Figure 5
GSAO+ platelets form in occluding murine thrombi and are attenuated with megakaryocyte-directed deletion of the cyclophilin D gene. (A-B) Dylight 649-conjugated antiplatelet CD42b antibody and GSAO-Oregon Green or control compound GSCA-Oregon green were injected into the murine circulation and thrombus initiated in the cremaster muscle arterioles by FeCl3 injury. Thrombi were captured by confocal intravital video microscopy, and images are displayed as 3-dimensional reconstructions (maximum intensity projection mode). Occlusive thrombi incorporate GSAO-Oregon green, but not the control compound. (C-E) Dylight 649-conjugated antiplatelet CD42b and GSAO-Oregon Green were injected into the murine circulation and thrombus initiated in the cremaster muscle arterioles by either laser (C) or FeCl3 (D) injury. Thrombi were captured by confocal intravital video microscopy and images displayed as cross-sectional orthogonal views for single-plane colocalization. There was minimal GSAO signal in the nonocclusive thrombus initiated by laser injury, but extensive signal in the occlusive thrombus initiated by FeCl3 injury (C; n = 6-8 in 6-8 different mice; Manders correlation coefficient, **P < .01). (F) Dylight 649-conjugated anti-platelet CD42b antibody, GSAO-AF546, and AF488-conjugated anti-fibrin antibody were injected into the murine circulation of wild-type (WT) or platelet-specific cyclophilin D-deficient (PF4Cre+ CypDFl/Fl) mice and thrombus initiated in the cremaster muscle arterioles by FeCl3 injury. Thrombi were captured by confocal intravital video microscopy and images analyzed for integrated GSAO, platelet, and fibrin fluorescence (WT n = 4, 29 thrombi; PF4Cre+ CypDFl/Fl n = 3, 15 thrombi; *P < .05; **P < .001). (G) Dylight 649-conjugated anti-platelet CD42b antibody, GSAO-Oregon Green, and calcium-sensing dye rhodamine 2 were injected into the murine circulation and thrombus initiated in the cremaster muscle arterioles by FeCl3 injury. Thrombus was visualized 10 minutes after injury. Images are represented as cross-sectional orthogonal views of single confocal plane displaying platelets and GSAO (left) with rhodamine signal (right). Persistence of high calcium signal is demonstrated in the GSAO+ platelets consistent with necrotic cell death.

GSAO+ platelets form in occluding murine thrombi and are attenuated with megakaryocyte-directed deletion of the cyclophilin D gene. (A-B) Dylight 649-conjugated antiplatelet CD42b antibody and GSAO-Oregon Green or control compound GSCA-Oregon green were injected into the murine circulation and thrombus initiated in the cremaster muscle arterioles by FeCl3 injury. Thrombi were captured by confocal intravital video microscopy, and images are displayed as 3-dimensional reconstructions (maximum intensity projection mode). Occlusive thrombi incorporate GSAO-Oregon green, but not the control compound. (C-E) Dylight 649-conjugated antiplatelet CD42b and GSAO-Oregon Green were injected into the murine circulation and thrombus initiated in the cremaster muscle arterioles by either laser (C) or FeCl3 (D) injury. Thrombi were captured by confocal intravital video microscopy and images displayed as cross-sectional orthogonal views for single-plane colocalization. There was minimal GSAO signal in the nonocclusive thrombus initiated by laser injury, but extensive signal in the occlusive thrombus initiated by FeCl3 injury (C; n = 6-8 in 6-8 different mice; Manders correlation coefficient, **P < .01). (F) Dylight 649-conjugated anti-platelet CD42b antibody, GSAO-AF546, and AF488-conjugated anti-fibrin antibody were injected into the murine circulation of wild-type (WT) or platelet-specific cyclophilin D-deficient (PF4Cre+ CypDFl/Fl) mice and thrombus initiated in the cremaster muscle arterioles by FeCl3 injury. Thrombi were captured by confocal intravital video microscopy and images analyzed for integrated GSAO, platelet, and fibrin fluorescence (WT n = 4, 29 thrombi; PF4Cre+ CypDFl/Fl n = 3, 15 thrombi; *P < .05; **P < .001). (G) Dylight 649-conjugated anti-platelet CD42b antibody, GSAO-Oregon Green, and calcium-sensing dye rhodamine 2 were injected into the murine circulation and thrombus initiated in the cremaster muscle arterioles by FeCl3 injury. Thrombus was visualized 10 minutes after injury. Images are represented as cross-sectional orthogonal views of single confocal plane displaying platelets and GSAO (left) with rhodamine signal (right). Persistence of high calcium signal is demonstrated in the GSAO+ platelets consistent with necrotic cell death.

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