Secretion of TGFBIp from activated human platelets and TGFBIp binding on activated platelet surfaces. (A) TGFBIp exists in platelets. Washed platelets were allowed to adhere on fibronectin and were stained with anti-CD41 (red) and anti-TGFBIp (green) antibodies. Attached platelets and TGFBIp expression were observed under a fluorescence microscope. (Top) Low magnification (×400; white bar represents 10 μm). (Middle and bottom) High magnification (×1000; white bar represents 5 μm). (B) TGFBIp on resting (left) and thrombin-activated (right) platelet surfaces was detected by FACS analysis. Black shading denotes immunoglobulin G control, and the gray line denotes TGFBIp. Presence of TGFBIp in resting and activated platelets (thrombin activation) was analyzed by immunoblotting. Washed platelets were incubated with (C) or without stirring (D), during which they were activated by thrombin. Cell (cell lysate) and supernatant (sup) were separated by centrifugation at 18 000g and were immunoblotted by anti-TGFBIp antibody. β-actin was loaded as a control. (E) TGFBIp mRNA exists in platelets. Total RNA was isolated and subject to RT-PCR for the analysis of TGFBIp, VWF, and GAPDH mRNA expression. (F) Measurement of the amount of TGFBIp in platelets. The amount of TGFBIp in platelets was semiquantitatively measured by Western blot analysis.