Angiogenesis is regulated by a novel mechanism: pro- and antiangiogenic proteins are organized into separate platelet alpha granules and differentially released

Abstract

Platelets, in addition to their function in hemostasis, play an important role in wound healing and tumor growth. Because platelets contain angiogenesis stimulators and inhibitors, the mechanisms by which platelets regulate angiogenesis remain unclear. As platelets adhere to activated endothelium, their action can enhance or inhibit local angiogenesis. We therefore suspected a higher organization of angiogenesis regulators in platelets. Using double immunofluorescence and immunoelectron microscopy, we show that pro- and antiangiogenic proteins are separated in distinct subpopulations of alpha-granules in platelets and megakaryocytes. Double immunofluorescence labeling of vascular endothelial growth factor (VEGF) (an angiogenesis stimulator) and endostatin (an angiogenesis inhibitor), or for thrombospondin-1 and basic fibroblast growth factor, confirms the segregation of stimulators and inhibitors into separate and distinct alpha-granules. These observations motivated the hypothesis that distinct populations of alpha-granules could undergo selective release. The treatment of human platelets with a selective PAR4 agonist (AYPGKF-NH(2)) resulted in release of endostatin-containing granules, but not VEGF-containing granules, whereas the selective PAR1 agonist (TFLLR-NH(2)) liberated VEGF, but not endostatin-containing granules. In conclusion, the separate packaging of angiogenesis regulators into pharmacologically and morphologically distinct populations of alpha-granules in megakaryocytes and platelets may provide a mechanism by which platelets can locally stimulate or inhibit angiogenesis.

Figure 1

Pro- and antiangiogenic regulators organize into separate, distinct α-granules in resting platelets. Double immunofluorescence microscopy of resting platelets using antibodies against VEGF (A) and endostatin (B) and an overlay (C). Double immunofluorescence microscopy of resting platelets using antibodies against bFGF (D) and TSP-1 (E) and an overlay (F). Double immunofluorescence microscopy of resting platelets using antibodies against fibrinogen (G) and von Willebrand factor (H) and an overlay (I). Double immunofluorescence microscopy of resting platelets using antibodies against von Willebrand factor (J) and endostatin (K) and an overlay (L). Double immunofluorescence microscopy of resting platelets using antibodies against VEGF (M) and fibrinogen (N) and an overlay (O)..

Figure 2

Pro- and antiangiogenic regulatory proteins are segregated into separate, distinct α-granules in megakaryocyte proplatelets. Double immunofluorescence microscopy of proplatelets using antibodies against VEGF (A) and endostatin (B) and an overlay (C). Double immunofluorescence microscopy of proplatelets using antibodies against bFGF (D) and TSP-1 (E) and an overlay (F). Double immunofluorescence microscopy of proplatelets using antibodies against fibrinogen (G) and von Willebrand factor (H) and an overlay (I). Double immunofluorescence microscopy of proplatelets against VEGF (J) and fibrinogen (K) and an overlay (L).

Figure 3

Localization of proteins in resting, human platelets using immunoelectron microscopy of ultrathin cryosections. Single immunogold labeling on ultrathin platelet sections was performed with anti-VEGF (A) and antiendostatin (B) antibodies. Double immunogold labeling on platelet sections was performed with the use of anti-VEGF antibody and antiendostatin antibodies. Large gold particles representing anti-VEGF staining (15 nm, arrows) are evident on one population of α-granules and small gold particles (5 nm) representing endostatin staining are abundantly present on a different population of α-granules (arrowheads) (C). Single immunogold labeling on ultrathin platelet sections was performed with antifibrinogen (D) and anti-VWF (E) antibodies. Double immunogold labeling on platelet sections was performed with the use of antifibrinogen antibody, which was revealed with a 15-nm, gold conjugate (arrows) and then with an antibody to VWF, which was revealed with a 5-nm, gold conjugate (arrowheads) (F). Single immunogold labeling on ultrathin platelet sections was performed with anti–P-selectin antibody (G). Gold particles representing P-selectin staining are abundantly present on the α-granules as well as the cell-surface membrane. Bar represents 300 nm. (H) The bar graph shows the quantitation of the percentage of α-granules positive (via immunogold staining) for specific factors. The data represent 3 separate experiments; error bars represent SD. More than 100 granules were scored for each study.

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.