Figure 1.

Transglutaminase activity confined to a subpopulation of phospholipid phosphatidylserine (PS)-exposing platelets. Washed human platelets (5×10⁷/mL) were stimulated with convulxin (Cvx, 100 ng/mL) plus thrombin (Thr, 4 nM) in the presence of 0.2 mM GPRP and 2 mM CaCl₂. Samples, taken at given time points, were labeled for 5 min with Rhod-A14 and AF647-annexin A5. Platelets were pre-treated with K9-DON (100 μM), where indicated. (A) Representative histograms obtained by flow cytometry. (B) Platelet subpopulations staining with Rhod-A14 (white bars) or AF647-annexin A5 (black bars) after 60-min stimulation with Cvx (100 ng/mL), Thr (4 nM), SFLLRN (15 μM) and/or ionomycin (10 μM), as indicated. (C–E) Effects of factor XIII and transglutaminase inhibitors on platelet subpopulations staining with Rhod-A14 (C) or AF647-annexin A5 (E) after stimulation with Cvx (100 ng/mL) plus Thr (4 nM). Platelets were pre-incubated with vehicle (○, solid line), 100 unit/mlLfactor XIII (●, dotted line), 100 μM K9-DON (■, dotted line), or 200 μM Boc-DON (♦, dotted line). Solid gray line, unstimulated platelets. (D) Dose-dependent inhibition of Rhod-A14 binding (60 min) by K9-DON or Boc-DON. Mean ± SEM (n=3–6); *P<0.05.