Fig. 5

Effects of ZW on gill bleeding, thrombocyte activation, and PAR-induced signaling.

(A) The zebrafish were subjected to the gill bleeding assay as described in Materials and Methods with the following conditions: 1, distilled water (DW); 2, 70 ng ZW; 3, 70 ng of ZW with 140 ng of PRAHT; and 4, 70 ng of ZW with 140 ng of control IgG. (B) Plate tilt assay measuring thrombocyte function: 1, PBS; 2, 7 ng ZW; 3, 7 ng ZW with 14 ng control IgG;and 4, 7 ng ZW with 14 ng PRAHT. Note the aggregated blood in 2 and 3 is more firm than the control blood 1 and blood 4. (C) Measurement of the functional activity of ZW on thrombocytes by annexin V binding. 7 ng of ZW, 14 ng of PRAHT and 14 ng of control IgG were used. The intensity of images from FITC-annexin fluorescence (green) was measured as mean pixels using Adobe Photoshop software version 7.0. (T test, n = 12). (D) ZW-induced ⁴⁵Ca²⁺ release from Xenopus oocytes microinjected with PARs mRNA. The data is presented as the mean value and standard error for 12 oocytes for each PAR mRNA.