It remains unknown how abnormal blood-lymphatic vascular connections arise in the absence of CLEC-2-PDPN signaling. Platelet-LEC interactions can be seen in the cardinal vein during the specification of PDPN-expressing LECs from venous endothelial cells,18, 20
but similar interactions have not been detected in the intestine where large numbers of blood-lymphatic connections are formed.18
Since both PDPN and CLEC-2 are transmembrane proteins, the mechanism requires direct contact between LECs and platelets. The use of platelets as a means of sensing blood vessel contact by LECs makes sense, as platelets are one of the few blood cell types that cannot extravasate and enter lymphatic vessels, even following trauma. Precisely how platelet activation by LECs negatively regulates the formation of LEC connections to blood vessels remains speculative. The requirements for SYK and SLP-76 indicate a need for platelet activation downstream of the PDPN-CLEC-2 interaction. The fact that mice lacking platelet integrins required for platelet aggregation do not form blood-lymphatic connections suggests that the formation of a platelet plug, the hallmark of platelet-mediated hemostasis, may not be the key step downstream of platelet activation. Instead, it is tempting to speculate that platelet degranulation may release regulators of LEC growth that inhibit the formation of blood-lymphatic connections. Platelet alpha granules are known to contain numerous angiogenic regulators, supporting the hypothesis that degranulation is the mechanism by which platelet activation controls lymphatic growth. Further genetic studies will be required to test the effects of such agents on LEC growth and lymphatic development.
The studies described above are also significant because they describe a clear embryonic role for platelets that is not connected to hemostasis. Platelet activation has been associated with many non-hemostatic roles in mature animals, including inflammation,28, 29
wound healing,30, 31
primary tumor growth,32
and tumor metastasis,33
but few embryonic roles have been defined. The lack of an embryonic phenotype in NF-E2-deficient mice lacking most but not all platelets 34, 35
has suggested that platelets do not have such roles, but this is clearly not the case and it seems likely that future studies will identify other roles for platelets in regulating development of the cardiovascular and other systems.
Finally, it remains unclear whether PDPN and/or CLEC-2 play roles outside of lymphatic vascular development. The number of CLEC-2 receptors on the surface of human or mouse platelets has not yet been determined. However, comparison of hematopoietic and megakaryocyte gene analysis libraries, as well as in situ hybridization studies of mouse megakaryocytes and binding of anti-CLEC-2 antibodies and PDPN-Fc fusion proteins suggests that CLEC-2 may be one of the most highly expressed activating receptors on the platelet surface.18, 23
In vitro studies of Clec-2−/−
platelets have suggested that CLEC-2 deficiency does not affect collagen-induced aggregate formation.36
Furthermore, tyrosines in the YxxL motif of platelet CLEC-2 receptors are not phosphorylated upon flow over collagen,36
suggesting that CLEC-2 is not involved in platelet activation by classical hemostatic stimuli. The existence of a PDPN-independent role for CLEC-2 remains unclear and is an open question in the field. Future studies addressing the hemostatic role(s) of CLEC-2 and PDPN are likely to yield new insights into platelet biology.