SLPI does not colocalize with known platelet granule markers but is partially released after thrombin stimulation. (A) Platelets were stained with SLPI antibody and with antibodies specific for markers of α-granules (von Willebrand factor [vWF]), dense bodies (serotonin), or lysosomes (LAMP-2) and examined by deconvolution IF microscopy. The SLPI antibody again identifies bicompartmental expression, but the central punctate distribution failed to colocalize reliably with any of the tested granule markers. Scale bars, 2 μm. (B) Washed platelet suspensions were stimulated with thrombin (Thr, +) or phosphate-buffered saline (-) and centrifuged, and the pellet (P) and supernatant (S) fractions were analyzed by immunoblotting. Activation achieves partial release of SLPI and the α-granule protein platelet factor 4 (PF4). Thrombin-stimulated SLPI and PF4 release is impaired in β1-tubulin^-/- platelets (right). (C) Control immunoblotting of platelet fractions with rabbit antimouse platelet serum (RAMPS) confirms that thrombin stimulation did not induce platelet lysis: only a few proteins, indicated by dots, transfer to the S fraction.