undesired, hypotension. The aims of this study were firstly to check the action of various prostanoids on thrombocyte function and erythrocyte fluidity and secondly to investigate whether the development of stable PGI2 analogues makes it possible to separate the structure related PGI effects. We tested PGI2 (Upjohn Co., USA), the stable oxacyclic PGI2 analogue CG 4203 and the stable carbacyclic PGI0 analogue CG 4305 (both Gruenenthal GmbH, FRG), the stable carbacyclic PGI2 analogue Iloprost (Schering AG, FRG) and PGE. (Sanol Schwarz GmbH, FRG). In vitro measurements were made in blood from 15 healthy volunteers. Thrombocyte activity was assessed by PA after stimulation with ADP (1 pmol/1) and by pseudopod formation. Erythrocyte fluidity was determined by the micropore filtration method. 50% inhibition of the ADP-induced PA occurred at the following concentrations: PGI 10 nmol/1, CG 4203 50 nmol/1, CG 4305 75 nmol/1, Iloprost 4 nmol/1, PGE 50 nmol/1; 50% inhibition of pseudopod formation: PGI2 1 nmol/1, CG 4203 75 nmol/1, CG 4305 75 nmol/1, 2 Iloprost 1 nmol/1, PGE. 10 nmol/1. The substantial variation in PA inhibition potency is conspicuous. Also, each single substance has effects of different strength on the two thrombocyte function parameters here tested. On contrast to the other substances here tested, which siqnificantly improve erythrocyte fluidity to an extent depending on concentration, CG 4203 and CG 4305 have no such effect. It should therefore be possible still better to separate desired from undesired PGI actions bv making further structural modifications.