NAADP regulates human platelet function

Platelets play a vital role in maintaining haemostasis. Human platelet activation depends on Ca2+ release, leading to cell activation, granule secretion and aggregation. NAADP (nicotinic acid-adenine dinucleotide phosphate) is a Ca2+-releasing second messenger that acts on acidic Ca2+ stores and is used by a number of mammalian systems. In human platelets, NAADP has been shown to release Ca2+ in permeabilized human platelets and contribute to thrombin-mediated platelet activation. In the present study, we have further characterized NAADP-mediated Ca2+ release in human platelets in response to both thrombin and the GPVI (glycoprotein VI)-specific agonist CRP (collagen-related peptide). Using a radioligand-binding assay, we reveal an NAADP-binding site in human platelets, indicative of a platelet NAADP receptor. We also found that NAADP releases loaded 45Ca2+ from intracellular stores and that total platelet Ca2+ release is inhibited by the proton ionophore nigericin. Ned-19, a novel cell-permeant NAADP receptor antagonist, competes for the NAADP-binding site in platelets and can inhibit both thrombin- and CRP-induced Ca2+ release in human platelets. Ned-19 has an inhibitory effect on platelet aggregation, secretion and spreading. In addition, Ned-19 extends the clotting time in whole-blood samples. We conclude that NAADP plays an important role in human platelet function. Furthermore, the development of Ned-19 as an NAADP receptor antagonist provides a potential avenue for platelet-targeted therapy and the regulation of thrombosis.     

Purification of human platelet calcium-activated protease. Effect on platelet and endothelial function

Calcium-activated protease (CAP) was purified from the cytosol fraction of homogenized human platelet concentrates using a combination of gel filtration chromatography and affinity chromatography on antipain aminohexyl-Sepharose and activated thiol-Sepharose 4B. Purified CAP is composed of two different polypeptides of Mr = 80,000 and 27,000. Half-maximal protease activity was observed at 0.52 mM Ca2+, and all activity was inhibited by antipain, leupeptin, and N-ethylmaleimide. Activated CAP showed a time-dependent inactivation in the presence of 1 mM Ca2+ with only 5% of the control protease activity remaining after a 1-h exposure to calcium. Preincubation of washed platelets with varying amounts of CAP (0.2-0.4 units) significantly interfered with thrombin-induced platelet aggregation. In addition, ristocetin-induced platelet agglutination in the presence of von Willebrand factor was completely inhibited by 0.4 units of CAP. Concomitant with these protease-induced changes in platelet function, a decrease was observed in a major glycoprotein band of Mr = 150,000 present in platelet membranes and presumed to be glycoprotein Ib. In addition to these effects on platelets, CAP inhibited thrombin-induced production of prostacyclin by cultured human endothelial cells in a dose-dependent manner when the cells were pretreated with CAP. Thus platelet CAP can modulate membrane functions in both platelets and endothelial cells and may thus contribute to the regulation of hemostasis.     

Effects of atrial natriuretic factor on human platelet function

We examined the hypothesis that atrial natriuretic factor (ANF), a substance with known vasorelaxant activities, shares with other vasodilators the property of inhibiting platelet function. Aggregation of citrated platelet-rich plasma (PRP) from 23 healthy volunteers induced by ADP, adrenaline, arachidonic acid, collagen, gamma-thrombin, the endoperoxide analogue U-44069, serotonin, the calcium ionophore A-23187 or platelet aggregating factor was measured after incubation of PRP with ANF for 3 minutes at concentrations of 4 X 10(-9), 4 X 10(-8) and 4 X 10(-7) M or vehicle as control. ANF decreased ADP-induced aggregation significantly (P less than 0.02), but only at the highest concentration used and to a minor extent (control: 73.6 +/- 11.2%; after ANF 4 X 10(-7) M: 60.0 +/- 17.1%, mean +/- S.D., n = 39) by a selective inhibitory effect on the secondary wave; neither aggregation by all other agents tested nor thromboxane B2 generation induced by ADP and adrenaline was altered by incubation with ANF. Although ANF thus has detectable effects on ADP-induced platelet aggregation in vitro, these data suggest that ANF is unlikely to be a physiologically significant modulator of platelet function.     

Stereostructure and anti-inflammatory activity of three diastereomers of ocobullenone from Ocotea bullata

A novel diastereomer of ocobullenone. designated as sibyllenone, was isolated from the stem bark of mature Ocotea bullata in the course of a search for anti-inflammatory compounds from this plant. The stereostructure was established by X-ray crystallography and corroborated by NOESY analysis. Ocobullenone, obtained previously, was re-isolated and crystallised successfully for X-ray analysis, thus making possible an accurate spatial comparison of ocobullenone, iso-ocobullenone and the new stereoisomer. Tested pharmacologically for cyclooxygenase-1 and 2, and 5-lipoxygenase inhibition, sibyllenone was the only compound from O. bullata which showed good inhibitory activity towards 5-lipoxygenase.     

Enteric-coated aspirin,platelet cyclooxygenase activity and function

Recent studies evaluating the effect of slow releasing enteric-coated aspirin formulations have reported contradictory findings regarding the bioavailability of the active molecule in the circulating blood and the length of duration of the inhibitory effect on platelet function. In the present study, we have evaluated the effect of a single dose of two commercially available enteric-coated aspirins on platelet arachidonic acid metabolism and function. A single dose of slow releasing aspirin was as effective as fast acting regular aspirin in its effect on cyclooxygenase activity and platelet function in both human and canine platelets. However, in view of its slow releasing property, the onset of inhibition was considerably delayed compared to the action of fast acting aspirin in those subjects who ingested enteric-coated aspirins.     

Inhibition of 2-P-mercaptophenyl-1,4-naphthoquinone on human platelet function.

As widely assumed, platelets and coagulation system heavily influence the pathogenesis and progression of cardiovascular diseases. Some 1,4-naphthoquinone derivatives, such as vitamin K3, have been reported to increase the synthesis of coagulation proteins. In this study, we examine how 2-p-mercaptophenyl -1,4-naphthoquinone (NTP), a newly synthesized 1,4-naphthoquinone derivative, affects the platelet function in humans. A tapered parallel plate chamber which provided a range of shear stress covering the entire physiological range in human circulation is used to assess platelet adhesiveness on fibrinogen coated-surface. In addition, platelet aggregation and thromboxane B2 (TXB2) production by inducers are evaluated by the turbidimetric method and enzyme immunoassay kit, respectively. Moreover, platelets [Ca2+]i are measured using a dual-wavelength fluorescence spectrophotometer. Analysis results indicate that 1) NTP decreases the percentages of attached platelets at the locations in various shear stresses and the levels of platelet adhesiveness, denoted as the slope; 2) NTP can inhibit the platelet aggregation by ADP (2 microM) and collagen (25 microg/ml), and the IC50 are: 0.32 and 26.83 microg/ml, respectively; and 3) NTP markedly inhibits TXB2 formation and platelet [Ca2+]i elevation caused by ADP and collagen. Therefore, we conclude that NTP may inhibit platelet adhesiveness on fibrinogen coated-surface, aggregability, [Ca2+]i, and thromboxane production, and that it may be used as an antiplatelet agent.     

O-Methylated and sulfoconjugated catecholamines: differential activities at human platelet alpha 2-adrenoceptors.

The physiological effects of the sulfoconjugates of epinephrine, norepinephrine, and the 3-O-methylated catecholamines, metanephrine, normetanephrine, and methoxytyramine were examined with regard to their alpha 2-adrenoceptor binding properties and aggregation activity in human platelets. Sulfoconjugation of catecholamines resulted in the loss of both their competitive potency for [3H]yohimbine binding and their influence on platelet aggregation. O-Methyl substituted catecholamines showed attenuation of their alpha 2-adrenoceptor binding affinities when compared with those of the corresponding non-esterified amines. Unlike the free amine epinephrine, which stimulated platelet aggregation, the O-methylated catecholamine derivatives inhibited aggregation. Inhibition was dose-dependent and restricted to the alpha 2-adrenoceptor mediated aggregation response stimulated by epinephrine (1 microM) or potentiated by subthreshold concentrations of epinephrine (30-300 nM) in the presence of subaggregatory doses of vasopressin (10-30 nM). Collagen- and ADP-induced platelet aggregation was not affected. The hydrophilic beta-antagonist CGP 12177 displayed no effects. However, high concentrations (0.1 mM) of both isomers of the strongly lipophilic beta-adrenoceptor antagonist propranolol inhibited the actions of all aggregators by stabilizing the membrane. Such a nonspecific membrane interaction of the methylated catecholamines could be excluded because of their low lipid solubility calculated in a n-octanol-phosphate buffer system at pH 7.4. We suggest therefore that methylated catecholamines are biological alpha 2-adrenoceptor antagonists acting on alpha 2-adrenoceptor stimulated reactions of human platelets. Whether this receptor antagonism is relevant to other human tissues needs clarification. Sulfated catecholamines, however, are wholly ineffective at this receptor site and may constitute a pathway to control the concentration of the active free catecholamines.     

Effects of Welsh onion extracts on human platelet function in vitro

Welsh onion has been consumed for prevention of cardiovascular disorders. However, its underlying mechanisms are still unclear. This study investigated whether Welsh onion extracts can alter human platelet function (ie, platelet adhesion, aggregation, and thromboxane release). To clarify the underlying mechanisms, we also measured the intracellular calcium ([Ca2+]i) and cyclic nucleotide levels in platelets. Our results showed that 1) boiled extracts directly induced platelet aggregation in a dose-dependent manner; 2) raw extracts inhibited platelet adhesion and ADP-evoked platelet aggregation, while boiled extracts enhanced them; 3) raw green extract suppressed ADP-stimulated platelet [Ca2+]i elevation and thromboxane production, whereas boiled green extract enhanced them; 4) raw green extract elevated platelet cAMP level, whereas boiled green extract had no effect on cAMP level. Furthermore, the boiled green extract, but not the raw extract, induced pronounced platelet morphological changes. In conclusion, raw extracts of Welsh onion inhibit platelet function in vitro while boiled extracts activate platelets.     

The effects of some salicylate analogues on human blood platelets. 1. Structure activity relationships and the inhibition of platelet aggregation

Utilizing a turbidometric technique and human platelet-rich plasma (PRP) at 37°C, aspirin, 2-propionyloxybenzoic acid, 2,3-diacetoxybenzoic acid, sodium salicylate and 4-aminosalicylic acid, at suitable final concentrations and without prior incubation in PRP, prevented adenosine diphosphate (ADP)-induced second phase platelet aggregation and inhibited collagen-induced aggregation. The minimum concentrations of the latter four compounds which inhibited second-phase ADP aggregation were respectively, 15, 43, 60 and 100 times the minimum inhibitory concentration of aspirin. Without prior incubation, 2,6-diacetoxybenzoic acid and 3-propionyloxybenzoic acid potentiated the second phase of ADP aggregation while 3-acetoxybenzoic acid, 4-acetoxybenzoic acid and 2,4-diacetoxybenzoic acid had no effects.Aspirin, 2,3-diacetoxybenzoic acid, 2,6-diacetoxybenzoic acid and 2-propionyloxybenzoic acid, incubated in PRP at 37°C for 5 and 10 min, inhibited collagen-induced platelet aggregation in a concentration dependent manner. Aspirin was most potent, followed by 2-propionyloxybenzoic acid, 2,3-diacetoxybenzoic acid and 2,6-diacetoxybenzoic acid. Inhibition increased with the time of incubation in all cases. The results indicate that structural specificity (the presence of an acyl group in the 2 position of the benzene ring) is important for the aggregation inhibiting activity of aspirin, but do not support the contention that such inhibition is dependent upon the availability of an acetyl radical.     

Surface activity and human blood platelet aggregation-inhibitory potency

Surface and interfacial activity is correlated with molecular constitution and inhibitory potency of mono- and bis(carbamoylpiperidino)alkanes and aralkanes, and of some corresponding quaternary pyridinium congeners, in ADP-induced human blood platelet aggregation. The measurements of surface and interfacial tension were carried out at concentrations and pH-values approximating those employed in the hemodynamic study. The effect of changes in chemical structure, ranging from relatively minor variations in a specific functional group to the alteration of major components in molecular constitution, was examined and interpreted in terms of contemporary theoretical chemistry.     

Effects of lipid-esterified conjugated linoleic acid isomers on platelet function: evidence for stimulation of platelet phospholipase activity

The effects of four conjugated linoleic acid (CLA) isomers on in vitro collagen-induced human platelet aggregation and thromboxane (TXB(2), the inactive metabolite of the proaggregatory TXA(2)) production were examined. As the free fatty acid (FFA), 9t, 11t-CLA was the most effective inhibitor of these two processes (I(50)s of 2.2 and 4 microM, respectively) and the 9c, 11c-CLA was the least effective (I(50)s of 8.3 and 37 microM) of the isomers tested. When platelets were preesterified with either 25 microM 9t, 11t-CLA or 9c, 11c-CLA, CLA incorporation in total platelet lipids increased from 0.24% to 0.31% and 0.38%, and most of this increase was found to be in the phosphatidyl choline and phosphatidyl ethanolamine subclasses. The decrease in arachidonic acid (AA) content in total fatty acids or phospholipids was an order of magnitude greater. Furthermore, no significant differences between platelets prelabeled with either 9t, 11t- or 9c, 11c-CLA in the inhibition of collagen-induced aggregation and TXB(2) formation were observed. However, platelets prelabeled with 9c, 11c-CLA stimulated basal TXB(2) production (4-fold) which was not observed with platelets pretreated with either 9t, 11t-CLA, linoleic acid or stearic acid. This enhancement was associated with a 2.4-5-fold increase in the release of endogenous AA. Our results suggest that the presence of a conjugated cis, cis double bond appears to change the lipid environment sufficiently to stimulate the basal platelet phospholipase activity, which in turn increases the formation of TXB(2).     

Structure and protein design of a human platelet function inhibitor

Hematophagous arthropods secrete a salivary apyrase that inhibits platelet activation by catabolizing ADP released from damaged tissues and blood cells. We report the X-ray crystal structures of a human enzyme of the soluble apyrase family in its apo state and bound to a substrate analog. The structures reveal a nucleotide binding domain comprising a five-blade beta propeller, binding determinants of the substrate and the active site, and an unusual calcium binding site with a potential regulatory function. Using a comparative structural biology approach, we were able to redesign the human apyrase so as to enhance its ADPase activity by more than 100-fold. The engineered enzyme is a potent inhibitor of platelet aggregation and may serve as the basis for the development of a new class of antithrombotic agents.