Increase of intracellular Ca2+ concentration by Vibrio vulnificus cytolysin in rat platelets; triggering mechanism of platelet cytolysis

ABSTRACT

Vibrio vulnificus cytolysin caused platelet cytolysis and increased intracellular calcium concentration ((Ca2+))i) of rat platelets in a concentration-dependent manner. In the presence of V. vulnificus cytolysin (3 HU/ml), lactate dehydrogenase (LDH) activity was increased from 1.3+/-0.4% of control to 64.3+/-3.4% in platelet suspension buffer. In Ca2+-free platelet suspension buffer, however, V. vulnificus cytolysin did not induce (Ca2+)i increase and LDH release. Addition of EGTA (2 mM) to suspension buffer after the initial Ca2+ influx reversed (Ca2+)i to the control level. However, a Ca2+ channel blocker verapamil (20 muM) or mefenamic acid (20 muM) did not inhibit V. vulnificus cytolysin-induced (Ca2+)i increase and LDH release. Divalent cations such as Co2+, Cd2+ or Mn2+ (2 mM each) also did not alter V. vulnificus cytolysin-induced (Ca2+)i increase and LDH release. V. vulnificus cytolysin (3 HU/ml)-induced calcium influx was completely blocked by lanthanum (2 mM). Lanthanum (2 mM) also completely blocked V. vulnificus cytolysin (3 HU/ml)-induced LDH release. Osmotic protectants such as, raffinose, sucrose or PEG600 (50 mM each) did not inhibit the lytic activity of V. vulnificus cytolysin. In conclusion, lanthanum sensitive Ca2+ influx plays a significant role in Vibrio vulnificus cytolysin-induced platelet cytolysis and thrombocytopenia in V. vulnificus infection.