Fig 2: Differential BDNF protein levels in mouse, rat, and human megakaryocytes and platelets. Western blot lysates of cultured Mks (A) and blood platelets (B) are shown. Eighty micrograms of protein per lane were loaded, and the blotting membrane was incubated with the mouse monoclonal antibody 3C11 developed by Icosagen (Tartu, Estonia). Recombinant BDNF and pro-BDNF were used as molecular mass markers and antibodies to ß-actin as loading controls. Asterisks (top right panels) point to a band unrelated to BDNF likely corresponding to immunoglobulin light chains in the mouse sample. Note the absence of BDNF in mouse Mks and platelets. C, antibodies to BDNF 9 (green) (15) and PF4 (red) reveal expression of both antigens in mature rat and human Mks. Note that unlike PF4, BDNF is not detectable in mouse Mks. The co-localization of BDNF with PF4 in rat and human Mks was quantified using the pixel intensity specifically generated by each channel. In humans, 83% and in rats 86% BDNF-positive granules were also PF4-positive. Blue, DAPI staining. D, immunofluorescence staining of F-actin (red) and BDNF (green) in proplatelet-forming cultured human Mks. Arrows indicate BDNF accumulation in proplatelet buds.

Fig 3: Platelet-specific proteins are abundantly detected in sEV fractions derived from serum of mouse blood.(A-D) Western blot analysis of the sEV fractions derived from plasma and serum using antibodies against EV marker proteins and platelet-specific proteins (A) and their densitometric analyses (B-D). Equal amounts of proteins were loaded onto each lane and analyzed (n = 3). The level of CD63 (B) and the levels of EV markers CD9 and Hsc70 relative to CD63 (C) showed no statistical difference between plasma and serum EVs. In contrast, platelet-specific GPIIb, GPIIIa, and PF4 were significantly increased in the serum sEV fractions (D). The asterisk in (A) indicates a nonspecific signal. (E) Optiprep density-gradient centrifugation analysis of the serum sEV fraction with EV marker proteins and platelet-specific proteins. GPIIb and GPIIIa were both detected in the fractions at the same density as the sEV, and cofractionated with EV markers. Data are shown as the average ± SEM. *P < 0.05, **P < 0.01, Student t-test. n.s., not significant.

Fig 4: Serum EVs contain high levels of platelet proteins in human blood.(A) Western blot analysis of the sEV fractions derived from plasma and serum of 5 individuals, using antibodies against EV markers and platelet-specific proteins. Equal amounts of proteins were loaded onto each lane and analyzed. (B) Bar graph showing relative protein levels of CD63 that were calculated from the band intensity of the Western blot image in (A). (C-D) Bar graphs showing the levels of the EV marker CD9 (C), and platelet-specific markers GPIIb, GPIIIa and PF4 (D), relative to that of CD63. Whereas the level of CD9 was comparable between the sEV fractions from plasma and serum, all the platelet markers showed a clear tendency of increased levels in serum EVs. (E) NTA results showing the numbers (left) and median diameters (right) of the particles in the sEV fractions derived from human plasma and serum. (F) Comparison of the sEV fractions derived from mouse and human blood by Western blot analysis using antibodies against EV markers and lipoprotein ApoA1. The sEV fractions isolated from human blood contain very low levels of the EV markers CD63 and Hsc70, but a high level of ApoA1, implying a large difference in particle population between human and mouse blood. Data are shown as the average ± SEM. Student t-test. n.s., not significant.

Fig 5: Validating CLAD-associated proteins in recipient rat liver allografts. (a) Immunohistochemistry analysis showed that CXCL4 detected on POD 60 was significantly expressed in CLAD liver allografts, compared with the control. (b) Validation by Western blot analysis on POD 60 showed that CXCL4, CXCR3, EGFR, JAK2, STAT3, and Collagen IV were significantly overrepresented in all CLAD liver grafts as compared with the control.