Identification of thrombin activatable fibrinolysis inhibitor (TAFI) in human platelets by Laurent O. Mosnier, Paula Buijtenhuijs, Pauline F. Marx, Joost C. M. Meijers, and Bonno N. Bouma Blood Volume 101(12):4844-4846 June 15, 2003 ©2003 by American Society of Hematology

Identification of TAFI in platelets. Identification of TAFI in platelets. TAFI was identified in cytospins of gel-filtered platelets by immunofluorescence confocal microscopy (A), using a monoclonal antibody against TAFI (9H10) and an FITC-labeled secondary antibody. The scale bar represents 5 μm. TAFI secretion by stimulated platelets (B). Gel-filtered platelets (2.8 × 107) were incubated with a high (dark bars) or a low (light bars) concentration of platelet agonist for 15 minutes at 37°C, and the secretion of TAFI was determined by ELISA. The agonists used were thrombin (IIa; 5 or 0.5 U/mL), thrombin receptor–activating peptide (TRAP; 100 or 10 μM), adenosine 5′-diphosphate (ADP; 20 or 2 μM), collagen (COL; 10 or 1 μg/mL), and epinephrine (EPI; 10 or 1 μM). Each bar represents the mean ± SEM of 5 independent experiments using different donors. Laurent O. Mosnier et al. Blood 2003;101:4844-4846 ©2003 by American Society of Hematology

Analysis of platelet-derived TAFI Analysis of platelet-derived TAFI. Platelet-derived TAFI and TAFI purified from plasma (lane 1; 300 pg) were analyzed on Western blot (A). Analysis of platelet-derived TAFI. Platelet-derived TAFI and TAFI purified from plasma (lane 1; 300 pg) were analyzed on Western blot (A). Gel-filtered platelets (3 × 107 cells) were incubated with either 0.5% triton (lane 2), buffer (lane 3), or 0.5 U/mL thrombin (lane 4) 15 minutes at 37°C. Deglycosylation of platelet-derived TAFI (B). Purified TAFI (16 ng, lane 1, or 1.6 ng, lane 2) and gel-filtered platelets (7 × 107 cells) incubated with thrombin (0.5 U/mL) for 15 minutes at 37°C (lane 3) were subjected to PNGase F treatment. TAFI was detected with a monoclonal antibody against TAFI (9H10). Approximate molecular masses (kDa) are shown on the left. Laurent O. Mosnier et al. Blood 2003;101:4844-4846 ©2003 by American Society of Hematology

Activation of platelet-derived TAFI Activation of platelet-derived TAFI. (A) Gel-filtered platelets (3 × 108) were either incubated with thrombin (50 nM), thrombomodulin (10 nM), and/or CPI (10 μg/mL) as indicated. Activation of platelet-derived TAFI. (A) Gel-filtered platelets (3 × 108) were either incubated with thrombin (50 nM), thrombomodulin (10 nM), and/or CPI (10 μg/mL) as indicated. As a control, platelets were removed by centrifugation before addition of thrombin-thrombomodulin to the supernatant (SUP). The inactivation of platelet-derived TAFIa was analyzed by incubation of the activated platelet mixture for 30 minutes at 37°C after the addition of PPACK but before addition of the substrate (hippuryl-Arg). (B) Thrombin (50 nM)-thrombomodulin (10 nM), activated platelets (2 × 108) or plasma TAFI were incubated for 1 hour at room temperature with immobilized minimally degraded fibrin (Desafib X), after which binding of plasminogen was determined. In both panels, each bar represents the mean ± SEM of 3 independent experiments. Laurent O. Mosnier et al. Blood 2003;101:4844-4846 ©2003 by American Society of Hematology