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Receptor mediated calcium entry (RMCE) is a now recognised mechanism by which the influx of Ca 2+ into cells can occur. Agents which antagonise or block this mechanism are expected to possess anti-platelet aggregation and anti-inflammatory activities. The first series of RMCE blockers based upon the lead compound SC 38249, has been prepared. Their synthesis, activity against ADP evoked Ca 2+ signals in human platelets and their anti-platelet aggregation activity is described. A trend between the c log P of these compounds and their activity as inhibitors of Ca 2+ influx into human platelets stimulated by ADP was observed. This SAR study led to a 40-fold increase in activity over the initial lead compound.

A description is given of the methodology, and problems encountered, for the use of a new fluorescent Ca2(+)-indicator dye, fluo-3, in neutrophils and platelets. The higher Kd and longer excitation wavelength of fluo-3 can have significant advantages over fura-2. Although neutrophils and platelets are used as examples, these observations will be applicable to other cell types. The Kd of fluo-3 for binding Ca2+ at 37 degrees C was measured and found to be 864 nM; the previously published value was 400 nM at 22 degrees C. The Kd of fluo-3, like that of fura-2, is therefore very temperature-dependent. Protocols for loading cells, and preventing leakage of fluo-3, are described; probenecid, known to inhibit fura-2 leakage from cells, was found to be essential to get good fluo-3 signals from platelets. Calibration of fluo-3 fluorescence signals to [Ca2+] and methods for obtaining maximum and minimum fluorescence signals are described; these methods differ from those used with fura-2. Agonist-stimulated responses of fluo-3-loaded neutrophils and platelets are shown, and the calculated cytosolic [Ca2+] is comparable with that previously obtained with fura-2. Responses of cells in the presence of plasma are also shown; such measurements, unobtainable with quin2, fura-2 or indo-1, are possible with fluo-3, owing to its longer excitation wavelengths. Co-loading of cells with bis-(o-aminophenoxy)ethane-NNN′N′-tetra-acetic acid and fluo-3 is included as an example of how cytosolic [Ca2+] can be buffered and manipulated. Many of these observations will be of value when using fluo-3 (or other Ca2(+)-indicator dyes) in most cell types.

A novel and potent azetidinone inhibitor of the lipoprotein-associated phospholipase A2 (Lp-PLA2), i.e. platelet-activating factor acetylhydrolase, is described for the first time. This inhibitor, SB-222657 (Ki=40+/-3 nM, kobs/[I]=6. 6x10(5) M-1.s-1), is inactive against paraoxonase, is a poor inhibitor of lecithin:cholesterol acyltransferase and has been used to investigate the role of Lp-PLA2 in the oxidative modification of lipoproteins. Although pretreatment with SB-222657 did not affect the kinetics of low-density lipoprotein (LDL) oxidation by Cu2+ or an azo free-radical generator as determined by assay of lipid hydroperoxides (LOOHs), conjugated dienes and thiobarbituric acid-reacting substances, in both cases it inhibited the elevation in lysophosphatidylcholine content. Moreover, the significantly increased monocyte chemoattractant activity found in a non-esterified fatty acid fraction from LDL oxidized by Cu2+ was also prevented by pretreatment with SB-222657, with an IC50 value of 5.0+/-0.4 nM. The less potent diastereoisomer of SB-222657, SB-223777 (Ki=6.3+/-0.5 microM, kobs/[I]=1.6x10(4) M-1.s-1), was found to be significantly less active in both assays. Thus, in addition to generating lysophosphatidylcholine, a known biologically active lipid, these results demonstrate that Lp-PLA2 is capable of generating oxidized non-esterified fatty acid moieties that are also bioactive. These findings are consistent with our proposal that Lp-PLA2 has a predominantly pro-inflammatory role in atherogenesis. Finally, similar studies have demonstrated that a different situation exists during the oxidation of high-density lipoprotein, with enzyme(s) other than Lp-PLA2 apparently being responsible for generating lysophosphatidylcholine.

A new CC chemokine, designated CKbeta-8 or myeloid progenitor inhibitor factor-1, was recently identified in a large scale sequencing effort and was cloned from a human aortic endothelial library. CKbeta-8 cDNA encodes a signal sequence of 21 amino acids, followed by a 99-amino acid predicted mature form. CKbeta-8 was expressed and purified from a baculovirus insect cell expression system, which resulted in the identification of different N-terminal variants of the secreted chemokine. The three major forms (containing amino acids 1-99, 24-99, and 25-99 of the secreted chemokine) showed a large variation in potency. CKbeta-8 activated both monocytes and eosinophils to mobilize intracellular calcium; however, the shortest form of CKbeta-8 (25-99) was2 orders of magnitude more potent than the longest form. Cross-desensitization experiments in both monocytes and eosinophils suggested that the CCR1 receptor was probably the predominant receptor that mediates this chemokine's physiologic response. However, incomplete desensitization was encountered in both cell systems, suggesting involvement of an additional receptor(s). Interestingly, the short form of CKbeta-8 was the most potent chemotactic chemokine that we have ever evaluated in the monocyte system (EC50 = 54 pM). However, in contrast to its action on monocytes, CKbeta-8 was a very poor chemotactic factor for eosinophils.

Lipoxin A4 (LXA4), a lipoxygenase-derived metabolite of arachidonic acid, stimulated a dose-dependent elevation in cytosolic free Ca2+ concentration, [Ca2+]i, in fura-2-loaded human neutrophils, with an EC50 of 0.4-0.5 microM. The time for [Ca2+]i to peak was also dose-dependent. In the presence of extracellular Ca2+ (CaDT-PA added), the rise in [Ca2+]i was due to a combination of Ca2+ release from internal stores and influx of extracellular Ca2+. In the absence of extracellular Ca2+, the rise in [Ca2+]i was due to release from internal stores, which then became depleted. No response to LXA4 was seen in the absence of divalent cation chelators (EGTA or DTPA); this is presumably because LXA4 forms an inactive complex with heavy metal cations. In the presence of extracellular Ca2+, LXA4 had no effect on the subsequent response of neutrophils to the chemotactic peptide fmetleu-phe (fmlp). In the absence of extracellular Ca2+, LXA4 dose-dependently reduced the subsequent response of neutrophils to fmlp; this is presumably because LXA4 discharges the store, and so reduces the amount of Ca2+ available for subsequent release by fmlp.

1. The effects of siguazodan (SK&F 94836) a selective phosphodiesterase (PDE) inhibitor with inotropic and vasodilator activity, were studied on human platelets. 2. Siguazodan selectively inhibited the major cyclic AMP-hydrolysing PDE in human platelet supernatants. The inhibited enzyme has been variously termed cyclic GMP-inhibited PDE or PDE-III. 3. In platelet-rich plasma (PRP), siguazodan inhibited U46619-induced aggregation more potently than that induced by ADP and collagen. Treatment of the PRP with aspirin had no effect on the potency of siguazodan. 4. In washed platelets, siguazodan increased cyclic AMP levels and reduced cytoplasmic free calcium [( Ca2+]i). ADP decreased the ability of siguazodan to raise cyclic AMP and this may explain its lower potency in inhibiting responses to ADP. 5. Siguazodan has anti-platelet actions over the same concentration range that it is an inotrope and vasodilator.