bluuebunny/crossref_metadata_2025 huggingface dataset:
SERUM PREPARED AFTER DAILY INTAKE OF FISH INHIBITS PGI-2 PRODUCTION BUT NOT PLATELET INHIBITORY ACTIVITY OF ENDOTHELIAL CELLS
Authors: Simonsen T , Vårtun Å , Lyngmo V , Hornstra G , Nordøy A
Published: August 2018

Healthy males were given dietary supplement of 100 g mackerelpasta for six weeks. Controlls were given meatpasta. Blood were collected before and at the end of the dietary intervention. Fatty acid composition of total phospholipids in serum and free fatty acids were measured by GLC

Human endothelium cells were grown in medium supplemented with 10% serum prepared from blood of the mackerel and controll groups. The medium was then examined for PGI-2 by RIA of 6-Keto-PGFIα , and for platelet aggregation inhibitory activity (PAIA) measured as inhibition of collagen induced platelet aggregation in platelet rich plasma.

Fatty acids of total phospholipids and FFA of serum showed a significant decrease in the ratio of Arachidonic acid (20:4 n-6) to Eicosapentaenoic acid (20:5 n-3) in the mackerel group.

The 6-Keto-PGFlα was significantly lower in the medium added serum from the mackerel group at the end of the period than at start. PAIA was not reduced by serum collected after fish consumption.

We conclude that dietary fish contribute to factors in serum, inhibiting PGI-2 production in endothelial cells in vitro. However, the ability of endothelial cells to inhibit platelet aggregation was not affected
TRYPTOPHAN PYROLYSIS PRODUCTS FOUND IN COOKED FOODS INHIBIT HUMAN PLATELET AGGREGATION BY INHIBITING CYCLOOXYGENASE
Authors: Manabe S , Yanagisawa H , Ishikawa S , Kitagawa Y , Tohyama K , Abe S , Wada O
Published: August 2018

Humans are exposed to numerous toxic compounds in foods. During the past decade, several carcinogenic heterocyclic amines have been reported to be present in the cooked foods. Recently, we reported that some of the carcinogenic heterocyclic amines isolated from foods were present in human plasma. In order to know the effects of the carcinogens isolated from foods on the cell function, we investigated the effects of the carcinogenic heterocyclic amines including Trp-P-1(3-amino-l,4-dimethyl-5H-pyrido❘4,3-b❘indole) and Trp-P-2(3-amino-1-methyl-5H-pyrido❘4,3-b❘indole) on human platelet aggregation and polymorphonuclear leukocyte aggregation. Only tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, had potent inhibitory effects on human platelet aggregation when platelets were preincubated with the carcinogens for 15 min. Other carcinogenic heterocyclic amines such as glutamic acid pyrolysates (Glu-P-1 and Glu-P-2) and 3H-imidazo ❘4,5-f❘quinoline-2-amines(IQ and MelQ) did show no effect on platelet aggregation even at 100 μM.

The autoradiogram demonstrated that Tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, dose-dependently inhibited the formation of HHT,PGD2,PGE2 and TXB2 induced by sodium arachidonate in human platelets labeled with ❘ 14c❘ arachidonic acid. Moreover, Trp-P-1 and Trp-P-2 did not show significant effects on leukocyte aggregation induced by sodium arachidonate (0.75mM) even at lOOnM. It is concluded that Trp-P-1 and Trp-P-2 isolated from cooked foodstuffs have potent inhibitory effects on the cyclo-oxygenase pathway of the platelet. Therefore, human platelet function might be affected with daily foods containing tryptophan pyrolysis products in vivo.
TRANSIENT AGGREGATION RESISTANCE OF HUMAN PLATELET-RICH PLASMA; A NEGLECTED IN VITRO PHENOMENON WITH PHYSIOLOGICAL IMPACT
Authors: Hardeman M R , Vreeken J
Published: August 2018

Due to instability in the first in vitro period, platelet-aggregometry is usually deliberately postponed until ca. 1 hour after venepuncture (VP) . At that time aggregability is fairly constant for 1 hour or more. Investigation of the period immediately followed VP, hcwever, revealed a high aggregation resistance - measured as the threshold ADP-concentration which the platelets just could resist before they aggregate maximally and irreversibly - which subsequently decreased exponentially with time. This “Transient Aggregation Resistance” (TAR) appeared to be superimposed on a stable, so called Baseline Aggregation Resistance (BAR) .The latter, measurable 60 min or more after VP, yields the “classical” threshold ADP-concentration.

Parallel aggregation-studies started 6 min after VP, subsequent studies were performed every 4 min. pH was controlled during storage of PRP at rocmtenperature. Extrapolation of the TAR-curve to t=0 (i.e. time of VP) yields the maximal value:TARmax Coefficients of variation for TARmax-method: 9.4% (n=6) ; intraindividial 15% (n=15, over 3 yrs); interindividual: 51% (n=16,wide range).

This TAR-phencmenon which is proven to be caused by a plasma-factor, can be influenced by dietary n-3 fattty acids and can be also inhibited by ASA,suggesting a prostanoid nature. The physiological significance of TAIfoax can be illustrated by the following findings:1. Patients with myocardial infarction, hyperlipoproteinemia, sickle cell anemia (i.e. diseases with a high risk for thrombotic complications) have low TARmax-values. 2.Individuals with “spontaneous platelet aggregation” in vitro,but asymptomatic, have positive TARmax-values. 3.There is a clear,reciproke age-dependency of TARmax

It is concluded that a technique is available measuring the effect of circulating,labile platelet-aggregation influencing plasma factor(s). Furthermore, using this technique,it was found that normal fresh plasma contains a labile aggregation-inhibiting factor which is several orders of magnitude more potent than other stabile factors either present in plasma or associated with platelets. This factor is probably of prostanoid nature and might have significance as a reflection of the antithrcmbotic potential of the endothelium.
PROTEOGLYCAN IN AORTIC WHITE MURAL THROMBUS AND IN RESULTING THROMBOATHEROSCLEROTIC LESIONS IN RABBITS
Authors: Weigensberg B I , Alavi M , Moore S , Lough J O , Riml H , Lee M H
Published: August 2018

Thromboatherosclerotic lesions in normolipidemic rabbits may contain variable amounts of lipid but the cause of this variable lipid content is not understood. Proteoglycan (PG) has been implicated in the trapping of lipoproteins in atherosclerosis. The object of this investigation was to study the quantity and quality of PG and glycosaminoglycan (GAG ) in early white mural thrombus induced with an indwelling catheter in the aorta of the rabbit as it evolves into thrombo atherosclerosis and to determine if the Alcian Blue stainable PG correlates with lipid accumulation. Lesions were compared with the normal aortic wall and a gut Alcian Blue standard embedded in the same block and cut and stained with Alcian Blue under the same conditions and analysed with a computerized color system that measures unstained lipid and Alcian Blue in a delineated lesion. The normal rabbit aorta had a mean ALCIAN BLUE COLOR VALUE “ ABCV ”of 10 equivalent to 14 ug GAG per ( mg dlt) mg delipidated tissue. Early mural thrombus in normolipidemic rabbits had virtually no Alcian Blue stainable proteoglycan and sin ABCV of 1 to 2 . After 12 weeks the resulting thrombo atherosclerotic lesions had a mean ABCV of 28±12 ( equivalent to 39±17 ug GAG per mg dlt. The ABCV in areas with large numbers of macrophages laden with lipid droplets and platelet debris ranged from 5 to 15. It is concluded that proteoglycans play a less significant role in the accumulation of lipid and platelet debris by the macrophages and this lipid and platelet debris may regress readily; but probably plays a more important role in the accumulation of interstitial lipid adjacent to smooth muscle cells. The interstitial lipid and PG deposits adjacent to numerous smooth muscle cells may be more resistant to regression. These two types of lipid deposits which are both variable determines the total lipid concentration in these lesions.
EFFECTS OF SIN 1 ON PLATELET ACTIVATION INDUCED BY THROMBIN IN HUMAN PLATELETS
Authors: SIMON M F , CHAP H , DOUSTE-BLAZY L
Published: August 2018

The mechanism of platelet activation is well known. The interaction of agonist such as thrombin, on specific membrane receptor induces phosphatidylinositol-specific phospholipase C activation, with a concomitant formation of two second messengers (from PIP2): inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 is able to induce a rapid discharge of Ca2+ from internal stores and Ca2+ influx through plasma membrane by unidentified Ca2+ channels linked to receptor activation. The increase of cytoplasmic free calcium concentration leads to the activation of the calcium calmodulin dependent myosine light chain kinase which phosphoryla-tes 20 kD proteins (myosine light chain). DAG is a potent activator of protein kinase C, which phosphorylates 40 kD proteins. These different pathways act in synergism.

Sin 1 is a platelet aggregating inhibitor. This compound is an active metabolite of molsidomine, which activates platelet guany-late cyclase, inducing a rapid rise in cyclic GMP level. The precise role of cyclic GMP in platelet activation is not yet known. In order to study the mechanism of action of this drug, we tried to determine the effect of Sin 1 on the different steps described above. We measured Ca2+ fluxes and phospholipase C activation in thrombin (0,5 U/ml) stimulated platelets in the presence of different doses of Sin 1 (10™7-10™3M). Serotonin secretion was inhibited by 30 % with Sin 1 (10™4M-10™5m). A parallel inhibition of phospholipase C was detected by measurement of [32P)-PA level. Platelets loaded with Quin 2 and stimulated by thrombin showed a 70 % inhibition of external Ca2+ influx as soon as a concentration of 10™7M of Sin 1 was added. A study on platelet loaded with [45Ca2+) and Quin 2 confirmed these results. On the contrary, discharge of internal Ca2+ store seemed to be unaffected.

In conclusion, the major effect of Sin 1 on platelet phospholipase C pathway is an inhibition of Ca2+ influx through plasma membrane. Some further experiments are necessary to shown whether this inhibition is correlated with cyclic GMP formation (the major effect of Sin 1) and try to establish a relation between this inhibition and that exerted on phospholipase C.

Sin 1 was a generous gift of Hoechst.
INHIBITORY EFFECT OF SIN 1 ON PLATELET FUNCTION:POSSIBLE INTEREFRENCE WITH PHOSPHOLIPASE C AND FIBRINOGEN BINDING
Authors: Levy-Toledano D , Weill D , Maclouf J , Rendu F , Soria C
Published: August 2018

The mode of action of SIN 1, the main metabolite of an antianginal drug (Molsidomine) was investigated in vitro on huma platelet functions. SIN 1 inhibited dose dependently platelet activation as reflected by aggregation and release of serotonin induced by arachidonic acid, prostaglandin endoperoxydes analogues (U 46619, U 44069), collagen, and ADP (figst and second wave). The maximal inhibition was reached at 10−5 M. The thromboxane (TX) synthesis was inconsis^antly impaired even at high concentrations of SIN 1 (104 M) suggesting a discrepancy between the inhibitory effect on platelet activation and TX formation. The main cofactor of ADP-stimulated aggregation is fibrinogen ; SIN 1 dose-dependently inhibited the fibrinogen binding to platelets thereby explaining it antiaggregatory properties.

In order to further investigate the mechanism of action of this drug on platelet activation we tested SIN 1 on the thrombin-induced phg^phosinositide metabolism and protein phosphorylation on 32P-prelabelled isolated platelets.P-phosphatidate (PA) formation was greatly inhibited. Phosphorylations of the myosin light chain (P20) and of 43 kDa protein (P43) were also reduced. These effects were accompanied by an inhibition of serotoninrelease, TXB2 synthsei^, and platelet aggregation.

SIN rl_would seem to act on early biochemical events and more especially at thelevel of the membrane phospholipase C.
MECHANISMS FOR THE IN VIVO ANTIPLATELET EFFECTS OF ORGANIC NITRATES
Authors: De Caterina R , Giannessi D , Bernini W , Mazzone A
Published: August 2018

Organic nitrates (nitroglycerin, isosorbide dinitrate) are inhibitors of platelet function more effective in vivo than in vitro (Am J Cardiol 1984; 53:1683), the in vivo effect requiring concentrations 10-100 times lower than in vitro. We have previously excluded that such difference is due to elicitation by nitrates of prostacyclin synthesis in human endothelial cells or vascular fragments (Circulation 1985; 71:176). In the present study we evaluated alternative explanations: that the difference is due (1) to generation of more active drug metabolites; (2) to synergism between nitrates and prostacyclin in the inhibition of platelet function. Isosorbide dinitrate (ISDN) and its two main in vivo metabolites, isosorbide-2-mononitrate (IS-2-MN) and isosorbide-5-mononitrate (IS-5-MN), were compared in their ability to inhibit platelet aggregation and thromboxane (TX) B2 formation (RIA) in respone to threshold doses of ADP, adrenaline, collagen, arachidonic acid and thrombin in citrated platelet-rich plasma. The same tests were performed in 10 healthy volunteers before, during (at 5, 15 and 30 min) and after infusion of the three drugs at 8 mg/h for 30 min in 3 different days. Finally, the concentration of prostacyclin (and its stable analogue Iloprost) added in vitro to platelets, and required to inhibit platelet aggregation by 50% (IC50) after 5 min pre-incubation of platelets with nitrates was determined. In vitro incubation of platelets with IS-2-MN resulted in greater inhibition of both aggregation and TX formation (by ADP and adrenaline) than with ISDN and IS-5-MN. At 10−7M, only IS-2-MN significantly inhibited aggregation (−12%, P< 0.05) and TX formation (from 9.2±1.8 to 5.9± 0.6 ng/ml) by ADP, while minimum effective concentrations were 10−7M for ISDN and 10−7m for IS-5-MN. These in vitro differences are unlikely to be the explanation of in vivo findings, since IS-2-MN, ISDN and IS-5-MN were equipotent when administered in vivo (complete abolition of secondary wave after ADP and adrenaline at 30 min of infusion). At supra-threshold doses of all the aggregating agents, all three drugs, at 10−7M, decreased IC50 for prostacyclin from 2.9± 1.3 to 0.32± 0.18 nM (P< 0.01). Synergim with prostacyclin is most likely to account, at least partially, for in vivo antiplatelet effects by organic nitrates.
HUMAN PLATELET ACTIVATION BY BACTERIAL PHOSPHOLIPASE C: MECHANISM OF INHIBITION BY FLURAZEPAM
Authors: Akbar Huzoor , Wallace David , Anwer Khursheed
Published: August 2018

We have shown earlier that flurazepam inhibits human platelet aggregation and serotonin secretion induced by bacterial phospholipase C (BPLC, Thromb. Res. 38, 361-374, 1985). This study was conducted to examine the mechanism(s) of inhibitory action of flurazepam. Only 15 uM and 11 uM flurazepam were required to inhibit platelet aggregation and serotonin secretion by 50%. In a platelet free system, BPLC hydrolyzed 14C-phosphatidylcholine (14C-PC> in a time- and concentration-dependent manner in the presence of calcium ions. Flurazepam had no effect on BPLC-induced hydrolysis of 14C-PC. Incubation of 14C-arachidonic acid labelled platelets with BPLC produced diacylglycerol(DAG) in a time- and concentration-dependent manner. Flurazepam did not inhibit DAG production by BPLC. However, prostaglandin E1 and paranitrophenolphosphorylcholine inhibited DAG production by 20% and 75% respectively. Platelet cytosolic fraction,containing phosphatidylinositol-specific PLC (PI-PLC), hydrolyzed 3H -phosphatidylinositol (3H-PI) in a concentration-dependent manner. Flurazepam did not inhibit hydrolysis:of 3H-PI by PI-PLC. BPLC caused phosphorylation of 47,000 Dalton protein (P47) in 32P-labelled platelets. Flurazepam did not inhibit phosphorylation of P47 in the first five minutes of incubation. However, flurazapam completely blocked phosphorylation of P47 by seven minutes. In Other experiments, flurazepam inhibited platelet aggregation induced by ionomycion, a calcium ionophore, in a concentration-dependent manner. These data lead us to suggest that flurazapam does not inhibit BPLC-ihduced platelet activation by inhibiting the action of BPLC or PI-PLC on platelet phospholipids or DAG production. However, the ability of flurazepam to inhibit ionomycin-induced platelet aggregation indicates that it may be blocking BPLC-induced platelet aggreagtion by interfering with the influx, of calcium ions into platelets. (Supported in part by the American Osteopathic Association, The Baker Award from Ohio University and the OUCOM).
EFFECT OF FUROSEMIDE ON PLATELET AGGREGATION AND ON ALPHA-ADRENOCEPTOR-DENSITY IN MAN.
Authors: Kribben A , Fritschka E , Sibold M , Fassbender M , Distler A , Philipp Th
Published: August 2018

Furosemide (FUR) reduces the ∝2-adrenoceptor- (∝2-R) mediated pressor effect of norepinephrine. Since it has been shown that FUR reduces platelet aggregation ( AGG) we studied the effect of FUR on α2-R as well as that on the ∝2-R-medi ated epinephrine ( EPI)-induced AGG a) ex vivo and b) in vitro. For comparison the effect of FUR on ADP-induced AGG was also studied. Methods: a) 8 normotensive men received FUR (30 mg b. i.d. ) for 3 weeks. EPI- (1 μmol/1) and ADP- (1 μmol/1) induced platelet AGG were measured before as well as after 3 weeks of FUR application with a semiautomatic device ( APACT) . Platelet ∝2-R-density was measured by 3H-yohimbine-binding and the fraction of high-affinity binding-sites was measured by competi tion of 3H-yohimbine with EPI. b) Platelet-rich plasma was incubated with FUR (1 mmol/1) for 10 min at 37 °C and AGG was measured (n=7) as described. Results: a) FUR decreased ∝2-R-densi ty from 293± 28 to 251±; 21 fmol/mg protein (p< 0.01) and high affinity binding sites from 64± 3 to 55± 5 % (p< 0.01). FUR inhibited ADP-induced AGG ex vivo while EPI-induced AGG did not change (table). b) In vitro FUR inhibited total ADP-induced AGG from 7 4 ± 4 to 45± 8 % (p< 0. 01) while again EPI-induced AGG did not change.

Conclusions: The results suggest separate effects of FUR on α2 R-density and on platelet AGG. Although FUR decreased < r2-R, EPI-induced platelet AGG remained unchanged. Since FUR inhibited ADP-induced AGG ex vivo as well as in vitro FUR may interfere directly with the mechanism of ADP-induced platelet AGG.
PULMONARY MICROEMBOLISATION IN SURGICAL SHOCK: THE EFFECT OF CYCLO-OXYGENASE INHIBITION
Authors: Backhouse C M , Meek A C , Poskitt K R , McCollum C N
Published: August 2018

Thromboxane release from platelet microemboli during major arterial surgery may mediate depression of cardio-pulmonary function. The effect of cyclo-oxygenase inhibition by aspirin has been studied in a porcine model of aortic surgery.

Following autologous platelet labelling with 111-lndium, 24 pigs (20-25kg) were randomised to low dose (LD) aspirin (0.5mg/kg), high dose (HD) aspirin (10mg/kg) or placebo.

Arterial and Swann Ganz catheters were inserted prior to surgery consisting of midline laparotomy, small bowel extériorisation, 1.5 hours of aortic clamping and 1 hour shock before resuscitation. On induction, during shock and at 3 days, platelet and leucocyte counts, lung platelet uptake (LPU), venous aggregates by screen filtration (SFP), mean arterial pressure (BP), cardiac output (CO), pulmonary shunt (PS) and alveolar-arterial pO2 difference (A-adO2) were measured.

During shock following aortic declamping aspirin preserved blood pressure by increasing vascular resistance rather than CO. Venous aggregates by SFP tended to be lower on aspirin with significantly reduced LPU, subsequent pulmonary shunting and A-adO2. The improvement in PS but not A-adO2 remained significant at 3 days (p<0.05).

Cyclo-oxygenase inhibition improved pulmonary function during surgical shock either by inhibiting platelet microemboli or by a direct effect on pulmonary arteriovenous shunts.
PREVENTION OF SYSTEMIC THROMBO-EMBOLISM IN PATIENTS WITH ATHEROSCLEROTIC INTERMITTENT CLAUDICATION
Authors: Boissel J P , Peyrieux J C , Destors J M , Lievre M , Moleur P
Published: August 2018

Ticlopidine (TI), an anti-aggregating agent which inhibits the ADP-pathway has been tested in patients with intermittent claudication (IC) in 11 randomized clinical trials (RTCs).

As expected, a significant reduction of cardio-vascular events (CVE) due to systemic thrombo-embolism was observed in the 2 larger. Reduction in the number of CVE due to systemic thromboembolism in any arterial bed was observed. This prompted us to confirm the hypothesis that TI was beneficial in preventing systemic thrombo-embolism in patients with IC. Four RCTs from the 11 were blindly selected on the basis on pre-set selection criteria : placebo controlled, more than 1 month duration, , less than 5 % lost-to-follow-up (index of quality), parallel groups, proven atherosclerotic disease. Meta-analysis was performed with 5 statistical methods which gave consistent findings : as compared to 311 patients on placebo, the 301 patients on TI have had a 66 % reduction in the number of CVE during the 6 months of follow-up (9.0 % to 3 % , p = 0.002). Walking distance, a secondary objective of meta-analysis, doubled in 42 % of the patients on TI as against 27 % (p = 0.0005).

It was concluded that TI 250 mg b.i.d. prevents CVE in patients with atherosclerotic IC.
GEMFIBROZIL HAS ANTI-PLATELET EFFECTS IN PATIENTS WITH HYPERCHOLESTEROLAEMIA DURING PHYSICAL STRESS
Authors: Laustiola K , Lassila R , Koskinen P , Manninen V
Published: August 2018

Earlier studies indicate an increased platelet reactivity in patients with hypercholesterolaemia. Physical and mental stress have also been reported to cause increased reactivity. The present study was undertaken to evaluate the effect of gemfibrozil (G) a new lipid lowering drug on platelet reactivity during physical stress. Ten otherwise healthy male subjects with serum cholesterol levels above 7 mmol/l were involved in a double-blind study. It consisted of two treatment periods of 8 weeks during which the patients were given either G (600 mg b.i.d.) or placebo (PI) and an 8 weeks wash-out period before the cross-over. At the end of the treatment periods an exercise test was carried out and platelet reactivity tested. Adrenaline, ADP and collagen were used to induce aggregation and 5-HT and T×B2 release measured. Plasma beta-TG and fibrinogen were also determined.

The treshold concentration of adrenaline necessary to evoke secondary aggregation was increased in 8/10 patients during exercise after G treatment and in 2/10 after PI. When the lowest ADP concentration to cause secondary aggregation (2-4 uM) was used there was a significant decrease in the 5-HT (− 44%) and T×B2 (− 48%) secretion and a significant decrease in the area under the aggregation curve (− 28%). A decrease in 5-HT secretion was also seen after G treatment when a fixed ADP concentration of 10 uM was used. During collagen stimulation no changes were seen between the two groups. Beta-TG remained unchanged irrespective of treatment and fibrinogen showed a modest increase during exercise in both treatment groups. These results indicate a new anti-platelet effect of gemfibrozil which might be of importance in prevention of acute thrombotic events in hypercho1estero1aemic patients.
PLATELET TXA2 SYNTHETASE INHIBITION AND TXA2/PROSTAGLANDIN ENDOPEROXIDE RECEPTOR BLOCKADE COMBINED IN ONE MOLECULE (R 68070)
Authors: De Clerck F , Van de Wiele R , Xhonneux B , Van Gorp L , Somers Y , Loots W , Beetens J , Van Wauwe J , Freyne E , Janssen P A J
Published: August 2018

F 68070, an oxime-alkane carboxylic acid derivative (Janssen Pharmaceutica), is a potent inhibitor of thromboxane A2 (TXA2) synthetase activity (IC50 in vitro against thrombin-stimulated human platelets in plasma : R 68070 : 2.9 x 10-8 M; CGS 13080 : 6 x 10-8 M; OKY-1581 : 8.2 x 10-8 M; dazmegrel : 2.6 x 10-8 M; dazoxiben : 2.3 x 10-8 M).

The compound specifically inhibits platelet TXA2 synthetase activity (14C-arachidonic acid metabolism by washed human platelets) without effect on the cyclo-oxygenase, lipoxygenase (platelets, RBL cells) or prostacyclin synthetase activities (rat aortic rings).

The inhibitory effect of R 68070 against human platelet TXA2 synthetase activity increases upon prolongation of the contact time (ICsg at 0.5 min of contact : 5.2 x 10-7 M; at 5 min : 8.3 x 10-8 M; at 30 min : 2.5 x 10-8 M) and is reversed by washing of the platelets.

In vivo, the compound has a comparatively strong inhibitory effect on platelet TXA2 synthetase activity after oral administration to rats (ED50 - 2 h : R 68070 0.013 mg/kg; CGS-13080 : 0.8 mg/kg; OKY-1581 : 0.61 mg/kg; dazmegrel : 1 mg/kg; dazoxiben : 4.1 mg/kg) and a protracted duration of action in rats and dogs (inhibition 8 h after 1.25 mg/kg orally > 80 %).

In vitro, R 68070 inhibits the aggregation of human platelets in plasma stimulated with collagen (IC50 : 4 x 10-6 M), but also with U 46619 (IC50 : 3.8 x 10-6 M) without affecting the primary aggregation reaction elicited by ADP, 5-HT or adrenaline. The compound thus also produces platelet TXA2/prostaglandin endoperoxide receptor blockade.

In rats and in dogs R 68070 (1.25 mg/kg I.V.) potently prevents thrombus formation in carotid and coronary arteries damaged by electrical stimulation.

The combination of platelet TXA2 synthetase inhibition with TXA2/prostaglandin endoperoxide blockade in one molecule thus might offer an improved anti-thrombotic effectiveness.
TREATMENT OF ADVANCED STAGES OF PERIPHERAL OBLITERATIVE DISEASE WITH THE THROMBOXANE RECEPTOR ANTAGONIST BM 13.177: A PLACEBOCONTROLLED DOUBLE BLIND STUDY
Authors: Hossmann V , Schäfer H J , Auel H , Etti H
Published: August 2018

Twenty patients (67.2 ± 12.3 yrs; aged 39-86 yrs, 10 males, 10 females) with stage IV of peripheral obliterative arterial disease received at random either a) BM 13.177, a thromboxane receptor antagonist, at a dose of 6 g/24 hrs for 7 days i.v., followed by oral treatment of 6.4 g/day in four doses for 2 weeks and a subsequent placebo week, or b) placebo alone with the same protocol. The clinical course was followed by measurement of blood pressure (by Riva-Rocci on the left brachial artery and by Doppler of the ankle artries), blood flow at restand after 3 min of tourniquet ischemia (by venous occlusion plethysmography), TcpCf2 at the wrist of the affected limb, and by subjective estimation of pain with visual analog scale before, at the end of the infusion period, as well as on day 7 and 14 of oral treatment, and 7 days after treatment while patients of both groups were on placebo. In addition spontaneous platelet aggregation by PAT III, induced platelet aggregation in whole blood by collagen and in PRP by ADP at different doses on the same days were measured on the same days as described above.

Results: BM 13.177 completely inhibited aggregation in whole blood induced by collagen 0.3 (ig/ml, however one week after treatment a rebound phenomenon was observed with 16.0 ± 3.8 OHM compared to pretreatment value of 11.0 ± 3.8 OHM (p < 0.01). At a higher dose of 1.2 |ig/ml the same inhibiting effect on platelet aggregation was observed. Spontaneous platelet aggregation as measured by PAT III was evident in only 2/10 pat. pre-treatment, was abolished in all patients on i.v. BM 13.177, returned in 1/10 pat. during oral treatment, but in 4/10 pat. on day 7 after treatment, while being on placebo, again indicating a rebound phenomenon (p < 0.03) ADP induced platelet aggregation was not significantly affected by BM 13.177. In the placebo group, too, no significant differences were observable between the different treatment regimens. Clinical data did not show any significant alteration in either verum or placebo group during the six week period, indicating no benificial effect of thromboxane receptor antagonists in advanced stages of peripheral obliterative arterial disease, although platelet inhibiting effects were clearly demonstable.
PLATELET FUNCTION AND LIPOPROTEINS IN PATIENTS WITH HYPOTHYROIDISM
Authors: Fujii S , Kariya T
Published: August 2018

Platelet function and serum lipoprotein levels were studied in ten patients (two males and eight females) with hypothyroidism. Platelet aggregation and ATP release were determined by Lumi-aggregometer using ADP , collagen and epinephrine as stimulants. Platelet factor 4 (PF4) and thromboxane B2 (TXB2) were determined by radioimmunoassay. High density lipoproteincholesterol (HDL-C) was determined by heparin-manga-nese method. HDL subfractions were separated by gradient gel electrophoresis (PAA 4/30). Apolipopro-teins were measured by single radial immunodiffusion. Platelet aggregation increased in those patients at stimulating by epinephrine. ATP release also increased at stimulating by epinephrine. PF4 increased at stimulating by epinephrine. TXB2 increased at stimu-lating--by ADP or epinephrine significantly (p<0.05), respectively. Platelet aggregation was not correlated with thyroid hormones or total cholesterol levels.

But it had a positive correlation tendency with HDL-C or HDL2-C and a negative one with HDL3-C levels.

These results suggested some relationships between platelet function and HDL metabolism in patients with hypothyroidism.
REDUCED THRESHOLD FOR COLLAGEN INDUCED PLATELET AGGREGATION IN WHOLE BLOOD FROM PATIENTS WITH SYSTEMIC SCLEROSIS
Authors: Goodfield M J D , Orchard M A , Rowell N R
Published: August 2018

Systemic sclerosis is a multi-system disorder characterised by skin lesions and narrowing of arterioles with reduction in blood supply to many organs. Previous reports have suggested that platelet aggregation is abnormal and could contribute to the pathogenesis. Platelet aggregation in whole blood was investigated in 10 patients with systemic sclerosis and 9 age matched controls. Subjects rested for 30 minutes before blood samples were taken without stasis, via a 19G needle into 3.8% trisodium citrate anti-coagulant. 0.5ml of blood was transferred to cuvettes, and equilibrated gt 22°C for 30 minutes, before being stirred at 750 r.p.m. at 37 C. Aggregation was induced by collagen (0.01 ug/1 to 1.0 ug/1), adrenaline (0,1,0.5 and 1.0 ug/1) and ADP (0.1,0.25 and 2.5 ug/1). Aliquots for counting free platelets were taken and assessed using the Ultraflow 100 particle counter.

Platelet aggregation was significantly increased in patients with systemic sclerosis at all doses of collagen (p<0.01), and the mean dose giving 50% aggregation was significantly lower in the disease group (0.026+/-0.06mg/l) than in the controls (0.23+/-0.06mg/l, p<0.01). Aggregation was not significantly different with the other aggregating agents.

Increased sensitivity of platelets to aggregation by collagen in whole blood may be of importance in the pathogenesis of the vascular and fibrotic lesions of this disease
PLATELET ACTIVATING FACTOR (PAF) AS A MEDIATOR OF PROTEINURIA IN ISOLATED PERFUSED KIDNEY (IPK).
Authors: Delani F , Tagliaferri M , Macconi D , Lupini C , Perico N , Rumuzzi G
Published: August 2018

PAF amplifies tissue damage in glomerulonephritis and can promote proteinuria stimulating platelet and neutrophil cationic protein release. We used IPK to establish whether PAF directly causes proteinuria. Kidneys were isolated from male Sprague-Dawley rats and perfused at constant pressure (100 mmHg) in a closed circuit with a Krebs-Henseleit buffer containing glucose urea creatinine, BSA (1%), Ficoll 70 (3.5%) and amino acids. After 25 min stabilization period, a basal 10 min clearance period was followed by PAF (1.8 nM f.c. n = 6) or vehicle (n = 5) injection into the renal artery. As seen in the figure PAF but not vehicle significantly (p<0.01) increased urine protein excretion. No significant changes in GFR (as creatinine clearance) were observed after PAF or vehicle injection (Basal: 0.786 ± 0.075 PAF: 0.658 ± 0.070. Basal 0.653 ± 0.081, vehicle 0.639 ± 0.074 ml/min/g kidney). The data indicate that PAF may directly increase glomerular permeability to proteins.
HEPARIN-RELEASED PLATELET FACTOR 4 (HR-PF4) IN DIABETIC MICR0VAS-CULAR DISEASE
Authors: Boschetti C , Vicari A , Cofrancesco E , Della Volpe A , Moreo G , Po-gliani E , Pozza G , Polli E
Published: August 2018

When heparin is injected i.v. as a bolus, PF4 but not (β-throm boglobulin ((βTG) is released immediately. HR-PF4 is not liberated from platelets but from the endothelial cells of vessels which serve as storage sites. The role of platelet activation in diabetic microvascular disease is still controversial, however there is experimental evidence of vascular injury and hemostatic activation preceding the appearance of microvascular disease. The contradictory results so far obtained in man may be partly attributed to the heterogeneity of the diabetic patients studied. We studied 20 insulin-dependent diabetics (age 21-40) in stable metabolic equilibrium (mean HbAlc=7.6%). 10 without fluoroangiographic evidence of retinopathy (Group l) and 10 with retinopathy (Group 2). None had signs or symptoms of macrovascular disease. The control group consisted of 10 healthy volunteers (age 22-39). No medication except insulin was taken for at least 10 days preceding the study. 12 h before the study all subjects received aspirin 500 mg p.o. Plasma (βTG and PF4 were determined before (basal) and 5,30,90 min after a heparin bolus i.v. (5000 U). Protein C, factor VIIIR:Ag and tissue plasminogen activator were also measured in plasma.
NEUTRALIZING EFFECT OF PLATELET FACTOR 4 OR HISTIGINE-RICH GLYCOPROTEIN AGAINST LOW MOLECULAR WEIGHT HEPARIN AND UNFRACTIONATED HEPARIN
Authors: Takahashi K , Niwa M , Sakuragawa N
Published: August 2018

Purpose: Low molecular weight(LMW) heparin shows stronger antifactor Xa(F-Xa) and weaker anti-thrombin(TH) activities compared with unfractionated(UF) heparin, and shows less bleeding tendency in the cases of clinical use. Platelet factor 4(Pf-4) and histidine-rich glycoprotein(HRG) neutralize heparin. We investigated on the heparin neutralizing effects of them to both kinds of heparin

Materials and methods: LMW heparin(Kabi and Pharmuka) and UF heparin(Novo) were used. Antithrombin III(AT-III), HRG(human origin ) and pf-4( bovine origin ) were purified by our methods

TH(Green-Cross) and F-Xa(Sigma) were used. Reaction mixtures for anti-TH or anti-F-Xa were as follows: 1 vol of AT-III( 0.1 U/ml)+
1 vol of heparin( 10 ug/ml)+l vol of pf-4 or HRG(varied)→incubated for 5 min→+l vol of TH(5 U/ml) or F-Xa( 7 nKat/ml)→incubated for 5 min→ + S-2238 or S-2222→ recorded at 405 nm.

Results: (1) Pf-4 showed the equivalent anti-TH effect on both kinds of heparin, and 3 ug of pf-4 neutralized 1 ug of heparin

On F-Xa neutralizing effect, 13 ug of pf-4 neutralized 1 ug of UF heparin, but could not neutralize LMW heparin. (2) HRG showed the same results on anti-TH effect of both kinds of heparin, but could not neutralize the anti-F-Xa effect of LMW heparin on the same amount of HRG which neutralized that of UF heparin. Conclusion: Anti-F-Xa effect of. LMW heparin could not be easily neutralized by pf-4 or HRG compared with that of UF heparin.
INTERACTION OF PLATELET FACTOR 4 WITH HEPATOCYTES AND ITS POSSIBLE SIGNIFICANCE IN HEMOSTASIS
Authors: Rucinski B , Stewart G J , Boden G , Niewiarowski S
Published: August 2018

Previous experiments have demonstrated the rapid clearance of himan platelet factor 4 (PF4) from rabbit and rat blood, its accumulation in the liver and its elimination of PF4 degradation products with urine. Injection of heparin resulted in a rapid loss of 125 i-pF4-radioactivity from the liver (Rucinski et al Amer. J. Physiol. 251, H800, 1986). Current experiments demonstrate the uptake of human 125i-pf4 by hepatocytes reaching maximum at 180 min. This uptake is 2-3 times greater at 37°C than at 4°C. At 37°C degradation of 125I-PF4 by hepatocytes was also observed as indicated by the increase of 125j_pf4 radioactivity soluble in 6% trichloracetic acid. By contrast, no uptake of 125I-β was observed. Autoradiography demonstrated association of l25I-PF4 with hepatocytes membranes while after longer incubation (20-60 min) radioactivity was also localized in endosanes. The heparin inhibited binding and uptake of PF4 by hepatocytes; accordingly the injection of heparin to or into rabbits within 10 min resulted in a rapid urinary clearance of 125I-PF4 and the injection of PF4 resulted in a rapid urinary clearance of 3H-heparin. We propose that 1) PF4 released to the circulating blood by activated platelets is bound to the surface of hepatocytes and that it is further processed by these cells over time; 2) the presence of PF4 in the liver may contribute to the regulation of hemostasis by neutralizing anticoagulant activity of endogenous heparin-like molecules; 3) in clinical situations PF4 may enhance clearance of injected heparin by accelerating its urinary excretion.
IN VIVO INTERACTION BETWEEN HUMAN PLATELET FACTOR 4 (PF4) AND PROTAMINE SULPHATE (PS) IN THE PRESENCE OF DIFFERENT GLYCOSAMI-NOGLYCANS (GAGs)
Authors: Celia G , Prosdocimi M , Sasahara A A
Published: August 2018

Anti-heparin substances, like PF4 or PS, have been studied largely in reference to their ability to neutralize the anticoagulant activity of heparin. On the other hand, few data are available concerning the relationship between GAGs and anti-heparin proteins clearance. We studied the action of PS on human PF4 kinetics in anesthetized rabbits pre-treated with heparin (H, 1000 I.U.), heparan sulphate (HS, 30 mg) and derma tan sulphate (DS, 30 mg). PF4 (given at a dose of 45 μgAg) disappearance reflected its different affinity for the GAGs, with the following half lives (min): control 2.09±0.28, H 14.80±1.47, HS 10.90±1.91, DS 6.87±0.68. Moreover, circulating PF4 (ng/ml) at 1 min was as follows: control 109±8, H 873±134, HS 751±34 and DS 473±15. In another group of H pre-treated rabbits, a bolus injection of PS (10 or 20 mg) caused an immediate disappearance in th.e circulating plasma PF4, from 900±23 ng/ml (1 min after PF4) to 75±11 ng/ml (1 min after 10 mg PS). However, a subsequent H injection 10 min after PS induced a peak release of PF4 (520±21 ng/ml). In a further group of animals pre-treated with HS, the interaction between PS and PF4 was similar to that observed after H treatment. In the last group, pre-treated with DS, the interaction between PF4 and PS was also similar, however, unexpectedly, when 20 mg of PS were given a subsequent bolus of H did not produce any increase of circulating PF4

We suggest that PS displaces PF4 from its binding sites on H or HS, thus allowing its uptake by the storage sites in the body, from where it can be harvested again after the subsequent H administration. In the presence of DS again PS is able to displace PF4, however the remaining excess of PS could neutralize the subsequent H injection, thus rendering it unable to induce PF4 release.
CHARACTERIZATION OF HUMAN PLATELET ClQ BINDING SITES
Authors: PEERSCHKE E IB , Ghebrehiwet B
Published: August 2018

We have recently shown that platelets possess specific binding sites for Clq, a subcomponent of the first component of complement, Cl, and that occupancy of these receptor sites correlates with the previously described inhibitory effect of Clq on collagen-induced platelet aggregation. To further characterize platelet Clq receptors, washed platelets were solubilized in 5 mM sodium phosphate buffer, pH 7.5 containing 10 mM EDTA, 150 mM NaCl, 10 mM EACA, 0.5 mM PMSF, and 1% Triton X-100. After dialysis against 5 mM phosphate buffer pH 7.5 containing 10 mM EDTA, 20 mM NaCl, 10 mM EACA,0.5 mM PMSF and 0.1% Triton X-100, the lysate was passed over Clq-Sepharose-4B affinity columns. A single protein peak eluted with buffer containing 300 mM NaCl. This peak was composed of two predominant molecular weight species (85-95K, 60-66K) as assessed by SDS-polyacrylamide gel electrophoresis under non-reducing conditions. When 125-iodine surface labeled platelets were solubilized and applied to Clq-Sepharose affinity resins, the same two molecular weight species eluted and could be visualized by autoradiography following SDS-polyacrylamide gel electrophoresis. Immunoabsorption studies performed under nondenaturing conditions using protein A and the IIl/Dl monoclonal antibody, which binds specifically to platelets and inhibits platelet-Clq interactions, revealed that the 85-95K molecular weight component was preferentially absorbed, but incomplete immunoabsorption of the 60-66K molecular weight constituent was also noted. Affinity purified Clq binding sites sedimented as a single peak during 5-40% sucrose density ultracentrifugation with an S value of approximately 2.4. In addition, both the 85-95K and the 60-66K molecular weight species coeluted in the void volume of Sephadex G-100 columns. The data suggest that the 85-95K and 60-66K molecular weight species represent platelet membrane Clq binding sites, and that these sites may form weak, noncovalently associated complexes.
HIP2: A NEW MONOCLONAL ANTI-HUMAN PLATELET ANTIBODY WITH STIMULANT FUNCTION
Authors: Chen W J , Chen Z , Bao C X
Published: August 2018

A monoclonal anti-human platelet antibody(MoAb), HIP2 (IgG3) which induced irrevesible aggregation of platelet in association with the release of serotonin and thromboxane B2 formation is described. Indirect immunofluorescence assay (IFS) showed that the antibody binded to platelets and megakryocytes, and gave a weak reaction with aortic, liver and capillary endothelial cells. Electrophoresis of radiolabelled antigen showed that HIP2 recognized platelet membrane glycoprotein lib (130KD). The purified HIP2 MoAb induced aggregation with normal PRP, not with thrombasthenia platelets, formalin-fixed platelets, washed platelets and EDTA-PRP. Washed platelet aggregation with HIP2 could be restored by adding normal plasma or serum into medium, but not by inactivated serum at 56°C for 30 minutes and fibrinogen, The results suggested that the aggregation induced by HIP2 needed Ca++ and complement in medium. HIP2-induced aggregation was completely inhibited by calmodulin inhibitor, compound 48/80, and partially inhibited by aspirin, apyrase, ATP, antimycin A and phentolamine. Anti-glycoprotein Ilia MoAb(SZ-21) inhibited HIP2-induced platelet aggregation, did not block HIP2 binding on platelet surface in IFS. HIP2 no affected platelet adhision on glass. HIP2 prolonged koalin cephalin clotting time in PRP and interruped whole blood retraction. Under electron microscopy, the fibrin formation of clots in the presence of HIP2 was much less than the control. Recently, we found that the platelets from patients with myelo-proligerative diseases had decreased response to HIP2 aggregation. So that, HIP2 MoAb may be a very useful tool not only for study of the platelet membrane structure associated with function of platelet and platelet physiology, but also of the pathogenesis of some platelet diseases.
EXTRACELLULAR CALCIUM AND PLATELET GLYCOPROTEINS
Authors: Jabbal-Gill I , Johnston G I , Heptinstall S
Published: August 2018

Platelet membrane glycoproteins lib and Ilia form Ca++-dependent heterodimer complexes that contain binding sites for fibrinogen and therefore are relevant to the ability of platelets to aggregate together. In this study we investigated the effects of extracellular Ca++ on the stability and expression of IIb-IIIa complexes using a IIb-IIIa complex-specific monoclonal antibody M148. Its specificity was examined using crossed immunoelectrophoresis: the antibody reacted only with intact IIb-IIIa complexes and not with either glycoprotein alone.

SDS-polyacrylamide gel electrophoresis of immunoprecipitates of soluble glycoproteins that interacted with Ml48 showed that lib and Ilia were present as complexes in Ca++-depleted media at 25°C, pH7.4. However, Ca++-depletion at 37°C, pH7.4 or 37°C, pH8.7 or 25°C, pH8.7 caused dissociation of the complex

The effect of extracellular Ca++ on the expression of IIb-Illa complexes on the surface of intact platelets was studied by a technique which is based upon indirect binding of M148 using a fluorescent- labelled second antibody (FITC-RAM) and measuring the fluorescence per platelet using the FACS IV cytofluorometer. Intact platelets were incubated in Ca++-depleted media at 25°C, pH7.4 or 37°C, pH7.4 either (i) prior to or (ii) after adding M148. At 25°C increased M148-binding was observed, compared to the value prior to Ca++-depletion. This increased binding could be reversed by adding Ca++ back to the preparation. Under condition (i) at 37°C a marked decrease in M148 binding was observed, which could not be reversed by restoring Ca++, while under condition (ii) at 37°C the results were the same as at 25°C.

Our studies demonstrate that (a) Ca++-depletion at 37°C and/or alkaline pH causes dissociation of the Ilb-IIIa complex (b) Ca++ depletion at 25°C possibly alters distribution of the complexes thereby increasing their availability to the antibody and (c) M148 prevents the dissociation of complexes in Ca++-depleted media at 37°C, possibly by holding lib and Ilia together
PLATELET MEMBRANE GLYCOPROTEINS ABNORMALITIES IN MYELOPROLIFERATIVE DISORDERS. STRUCTURE/FUNCTION RELATIONSHIP
Authors: Mazzucato M , Del Ben M G , Casonato A , De Angelis V , De Marco L
Published: August 2018

The platelet membrane glycoproteins (GP) lb and GPIIb/IIIa were investigated in 10 patients with myeloproliferative disorders. 2 patients had essential thrombocytemia (ET), 2 had chronic myeloid leukemia (CML) and 6 policytemia vera (PV). The number of GP molecules were detected by radiolabelled monoclonal antibodies anti GPIb and anti GPIIb/IIIa complex (gift of dr. Z.M. Ruggeri) and their function was evaluated by using, in a binding assay, purified radiolabelled asialo von Willebrand factor (1251 ASvWF) and purified radiolabelled fibrinogen (1251 F). Binding isotherms were evaluated by Scatchard type analysis using the computer assisted programLigand. The binding of 1251 anti GPIb to the platelets of the ten patients showed 14,955 ∓ 4,636 molecules/platelet (M/Plt) compared to 19,790 ∓ 3,791 M/Plt of 11 normals with a p value < 0.01. The binding of 125IAsvWF to the GPIb of nonstimulated platelets in platelet rich plasma (PRP) was then measured and ound to be decreased. The dissociation constants (Kds) were within normal values except in one patient. There was a good correlation (r = 0.91, p < 0.01) between the amount of 1251 ASvWF bound and GPIb molecules. The binding of radiolabelled anti GPIIb/IIIa to the platelets of six patients (4 with PV and 2 with CML) was measured and found to be constantly decreased in all patients with a mean value of 25,349 ∓ 2,077 M/Plt compared to 43,192 ∓ 6,354 M/Plt found in normals (p < 0.01). 1251 fibrinogen binding to the GPIIb/IIIa complex of ADP + adrenalin stimulated washed platelet was studied in 2 patients and we found 16,267 M/Plt and 14,752 M/Plt respectively, significantly diminished when compared to the mean value of 36,591 M/Plt found in 2 normal controls. The Kds were within normal values. Our studies demonstrate a significant decrease of GPIb and GPIIb/IIIa on the platelet membrane of patients with myeloproliferative disorders. Furthermore this decrease is accompanied by a diminished binding of both vWF and F to their platelets receptors. These findings may partly explain the hemorragic tendency often encountered in these patients.