xestospongin-a and lanthanum-chloride

We previously reported that only a subpopulation of PAR-1-stimulated platelets binds coagulation factor IXa, since confirmed by other laboratories. Since calcium changes have been implicated in exposure of procoagulant aminophospholipids, we have now examined calcium fluxes in this subpopulation by measuring fluorescence changes in Fura Red/AM-loaded platelets following PAR-1 stimulation. While fluorescence changes in all platelets indicated calcium release from internal stores and influx of external calcium, a subpopulation of platelets displayed a pronounced increase in calcium transients by 15 s and positive factor IXa binding by 2 min, with calcium transients sustained for 45 min. Pretreatment of platelets with Xestospongin C to inhibit IP3-mediated dense tubule calcium release, and the presence of impermeable calcium channel blockers nifedipine, SKF96365, or LaCl3, inhibited PAR-1-induced development of a subpopulation with pronounced calcium transients, factor IXa binding, and platelet support of FXa generation, suggesting the importance of both release of calcium from internal stores and influx of extracellular calcium. When platelets were stimulated in EDTA for 5-20 min before addition of calcium, factor IXa binding sites developed on a smaller subpopulation but with unchanged rate, indicating sustained opening of calcium channels and continued availability of signaling elements required for binding site exposure. While pretreatment of platelets with 100 microM BAPTA/AM (Kd 160 nM) had minimal effects, 100 microM 5,5'-dimethylBAPTA/AM (Kd 40 nM) completely inhibited the appearance and function of the platelet subpopulation, indicating the importance of minor increases of cytoplasmic calcium. We conclude that PAR-1-stimulated development of factor IXa binding sites in a subpopulation of platelets is dependent upon release of calcium from internal stores leading to sustained and pronounced calcium transients.

Topics: Blood Platelets; Calcium; Cytoplasm; Factor IXa; Humans; Imidazoles; Ion Transport; Lanthanum; Macrocyclic Compounds; Oxazoles; Protein Binding; Receptor, PAR-1; Thapsigargin

The contribution of intracellular calcium stores to Mannheimia haemolytica leukotoxin (LKT)-induced increase in cytosolic calcium concentration was studied by pharmacologically inhibiting transport of calcium across the plasma and endoplasmic reticulum membranes of bovine neutrophils exposed to LKT. Active intracellular storage of calcium by sarcoplasmic/endoplasmic reticulum calcium ATPase, influx of extracellular calcium across the plasma membrane, and release of stored calcium via inositol triphosphate receptors and ryanodine-sensitive calcium channels were inhibited using thapsigargin, lanthanum chloride, xestospongin C, and magnesium chloride, respectively. Pre-incubation with thapsigargin attenuated the increase in cytosolic calcium concentration produced by LKT, thus confirming the involvement of intracellular calcium stores. Inhibitory effects of lanthanum chloride, xestospongin C, and magnesium chloride indicated that the increase in cytosolic calcium concentration induced by LKT resulted from both influx of calcium across the plasma membrane and release of calcium from intracellular stores.

Topics: Animals; Calcium Signaling; Cattle; Cytosol; Exotoxins; In Vitro Techniques; Inositol 1,4,5-Trisphosphate; Intracellular Fluid; Lanthanum; Macrocyclic Compounds; Magnesium Chloride; Mannheimia haemolytica; Neutrophils; Oxazoles; Ryanodine Receptor Calcium Release Channel; Thapsigargin