Figure 4
Figure 4. Activation of specific protease activated receptors stimulates the selective release of α-granules containing either endostatin or VEGF. Platelets were treated with platelet buffer in the presence of agonists for 10 minutes with PAR4-activating peptide (A-D), and PAR1-activating peptide (E-H) and then fixed and processed for immunofluorescence microscopy. Cells were stained with either anti-VEGF antibodies (Alexa 488 green labeling; A,E) or antiendostatin antibodies (Alexa 568 red labeling; C,G,) to assay for granule retention or release. All micrographs were taken at the same exposure time. Corresponding staining with Alexa-phalloidin (B,D,F,H) in the bottom panels highlights the morphology of the platelets. Negative controls consisting of incubation with both secondary fluorescently labeled antibodies only or incubation with only primary antibodies failed to show appreciable fluorescence (data not shown). Images are representative of at least 10 high-power fields for each experiment, and each experiment was performed 3 times. Representative images of immunoelectron microscopy of platelets treated with either PAR4-AP (I) or PAR1-AP (J). Double immunogold labeling on platelet sections was performed with the use of anti-VEGF antibody and antiendostatin antibodies. In the PAR4-treated samples (I), large gold particles representing antiendostatin staining (15 nm) are evident on one α-granule (arrow) and small gold particles (5 nm) representing VEGF staining are abundantly present on separate population of multiple α-granules. In the PAR1-treated samples (J), large gold particles representing anti-VEGF staining (15 nm, arrow) are evident on one α-granule (arrow) and small gold particles (5 nm) representing endostatin staining are abundantly present on separate population of multiple α-granules. (K) A model illustrating the mechanism of differential granule release from platelets. A simplified summary of the pathway is shown. Resting platelets contain both proangiogenic (green) and antiangiogenic (red) granules. Selective activation of the PAR1 receptor causes release of granules containing proangiogenic factors, whereas selective activation of the PAR4 receptor causes release of granules containing antiangiogenic factors.

Activation of specific protease activated receptors stimulates the selective release of α-granules containing either endostatin or VEGF. Platelets were treated with platelet buffer in the presence of agonists for 10 minutes with PAR4-activating peptide (A-D), and PAR1-activating peptide (E-H) and then fixed and processed for immunofluorescence microscopy. Cells were stained with either anti-VEGF antibodies (Alexa 488 green labeling; A,E) or antiendostatin antibodies (Alexa 568 red labeling; C,G,) to assay for granule retention or release. All micrographs were taken at the same exposure time. Corresponding staining with Alexa-phalloidin (B,D,F,H) in the bottom panels highlights the morphology of the platelets. Negative controls consisting of incubation with both secondary fluorescently labeled antibodies only or incubation with only primary antibodies failed to show appreciable fluorescence (data not shown). Images are representative of at least 10 high-power fields for each experiment, and each experiment was performed 3 times. Representative images of immunoelectron microscopy of platelets treated with either PAR4-AP (I) or PAR1-AP (J). Double immunogold labeling on platelet sections was performed with the use of anti-VEGF antibody and antiendostatin antibodies. In the PAR4-treated samples (I), large gold particles representing antiendostatin staining (15 nm) are evident on one α-granule (arrow) and small gold particles (5 nm) representing VEGF staining are abundantly present on separate population of multiple α-granules. In the PAR1-treated samples (J), large gold particles representing anti-VEGF staining (15 nm, arrow) are evident on one α-granule (arrow) and small gold particles (5 nm) representing endostatin staining are abundantly present on separate population of multiple α-granules. (K) A model illustrating the mechanism of differential granule release from platelets. A simplified summary of the pathway is shown. Resting platelets contain both proangiogenic (green) and antiangiogenic (red) granules. Selective activation of the PAR1 receptor causes release of granules containing proangiogenic factors, whereas selective activation of the PAR4 receptor causes release of granules containing antiangiogenic factors.

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