bluuebunny/crossref_metadata_2025 huggingface dataset:
CHANGES IN ENZYMATIC ACTIVITIES IN BRAIN CAPILLARY ENDOTHELIAL CELLS INJURED BY PLATELET AGGREGATION IN VIVO
Authors: Fujimoto T , Djuricic B , Tanoue K , Fukushima Y , Yamazaki H
Published: August 2018

We have reported cerebrovascular injuries induced by platelet aggregation in vivo. Appearance of vacuoles in endothelial cells and eventual deendothelialization are characteristic in large cerebral arteries (Stroke, 16:245, 1985). Minor changes are observed in brain capillaries, but disturbances of blood-brain barrier (BBB) are seen. To analyse changes in BBB, enzymatic activities in capillary endothelial cells before and after ADP-induced platelet aggregation in vivo were investigated.

80 mg/kg of ADP was injected through a catheter into the right internal carotid artery of 32 rabbits. One hr later, right and left cortexes freed from pial membranes were homogenized and microvessels were isolated using discontinuous sucrose gradient ultracentrifugation. Purity of microvessel fraction was ascertained microscopically. The follwing enzymatic activities in these samples were measured.; cytochrome C oxydase (CCO), monoamine oxidase (MAO), p-nitrophenyl-phosphatase transferase (pNPPase, K-dependent component of Na, K-ATPase), gamma-glutamyl transferase (GT) and adenylate cyclase (AC). The enzymatic activities did not change after a vehicle-injection and did not show any differences between capillaries of both the cortexes before the ADP-injection. One hr after the ADP, GT and CCO activities decreased significantly in the capillaries of injection side. MAO activity also reduced without significance. The other enzymes did not show significant changes in their activities. Although pNPPase and AC which are associated with inner surface of plasma membrane were preserved well, activity of GT which is associated with outer portion of the membrane decreased significantly. It suggests superficial luminal injury and that plasma membrane might be affected from the side of vascular lumen. Reduced CCO activity suggests that disturbance in BBB is probably related to the increase in vesicular transportation and/or energy failure. Reduction of MAO activity indicates that damages to mitochondria exist in the capillaries. Cerebral blood vessels are prone to damage by released substances from activated platelet in vivo.
Production and characterization of a murine monoclonal antibody against human thromboxane synthetase
Authors: Wernet P , Haurand M , Nüsing W , Schneider E M , Jaschonek K , Ullrich V
Published: August 2018

Eicosanoids appear to have an important role in the actual momentary regulation of tissue blood flow. The function of constricting blood vessels by affecting the vascular tone has been assigned to thromboxane. Thromboxane synthetase, the enzyme responsible for the conversion of Prostaglandin-H2 into thromboxane A2, has been shown to be present in platelets, lung fibroblasts and the brain. Recently, thromboxane synthetase has been totally purified. The enzyme isolated from platelets appears to have a molecular weight of 58,800 Dalton and to belong to the group of cytochrome P450 proteins. In order to make a monoclonal antibody against thromboxane synthetase, BALB/c mice were injected four times i.m. with 10, 5, 5 and 4 μg of the platelet purified enzyme in complete Freund's adjuvant. The serum antibody titer against thromboxane synthetase in an ELISA was higher than 1:1000 after the second boost. One mouse received a fifth i.v. injection of 10 μg of the purified enzyme. One monoclonal antibody of the several hundreds of hybridomas screened in an ELISA revealed specific activity against thromboxane synthetase with a titer of 1:512 present in the culture supernatant. After recloning this reagent, called T0300, was used for the preparation of an immunoaffinity column, where it also reacted specifically. In immunoprecipitation experiments T0300 was able to precipitate a 58,000 D molecule. Also the biological activity of thromboxane synthetase could be blocked by monoclonal antibody T0300. In addition this reagent was employed in indirect immunofluorescence on leukemic cells employing a FACS IV cytofluorometer. Here specific staining of two megacaryocytic blast cell populations could be demonstrated. Thus T0300 appears to be a monoclonal antibody against human thromboxane synthetase.
EICOSANOIDS AND VASOSPASM
Authors: Wilkinson D , Vowden P , Latif A B , Rajah S M , Kester R C
Published: August 2018

To investigate the hypothesis that abnormalities in prostanoid metabolism may be an aetiological factor in Raynaud's syndrome (RS) we have measured the stable metabolites 6-keto-prostaglandin F1α (6-keto-PGF1α ) and thromboxane B2 (TXB2), using a radioimmunoassay. The table gives their levels (in ng/ml) and ratio (mean±SD) in healthy volunteers and in a group of patients with RS.

In the control group there was no significant correlation between prostanoid levels and age, sex or smoking habit. In primary Raynaud's TXB2- levels and the ratio tend to be elevated from our control values. Patients with Vibration White Finger Disease (VWF) have a similar eicosanoid profile to that of the primary Raynaud's group with elevated TXB2 levels. One patient in this group had markedly raised 6-keto-PGF1α and TXB2 levels and later proved to have a circulating auto-antibody. Menstruating females in our study showed both elevated TXB and ratios similar to the primary group. It is interesting to note that menstruating females have an increased sensitivity to cold with abnormal cold stress tests. Patients with Raynaud's syndrome secondary to a systemic disorder have markedly elevated 6-keto-PGF1α and TXB2 levels although the ratio of the two remains within our normal range. There is a significant difference between secondary Raynaud's and all other groups investigated in the study (Mann-Whitney U test p = 0.04). Further research into prostanoid metabolism may yield a greater understanding of the pathophysiology of Raynaud's syndrome. Therapy may best be aimed at altering the levels of these important local hormones.
THE RECOVERY OF ENDOTHELIAL CELL AND PLATELET PROSTAGLANDIN SYNTHESES AFTER ADMINISTRATION OF ASPIRIN AND INDOBUFEN
Authors: Sheehan S J , Latif A B , Bibby S R , Kester R C , Rajah S M
Published: August 2018

Suppression of endothelial prostacyclin (PGI2) by cyclooxygenase inhibition may contribute to the formation of neointimal hyperplasia. While aspirin (ASA) inhibits platelet function for several days, it returns to normal within 24 hours after indobufen (IDB), a reversible cyclooxygenase inhibitor, suggesting that prostacyclin may recover at an even faster rate. In a randomised, controlled study, recovery of platelet and endothelial cell prostaglandin synthesis was compared in 49 New Zealand white rabbits following indobufen (3.5 mg/kg) or aspirin (5 mg/kg). Prostacyclin in arterial incubates and platelet thromboxane A2 (TXA2) production in serum were measured by radioimmunoassay of their meta-bolities, 6Keto PGF1 and thromboxane B2. Platelet prostaglandin synthesis was also monitored by changes in serum malondiealdehyde (MDA).

At one hour, both ASA and IDB significantly inhibited PGI2 (p<0.005). TXA2 (p<0.005), and MDA (p<0.02). After IDB, TXA2 and MDA levels returned to normal by 24 hours while inhibition continued in the ASA group (p<0.01). PGI2 synthesis following IDB recovered at 8 hours against 24 hours in the ASA group. We conclude that while both drugs inhibit platelet and endothelial prostaglandins, the earlier recovery of prostacyclin synthesis following indobufen may allow the endothelium to resume its physiological function.
THE REDUCED CAPACITY OF THROMBOXANE FORMATION IN THROMBIN-PREACTIVATED PLATELETS IS NOT CAUSED BY DEPLETION OF THEIR ARACHIDONIC ACID POOL
Authors: Scharf R E , Stockschläder M , Reimers H J , Schneider W
Published: August 2018

Thromboxane (TX) synthesis of washed human platelets pretreated with high concentrations of thrombin (0.5-2.0 U/ml) for 20 sec is significantly reduced upon further thrombin stimulation. Compared to controls (tyrode-pretreated platelets), thrombin-preactivated platelets recover normal TX synthesis following exposure to exogenous arachidonic acid (AA) indicating that short-time thrombin treatment does not inactivate platelet cyclooxygenase or TX synthetase (Blood 63: 858, 1984). To evaluate whether the reduced TX synthesis upon -the second thrombin exposure is due to depletion of their AA precursor pool, thrombin-pretreated platelets and tyrode-pretreated platelets (5×108/ml) were resuspended in autologous ACD plasma and incubated at 37°C with 0.2 μCi 14C-AA (20 μM) for 60 to 90 min in the presence of PGE1 (10 μM). Mean platelet uptake of 14C-AA (disappearance of radioactivity from the supernatant) was 12+3 nmoles AA/109 platelets and did not differ significantly between thrombin-pretreated platelets and controls. Thrombin-pretreated platelets released 10% or 4.5% of their 14c-activity upon further exposure to thrombin (2 U/ml) or collagen (8 μg/ml), respectively. The release from control platelets (15% with thrombin, 6.5% with collagen) did not differ from that of thrombin-pretreated platelets. However, even after incubation in ACD plasma, thrombin-pretreated platelets continued to form significantly less TXB2 (5.0±1.6 nmoles/109 platelets) than controls (9.7±2.2 nmoles/109 platelets, p< 0.05). These data indicate that the reduced capacity of thrombin-pretreated platelets is due neither to a depletion of the endogenous AA pool nor to an inactivation of cyclooxygenase or TX synthetase. The reduced TX synthesis capacity may be caused by a modification, destruction or desensitization of the platelet thrombin receptor as a consequence of the preceding thrombin stimulation.
HYPERCOAGULABILITY AND REDUCED SENSITIVITY TO WARFARIN IN RATS FOLLOWING CAFFEINE INTAKE
Authors: Gerna M , Donati M B
Published: August 2018

The effect(s) of caffeine Intake on cardiovascular functions are still a matter of debate. We have considered here the level of the prothrombin complex activity as a parameter of blood coagulability following caffeine Intake. The activity of the four vitamin K-dependent clotting factors (II, VII, IX and X) was tested after acute and chronic administration of high doses of caffeine. With a single administration of 50 mg/kg caffeine a statistically significant increase in the activity of factors VII, IX and X was observed. The increase lasted for 4, 3 and 2 days respectively. Factor II was not affected by caffeine administration. The same effect was observed during chronic administration (50 mg/kg/day). With a lower dose of caffeine during chronic administration (5 mg/kg/day) only factor X showed a significant increase. A single dose of caffeine (50 mg/kg) given to animals 24h before-a dose of warfarin (0.4 mg/kg i.v.) markedly reduced the anticoagulant effect of warfarin, as measured by the thrombotest. During the latter experiment we measured also the rate of synthesis (Rsyn) of the prothrombin complex activity. Rsyn, during warfarin treatment, was significantly higher in the rats pretreated with caffeine than In controls. γ-Carboxylase activity in rat liver mlcrosomes was measured after administration of 50 mg/kg of caffeine as a single dose. The Incorporation of 14CO2 in the endogenous precursor was higher in the rats pretreated with caffeine than In controls (84,905 cpm/mg of protein versus 59,826 cpm/mg of proteins); this difference, however, was not statistically significant. In conclusion, caffeine jntake may affect the clotting system mainly by stimulating the synthesis of factors of the prothrombin complex and, as a consequence, their response to coumarin anticoagulation.
FATTY ACIDS AND PLATELET FUNCTION IN A SOUTH AMERICAN INDIAN GROUP WITH A HIGH DIETARY CONSUMPTION OF DOCOSAHEXAENOIC ACID
Authors: Bosch V , Bosch N , Valles M , Ortíz N , Gómez R
Published: August 2018

The effect of dietary polyunsaturated fatty acids on hemostasis has elicited much interest. We studied indians from the Orinoco river shore, whose main animal protein intake derives from river fishes with a high confceqt of 22:6n-3 (0.2g/100g)r We determined in 50 indians plasma phospholipid fatty acids (FApl) by gas/liq chromatography and bleeding time by Symplate I divice (BT), in 15 were analyzed platelet count, aggregation with collagen and ADP, platelet factor 3 availability (PF3), platelet phospolipid fatty acids (FApt) and plasma vWFAg.RA from human milk was also determined. Subjets from the city of Caracas served as control. Data on BT, FAlp and FApt are shown in table (X±SD).

FA Composition of milk showed that indians have 3 times more 22: 6n-3 than controls. Platelet studies shewed normal number and morphology. Percent platelet aggregation with collagen (4ug/ml) was below 50% in 4 of indians, 2 of them with a BT within the control range. Maximum slope of aggregation with ADP (4uM) was diminished in 2 cases. Diference in PF3 was not significant, VWFAg range from 50 to 100% and control from 53 to 127%. In conclusion we have found a population that shows an increased plasma and platelet 22:6n-3 and a prolonged BT most likely of dietary origen. Mechanism by wich n-3 FA modifies BT needs fur ther investigation.
SEX DIFFERENCE IN THE INHIBITORY EFFECT OF ASPIRIN UPON THE DEVELOPMENT OF OCCLUSIVE ARTERIAL THROMBOSIS IN RATS
Authors: Uzunova A
Published: August 2018

The aim of the study was to investigate the effect of aspirin upon the development of occlusive arterial thrombosis in rats of either sex. For this purpose 3 months old male and female Wistar rats were pretreated with three different doses of aspirin: 10, 30 and 60 mg/kg b.w., orally by stomach tube, 3 hours before the thrombosis procedure. Occlusive arterial thrombosis was induced by the method of Hornstra and Vendel-mans-Starrenburg (1973). The dry thrombus weight (TW) and the obstruction time (OT) of the bypass-cannula served as criteria for the degree of the development of thrombosis.

The results showed a dose-dependent statistically significant inhibition of arterial thrombosis in male rats by all doses of aspirin studied. TW was decreased 2, 2.5 and 9 times by 10, 30 and 60 mg/kg aspirin respectively. OT was significantly prolonged in male rats by all doses of aspirin studied. Inhibition of arterial thrombosis in female rats was achieved only by the largest dose of aspirin tested - 60 mg/kg: TW was decreased and OT was prolonged. Paradoxically, female rats treated with 10 mg/kg aspirin showed a tendency for augmentation of arterial thrombosis which contrasted to the significantly decreased TW and prolonged OT of male rats given 10 mg/kg aspirin. Female rats treated with 30 mg/kg aspirin showed only a tendency for inhibition of arterial thrombosis without any significant difference for both, TW and OT. The results were interpreted as suggestive the existance of sex differences in cyclooxygenase inhibition by aspirin and/or the prostaglandin system in rats. The data obtained were in agreement with epidemiological observation of uneffectiveness of aspirin treatment of female patients with stroke. They also showed the need for the most appropriate dose of aspirin for clinical purposes which might be effective in females too.
MODE OF ACTION OF A NOVEL ANTI-PLATELET AGENT (E-5510)
Authors: Saeki T , HARADA K , Youshimura T , Nakamura Y , Fujimori T , Katayama K E , Yamagish Y , Yamastu I , Ikeda I
Published: August 2018

A novel anti-platelet agent, 4-cyano-5,5-bis(4-methoxy-phenyl)-4-pentenoic acid(E-5510), has been shown to inhibit platelet aggregation and secretion induced by thrombin as well as by other inducers such as ADP, collagen and PAF. Although E-5510 can act as a cyclooxygenase inhibitor,inhibition of cyclooxygenase may not be the primary mode of action since this compound effectively inhibits thrombin-induced platelet activation. In this paper, effects^of E-5510 on arachidonic acid (AA) metabolism, intracellular Ca++ and cAMP in human platelets are examined.

(1) Effect on AA metabolism. Platelets prelabeled with [14 C]-AA were stimulated with thrombin. E-5510 inhibited not only thromboxane A2 and HHT generation but also 12-HETE generation in a dose-dependent fashion. The total AA released was also reduced by E-5510. An almost 50% reduction was obtained by 10 uM of this compound. On the other hand, a cyclooxygenase inhibitor such as U-53059 increased 12-HETE generation in a dose-dependent fashion. In addition to the inhibition of AA metabolism, E-5510 exerted inhibitory effects on phosphatidic acidgeneration, which suggests the possible inhibition of phospholipase C activity by this compound.

(2) Effect on intracellular Ca++ and protein phosphorylation. Intracellular Ca++ mobilization was examined using Fura-2 loaded human platelet suspension. The increase in intracellular Ca induced by thrombin was inhibited by E-5510 and the increase in phosphorylation of 40 K protein was also suppressed by this compound after the stimulation of human platelets by thrombin.

(3) Effect on cAMP. Platelets were incubated with 10-100 uM of E-5510 and the cAMP content in human platelets was measured. E-5510 increased the cAMP content in a dose-dependent fashion. In platelet homogenate, E-5510 inhibited phosphodiesterase activity with an IC50 of 10 uM.

These results suggest that E-5510 may inhibit platelet aggregation and secretion through the multiple modes of action, such as inhibition of phospholipase C, phosphodiesterase and cyclooxygenase, in the process of platelet activation.
ANTITHROMBOTIC OLIGOAMINES
Authors: Rehse K , Lukens U , Leibring S , Schein V , Kesselhut A
Published: August 2018

We have found that oligoamines of the general formula R2−3X(R=-(CH2)m-NH-(CH2)n-Y) in which X and Y may be aliphatic, alicyc-lic, aromatic or even heterocyclic moieties are a new class of compounds which exhibit platelet aggregation inhibiting and anticoagulant activities in vitro and have antithrombotic properties in vivo. The compound RE 1492 (N,N’,N1’-Tris-4-phenylbutylbenzene-1,3,5-trimethanamine) is chosen as example to demonstrate these effects. In PRP the following IC50 of RE 1492 (inductor in brackets) were measured: 3 ¼mol/L (Collagen), 1 ¼mol/L (ADP, 2ndphase), 7.5 μmol/L (ADP, lstphase), 2,5 μmol/L (A 23187, 2ndphase), 7,5 μmol/L (Ionophor A 23187, lstphase), 30 ¼mol/L (Thrombin). The inhibition of the aggregation Induced by ADP could as well be demonstrated in whole blood. The formation of fibrin was inhibited as shown by the prolongation of the thromboplastin time (Quick) and the partial thromboplastin time (PTT) the first being more sensitive (25% of normal at 50 ymol/L) than the latter (25% of normal at 100 ymol/L). The reason was the inhibition of coagulation factors in the following order: VII (25% of normal at 12.5 ¼mol/L) >WErwnr/IX (25 ¼mol/L) »X (200 μmol/L). The thrombin time remains normal. The antithrombotic properties of RE 1492 were investigated in an in vivo thrombosis model. The formation of platelet thrombi in mesenteric arterioles and venoles of rats (diameterM5 ym) was induced by a laser beam. In controls 1,76±1,14 (SD) shots (50 msec, 50 mW) on the arterioles were necessary for thrombus formation. Twenty minutes after i.v. application of RE 1492 this number rose to 3,18±2,08 (3 mg/kg, p ≤ 0,01, X2-test) and 4,59±1,93 (10 mg/kg, p ≤ 0,01) in arterioles. In venoles of the control animals 1,29±0,45 shots were necessary for thrombus formation. This number rose to 2,11±1,62 (p ≤ 0,05) after 3 mg/kg and 3,28±2,03 (p0,01) after 10 mg/kg. As the number of shots applied was limited to five an average shot number of 5± SD would indicate that no thrombus formation takes place at all. As RE 1492 does neither influence the metabolic pattern of arachidonic acid in platelets nor the activity of phosphodiesterase or adenylatcyclase it is supposed that the oligoamines exert their effects by interaction with phospholipids (PL) resulting in a “membrane stabilization” in platelets and inhibition of PL dependent coagulation factors during fibrin formation.
ORALLY ADMINISTERED VITAMIN B PROLONGS THE BLEEDING TIME AND INHIBITS PLATELET AGGREGATION IN HUMAN VOLUNTEERS.
Authors: Randi A M , Sacchi E , Cattaneo M
Published: August 2018

It is known that mM concentrations of vitamin B6 inhibit human platelet aggregation and fibrin formation in vitro. There are very few and controversial data on the ex vivo effects of vitamin B6 on hemostatic parameters. We evaluated the effects of oral administration of vitamin B6 on bleeding time (BT), fibrin formation, platelet aggregation (PA) and the release reaction (RR). Vitamin B6 300 mg/day p.o., was given to 18 healthy volunteers (8 M, 10 F, aged 23-35) for 8 days. BT was measured before the first dose (Baseline), 2 hr after the first (Day 1) and the last dose (Day 8). In 7 subjects BT was measured also 7 days after the suspension of the drug (Day 15). Before and 2 hr after the first dose, prothrombin time (PT), partial thromboplastin time (PTT), thrombin time (TT), PA and the RR induced by different concentrations of ADP, PAF-acether, collagen (coll), epinephrine (epi), arachidonate (AA) were studied. BT was significantly prolonged after vitamin B6 administration, and returned to baseline values 7 days after suspension of the drug. PA and the RR induced by 1 uM ADP, 1 ug/ml coll or 5 uM epi were significantly inhibited 2 hours after vitamin B6 administration. Vitamin B6, however, did not affect PA or the RR induced by 0.2-2 uM PAF-acether, 2 uM ADP, 1 mM AA, 2 ug/ml coll, nor did it affect PT, PTT or TT. These data show that orally administered vitamin B impairs primary hemostasis, but does not affect fibrin 6 formation, as measured with standard coagulation tests.
CORRELATION BETWEEN THE ANTICOAGULANT AND ANTIPLATELET EFFECT OF D-PHE-PRO-ARG-H (RG-2958).
Authors: Bagdy D , Bara-bás É , Sebestyén L , Diószegi M , Fittler Zs , Bajusz S , Széll E
Published: August 2018

Anticoagulants usually have no antiplatelet effect and platelet function inhibitors do not interact with the coagulation factors. Since thrombin has a decisive role in thrombus formation (growth and stabilization), inhibitors of the effect of thrombin on platelets may be of special importance in developing a novel type of anticoagulant with antiplatelet properties.

D-Phe-Pro-Arg-H /I/ designed and synthetized in our Institute was found to be a highly specific,reversible non-competitive inhibitorgOf thrombin,a specific platelet agonist. (K.= 1x10-8 M). /I/ was administered parenterally and orally to white New Zealand rabbits and to beagle dogs. The kinetics of action was recorded by measuring the WBCT, APTT, PT, TT,platelet count (PC) and platelet aggregation (PA). Optimum degree of anticoagulation was considered by the values proposed by Verstraete and Verwilghen. /I/ was shown to be a highly specific inhibitor of PA induced by thrombin.No direct interaction between the inhibitor and the platelet membrane could be detected. Aggregability of human platelets in citrated PRP and that of the gel-filtrated platelets induced by ADP or collagen did not change after incubation with /I/. The antiplatelet effect of /I/ was studied by ex vivo experiments where the inhibitor was the anticoagulant ( 30 ug/ml whole blood ) instead of citrate. Comparing the aggregability caused by several inducers in citrated human PRP with that of in /I/-PRP a significant difference was observed when epinephrine was the PA-inducer. /I/ acts via formation of an enzyme-inhibitor complex that inhibits the binding of thrombin on their receptor-sites at the platelet membrane. In vivo experiments showed a close correlation between TT and PA induced by thrombin. /I/ proved completely harmless to platelets and red blood cells. No significant change in PC could be detected.
DIFFERENTIAL INHIBITION OF THE PLATELET ACTIVATION SEQUENCE: SHAPE CHANGE, MICRO- AND MACRO- AGGREGATION, BY A STABLE PROSTACYCLIN ANALOGUE (IL0PR0ST)
Authors: Pedvis L G , Wong T , Wylie J , Frojmovic M M
Published: August 2018

The relative sensitivities of ADP-induced activation, and prostaglandin-mediated inhibition, were determined for rates of platelet shape change (SC), early platelet recruitment measured by electronic particle counting (PA), and turbidometrically-measured aggregation (TA). Studies were performed in stirred citrated platelet-rich plasma from 7 healthy human donors. The [ADP]½ ([ADP] giving half maximal rate) was determined for the sequence of activation steps expanding on Holmsen’s classical scheme: unactivated platelets → SC → PA → TA. Distinct ADP sensitivities were obtained from log dose-response studies, with a relative dose dependency in the order of [ADP] ½ TA → [ADP] ½ PA → [ADP]> SC of 4:3:1. Sex differences in ADP sensitivities ([ADP]½), for rates of early platelet recruitment measured at 3 seconds were studied from a pool of 20 females and 19 males. Values obtained between the two sexes were comparable (p > 0.05) and independent of hematocrit. Differential inhibition of the above activation scheme was evaluated with Iloprost (ZK 36 374), a stable carbacyclin analogue of prostacyclin (PGI2), with similar potency as PGI2 for the same platelet receptors. Log dose - response curves for inhibition were measured at one high [ADP] (> 1.5 μM) for all 3 parameters, or at respective [ADP]½ values for each parameter. IC5u values ([ZK J causing 50% of inhibition) for inhibition of TA:PA:SC were found in the relative ratios of ∽ 1:3:5, when normalized and expressed as nM ZK per pM ADP used as activator. Thus, ∽ 3x and ∽ 5x more ZK, and likely PGI2, is required to respectively inhibit PA and SC, than that needed to inhibit TA. As observed above for activation, no sex differences in ZK sensitivities were observed (p > 0.1) for 6 males and 6 females. The range of ZK used in this study was below the threshold (∽ 3 nM) generally reported for measurable increases in total basal cyclic 3’ ,5’ adenosine monophosphate (cAMP). This suggests that for each parameter, any increase in cAMP may be associated with selective intracellular pools. The relationship between ZK or PGI2 and intracellular signals remains to be determined.
PLATELET FUNCTION IN VASOSPASTIC DISORDERS
Authors: Wilkinson D , Vowden P , Gilka L , Rajah S M , Kester R C
Published: August 2018

If vasospastic disorders are associated with abnormal eicosanoid metabolism then disturbed platelet function may be observed. We have measured platelet aggregation (PAG) at 37°C to ADP (8 1:1 sequential dilutions from 10μM), collagen (2mg/l) and adrenaline (5μM) in 37 patients with Raynaud's syndrome (21 primary (RD), 9 secondary to scleroderma (RP) and 7 vibration white finger disease (VWF)) and 10 normal subjects (N). In addition we have determined the platelet aggregate ratio (PAR). PAG was expressed as the percentage fall in optical density after the addition of the aggregant. We also recorded the duration of the collagen lag phase and the concentration of ADP at which disaggregation occurred (DAC).

Patients with RP showed enhanced PAG compared to normals. This reached significance for collagen lag phase (p<0.001), percentage PAG at 3 minutes to ADP 1.25μM (p<0.05) and the DAC (p<0.01). Similar differences were observed between patients with RP and VWF. Patients with RP also showed significantly enhanced PAG with regard to ADP 1.25μM (p<0.01) and the DAC, (p<0.05) compared to patients with RD. There was no significant difference in platelet function between normal subjects and patients with VWF or between normal subjects and patients with RD. Only with regard to DAC did patients with RD differ from patients with VWF (p<0.05). The differences observed in PAG were not reflected by the PAR. Patients with RP have significantly enhanced PAG when compared to all other groups. This may relate to eicosanoid metabolism and provides a rationale for treatment.
INCREASED PLATELET AGGREGABILITY IN NATIVE WHOLE BLOOD IN THE ADULT RESPIRATORY DISTRESS SYNDROME (ARDS)
Authors: Mackie I J , Bihari D , Machin S J
Published: August 2018

The measurement of aggregation in whole blood allows the study of platelets in their natural milieu, but may still have anticoagulant induced artifacts; citrate decreases extracellular (Ca++) and heparin activates platelets. A technique for measuring aggregation in unanticoagulated (native) whole blood (NWB) was developed; blood is diluted in prewarmed saline with and without platelet agonists and aggregation is monitored by electrical impedance. Spontaneous and collagen induced aggregation were measured and the effect of prostacyclin analogue ZK 36,374 studied. The time until response (lag), aggregation rate, and clotting time were measured. Normal blood gave a cv of <7% for the NWB parameters; collagen gave a shorter lag and higher rate than in citrated whole blood (CWB). The lag and rate were inhibited in a dose dependent manner by ZK 36,374. 6 patients were studied on admission with ARDS and followed for several days. All had increased aggregation to collagen, which was more pronounced in NWB than CWB. A shortened lag was seen in some patients, but none showed spontaneous aggregation. ARDS patients showed no inhibition to ZK 36,374, and the heightened aggregation was not influenced by 2mM ASA, or normal plasma, while 2.5mM EDTA abolished all responses. Serum thromboxane B2 was normal, and increased flux through the cyclo-oxygenase pathway therefore unlikely. A parallel study has shown very low levels of antithrombin III (ATIII) in these patients, which may mean an increased thrombin generation rate with more thrombin available for platelet activation. This partly explains the difference in degree of hyperaggregability seen with NWB and CWB. During recovery from ARDS, collagen aggregation and the response to ZK 36,374 normalises at the same time as AT-III increases.
SYNTHESIS OF PLATELET ACTIVATING FACTOR BY HUMAN BLOOD PLATELETS
Authors: Struk A , Prins A , Schaap M C L , ten Cate J W , van den Bosch H
Published: August 2018

Synthesis of platelet activating factor (PAF) by human blood platelets is a controversial issue. Whereas some groups have reported the synthesis, induced by thrombin, collagen or Ca2--ionophore Azsio?, others were unable to obtain for instance the thrombin-induced PAF synthesis. Also, synthesis of only up to 6 pMoles PAF/10 platelets has been reported, but leucocytes may synthesize up to 6000 pMoles PAF/10 cells. Only an 0.1% leucocyte contamination would thereby explain the “ platelet PAF synthesis” . We therefore optimized the PAF synthesis by human blood platelets and leucocytes, induced by thrombin and A23i07. As platelets have been reported to show an increased PAF synthesis upon treatment with phenylmethylsulfonylfluoride (PMSF), this was also investigated.

Leucocytes were optimally stimulated with 10 uM Azale? (mean ± SD 4678 ± 2033, range 1698-7058 pMoles PAF/10 cells, n=6), but could not be stimulated by thrombin. PMSF treatment itself induced PAF synthesis by these cells, but this was not influenced by thrombin or A23ia7.

Platelet suspensions, with <0.005% leucocyte contamination as determined by light microscopy after Jenner-Giemsa staining, could synthesize PAF when treated with PMSF and stimulated with 2.5 IU/ml thrombin (mean ± SD 0.6 ± 0.3, range 0.3-1.0 pMoles PAF/10 platelets, n=6), but in these suspensions A23io7-induced synthesis could not be demonstrated.

The results indicate that synthesis of PAF by human blood platelets is not due to contaminating leucocytes, if thrombin is used as the stimulus. These results were confirmed by 3H-acetate uptake experiments.
SELECTIVE ANTAGONISTS OF PAF: INTERFERENCE WITH PLATELET FUNCTION AND EFFECT ON THROMBOGENESIS INDUCED BY SUBENDOTHELIUM.
Authors: Hadvary P , Baumgartner H R
Published: August 2018

Platelet activating factor (PAF) is a very potent excitatory agonist of blood platelets but the physiological importance of this mediator in platelet thrombus formation is not known. We investigated the effect of two chemically unrelated selective inhibitors of PAF-induced platelet aggregation on thrombogenesis induced by rabbit aorta subendothelium (SE) using an ex vivo perfusion system.

Ro 19-3704 is a highly potent inhibitor structurally related to PAF. This compound inhibits PAF-induced aggregation of rabbit platelets in platelet rich plasma in vitro competitively. Against 4 nM PAF, a concentration resulting in submaximal platelet aggre-gregation velocity, the IC50 was 70 nM. Inhibition was highly selective for PAF-induced aggregation, since aggregation induced by collagen (HORM, 5 yg/ml), ADP (1 yM) or thrombin (0.4 U/ml) was not inhibited even at a concentration as high as 10 yM. Bro-tizolam, a triazolobenzodiazepine reported to be a selective inhibitor of PAF-induced platelet activation, had in our system an IC50 of 200 nM. The selective benzodiazepine antagonist Ro 151788 was without effect on inhibition of PAF-induced platelet activation by brotizolam.

Ro 19-3704 was given intravenously to rabbits as a bolus of 0.2 mg/kg followed by constant infusion of 0.02 mg/kg/min. This dosage provoked ex vivo a constant right shift ratio of the dose response curve for PAF-induced aggregation (RSR[PAF]) by a factor of 25 to 35. Brotizolam was given orally at a dose of 100 mg/ kg together with 300 mg/kg of Ro 15-1788 (to antagonize the central effects) 90 minutes before starting the perfusion experiment, resulting in a RSR[PAF] of 35 to 135. ADP induced platelet aggregation was not impaired by either compound. SE was exposed to the non-anticoagulated blood withdrawn from the carotid artery for 3 min at 2600 s-1 and for 20 min at 200 s-1 shear rate. Quantitative morphometric evaluation showed that SE coverage by platelets and by fibrin, thrombus area and thrombus height were all unchanged by the PAF antagonists at low and at high shear rates despite a very substantial inhibition of PAF-induced platelet aggregation. Therefore a major role of PAF in SE-induced thrombogenesis seems unlikely.
THE SECRETORY PATHWAY IN PLATELETS STUDIED BY CRYO-FIXATION
Authors: morqenstern E , Patscheke H
Published: August 2018

It is widely held, that the constituents packed in the a -granules are released by stimulated platelets via the surface connected system (SCS). By means of the fast-freezing and freeze substitution technique (which allow the investigation of membrane fusion) we found a secretory pathway in platelets (compound exocytosis) without an involvement of the SCS during the release of a-granules. To study the process of a-granule secretion human platelets concentrated in citrated blood plasm were stimulated with thrombin or collagen. 20 - 120 seconds after stimulation the platelets were rapidly frozen with a metal-mirror attachment to the KF 80 cryofixation unit (REICHERT-JUNG). Using plastic spacers droplets of the PRP were slammed against a copper block at 80 K at a rate of 0.2 m/sec. After cryofixation the specimens were transferred (in liquid nitrogen) into a Cs-auto cryosubstitution unit (REICHERT-JUNG). Cryosubstitution was programmed for 48h at 193 K in acetone with 4% osmium tetroxide. The temperature went automatically up to room temperature at a rate of 10 K/h. The specimens were embedded in araldite. The analysis of serial ultrathin sections of platelets in different phases of exocytosis revealed the following. a -granules in apposition showed different stages of swelling and dispersal of their electron dense matrix. Membrane appositions were also found between a -granules. The contraction of a sphere of microfilaments and microtubules during stimulation seemed to support this process. On the other hand this internal contraction prevented most of the a-granules from contacting with the plasmalemma. We observed fusion between swollen -granules in apposition and the plasmalemma and swollen and unswollen a -granules. Thus, large compound granules were formed frequently before fusion of the secretory organelles with the plasmalemma took place. These observations suggested that a -granules in stimulated platelets performed a compound exocytosis after swelling. The process seemed to start with the apposition of a -granule membranes to the plasmalemma. It cannot yet be answered whether the swelling of the granules is due to an osmotically driven influx of water or due to an influx after microfusion.Supported by DFG, Grant Mo 124/2-4
THE EFFECT OF ENZYMATIC TREATMENT OF RABBIT PLATELETS ON THE BINDING OF IMMUNOGLOBULIN G TO THE SURFACE
Authors: Evans R M , Packham M A
Published: August 2018

As shown previously, cleavage of surface glycoproteins on the platelet membrane shortens platelet survival. Since immunoglobulins have been implicated in the condition of idiopathic thrombocytopenic purpura in which patients have an elevated level of platelet-associated immunoglobulin G (I g G) and shortened platelet survival, we measured the binding of IgG to rabbit platelets that had been treated with neuraminidase or plasmin, to determine whether increased IgG binding to these platelets might be the signal that leads to their accelerated clearance. Rabbit platelets were washed and resuspended in calcium-free Tyrode-albumin solution (1.0 x 106/ μL), pH 6.5, and then incubated with either neuraminidase (0.1 U/mL at 22°C) or plasmin (activity: 1.6 ymol BAEE/min/mL platelet suspension at 37¼C) for 30 min. The platelets were then centrifuged and resuspended in heat-inactivated rabbit serum for 30 min to allow IgG to bind to the surface. After additional washing steps to remove non-specifically bound IgG, the amount Qt IgG that associated with the platelets was measured using an 125I-labeled goat anti-rabbit IgG binding assay. Binding data were analyzed by plotting the calculated fg of IgG bound/platelet versus the amount of 125-1 goat anti-rabbit IgG added. The binding curves for both neuraminidase and plasmin indicated significantly increased amounts of specifically bound IgG compared to control platelets. At a concentration of 30 μg/mL of labeled goat antirabbit IgG added, the amount of label associated with untreated rabbit platelets had plateaued at 0.36± 0.11 fg/platelet whereas the amount associated with neuraminidase-treated platelets was 5 times greater and with pi asmin-treated platelets, 1.5 times greater, and neither had plateaued. The results obtained with plasmin are of particular interest since this proteolytic enzyme shortens platelet survival and is present in vivo during thromboembolic events. These results indicate that IgG binds specifically to the surface of rabbit platelets following enzymatic alterations of the membrane glycoproteins and therefore may represent at least one mechanism that determines which platelets are cleared by the reticuloendothelial system.
A GENETIC VARIANT OP PLATELET MEMBRANE GLYCOPROTEIN lb ASSOCIATED WITH A MILD BLEEDING DISORDER
Authors: Meyer M , Schellenberg I , Hofmann B , Vogel G
Published: August 2018

Mild bleeding symptoms in a female patient were shown to be related to defective platelet function: Aggregation induced by ADP and PAP was decreased and platelet spreading was disturbed. Platelet membrane glycoproteins were analyzed by various electrophoretic procedures (SDS electrophoresis, nonreduced-reduced 2-dimensional and high resolution 2-dimensional electrophoresis). These studies revealed a decreased concentration of normal glycoprotein lb (GP Ib) and the appearance of an additional glycopeptide with an apparent Mr of 160.000 under reducing conditions. This component was strongly labeled in intact cells by periodate-3H-sodium borohydride. Staining characteristics of the additional glycopeptide were also similar to those of GP lb. Immunobiotting using a monospecific anti-glycocalicin antiserum provided conclusive evidence for a structural variant of GP lb molecularly characterized by an increase of about 20.000 in apparent Mp of the large subunit. The abnormal glycopeptide is cleaved by the endogenous calcium-dependent neutral protease and the resulting glycocali-cin exhibits the same increase in Mr as intact variant GP lb. The abnormal glycopeptide was also detected in 3 relatives of the proposita. All of the carriers are obviously heterozygous for the underlying mutant gene.
A MONOCLONAL ANTIBODY AGAINST AN ERYTHROID ONTOGENIC ANTIGEN IDENTIFIES GP IV ON HUMAN PLATELETS
Authors: Kieffer N , Edelman L , Edelman P , Legrand C , Breton-Gori us J , Vainchenker W
Published: August 2018

A murine monoclonal antibody (FA6-152), obtained by immunizing mice with fetal human erythrocytes agglutinated fetal but not adult erythrocytes and bound to both adultand fetal monocytes, platelets andreticulocytes. The antibody did not react with lymphocytes or granulocytes (P. Edelman et al., Blood, 1986, 67, 56). Fluorescent labeling of marrow cells and of in vitro BFU-E, CFU-GM and CFU-MK derivedcolonies revealed that the antigen defined by FA6 was absent from the granulocytic precursors and was detected on the megakaryo-cytic lineage at a later stage of differentiation than major platelet membraneglycoprotein markers. In contrast,the antigen appeared as a very early marker of erythroid differentiation.

In the present report, we performed immunoprecipitation experiments on surface labeled platelets toidentify the platelet FA6 antigen.A band of apparent Mr - 85,000 wasimmunoprecipitated from iodinated platelets. This band was also revealed after periodate/Na[3H]BH4 orneuraminidase galactose oxidase/Na[3H]BH4 surface labeling of the platelets, providing evidence that the FA6 antigen corresponds to platelet GP IV. Preliminary studies revealed that FA6-IgG inhibited platelet aggregation induced by low doses of thrombin without affecting the platelet release reaction. Our results therefore suggest that GP IV, which isa multi-lineage hematopoietic differentiation marker, could play a role in cell-cell or cell-substrateinteractions common to different hematopoietic cell types.
A MORPHOLOGICAL APPROACH TO THE MECHANISMS OF RISTOCETIN INDUCED PLATELET AGGLUTINATION(RIPA)
Authors: Escolar G , Monteagudo J , Villamor N , Garrido M , Castillo R
Published: August 2018

Changes in the morphology of human platelets induced by Ristocetin (RIPA) have been analyzed at ultraestructural level by means of a tannine acid procedure. Studies were completed with aggregation and binding experiments. Modificationsof these tests induced by apyrase,a monoclonal antibody (Mab) to GPIIb/IIIa and EDTA were also investigated.

Transmission electron microscopy reveals that ristocetin precipitates adhesive proteins on plateletmembrane. An electron-dense deposit was noticeable within 20 secondsafter ristocetin was added. When experiments were carried out in theaggregometer cuvette under stirring, groups of platelets become activated, change shape, and finally aggregate releasing part of their content. The morphology of aggregates did not differ from those formed in the presence of ADP.

Aggregation studies demonstrated that a Mab to GPIIb/IIIa modifies the extent and the rate of the aggregation curve when RIPA was performed in citrated platelet rich plasma (c-PRP). Apyrase modified the extent but not the slope of the curve. Neither the antibody nor apyrase modified RIPA when it was performed in PRP obtained in presence of EDTA. Binding experiments confirmed that I-vWF bound to platelets in presence of ristocetin was not modified by apyrase or anti-GPIIb/IIIa Mab. All these facts together suggest that RIPA,when performed in c-PRP besides reflecting the interaction of GPI with vWF, is also testing other mechanisms of the platelet function including exposure of GPIIb/IIIa complex, interaction of fibrinogen with this glycoprotein, and the contribution of the release reaction.
THE DENSE TUBULAR SYSTEM IS PRESENT IN BOVINE PLATELETS
Authors: White James G , Rao Gundu H R
Published: August 2018

Human platelets contain two channel systems. The open canalicularsystem (OCS) consists of tortuous channels, continuous with the cellsurface and each other, and spreadthroughout the platelet cytoplasm.The dense tubular system (DTS) derives from rough endoplastic reticulum of the parent megakaryocyte.

It is also spread randomly through the platelet and forms specialized membrane complexes with elements of the OCS in one or two areasof the cytoplasm. The OCS serves as the conduit for discharge of granule products during the platelet release reaction. Elements of the DTS contain enzymes involved in prostaglandin synthesis and serve asreservoirs for calcium involved inplatelet physiological activities.Recent studies have shown that bovine platelets lack the OCS and usethe surface membrane as a conduit for the release of granule associated products.

The existence of a DTS and its functional role in bovine platelets has not been examined previously. Bovine platelets converted 14C-arachidonic acid to thromboxane B2 as efficiently as human cells. Fura 2 loaded bovine platelets stimulated by thrombin in the presence of EDTA developed cytoplasmic levels of calcium flux comparable to human cells, suggesting that bovine platelets must have a DTS. Ultrastructural procedures for cyto-chemical demonstration of platelet peroxidase and glucose 6 phosphatase resulted in selective deposition ofreaction product in channels of a DTS in bovine cells. Membrane complexes formed by interaction of theOCS and DTS in human platelets were absent. However, some elements of the DTS in cattle platelets are located very close to the cell surface. Results of these studies demonstrate that bovine platelets possess a functional DTS involved in prostaglandin synthesis and calcium flux.
REORGANIZATION OF ACTIN AND MYOSIN IN THE ACTIVATED PLATELETS
Authors: Shibatay N , Tanaka K , Okamoto K , Onji T
Published: August 2018

This study was done to clarify the intracellular dynamic arrangements of myosin(My) and actin(Ac) in activation process of human platelets (PLs) from unactivated to activated stage (clot retraction) in electron microscopy. The observation of unactivated PLs was done either in the fresh whole blood fixed directly with 0.1 % glutaraldehyde or in PLs isolated by gel filtration of platelet rich plasma(PRP) containing prostaglandin I2 (10 ng/ml). The isolated PLs mounted on a glass cover slip were used as activated PLs (adrerent ones). The contracted PLs were prepared in PRP incubated with thrombin (0.5 u/ml) and 20 mM CaCl- for 10-60 min. Treating PLs with 0.15 % Triton X-100 containing 0.05 % glutaraldehyde produced cytoskeleton. My and F-Ac were identified by an indirect immuno-cytochemical method using the specific antibody (rabbit IgG) against PL-My and protein A-gold and by demonstration of in “arrow-head” decoration by Ishikawa's method using skeletal meromyosin (HMM), respectively. [Results] (1) Unactivated PLs. Mys in monomer or oligomer distributed homogenously in scare association with cytoskeleton. Cytoskeletons were exclusively composed of F-Ac networks of crossolinked short filaments which were thinly distributed in the cytoplasm with partial connection to the cell membrance. (2) Surface activated spreading PLs. PLs adhered to the glass cover slip in dendritic forms. Mys were densely located around granulomere and formed linear arrays associated with F-Ac filaments of the cytoskeleton surrounding the granulomere and running straightly in cytoplasm. (3) Contracted PLs. Activated PLs protruded several filopodia in which networks or bundles of F-Ac filaments were found connecting to extracellular fibrin strand through cell membrene. Microfilaments formed arrow-head decoration with HMM pointing toward the cell body. The cytoskeleton in contracted PLs contained thick filaments of My-polymers attaching to F-Ac filaments end by end. It is concluded that the reorganization of Ac-My is the basis for the shape change, secretion and clot retraction of activated PLs.