Microvesiculation of FlnA-null platelets. (A) Control or FlnA-null platelets were stored at 37°C for 24 hours with or without metalloproteinase inhibitor GM6001 (100μM), calpain inhibitor calpeptin (20 μg/mL), or DMSO as control, and analyzed by flow cytometry. Forward/side scatter dot plots shown are representative of 4 independent experiments. FlnA-null platelets release microvesicles after 24 hours of storage at 37°C (arrow). (B) Quantification of microvesicle formation. Results represent the percentage of microvesicle events over all events (microvesicles and platelets) and are mean ± SD (n = 4). *P < .05. (C) Expression of GPIbα on the surface of control and FlnA-null platelets stored for 24 hours at 37°C was analyzed by flow cytometry. GPIbα expression decreased on both control and FlnA-null platelets. (D) Effect of GM6001 and calpeptin on the surface expression of GPIbα on control and FlnA-null platelets. Data are expressed as percentage of positive platelets and are mean ± SD (n = 4). #P < .05 versus 0 hour control platelets. *P < .05 versus 0 hour FlnA-null platelets. (E) PS exposure was evaluated on control and FlnA-null platelets stored for 24 hours at 37°C by annexin V binding assay in flow cytometry. Results are expressed as MFI and are mean ± SD (n = 4). (F) FlnA-null microvesicles were analyzed for glycoprotein expression (GPIbα, GPIbβ, and CD61) and PS exposure (annexin V) at 24 hours. Results are expressed as percentage of positive microvesicles and are mean ± SD (n = 4).