Healthy blood donors and patient donors had abstained from aspirin in the last two weeks, and consent was obtained according to GT IRB H15258. Blood was drawn by median venipuncture into acid-citrate-dextrose (ACD) solution 2. The sample was subsequently centrifuged at 150 G for 15 min, and the resulting platelet rich plasma was gel filtered into HEPES modified Tyrodes buffer as described previously 35 . Platelets were diluted to a final concentration of 4 × 106/mL in Tyrodes buffer to minimize potential paracrine signaling. This equated to an average distance between microdot pairs of 30–50 microns depending on the donor. In some experiments, platelets were incubated for 1 hour with vehicle (dimethyl sulfoxide, DMSO); ROCK inhibitor Y-27632 at 50 μm (Sigma Y0503), or MLCK inhibitor at 10 μm (Sigma I2764).
For bulk contraction studies, blood was drawn by median antecubital venipuncture into acid-citrate-dextrose (ACD) solution 2. The sample was centrifuged 150 G for 15 min and the resulting platelet rich plasma was collected, and centrifuged with an additional 10% ACD by volume at 900G for 5min. The supernatant, platelet poor plasma, was discarded and the platelets were resuspended into HEPES modified Tyrodes buffer.
For bulk contraction studies, blood was drawn by median antecubital venipuncture into acid-citrate-dextrose (ACD) solution 2. The sample was centrifuged 150 G for 15 min and the resulting platelet rich plasma was collected, and centrifuged with an additional 10% ACD by volume at 900G for 5min. The supernatant, platelet poor plasma, was discarded and the platelets were resuspended into HEPES modified Tyrodes buffer.