Active TFPI is expressed on the surface of coated platelets and secreted. (A) Platelets were simultaneously activated with convulxin plus thrombin (top 4 panels) or calcium ionophore and thrombin (bottom 2 panels) to make coated platelets and analyzed for surface TFPI expression using flow cytometry. Fibrinogen staining (y-axis) identifies the coated platelets. Staining on the x-axis is unstained cells (top left panel), MOPC (top right panel; negative control), factor V (middle and lower left panels; positive control), and TFPI (middle and lower right panels). (B) The relative fluorescence intensity of platelets determined by flow cytometry of platelets stained for TFPI following stimulation with different single agonists as indicated. Error bars represent the standard deviation of 3 or more experiments. (C) Western blot analysis of TFPI in quiescent and coated platelets produced by stimulation of platelets with thrombin and calcium ionophore following differential centrifugation. Lane 1, 1500g pellet; lane 2, 10 000g pellet; lane 3, 200 000g pellet; lane 4, 200 000g supernatant. Western blots from 3 coated-platelet preparations are shown to indicate the variable amounts of TFPI found in the 10 000g pellet and 200 000g supernatant. (D) Comparison of TFPI activity (surface and secreted) in quiescent platelets, platelets stimulated with 8 μM TRAP, 20 μM TRAP or coated platelets produced by stimulation with thrombin and calcium ionophore. TFPI activity was measured by inhibition of factor Xa generation by factor VIIa/TF. The number of persons tested in each category is indicated. Error bars represent the standard deviation of 3 or more experiments. (E) TFPI activity is reversed by a monoclonal antibody directed against the first Kunitz domain of TFPI. (•) No platelets; (▪) calcium ionophore plus thrombin activated platelets; (▴) calcium ionophore plus thrombin activated platelets with 100 μM anti-TFPI antibody.