Effects of ebelactone B on cathepsin A activity in intact platelets and on platelet activation

Previous in vitro studies have demonstrated that a potent antihypertensive agent ebelactone B inhibits cathepsin A/deamidase activity. The aim of our studies was to assess the effects of this inhibitor on cathepsin A activity in intact platelets and on platelet activation events.

PRP or washed human platelets from healthy volunteers were pre-incubated with different concentrations of ebelactone B (1-10 microM) for 10-60 min. Cathepsin A activity in platelets was assayed colorimetrically using Cbz-Phe-Ala at pH 5.5. Expression of platelet activation markers GpIIb/IIIa and P-selectin on non-activated or agonist-activated platelets (ADP, TRAP) was measured by flow cytometry.

Pre-treatment of platelets for up to 60 minutes with 10 mumol/l ebelactone B, that effectively inhibits cathepsin A activity in platelet lysate, did not affect this activity in intact platelets. Exposure of PRP to 10 mumol/l ebelactone B alone, or before platelet activation with ADP or TRAP caused only a small but non-significant increase in P-selectin and GpIIb/IIIa expression on the platelet surface, as demonstrated by flow cytometry analysis.

The lack of cathepsin A inhibition by ebelactone B in intact platelets indicates that this inhibitor does not enter cells. Therefore, a potential antihypertensive significance of this compound may be through the inhibition of cathepsin A/deamidase released from activated or damaged cells. In vitro ebelactone B seems to exert no effect on platelet activation. Further studies are underway to determine whether ebelactone B administration affects platelet activation events in experimental model of hypertension in rats.