Flow cytometry analyses of unstimulated and agonist-treated platelets from control and affected dogs labeled to detect surface exposure of PS.

Dot plots depict platelet suspensions from a healthy dog (control) and an affected GSD labeled with annexin V-FITC, a fluorescein-conjugated protein with high affinity for membrane surface aminophospholipid. The platelet suspensions were stirred in a buffer containing 2 mM CaCl₂, in the absence of agonist compounds (A,B) or in the presence of one of the following treatments: A23187 (3 μM A23187, 25 minutes at room temperature); Th/Coll (0.5 U/mL thrombin plus 24 μg/mL collagen, 25 minutes at room temperature); tetracaine (2 mM tetracaine, 60 minutes at 37°C). Unstimulated control and GSD platelets are not labeled, indicating a lack of surface PS. All 3 treatments induced surface PS exposure in control dogs, denoted by the appearance of high-intensity fluorescence. The ionophore A23187 and thrombin plus collagen also induced a marked decrease in forward scatter for all or a portion of the labeled control platelets. The GSD platelets demonstrated virtually no increase in surface PS in response to A23187 and a relatively minor increase in PS exposure in response to thrombin plus collagen. Exposure of PS in response to tetracaine was equivalent to that of control dogs. Arrows indicate increasing forward light scatter intensity and increasing green fluourescence intensity. Data were collected without gating. The horizontal quadrant marker is at the lower boundary of forward scatter for unstimulated platelets, and the vertical marker is set at the upper boundary of fluorescence for more than 95% of the unstimulated platelets (indicating nonspecific fluorescence). Particles in the lower left quadrant of each plot represent debris and machine noise. These plots correspond to a single analysis, representative of results obtained from agonist treatment of all 5 affected GSDs.