Jacques Benveniste

Platelet activating factor (PAF-acether): total synthesis of 1-O-octadecyl 2-O-acetyl sn-glycero-3-phosphoryl choline

Platelet-activating factor (PAF), a mediator of anaphylaxis and inflammation, is released from IgE-sensitized antigen-stimulated basophils [l-3]. PAF is a low molecular weight phospholipid which is sensitive to hydrolysis by phospholipases AZ, C, and D, but not by lipase from Rhizopus arrhizus or sphingomyelinase C [4]. PAF appeared to be a glycerophospholipid with a choline polar head group devoid of an ester function at position 1 [4,5]. The presence of PAF has been demonstrated in cells from several species including man [3,6,7], rabbit [l-3], rat [8] and pig [4]. PAF has been obtained from rat and mouse peritoneal macrophages upon stimulation by the ionophore A23187 or exposure to phagocytable particles [9]. PAF has been shown to induce aggregation of blood platelets from rabbit [1,3],man [lO],rat [ll] andguineapig [12].The structure of the molecule responsible for PAF activity has been elucidated [13,14]. This was obtained by acetylation of 2-lyso plasmalogens possessing a 30phosphoryl choline group [ 131 or a 3-O-phosphoryl ethanolamine group followed by reaction with diazomethane and quaternization with methyl iodide [ 141. This result lead us to propose a 1 -O-alkyl 2-0-acetyl sn-glycero3-phosphoryl choline structure and the terminology ‘PAF-acether’ for this substance. However, total synthesis was necessary to confirm the structure of PAF-acether. Here, we report the total synthesis of l-O-octadecyl20acetyl sn-glycero3-phosphoryl choline, which has the same biological activity as the natural PAF-acether.

Structural analogs of platelet-activating factor (PAF-acether)(⋆)

Platelet-activafing factor (PAF-acether) is a potent mediator of anaphylaxis and inflammation. It induces aggregation of platelets mad releases their vasoactive amines. PAF-acether has been shown to be produced by basophils [1, 2], macrophages [3, 4], neutrophils [51, and platelets themselves [6]. This mediator, partially characterized as a 2acylglycero-phosphorylcholine (2-acyl-glyceroPC) [7], was fully identified as a 1-O-alkyl-2-Oacetyl-sn-glyceryl-3-PC by synthesis from natural ptasmalogens [8, 9] and by total synthesis of the 1-O-octadecyl-ether 2 (see table I and fig. 1) [101. The structure for natural platelet-activating factor released from antigen stimulated IgE-sensitized basophils was confirmed by Hanahan et al. [11]. Recently, 1-O-alkyl-2-Iyso-glyceryl-3-PC has been isolated from hog leucocytes; this precursor (or

Inhibition of rabbit platelet aggregation by the Ca2+-antagonists verapamil and diltiazem and by trifluoperazine

Abstract In rabbit platelets, the Ca2+-antagonists, Verapamil and Diltiazem, and the calmodulin inhibitor, Trifluoperazine, inhibited platelet aggregation induced by the mediators of the three pathways platelet-activating factor (Paf-acether), arachidonic acid (AA) and adenosine diphosphate (ADP). The Paf-acether pathway was the most markedly influenced by Verapamil (IC50 = 23 ± 12 μM) and it was the only one to be inhibited by Diltiazem (IC50 = 13 ± 6μM). Trifluoperazine inhibited predominantly the ADP pathway (IC50 = 1 ± 5 μM). Inhibitions were not reversed by increasing Ca2+ concentrations from 1.4 to 14.2 mM but in the case of Paf-acether were suppressed after removal of Verapamil and Diltiazem but not of Trifluoperazine. Platelets aggregated by Paf-acether, AA and ADP were immediately desaggregated upon addition of each drug. The potency of their anti-aggregating activity was close to that of their vasodilator activity suggesting that Ca2+-antagonism is the cause of the platelet aggregation inhibition.

BN 52021 displaces [3H]paf-acether from, and inhibits its binding to intact human platelets

Intact platelets incubated at 20 degrees C in the presence of 0.25% (w/v) bovine serum albumin (BSA) bound [3H]paf-acether in a concentration-dependent (0-6.5 nM) manner. Specific [3H]paf-acether binding in the presence of unlabelled paf-acether or BN 52021, a chemically defined ginkgolide, reached a plateau of 14.5 +/- 5 or 17.5 +/- 7.0 fmol at concentrations higher than 0.65 nM [3H]paf-acether. Unlabelled paf-acether or BN 52021 inhibited and displaced [3H]paf-acether binding in a concentration- and time-dependent manner reaching a plateau at 5 nM or 5 microM. Unlabelled paf-acether inhibited [3H]paf-acether binding (75.1 +/- 8%) more strongly if labelled and unlabelled ligands were incubated together than after 15 min preincubation with [3H]paf-acether alone (32.2 +/- 8.6%). The enantiomer of paf-acether (50 nM) failed to displace platelet-bound [3H]paf-acether and the possibility of degradation of [3H]paf-acether by platelets or BN 52021 was excluded under binding conditions. The results demonstrate the presence of specific binding sites for paf-acether in human platelets and the direct competition by BN 52021 for these sites. Serum albumin inhibited total and enhanced specific [3H]paf-acether binding. Radiolabelled albumin itself did not bind to human platelets at 20 degrees C and preincubation of platelets with antibodies to human serum albumin did not inhibit specific paf-acether binding but increased total [3H]paf-acether binding slightly. Serum albumin appears as a necessary phospholipid carrier for specific [3H]paf-acether binding.

Hispidulin, a natural flavone, inhibits human platelet aggregation by increasing cAMP levels

Hispidulin, a natural flavone, and theophylline inhibited platelet aggregation triggered by adenosine-5-monophosphate, arachidonic acid, paf-acether and collagen. Hispidulin was 100-fold more potent than theophylline. A threshold concentration of PGE1 did not modify the anti-aggregatory effect of hispidulin but potentiated the effect of theophylline. A threshold concentration of hispidulin had no effect on the inhibitory action of theophylline. Hispidulin (100 microM) and theophylline (10 mM) increased the control cAMP level in platelets 4-fold. A threshold concentration of PGE1 had a small effect on hispidulin-induced cAMP levels but increased the theophylline-induced cAMP levels 3-fold. Theophylline (10 mM)-induced cAMP levels were not modified by hispidulin. We demonstrate a correlation between the inhibition of platelet aggregation and the increase in cAMP levels induced by hispidulin. These data suggest that hispidulin could inhibit platelet aggregation by elevating cAMP levels by a mechanism different from that of theophylline or PGE1.

Comparison between Paf-Acether Receptors on Intact Washed Human Platelets and Human Endothelial Cells in Culture

Paf-acether (paf, formerly platelet-activating factor) (Benveniste et al., 1972), with the structure 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (Benveniste et al., 1979; Demopoulos et al., 1979; Blank et al., 1979), is a biologically active phospholipid which is released by several cell types upon stimulation (for review Pinckard et al., 1982; Vargaftig et al., 1981). The mediator activates platelets, polymorphonuclear neutrophils, macrophages and endothelial cells most probably via a receptor-dependent pathway (Valone et al., 1982; Valone and Goetzl 1983; Lambrecht and Parnham, 1986; Korth et al., 1987; Valone, 1988). Serum albumin acts as a necessary phospholipid carrier for both paf release (Benveniste, J. et al., 1972; Benveniste, J., 1974) and specific paf binding (Korth and Benveniste, 1987) and seems to inhibit the pafdegrading enzyme, acetylhydrolase (Wardlow et al., 1986). Different specific antagonists, such as the paf analogue CV 3988 (Terashita et al., 1983), the naturally occurring kad-surenone (Shen et al., 1985) and Ginkgolides (Nunez et al., 1986; Korth and Benveniste, 1987; Korth et al., 1988 c; Braquet and Godfroid, 1987), as well as synthetic hetrazepines and calcium-blocking agents, inhibited platelet activation and paf binding (Casals-Stenzel and Weber, 1987; Korth et al., 1987 and 1988 b; Wade et al., 1986). Binding studies with radiolabeled antagonists supported the existence of paf receptors in platelets (Hwang et al., 1986 a). A close correlation between inhibition of paf-induced platelet aggregation and binding of labelled paf suggested the importance of the putative paf receptor for platelet activation (Korth et al., 1988 c). Paf receptors were coupled with modulation of phospholipase C activity (Morrison and Shukla, 1988).

Inhibitory effects of three new synthetic compounds on human platelet aggregation

Three compounds of the AQ series (benzothienyl-aminoethyl ketone derivatives), i.e. 3178 (benzothienyl-2 N,N-diallyl amino ethyl cetone), 1994 (alpha-benzothienyl-beta-N-morpholino ethyl cetone), and 1989 (benzothienyl-2-beta-N,N-dimethyl amino ethyl cetone) were tested against aggregations triggered by adenosine 5-diphosphate (ADP), arachidonic acid (AA), paf-acether, thrombin or collagen under different experimental conditions. None of them exhibited a specific inhibitory effect on washed platelets prepared so as to render them specifically sensitive either to ADP, AA or paf-acether. Thus for compound 3178 AQ, the most potent of the three, IC50 values were 2.9 +/- 0.6, 2.9 +/- 1.0 and 4.3 +/- 0.9 uM (means +/- 1 SD of 4 experiments) against ADP, AA or paf-acether respectively. Aggregations triggered by subthreshold concentrations of thrombin were also inhibited by compound 3178 AQ (50 uM) even after washing, showing the persistence of the inhibitory effect. Inhibition was surmountable since addition of a 10 fold greater concentration of thrombin than the subthreshold one induced a full aggregation. When tested on platelet-rich plasma (PRP) higher concentrations of the inhibitors than those used on washed platelets were needed in order to counteract ADP, AA or paf-acether effects. Collagen-induced aggregation was also inhibited by the AQ compounds when tested either in PRP or in whole blood although, in the latter case, high concentrations of the antagonists had to be used. These data show that compounds of the AQ series bear a wide spectrum of activity which makes them potential anti-thrombotic agents.