bluuebunny/crossref_metadata_2025 huggingface dataset:
RELEASE AND TRANSACYLATION OF ARACHIDONATE FROM A COMMON POOL OF 1-ACYL-2-ARACHIDONOYL GLYCEROPHOSPHOCHOLINE IN HUMAN PLATELETS
Authors: Purdon A D , Smith J B
Published: August 2018

We have previously shown that the main source of arachidonate in thrombin-stimulated human platelets is 1-acyl-2-arachidonoyl (AA) glycerophosphocholine (GPC) and release of 3H-AA from this phospholipid also was correlated with increased 3H-AA in ether phospholipid. This ATP independent transfer of 3H-AA from 1,2 diacyl GPC to ether phospholipid (transacylation) also occurs in resting cells. Human platelets in 1/10 volume of plasma (ACD anticoagulant, pH 6.5) were radiolabelled with 3H-AA for 60 min at 37°C and then exogenous 3H-AA was removed by gel filtration into Tyrode's buffer, pH 7.4, 0.2% albumin. These radiolabelled cells were incubated in the absence of exogenous 3H-AA for four hours followed by Bligh and Dyer extraction and thin layer chromatography purification of phospholipids. 3H-AA in 1,2 diacyl GPC was found to decrease by over 20% and increase substantially in 1-0-alkyl-2-acyl GPC and 1-0-alk-1'-enyl-2-acyl glycerophospho ethanolamine (GPE), In this same time interval the mass of AA released by thrombin (5 U/ml, 10 min, 37°C, no stirring)in the presence of BIT 775C and measured by GLC, stayed the same (30 nmoles/109 cells), however, the specific activity decreased. Using reverse phase HPLC to resolve diradylglycerobenzoate derivatives of phospholipids: acylation, deacylation, and transacylation were observed for individual AA-containing molecular species of phospholipid, including those with an unsaturated fatty acid at sn-1. In particular the radiolabellinq of the 1-unsaturate-2-arachidonoyl GPC correlated with the specific activity of the 3H-AA released by stimulation with thrombin. Furthermore, 1-arachidonoyl-2-3H-arachidonoyl GPC was completely deacylated while 50 % of its mass remained. This contrasted with 16:0, and 18:0-2-arachidonoyl GPC in which the specific activity remained the same before and after deacylation. We conclude that deacylation of AA-containing molecular species of 1,2 diacyl GPC in stimulated cells includes molecular species which are also a source of arachidonic acid for transacylation reactions.
PROTECTION FROM ARACHIDONIC ACID-INDUCED SUDDEN DEATH BY INJECTION OF TRIDOCOSAHEXAENOYL-GLYCEROL EMULSION INTO RABBITS
Authors: URAKAZE M , HAMAZAKI T , SAWAZAKI S , YAMAZAKI K , FUJIKAWA M , YANO S
Published: August 2018

An injectable emulsion of docosahexaenoic acid (DHA) was prepared. One hundred ml of the emulsion contained 3g of 93%-pure 1,2,3-tridocosahexaenoy1-glycerol (DHA-TG), 1.2g of 93%-pure 2-docosahexaenoyl-phosphatidylcholine as an emulsifier and 2.5g of glycerol. Thirty ml of the emulsion of DHA-TG was injected into rabbits on days 1 and 4 of the study. Blood was taken on day 0, on day 4 just before the second injection, and on day 7. The percent of DHA in the total phospholipid fraction of platelets was increased from 0.46% (day 0) to 1.88% (day 4, p<0.05) and 3.66% (day 7; p<0.02 vs day 0); that of eicosapentaenoic acid (EPA) was increased from 0.46% (day 0) to 1.03% (day 4, p<0.02) and 1.63% (day 7; p< 0.05 vs day 0); that of arachidonic acid (AA) was decreased from 9.45% (day 0) to 4.31% (day 4, p<0.05) and 6.68% (day 7; p<0.02 vs day 0). The percent of DHA in the total phospholipid fraction of erythrocyte membranes was increased from 0.23% (day 0) to 0.91% (day 4, p<0.05) and 1.52% (day 7; p<0.005 vs day 0); that of EPA was increased from 0.21% (day 0) to 0.34% (day 4, p<0.005) and 0.52% (day 7, p<0.01 vs day 0); that of AA was unchanged at all. Blood lipids were the same before and after the two injections of the emulsion, except that free fatty acids decreased markedly from 0.32 to 0.06 mEq/1 (p<0.02). On day 8, free AA (2 mg/Kg) was injected into ear veins of the treated three rabbits and also into those of four control rabbits, which were not treated with DHA-TG. All the control rabbits died a few minutes after the AA injection but none of DHA-treated rabbits died after AA injection (p<0.01). An emulsion of DHA-TG may be useful for patients having immediate risk of thrombosis or for those who need DHA but cannot take it orally.
DIFFERENTIAL EFFECTS OF ORAL ADMINISTRATIONS OF ACETYL SALICYLIC ACID, INDOMETHACIN AND SODIUM SALICYLATE TO HUMAN SUBJECTS ON THE FORMATION OF 12-HYDROXYEICOSATETRAENOIC BY PLATELETS
Authors: Tremoli E , Caruso D , Maderna P , Galli G , Paoletti R
Published: August 2018

The effects of a single oral administration of acetylsalicylic acid (ASA, 500 mg), indomethacin (Indo, 50 mg) and sodium salicylate (SA, 400 mg) to healthy volunteers on platelet formation of 12-hydroxyeicosatetraenoic acid (12-HETE) were evaluated. Blood was collected before, 2, 6 and 24 hours after drug administrations. Platelet rich plasma (PRP) samples were stimulated with 20 μg/ml collagen and 12-HETE levels were determined by selective ion monitoring. The effects of ASA on the same parameter were also evaluated in vitro in PRP and in washed platelet (WP) samples in the absence and in the presence of platelet poor plasma (PPP, 25-100%) or bovine serum albumin (BSA, 10-40 mg/ml). In subjects who ingested ASA, the formation of 12-HETE in PRP stimulated with collagen was significantly inhibited 2 and 6 hours after the drug administration. At 24 h 12-HETE synthesis tended to return toward basal values. In contrast the administration of a single dose of Indo or of SA did not significantly affect 12-HETE synthesis by stimulated PRP. ASA (3 mM) added in vitro inhibited 12-HETE formation in PRP but not in WP. In addition ASA inhibited 12-HETE synthesis in washed platelets resuspended either in PPP (25-100 %) or in BSA (10-40 mg/ml). It is concluded ASA, but not Indo or SA, orally administered to normal subjects inhibits 12-HETE synthesis in collagen stimulated PRP. The results obtained in vitro suggest that albumin and/or some albumin component may be responsible for the inhibitory effect of ASA on platelet 12-HETE syntesis.
NORMAL LIPOXYGENASE ACTIVITY IN ERYTHROMELALGIC THROMBOTIC THROMBOCYTHEMI
Authors: Michiels J J , Haddeman E
Published: August 2018

It has been suggested that a selective lipoxygenase deficiency may be a mechanism for hyperfunction of platelets in myeloproliferative disorders. 24 out of 60 patients with one of the myeloproliferative disorders polycythemia vera, myeloid metaplasia and myelofibrosis, essential thrombo-cythemia or chronic myeloid leukemia have been reported to be deficient for platelet lipoxygenase activity (NEJM 1982; 306:381). These patients paradoxically tended to have a greater hemorrhagic tendency rather than thrombotic episodes. We investigated platelet lipoxygenase activity in clearly defined patients with erythromelalgic, thrombotic thrombocythemia (til) (Ann IntMed 1985; 102:466) while on treatment with low dose aspirin in order to prevent platelet activation in vivo or in vitro. The production of hydroxyheptodecatrienoic acid (HHT) by cyclo-oxygenase and 12-hydroxy-eicosatetraenoic acid (12-HETE) by lipoxygenase in 14C labeled platelets after stimulation with thrombin or arachidonic acid were estimated in 12 ETT patients and in 3 patients with reactive thrombocytosis (RT). HHT and 12-HETE were measured with the technique of High Performance Liquid Chromatography. Platelet lipoxygenase activity was completely normal in the 3 patients with RT and in 9 of 12 patients with ETT. Three ETT patients were severely deficient for platelet lipoxygenase activity. Two of them had progressive myeloproliferative disease with very high platelet counts, many large and some giant platelets, a few megakaryoblasts and chromosomal abnormalities of bone marrow cells (trisomy of chromosome 9 and 18p+, 1P-, 11a- respectively). Normal bone marrow karyograms were found in the 9 ETT patients with normal platelet lipoxygenase activity and stable disease. In conclusion platelet lipoxygenase deficiency is not a feature of ETT, but may reflect abnormal megakaryopoesis in progressive myeolo-proliferative disorders.
COVALENT BINDING OF CYCLOOXYGENASE AND LIPOXYGENASE PRODUCTS TO HUMAN PLATELET PROTEINS
Authors: Lecomte M , Boeynaems J M
Published: August 2018

Several studies in acellular systems have shown a covalent binding of eicosanoids to proteins (1). We have therefore investigated whether eicosanoids bind covalently to proteins in intact platelets. After incubation of washed human platelets with 14C-arachidonic acid, ethanol precipitation followed by extractions, a small fraction of the radioactivity (0.3%) was tightly bound to the protein pellet. Four criteria suggest the covalent nature of this binding. The radioactivity remained bound after exhaustive extractions with solvents of various polarities, and was not removed by dialysis against SDS-buffer. 15 labelled protein bands could be separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Finally, exhaustive enzymatic hydrolysis of platelet proteins by several proteases liberated an amphipathic radioactive material which had a chromatographic behaviour similar to that of a known peptidolipid, leuko-triene c4 This covalent binding involved products of both cyclooxygenase and lipoxygenase pathways : it was partially inhibited by indomethacin (±40%) and completely abolished by eicosatetraynoic acid. The covalent binding was increased five-fold by dazoxiben, suggesting attachment of prostaglandin endoperoxydes , and by diamide (a glutathione depleting agent) (2) suggesting the involvement of 12-hydro-peroxyeicosatetraenoic acid. Dazoxiben and diamide intensified selectively the labelling of distinct protein bands, separated by SDS-PAGE. Further studies will be needed to evaluate the possible physiological significance of this covalent modification.

(1) Wilson, A.G.E. et al. Prostaglandins 18: 409-422, 1979.

(2) Bryant, R.W. et al., J. Biol. Chem. 257: 14937-14943, 1982
FISH AND PLATELETS
Authors: VREEKEN J , HARDEMAN M R
Published: August 2018

The effect of two different amounts of a fish oil, corresponding to 0.75 g (2.5 mmol) and 1.5 g (5 mmol) of eicosapentaenoic acid respectively, added in a cross-over design to the normal diet of 16 healthy male volunteers, was studied. Of the various parameters investigated, the most important appeared to be a new test: “Transient Aggregation Resistance” (TAR) of platelets, a phenomenon which, due to its short half life, is hardly measured when platelet aggregation is studied according to the classic method (see M.R.Hardeman, TAR determination, this congress). Under the influence of fish oil, the half life of TAR was found significantly prolonged. This prolongation, however, was not related to the amount of fish oil used. A highly significant decrease of triglycerides was found, the effect being most pronounced in subjects with triglycerides starting values >1.0 uM. This decrease was related to the amount of fish oil used. These.results may cast light on controversies found in literature concerning the effect of fish oil on platelet aggregation . They can also help to clarify controversies about the effect of fish consumption on cardiac mortality.
THE EFFECT OF COD LIVER OIL INTAKE ON THE STIMULATION OF BLOOD CELLS
Authors: Hansen J B , Olsen J O , Wilagård L , Østerud B
Published: August 2018

In an in vitro model, stimulation of blood cells with a low concentration of lipopolysaccharides (LPS) revealed differences between women and men that possibly could be an explanation to why young women have less coronary heart disease than men (see abstract Hansen et al. “A model to--”).

This model was also used to study the effect of intake of cod liver oil (CLO). 40 students (20 men and 20 women) were tested followed by an intake of 25 ml CLO daily for 2 months by 20 of the students.

Heparinized blood samples were incubated with 2 ng LPS/ ml for 2 hours followed by isolation of plasma for thromboxane B2 and 6-keto-PG 1α quantitation.

After the first 2 months period of CLO drinking we have the following results:

The two months of CLO intake had no significant effect pn the thromboplastin induced synthesis in monocytes. In addition platelet aggregation was tested in a whole blood aggregometer using ADP addition to heparinized blood or collagen induced platelet aggregation in citrated whole blood. ADP aggregation was reduced from 75.9 ± 16.8% to 55.4 ± 19% in the CLO group of women, whereas the reduction in the CLO group of men was 70.1 ± 17.1% to 60.9±18.6%. Similar result were found with collagen aggregation (57% to 33% for women and 48% to 30% for men).

It is concluded that CLO intake reduces TxA2 production and plateletaggregation without having reduced effect on PGI2 production in whole blood.
EFFECT OF POLLEN TYPHAE ON 6-KETO-PGF , TXB2, TOTAL CHOLESTEROL AND HDL-C IN RABBITS WITH CHRONIC HYPERLIPIDEMIA
Authors: Wang Z Y , Zhang L , Su Y L
Published: August 2018

62 NZW rabbits were divided into 4 groups: (1) Control (C), (2) rabbits fed with high lipids diet (H), (3) H+pollen Typhae (H+P), (4) H+VitE (H+E). The following parameters were determined: (1) total cholesterol of aortic wall (WTC), (2) Serum TC (STC), High density lipoprotein cholesterol (HDL-C), (3) Plasma 6-keto-PGF1 (6-keto) and thromboxane B2 (TXB2), (4) 6-keto of aortic wall, (5) ratio of STC/HDL-C and plasma TXB2/6-keto

The results showed that: (1) the level of WTC, STC and STC/HDL-C was significantly higher in the group H than in the group C, while that of 6-keto was lower (P < 0.01), (2) STC, WTC and plasma TXB2, TXB2 /6-keto all were significantly lower in the group H+P than m the group H, while wall's 6-keto was higher (P < 0.01).

There was inverse correlation between plasma 6-keto and STC/HDL-C (P < 0.01), while positive correlation has been found between STC/HDL-C and plasma TXB2/6-keto (P < 0.05).

In the group H+E, the plasma TXB2/6-keto, was lower than in the group H, the wall's 6-keto was higher, but there was no difference between the groups H+E and H+P. The STC and WTC were higher in the group H+E than in the group H+P, although lower than in the group H.

It has been demonstrated in our Department that the Chinese traditional medicine Pollen Typhae had preventive effect on the experimental atherosclerosis in rabbit fed with high lipids diet, the mechanism would not only due to its effect lowering the STC, WTC and STC/HDL-C, but also to its action on the metabolism of prostaglandins. Its action on lowering STC and WTC appeared to be better than VitE, however, the latter had similar effect on the metabolism of prostaglandins as compared with Pollen Typhae.
LIPID ACCUMULATION AND METABOLISM IN CARRAGEENAN-INDUCED GRANULOMAS COMPARED TO BLOOD MONOCYTES AND THE AORTA
Authors: Schaub R G , Bell F P
Published: August 2018

Arteries undergoing atherogenic change show an increase in cholesteryl esterifying activity by acylCoA:cholesterol acetyl-transferase (ACAT) and a progressive accumulation of cholesterol esters within monocyte derived foam cells. The study of these factors, however, is limited by the necessity of obtaining artery tissues for analysis. In this study, an in vivo model (Am J Path 118:134 and 120:391, 1985) which permits the analysis of foam cell development without requiring collection of aortas was examined in more detail. New Zealand rabbits (6 each) were either maintained on a 1% cholesterol/peanut oil diet (HD) or a regular chow diet (RD) for 2 weeks after which each had 15 ml of a 1% carra-geenan gel (Marine Colloids) injected subcutaneously into the mid-abdominal area. The rabbits were maintained on their respective diets for an additional 4 weeks. At sacrifice, blood was collected for both serum and monocyte isolation. Granulomas and aortic arches were also excised. Tissues were assayed for lipid accumulation and metabolism. Electron and light microscopy was also performed on immersion fixed (1% glutaraldehyde) granuloma tissue. Granulomas of HD rabbits were pale yellow and averaged 36 grams, while RD granulomas were a pale red and averaged 11 grams (p less than 0.05). RD granulomas did not stain with oil red 0. HD granulomas had homogenous oil red 0 staining which indicated lipid accumulation. Both RD and HD granulomas had large numbers of macrophages. RD macrophages accumulated follicular carrageenan, but not lipid. In HD granulomas, foam cell development was observed. Granuloma lipid content and metabolism paralleled the aorta and blood monocytes. The HD tissue had increased ACAT activity and lipid composition changes indicative of atherosclerosis. RD granulomas had no elevation of lipid content or ACAT activity. The results suggest that the carrageenan-induced granulomas provides a useful model for studying the biochemical and morphologic changes characteristic of aortic monocyte-derived foam cells and the early arterial atherosclerotic process.
SHORT TERM HIGH CHOLESTEROL DIET CAUSES CHANGES IN MEGAKARYOCYTE SIZE AND IN VASCULAR ULTRASTRUCTURE
Authors: Dalby Kristensen S , Roberts K M , Lawry J , Martin J F
Published: August 2018

Platelets produced by megakaryocytes (MK) have a role in atherogenesis. Six pairs of male litter mate rabbits were randomised to feeding with either 2g of cholesterol daily in addition to their normal diet or normal diet alone. After seven days the animals were killed and serum cholesterol, platelet count, MK total, cytoplasmic and nuclear area (microscopic planimetry) and MK DNA content cell distribution (fluorescent activated cell sorting) were measured and compared between the two groups. The results are given in the table as medians with range values in brackets.

After perfusion-fixation the aortas were examined by transmission electron microscopy. In the aortas from the animals on high cholesterol diet cells with ultrastructural features resembling smooth muscle cells were found in the intima. Changes in megakaryocyte size are associated with the occurrence of smooth muscle cell proliferation and migration. The platelet-megakaryocyte axis may be activated in early atherogenesis.
INCREASE IN MEGAKARYOCYTE SIZE AND PLOIUY PRECEDES ACCELERATION OF ATHEROGENESIS IN THE HYPERCHOLESTEROLAEMIC RABBIT
Authors: Dalby Kristensen S , Roberts K M , Martin J F
Published: August 2018

Platelet derived growth factor(s) probably synthesized by the megakaryocyte are important in atherogenesis. In a pilot study destruction of the circulating platelets was induced by injection of goat serum containing a specific platelet antibody (APS) to rabbits fed a high cholesterol diet (2g per day) for 12 weeks. Seven days after APS the percentage of atheroma in the aorta measured by planimetry was increased in these animals compared to control animals on the same diet that had been injected with goat serum (GS) 7 days before. In a new study 15 pairs of male litter mate rabbits on high cholesterol diet were randomised in pairs to treatment with either APS or GS. Five pairs of animals were killed 18 hours after the injection, 5 pairs 4 days after and 5 pairs 7 days after the injection and the platelet count, mean platelet volume, megakaryocyte nuclear, cytoplasmic and total size, megakaryocyte DNA distribution and the percentage of atheroma in the aorta were measured. Comparison of these variables between the 2 groups revealed the following statistically significant findings (p<0.05) : 18 hours after the injection the platelet count was decreased and the mean platelet volume increased in the APS group. At day 4 the platelet count, megakaryocyte nuclear, cytoplasmic and total size and the megakaryocyte DNA content were increased in the APS group. At day 7 the platelet count and the percentage of the atheroma were higher in the APS group. Since platelets produced by big megakaryocytes with high DNA content are more reactive than normal platelets, we suggest that the acceleration of atheroma formation demonstrated 7 days after APS is caused by the large number of platelets with possible high concentrations of growth factor(s) derived from the large megakaryocytes with increased DNA content.
EXTRAVASCULAR INJURY CAUSES FOAM CELL FORMATION, ACCUMULATION OF CHOLESTEROL (C) and CHOLESTEROL ESTER (CE) IN THE CAROTID ARTERY
Authors: Hassall D C , Booth R F G , Honey A C , Martin J F
Published: August 2018

In atherosclerotic arterial tissue, cholesterol is delivered to smooth muscle cells by low-density lipoprotein (LDL) and to macrophages via modified LDL. The reasons for accumulation of excess lipid are unknown although increased uptake of C and CE occurs in regions of arterial de-endothelialisation. We now report that the positioning of a silastic collar containing saline around the outside of arteries induces accumulation of C and CE within those tissues. 9 rabbits were separated into 3 groups, each group was fed a normal laboratory chow, 2 groups were supplemented with lg/day cholesterol; these rabbits were used for C and CE determinations. Three parallel groups of rabbits were set up for histological analysis. At day O, under anaesthesia, a silastic collar was* placed around the left carotid artery. The collar was filled with saline and carefully sealed without causing constriction of the vessel. The vessels were replaced and the animals allowed to recover. After 14 days the carotids from animals for C and CE determination were rapidly removed and divided up into a region from the middle of the collar, a region proximal to the collar and a distal region. The tissues were freeze clamped in liquid nitrogen and lipids extracted with C and CE determined for each region. The carotids for histological analysis were perfuse-fixed in situ and similarly subdivided.

We conclude 1) Animals fed high cholesterol accumulate more C than CE within arterial tissue, furthermore this accumulation is greater than in animals fed a normal diet 2) In both normal diet and high cholesterol fed animals an enhanced accumulation of C + CE occurs within arteries with a collar 3) CE appears to be preferentially accumulated within the collar region.
EFFECTS OF ORAL ASPIRIN ON PLATELET AND VASCULAR CYCLOOXYGENASE ACTIVITY IN RATS WITH PORTACAVAL SHUNT
Authors: Cerletti C , Gombino M C , Possaghe S , Bucchi F , Chen Z M , de Gaetano G
Published: August 2018

Oral aspirin can be extensively hydrolysed to salicylate in stomach and liver before entering the systemic circulation. “Pre-systemic” acetylation of the platelets may thus occur during aspirin absorption. This may result in concomitant sparing of peripheral vascular cyclooxygenase mainly exposed to salicylate. We tested whether the “biochemical selectivity” of oral aspirin as an inhibitor of platelet vs. vascular cyclooxygenase would be reduced by elimination of the “first-pass” hepatic metabolism. A portacaval shunt was inserted in anaesthetized rats by connecting the portal vein to the inferior vena cava through a “Y” heparinized polyethylene PE 60-160 cannula. Sham operated rats acted as controls. 90 min after recovery from anaesthesia rats were given aspirin orally (10 mg/kg) and 45 min later serum TxB2 and 6-keto-PGF la formation by vascular rings were evaluated by radioimmunoassay. Serum TxB2 was completely suppressed in all animals; in contrast, vascular 6-keto-PGF la was significantly reduced (by 40-60* in aorta and vena cava) in rats with portacaval shunt but not in controls. The results in rats with portacaval shunt were similar to those previously obtained after i.v. aspirin. 15 min after aspirin administration, plasma levels of unmetabolized drug measured by HPLC were significantly higher in rats with portacaval shunt (0.56±0.16 μg/ml; n= 5) than in sham operated controls (0.16±0.22 }μg/ml; n= 5). This study directly supports the role of “ first-pass” hepatic metabolism in determining the “biochemical selectivity” of oral aspirin.
TICLOPIDINE AND PREVENTION OF ARTERIAL REOCCLUSION AFTER DIRECT PERIPHERAL VASCULAR SURGERY
Authors: Castelli P , Basellini A , Agus G B , Martelli E , Zucca R
Published: August 2018

Late thrombosis after arterial revascularization surgery is a relatively frequent complication especially when the more peripheral districts are involved. Among the causes of these failures, in addition to progression of the atherosclerotic disease, the behavior of platelet aggregation is of particular importance. With the aim of preventing this pathology we performed two controlled clinical 6-month studies on patients undergoing thrombo-endoarterectomy (TEA) of the femoropopliteal district and on patients undergoing aortobifemoral bypass.

In the first study 47 patients were randomly allocated to receive ticlopidine, 500 mg/day, (23 patients) or placebo (24 patients), and in the second study 125 patients were evaluated (25 treated with ticlopidine, 500 mg/day, and 100 controls).

Physical examination, doppler sonography, angiography, and blood tests were performed on all patients at baseline, during and at the end of treatment.

In the patients who underwent TEA of the femoropopliteal district doppler sonography revealed 3 reocclusions in the ticlopidine-treated patients versus 6 reocclusions and 6 significant stenoses in the placebo group (p = 0.007).

Furthermore, clinical symptomatology persisted or reappeared in 5 patients in the ticlopidine group versus 15 in the placebo group (p = 0.003).

The active treatment significantly reduced the incidence of acute ischemia. In the. patients who underwent a bypass 5 reocclusions were observed in the control group, whereas the bypass remained patent in all the patients who received the active treatment. Ticlopidine was well tolerated in all the patients and no alterations of blood composition or other side effects were observed.
ANTIPLATELET ACTIVITY OF DYPIRIDAMOLE IN HUMAN WHOLE BLOOD WITH PHYSIOLOGICAL LEVELS OF IONIZED CALCIUM
Authors: Pérez-Requejo J L , Aznar J , Santos T , Vallés J
Published: August 2018

In spite of the windespread use of dypiridanole (DP) as an anti thrombotic drug, its usefullnes has not been generally proved. The clinical trials have been somewhat inconclusive or challenge and most of the studies were not able to snow a clear inhibitory action of DP on platelet aggregation. Using the impedance aggre gometer it has been shewn that the antiplatelet action of DP can be demonstrated in whole blood (WB) ex vivo an it was suggested that this was due to the inhibition of adenosin reuptake by the red cells. We have recently described (Thrombos & Haerostas 54: 799, 1985) a method, that detects the early platelet-collagen interaction in whole blood, which has been called BASIC wave. Using slight modifications of the BASIC wave method, we were able to study the platelet-collagen interactions in native WB with physio logical levels of calcium and its inhibition by DP ex vivo. We performed the BASIC wave in 25 human healthy volunteers in WB without sodium citrate, WB with citrate and citrated platelet rich plasma (PRP) before and 2 hours after the oral administration of DP 3mg/kg. We observed that in WB with citrate, DP produced a 66% inhibition of the BASIC wave (p<0.005) in PRP the inhibition was 40.7% (p< 0.005) but it was a non-significant 21.7% in WB without citrate. Using hirudin 40U/ml as anticoagulant, the inhi bition was the same as in WB without citrate. There were no correlation between the oral dose and the DP plasma levels or with the observed inhibition of the BASIC wave. We postulate that the antiplatelet action of DP is only demonstrable in citrated samples, beer negligible in native blood with physiological levels of calcium. The present report could explain the lack of firm da ta supporting the antithrombotic action of DP in clinical trials. Furthermore, the method here presented allows the study of anti -thrombotic drugs ex vivo in whole blood in its normal calcium environement.
THE EFFECT OF PIRACETAM IN ISCHEMIC FLAP VASCULARISATION
Authors: Rossillon O , Bayet B , Huymans M , Englebert P , Stren Ides , Calteux N , Vanwyck R
Published: August 2018

Piracetam. a drug traditionally used for stimulating the telencephalon has a strong anti-aggregant, anti spasmodic and rheological action, due to its antisludge effect and the increase of the red cell deformability capacity. The authors show the interest of this substance in reconstructive surgery : Piracetam significantly increases the circulation in the flaps, reduces the zones prone to necrosis and allows for a faster skin expansion. The first three experiments have been performed on rats - on an epigastric flap with thoracic pedicle-. In the first one. we operated on 150 rats divided into 5 groups: a control series, and 4 series which received the following doses of Piracetam per os : 20.40.80 and 100 mg/100 g/day. The necrotic area, as measured daily, represents 13.8%+−2.6 (+− SEM) in the control series. 6% +− 0.8 in the series with 20 mg/100 g. 2.53% +− 0.66 in that with 40 mg/100 g. 1.67% +− 0.4 in thatwith 80 mg/100 g and 2.77% +− 1.96 in that with 100 mg/100 g. (all these percentages are expressed versus the overall surface). A thorough statistical study including the AN0VA table.“t” and regression tests shows that these differences are significant at 0.0001 for the 40 mg/100 g dosis and 0.0000 for the 80 mg/100 g dosis. A second experiment measured the local blood flow in 4 areas of the flap from its thoracic basis towards its epigastric point thanks to the radio-active microsphere technique described 1n rats by He Oevitt and Mallck. Measures were carried out on 60 rats divided into 3 groups : control series without flap, series with a flap at the second post-operative day without Piracetam. series with a flap at thesecond post-operative day with Piracetam.With Piracetam. the increase of the bloodflow in the thoracic part proximal to th flap (zone 1) is of 60% +− 13 (p < 0.0000). that of zone 2 of 72% +< 20 (p < 0.0008). that of zone 3 of 92% +< 23 (p < 0.0003), and finally that of the epigastric zone 4 of 117% +− 40 (p <0.004) (vs the overall surface). A thirdexperiment measured the temperature difference of these 4 zones by means of a, thermocouple according to the method described by Jones in 1983 and it compared the results obtained with the flow measured by microspheres. Finally, in a last experiment, the blood flow in expanded tissueswith- and without- piracetam 40 mg/100 gwas measured with a laser-doppler.

Therefore it was concluded that piracetam is a useful drug to prevent necrosis in flaps. It can also be used to expand tissues more quickly.
TREATMENT OF RAYNAUD SYNDROMES WITH PIRACETAM(NOOTROPIL R),NOOTROPIC AND ANIPLATELET AGENT
Authors: Moriau M , Lavenne-Pardonge E , Col-De Beys C H
Published: August 2018

Three prospective studies have been performed with piracetam (Nootropil ®-) in the treatment of Raynaud disease and syndromes.

The first one, realized in 20 cases of Raynaud disease established that 8 g piracetam daily was the optimal dosage necessary to obtain a significant clinical, ultrasonic and biologic improvement.

In the second one, 58 cases of Raynaud syndromes (47 idiopathic and 11 associated with systemic disease) were treated with 8 g daily piracetam during 6 to 12 months. A clinical and ultrasonic improvement was observed in 75 and 80 % of the cases and a normalisation of the disturbed platelet functions (36/58) in 88 %. Moreover a benefic rheologic effect was noted and related to the membrane deformability, the antiplatelet and the Von Willebranc factor synthese or release inhibiting activity of piracetam

The third one realized in cross over in 30 cases of Raynaud syndromes compare the effects of piracetam alone (dosage 8 g daily) with the combination piracetam (4 g daily) - aspirin (100 mg daily) or with other drugs like buflomedil, calcium antagonists and ketanserine.

A synergic effect was obtained with the combination piracetam -aspirine and piracetam alone seems to be more efficient than the other drugs.

A fourth double blind study is actually performed
INHIBITION OF HUMAN PLATELET FUNCTION BY PCA-4230
Authors: Ortega M P , Sunkel C , Priego J G
Published: August 2018

PCA-4230 (3-{2-(N-l,2-benzisothiazolyl-3(2H)one-1,1-dioxide) ethoxycarbonyl}-2,6-dimethyl-5-ethoxycarbonyl-4-methyl-l,4-dihy-dropyridine) is a new synthetic compound which has been selected after evaluation of .several series of molecules included in an ex tensive program of synthesis and biological screening.The purpose of this study was to investigate the In vUjiO effects of PCA-4230 on human platelet function.

Platelet aggregation (PA) was measured, in platelet rich plasma (PRP) or washed platelets, according to Born’s technique. Release reaction (RR) was measured by the luminiscence method as adenosine triphosphate (ATP) release in response to stimulation. Aggregating agents were adenosine diphosphate (ADP), epinephrine (Epi), collagen (Col), thrombin (Thr), calcium ionophore (A23187) arachidonic acid (AA), thromboxane agonist (U46619) or platelet activating factor (PAF). Incubation with PCA-4230 (1 to 10 μM) we re carried out at 37°C for 15 minutes. PCA-4230 potentiation of Prostacyclin (PGI2) anti-aggregatory activity was also studied by addition to PRP of PGI2, PCA-4230 or both, and PA by ADP.PCA-4230 inhibited PA and RR in PRP in a concentration-dependent fashion when Col, Epi, U46619 or PAF were used as agonists. AA-and Thr-induced aggregation were only slightly impaired and no in hibition was shown on ADP or A23187-triggered activation. A23187-induced aggregation and RR were inhibited only in the absence of extracellular Ca++ in washed platelets. This effect was overcome by addition of Ca++ 1 mM. Additionally, the inhibitory effect of PGI2 on ADP-induced PA, was synergistically potentiated by PCA-4230, suggesting inhibitory activity of the compound on cjy clic nucleotide phosphodiesterase.

Since PCA-4230 inhibited PA and RR induced by Epi, U46619 and PAF, mediated viareceptor-agonist binding, and Col-inducedactivation, it is reasonable to suspect that common process(es) may be involved. Recently, it has been suggested that intraplatelet Ca++ levels, actingas a second messenger, are directly linked to the d<2 gree of platelet activation.Therefore, the ability of PCA-4230 to modulate platelet function appears, at leastin part, to be due to regulation of cytosolic Ca++ levels. This hypothesis is confii: med by the results with A23187-induced aggregation in absence of extracellular Ca++.
CORRELATION BETWEEN INHIBITION OF PLATELET PHOSPHODIESTERASES (PDEs) AND PLATELET FUNCTION TESTS.
Authors: Weisenberger H , Haarmann W
Published: August 2018

A systematic investigation of over 100 PDE inhibiting pyrimidopyrimidines was performed regarding a possible correlation between influence on platelet function tests and inhibition of platelet PDEs. Basically, the compounds tested were congeners of Dipyridamole but lack substitution in position 6. The concentration necessary for a 50% inhibition of PDE activity in platelet homogenates ranged between 0.000045 and 35 μmoles/L.

The inhibition of platelet PDEs was measured in freeze-thaw homogenates of human platelets using 3H-cAMP as substrate. Intraplatelet cAMP changes were measured by prelabelling the ATP pool with 3H-adenine and isolation of 3H-cAMP. Inhibition of platelet retention was determined using Morris' method (rotating citrated whole blood with glass beads) and the aggregation tests were performed according to Born and Cross in citrated platelet rich plasma.

The rather large number of compounds allowed the application of correlation analysis. The results of linear regression tests (IC50 and EC200 values) are shown in the table.

It appears that increased intracellular cAMP caused by PDE inhibition shows a strong correlation to the inhibition of the above mentioned platelet function tests. A weaker relation exists between inhibition of isolated PDEs and platelet function. Thus, determination of intrinsic PDE inhibitory potency is not sufficient to predict the influence of a given compound on platelet function tests.
ADDITIVE EFFECT OF DILTIAZEM (DIL) ON THE INHIBITION OF PLATELET AGGREGATION PRODUCED BY ASPIRIN (ASA).
Authors: Altman R , Scazziota A , Windor S , Dujovne C A
Published: August 2018

Platelet activation in vivo occurs by the action of several stimuli. It is generally agreed that actives products of arachidonic acid derived via the cyclooxy-genase pathway can stimulate platelet aggregation. ASA decrease thromboxane A2 generation and thereby inhibit platelet aggregation produced by AA and, partially, by others agonists. Nevertheless, the antiaggregating effect of ASA can be overcome by the conjointly activity of arachidonic acid(AA) and platelet activating factor (PAF). The inhibition of this cooperative aggregating effect can be important in platelet function suppressive therapy. The effect of DIL was tested in this sys tern. DIL was added in vitro to platelet rich plasma oS talned from volunteers before and after ASA(100mg/dayT intake for 7 days. DIL (2ug/ml) inhibited 50% platelet aggregation induced by AA (0.75mM) in non aspiri-nated volunteers. At even lower concentrations of DIL (0.4-lug/ml) an inhibition of aggregation induced by 300nM of PAF was also observed. After ASA, no aggregation by AA, only a first wave followed by disaggregation when PAF (30nM) was used and a full response when this pair of agonists were added together was obtained. DIL (0.lug/ml) added in vitro, produced significant inhibition of the synergism.

The effect in vivo of DIL plus low dose of ASA was also explored. In vivo administration of therapeutic dose of DIL (60mg T.I.D.) and low dose of ASA (75-100 mg/day), prevented the synergistic activity of AA plus PAF on platelet aggregation. In conclusion, DIL may enhance the effectiveness of low dose of ASA in the prevention of arterial thromboembolism.
EFFECTS OF DILAZEP ON CYTOPLASMIC CALCIUM ION CONCENTRATION AND ARACHIDONIC ACID METABOLISM IN HUMAN PLATELETS AND NEUTROPHILS.
Authors: Okuma M , Kanaji K , Sensaki S , Uchino H
Published: August 2018

We studied effects of dilazep(DZ), an antiplatelet drug, on the cytoplasmic Ca2+ concentration ([Ca2+]-i) and arachidonic acid (AA) metabolism in human platelets and neutrophils. [Ca2+]i of aequor-in-loaded platelets was estimated by using the platelet ionized calcium aggregometer. AA metabolism was studied by the determination of AA metabolites including hydroxyheptadecatrienoic acid (HHT), hydroxyeicosatetraenoic acid (HETE) and leukotriene B4 (LTB4) by reversed-phase high-performance liquid chromatography. When aequorin-loaded platelets were preincubated with DZ (0−0.5 mM) for 1 min at 37°C, both platelet aggregation and [Ca2+]i elevation induced by thrombin, AA and collagen were inhibited by DZ in a concentration dependent manner, while only aggregation was inhibited after stimulation by the calcium ionophore A23187 (1 yM). Both influx and release of Ca2+ into platelet cytoplasm induced by thrombin or AA were inhibited by DZ, while neither of them was affected when induced by A23187. The production of HHT and 12-HETE by the reaction of 100 yM AA with platelets preincubated with DZ (0−1 mM) was not inhibited, whereas their production by throirfbin (10 u/ml) was remarkably inhibited by DZ in a concentration dependent manner. When DZ (0−0.3 mM)-treated neutrophils were incubated with 5 μM A23187 in the presence or absence of 20 μM AA, the production of LTB4 and 5-HETE was increased by DZ in the presence of AA, whereas their production was inhibited by DZ in its absence. When platelet/neutrophil mixtures preincubated with DZ (0−0.3 mM) and cytochalasin B were stimulated by thrombin (5 u/ml) in the presence of FMLP, DZ inhibited LTB4 production in a concentration dependent manner, while 5S,12S-diHETE synthesis was enhanced by lower concentrations of DZ and inhibited by its higher concentrations (> 0.1 mM).

Thus, DZ inhibits platelet aggregation induced by any agonist including A23183, while [Ca2+]i elevation is inhibited by the drug only when it is induced by the receptor-mediated agonist. Furthermore, it was suggested that AA liberation from phospholipids in platelets and neutrophils was inhibited by DZ, leading to reduced production of all endogenous AA metabolites by these cells after appropriate stimulation, although lipoxygenase metabolites of liberated or exogenous AA could be increased.
INFLUENCE OF L0MUSTIN ON SOME PLATELET FUNCTIONS IN VITRO
Authors: Kubisz P , Brahimi S , Cronberg S
Published: August 2018

Lomustin (CCNU) is a cytostatic drug that is used in the trea-tement of some forms of solid tumors and hemoblastosis. The most important form of toxicity associated with its use consists of thrombocytopenia often associated with hemorrhagic syndrome. Lomustin may exert its action on the formation of platelets, but it may also interfere with platelets themselves.

In the present study its influence on the platelet function was investigated using a wide battery of tests.

Platelet-rich plasma was incubated with lomustin (1,5 to 15 jjg/ml and 30 minutes at 37°C) in vitro.

This inhibited strongly aggregation by ADP (2 × 10-6 M), adrenaline (2×10−6 M) and collagen (15 ¼g/ml). Platelet factor 4 release was almost completly inhibited independently of the inducer used. 14C-serotonin release was decreased by approximately 70% when incubation proceeded in the presence of 15 ¼g/ml of lomustin, and completly at higher concentration. Platelet factor 3 availability was also significantly impaired. Reptilase clot retraction was diminished, regardless of inducer used. In the presence of N-ethyl maleimide (1 mmol) significantLy lower malondialdehyde was produced in platelets incubated with lomustin, than in that from normal controls. After addition of arachidonic acid (1 mmol), the platelet synthesized Less thromboxane B2. Lomustin did not interfere with coagulation factors and did not induce overt fibrinolysis.

Its is concluded that lomustin acts as an inhibitor of platelet functions and can induce an acquired thrombocytopathy.

Thus impairement of platelet functions might play a part in hemorrhagic complications accompanying in some cases lomustin therapy.
SURPRISING EFFECTS OF THE CONSECUTIVE ADMINISTRATION OF PENTOXIFYLLINE AND LOW DOSE ACETYLSALICYLIC ACID ON THROMBUS FORMATION.
Authors: SEIFFGE D , Weithmann U
Published: August 2018

The effect of the combined oral administration of pentoxifylline (pof) and low dose acetylsalicylic acid (ASA) was evaluated with the help of the laser-induced thrombosis model in rat mesenteric arterioles. Laser-induced thrombosis is inhibited in a dose-dependent way by both drugs. The administration of ASA, either simultaneously with or 1 hour prior to pof, does not show any effects in the laser model. On the contrary, the administration of pof followed 1 h later by ASA not onyl exhibited a significant effect but also produced a supraadditive inhibition of the laser-induced thrombus formation. Specific investigations concerning the time interval between the administration of both drugs determined that a significant effect can be achieved only after an interval of 30 to 90 minutes (principle of HWA 5112).

The striking results could also be shown in diseased animals after consecutive chronic administration of pof 1 h prior to ASA. HWA 5112 exhibits significant effects on laser-induced thrombus formation in the following chronic animal models: 1. adjuvant arthritic rats, 10 → 1 mg/kg for 21 days; 2. spontaneously hypertensive stroke-prone rats, 10→ 1 mg/kg three times within 24 h; 3. cholesterol-induced atherosclerosis in rabbits, 10→ 1 mg/kg for 14 days. The reported data clearly demonstrate that the sequential drug administration of first pentoxifylline followed 30 to 90 min later by ASA exhibits a supraadditive antithrombotic effect.
IN VIVO SELECTIVITY BETWEEN HYPOTENSIVE AND PLATELET ANTIAGGREGATING ACTIONS OF ILOPROST AND PGI2 IN BEAGLE DOGS
Authors: Hermán F , Hadházy P , Magyar K
Published: August 2018

Iloprost (Schering A.G.) is a chemically stable derivative of prostacyclin. We compared the hypotensive and antiaggregatory effects of PGI2 and Iloprost. The concentration producing 50% inhibition (IC50) of ADP-induced platelet aggregation in vitro was 0.35±0.15 nmol/1 for PGI2 and 0.56±0.2 nmol/1 for Iloprost (n=5). The in vivo antiaggregatory activity was measured with a modified filtration pressure technique (F.Hermán et al.Thromb. Res.44 /1986/, 575) in anaesthetized beagle dogs; the change in arterial blood pressure was recorded simultaneously. Using this technique, the dose-response relationship and the duration of action of prostacyclin and Iloprost following bolus administration have been determined. PGI2 was equipotent with Iloprost in inhibiting platelet aggregation in vivo (ED25: 0.25±0.04 nmol/kg; 0.28±0.05 respectively). At the same time PGI2 was two times as potent as Iloprost in decreasing the mean arterial blood pressure (ED25: 0.41±0.12 nmol/kg; 0.87±0.14 nmol/kg respectively). The antiaggregatory and hypotensive effects of Iloprost last longer in each experiment than that of PGI2, but did not reach the level of significance probably due to the considerable interindividual differences. The in vivo selectivity ratios (hypotensive potency/antiaggregatory potency) of Iloprost and PGI2 were 0.32 and 0.6 respectively. These results show that in anesthetized beagles Iloprost is somewhat more selective than PGI2 in inhibiting platelet aggregation.
EFFECT OF A SYNTHETIC ANTICOAGULANT (MD 805) ON PLATELETS IN HUMAN VOLUNTEERS AND HEMODIALYSIS PATIENTS.
Authors: Matsuo T , Yamada T , Nakao K
Published: August 2018

Twelve normal subjects were injected with 5000 U of commercial mucous heparin with or without preloading of 1.0 g aspirin, and 0.2 mg/kg MD 805, an arginine derivative, which is a new synthetic compound with an extremely strong affinity for thrombin, at an interval of 4 weeks after each injection. Heparin injection with or without aspirin significantly increased platelet factor 4 release. In contrast, the preloading of aspirin significantly inhibited the decrease of platelet count and the elevation of /8 thromboglobulin induced by heparin. However, MD 805 had no effect on platelet release proteins, and adequate anticoagulation by APTT was still present 60 min after the injection. MD 805 shows no stimulative effects on platelets such as with heparin.

In the case of the patient's study, three patients complicated with heparin induced thrombocytopenia plus thrombus formation in the extracorporeal circulation during hemodialysis, and were treated with MD 805 instead of heparin. The platelet counts in those patients quickly returned to within the normal range, and adequate anticoagulation was obtained in the following hemodialysis sessions and no further bleeding or clot formation was noted.

In conclusion, MD 805 may represent a useful alternative anticoagulant in patients with heparin induced thrombocytopenia.
BLOOD PLATELET FUNCTION OF HYPERLIPIDEMIC PATIENTS TREATED WITH BEZAFIBRATE
Authors: Borowska A
Published: August 2018

Platelets from patients with hyperlipoproteinemia (HLP) are more sensitive to some aggregating agents than platelets from normal persons.On the other hand, it is known that from 15% to 20% of patients with coronary heart disease have primary or secondary HLP.The progress of the knowledge in this field has been expressed in production of the new drugs diminishing HLP.Be-zafibrate is a new derivative of clofibric acid,which has been used in the treatment of HLP.The purpose of our study was and assessment of the effect of bezafi-brate on platelet aggregation and thromboxane (TXB2) generation.The experiments were carried out in 18 patients (7 women and 11 men),aged 32-60 (mean 46 years) with type Ila HLP.The control group of consisted of 10 healthy volunteers.For 6 weeks the patients with HLP were given bezafibrate (Bezalip-Boehringer Mannheim) 600mg daily in divided doses and were taking the same diet as before the treatment.Blood platelet aggregation and 14C arachidonic acid (AA) conversion to thromboxane in washed platelets (using thin-layer-chromatography) were determined before and after bezafibrate administration. The obtained results are presented in the table (mean±S.E.).

It is concluded that the main arteriosclerosis-protecting bezafibrate action lies not only in decreasing of lipid levels in serum, but also in normalization of platelet function.