Platelet Activation Receptor CLEC-2 Regulates Blood/Lymphatic Vessel Separation by Inhibiting Proliferation, Migration, and Tube Formation of Lymphatic Endothelial Cells

Makoto Osada,Osamu Inoue,Guo Ding,Toshiaki Shirai,Hirotake Ichise,Kazuyoshi Hirayama,Katsuhiro Takano,Yutaka Yatomi,Masanori Hirashima,Hideki Fujii,Katsue Suzuki-Inoue,Yukio Ozaki

doi:10.1074/jbc.m111.329987

Abstract

The platelet activation receptor CLEC-2 plays crucial roles in thrombosis/hemostasis, tumor metastasis, and lymphangiogenesis, although its role in thrombosis/hemostasis remains controversial. An endogenous ligand for CLEC-2, podoplanin, is expressed in lymphatic endothelial cells (LECs). We and others have reported that CLEC-2-deficiency is lethal at mouse embryonic/neonatal stages associated with blood-filled lymphatics, indicating that CLEC-2 is essential for blood/lymphatic vessel separation. However, its mechanism, and whether CLEC-2 in platelets is necessary for this separation, remains unknown. We found that specific deletion of CLEC-2 from platelets leads to the misconnection of blood/lymphatic vessels. CLEC-2(+/+) platelets, but not by CLEC-2(-/-) platelets, inhibited LEC migration, proliferation, and tube formation but had no effect on human umbilical vein endothelial cells. Additionally, supernatants from activated platelets significantly inhibited these three functions in LECs, suggesting that released granule contents regulate blood/lymphatic vessel separation. Bone morphologic protein-9 (BMP-9), which we found to be present in platelets and released upon activation, appears to play a key role in regulating LEC functions. Only BMP-9 inhibited tube formation, although other releasates including transforming growth factor-β and platelet factor 4 inhibited proliferation and/or migration. We propose that platelets regulate blood/lymphatic vessel separation by inhibiting the proliferation, migration, and tube formation of LECs, mainly because of the release of BMP-9 upon activation by CLEC-2/podoplanin interaction.

Highlights

Mice deficient in the platelet receptor CLEC-2 for podoplanin showed impaired blood/lymphatic vessel separation
We have proposed that podoplanin in lymphatic endothelial cells (LECs) activates platelets by binding to CLEC-2 in the connection between lymph sacs and veins during the developmental stages and that released Bone morphologic protein-9 (BMP-9) from activated platelets, in conjunction with other releasates, inhibits the migration, proliferation, and tube formation of LECs, which facilitates blood/lymphatic vessel separation
Subsequent studies revealed that the surface expression of CLEC-2 is limited to in platelets/megakaryocytes [1, 32] in human blood cells, it is expressed in the liver sinusoid [32, 33]

Summary

Background

Mice deficient in the platelet receptor CLEC-2 for podoplanin showed impaired blood/lymphatic vessel separation. We propose that platelets regulate blood/lymphatic vessel separation by inhibiting the proliferation, migration, and tube formation of LECs, mainly because of the release of BMP-9 upon activation by CLEC-2/podoplanin interaction. CLEC-2-deficient mice show embryonic/neonatal lethality, and almost all of the CLEC-2Ϫ/Ϫ pups die shortly after birth, probably because of respiratory failure These studies revealed that the CLEC-2/ podoplanin interaction facilitates blood/lymphatic vessel separation in the developmental stages when primary lymph sacs are derived from the cardinal vein. Platelet aggregate formation, which is an indicator of platelet activation, was observed in the connection between the cardinal vein and primary lymph sacs in normal mice but not in SLP-76- or podoplanin-deficient mice [6, 10] These findings suggest that platelet activation is necessary for blood/lymphatic vessel separation. We reported some of these results previously in abstract form following the 52nd Annual Meeting of the American Society of Hematology [11]

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION