At low extracellular calcium concentration platelet activating factor induces beta-thromboglobulin release from human platelets through thromboxane-endoperoxides formation

Abstract

The aim of this study was to investigate the mechanism involved in beta-thromboglobulin (BTG) release induced by platelet activating factor (PAF) in human platelet-rich plasma (PRP) and washed platelets (WP) during aggregation. PAF was used in PRP at increasing concentrations starting at its threshold concentration for irreversible aggregation (TAC: 90-150 nM). In citrated PRP, PAF induced release of BTG (80-95% of total content) and thromboxane B2 (TXB2) formation (30-40 pmol/ml). At low PAF concentrations aggregation and BTG release were blocked by apyrase (a scavenger of ADP), by ASA (an inhibitor of cyclooxygenase) and by BM 13177 (a thromboxane receptor antagonist). Higher concentrations of PAF overcame the effect of apyrase, but only induced reversible aggregation and minor release in the presence of ASA or BM 13177. In heparinized PRP, PAF induced full irreversible aggregation, but only very low BTG release (about 25% of total content) and thromboxane synthesis (2-3 pmol/ml). WP resuspended in the presence of 2 mmol/l Ca2+ seldom responded to PAF alone, as previously shown by others, but full aggregation could be induced by concomitant addition of subthreshold concentrations of PAF (25-50 nM) and epinephrine (1 microM). In these conditions average BTG release from WP was less than 20% of the amount releasable by thrombin. In contrast, when WP were resuspended in the absence of Ca2+, stimulation by PAF + EPI induced sustained BTG release (40-50% of total content) and TXB2 synthesis (15-20 pmol/ml). We conclude that at low Ca2+ concentration PAF induces BTG release mainly through thromboxane-endoperoxides formation. In contrast, when [Ca2+] is normal, PAF does not or weakly induces thromboxane formation and BTG release.

Neutralization of the Antiheparin Activity of Platelet Factor 4 by a Monoclonal Antibody

“…Woshed platelets and platelerpoor plasmas. Washed platelets were obtained as previously described (25) using either blood drawn from human volunteers who had taken no drug for at least 15 days before the blood collection, or from the ear artery of New Zealand White rabbits. Platelet-poor plasmas (PPP) were prepared from human or rabbit freshly drawn blood, anticoagulated with 3.8% citrate (1 :10).…”

“…However, an appropriate control, carried out with no coating, showed no significant differences between these two methods. Zatta et al (25). The aggregated platelets were pelleted by centrifugation.…”

“…'s sample buffer to which DTT was added at25 mM final concentration. The sample was boiled for 3 min in a waterbath and then divided into 2 parts.…”