Flow cytometric analysis of shear-induced generation of procoagulant microparticles. (A) Increased microparticle but not platelet counts in citrated whole blood flowing out of a chamber after perfusion over dVWFA1 at γ_w of 30 000 s^–1. Flow cytometry was performed after centrifugation to PRP. Microparticles were identified by the presence of integrin αIIbβ3 (n = 5), GPIbα (n = 5), or tissue factor (TF; n = 9). Note that the microparticle counts with a positive TF signal were approximately half of the GPIb or αIIbβ3 positive ones. (B-C) Microparticle counts in citrated PRP exposed to different shear rates in a cone-and-plate viscosimeter (n = 4, respectively). Results obtained in control PRP (B) or PRP treated with cytochalasin D, which disrupts the actin cytoskeleton (C). In either case, the increase in microparticle generation was significant when γ_w of 10 000 s^–1. Note that the increase of microparticle counts in the cytochalasin D–treated PRP was approximately half that seen in control PRP. The counts reported in panels A to C are for events acquired over a 1-minute period (A) or a 3-minute period (B-C). (D) Clotting time obtained after addition of calcium ions (25 mM) and tissue factor (1.44 ng/mL) to PRP exposed to different shear rates (n = 8). A significant reduction was observed in PRP exposed to a shear rate of 10 000 s^–1. (E) Significant reduction of the clotting time obtained after addition of calcium but not tissue factor to PRP exposed to the shear rate of 10 000 s^–1 (n = 3). PGE₁, apyrase, tirofiban, and CTI were present in the PRP. (F) Change in the number of microparticles positive for αIIbβ3 (CD41) or phosphatidylserine, measured by annexin (anx) V binding, after exposure of PRP to a shear rate of 10 000 s^–1 (n = 5). The detected increase was more pronounced when the flow cytometric analysis after shearing was performed in PPP as compared with PRP. All results are shown as mean ± SEM. ***P < .01; **P < .05.