Interaction of the Paf antagonist WEB 2086 and its hetrazepine analogues with human platelets and endothelial cells.

1. Intact platelets and confluent human umbilical vein endothelial cells bound [3H]-Paf-acether (platelet activating factor, [3H]-Paf) at 20 degrees C in the presence of 0.25% (w/v) bovine serum albumin (BSA). 2. [3H]-Paf binding to platelets was inhibited in a concentration-dependent manner by WEB 2086. An excess of WEB 2086 indicated the presence of specific, saturable Paf binding which reached a maximum of 28.3 +/- 3.7 fmol [3H]-Paf per 5 x 10(7) platelets. In platelets, different hetrazepines (WEB 2098, 2105, but not 2118) also inhibited [3H]-Paf binding in a concentration-dependent manner. 3. WEB 2086 partially displaced platelet-bound [3H]-Paf in a concentration-dependent manner reaching a plateau at 400 nM WEB 2086. No further displacement was observed when WEB 2086 and an excess of unlabelled Paf were added together. 4. The hetrazepines inhibited platelet aggregation. Platelet aggregation IC50 values correlated well with the IC50 values of the hetrazepines against [3H]-Paf binding (r2 = 0.99). WEB 2086 shifted the Paf dose-response curve rightwards in a parallel manner. Tested against platelet aggregation the pA2 obtained for WEB 2086 was 7.9. 5. WEB 2086 inhibited [3H]-Paf binding to endothelial cells in a concentration-dependent manner. WEB 2086 also inhibited the Paf-mediated cytosolic calcium increase in endothelial cells with an IC50 value of 23.1 +/- 10.4 nM as compared with an IC50 of 21.6 +/- 10.4 nM WEB 2086 for platelet aggregation. 6. These results demonstrate an inhibition of [3H]-Paf binding to platelets and endothelial cells by different hetrazepines, most probably at the Paf receptor level.

The role of extracellular Ca2+ and Na+ in paf-acether-induced human platelet activation.

The dependence of paf-acether (paf)-induced human platelet activation on extracellular Ca2+ and Na+ was examined by quantitating aggregation, secretion and thromboxane (Tx) formation in the presence of physiological and/or low concentrations of Ca2+ and/or Na+. In the presence of 2 mM Ca2+ and 140 mM Na+, paf induced a dose-dependent reversible aggregation and less than 25% of [14C]serotonin release. These responses were insensitive to aspirin or Tx antagonist SQ 29548 treatment and negligible amounts of Tx were formed. In low Ca2+ buffer, paf induced irreversible aggregation and the 14C-serotonin release could exceed 60%. These increases in platelet response were associated with the formation of Tx and were suppressed by aspirin and SQ 29548 treatments, or by substituting NaCl with N-methylglucamine hydrochloride. Thus, in low Ca2+ medium, Tx synthesis is favored during platelet activation and is dependent on Na+ concentrations. A decrease in extracellular Na+ inhibited the paf-acether-induced Tx synthesis observed in low Ca2+ medium but not that induced by the Tx direct precursor, arachidonic acid (AA). Therefore, the increase observed in low Ca2+ medium, no longer seen when the Na+ level is decreased is not related to an impairment of the cyclooxygenase activity but rather implicates an effect on the activity of phospholipase A2. A decrease in extracellular Na+ (2 mM Ca2+ present), inhibited [14C]serotonin release induced by paf from platelets which had, or had not been, treated with aspirin. In this medium, the AA-induced release reaction was also affected whereas Tx formation was not altered, thus suggesting that other mechanisms involved in platelet response apart from Tx synthesis are dependent on extracellular Na+.

Effect of structural analogues of PAF-acether on platelet desensitization.

The 1-O-alkyl-2-O-acetyl-sn-glyceryl-3-phosphorylcholine (PAF-acether) aggregates rabbit platelets and desensitizes them to a second challenge with the same agonist but not to arachidonic acid. The desensitizing activities of 14 analogues of PAF-acether were explored with particular attention to the dose-response dependency of the desensitization process. PAF-acether was 500-fold more active than its 1-O-acyl analogues. The 2-lyso PAF-acether was inactive and the PAF-acether enantiomer 2000 times less effective than the natural isomer, thus confirming the importance of the presence and steric position of the 2-acetate group. The desensitizing activities of the 2-propionyl and the 2-butyryl analogues were close to that of PAF-acether. Substituting an ether to an ester bond at the 2-position indicated that the number of carbon of the 2-substituant seems more determinant than the nature of the linkage for the desensitizing process. Indeed, the 2-ethoxy and the 2-methoxy analogues were 87 and 5000 times less active than PAF-acether respectively. The presence of methyl groups on the nitrogen base is also critical to desensitize platelets. The desensitizing potency of the tested phospholipids was always identical to their aggregating efficiency. It is suggested that these compounds activate cells through a common mechanism.

Specific desensitization of rabbit platelets by platelet-activating factor (PAF-acether) and derivatives.

PAF-acether is a 1-O-alkyl-2 acetyl-sn-glycerol-3-phosphorylcholine which triggers platelet aggregation independently from ADP release and thromboxane A2 formation. PAF-acether-aggregated rabbit platelets did not respond to a second challenge with the same agonist, but still aggregated to arachidonic acid. This desensitization to PAF-acether was a dose-dependent phenomenon. Specific cross-desensitization to PAF-acether was achieved using PAF-acether from four cellular origins, synthetic PAF-acether and its 2-propionyl analogue. Conversely, 2-lyso-PAF-acether and PAF-acether enantiomer were unable to induce specific desensitization. These results suggest that PAF-acether, whatever its origin, exhibits identical active molecular site, and that the presence and the stereospecific position of the 2-acetyl group are critical for the interaction of PAF-acether with platelets.