Our studies using a novel method to generate mice with multiple platelet-specific signaling defects identified platelet ITAM signaling as a critical event in the maintenance of vascular integrity at sites of inflammation. Platelets defective in ITAM signaling were unable to prevent hemorrhage at sites of immune complex–induced inflammation in the skin as well as LPS-induced inflammation in the lung. In contrast, platelets defective in GPCR signaling were indistinguishable from WT platelets in their ability to support vascular integrity in both models. Our findings provided evidence that the platelet signaling response required to prevent hemorrhage in inflammation can be generalized for different triggers and vascular beds, although this conclusion will need to be confirmed in other models of inflammation.
The central finding of our work was that the platelet ITAM signaling pathway is critical for the maintenance of vascular integrity in inflammation. Mouse platelets express 2 ITAM receptors, GPVI and CLEC2. Inhibition of GPVI or deficiency in CLEC2 partially reduced the ability of platelets to maintain vascular integrity in immune complex–induced inflammation in the skin or LPS-induced inflammation in the lung. A defect in both GPVI and CLEC2 signaling, or genetic deletion of the downstream adapter protein SLP-76, completely abolished the positive effect of platelets on vascular integrity. GPVI signaling is likely activated at sites of inflammation by collagen and/or laminin, the 2 physiological ligands for GPVI found in the vessel wall (38
). More difficult to explain, however, is how CLEC2 on platelets is activated in these situations. Biochemical and genetic studies have established the transmembrane protein podoplanin as the major CLEC2 ligand. Podoplanin expression was first documented in glomerular podocytes, lymphatic endothelium, and the brain (39
). However, podoplanin was not found in blood endothelial cells or pericytes, and smooth muscle podoplanin was identified in advanced, but not early, atherosclerotic lesions (40
). Thus, podoplanin is not constitutively expressed in the vessel wall. In LPS-induced inflammation in the lung, platelet CLEC2 may engage podoplanin expressed at high levels in type I epithelial cells in alveolae (41
). However, a cellular source for podoplanin has not yet been identified in the skin. Alternatively, podoplanin could be “delivered” to sites of inflammation by infiltrating macrophages, a recently identified source of this molecule (42
). Our observations that both GPVI and CLEC2 contributed to a similar extent to platelet-dependent vascular integrity in the lung and skin are consistent with a tissue-independent source of podoplanin as the trigger of platelet CLEC2 signaling. Finally, it is also possible that platelet CLEC2 receptors respond to an as yet unidentified ligand expressed in these tissues or in the vessel wall. Future studies in mice with conditional deletion of podoplanin in various tissues/cell types, such as alveolar cells, lymphatic endothelial cells, monocytes/macrophages, or smooth muscle cells, will be required to distinguish among these possible mechanisms.
Our results also provided evidence that platelet activation via soluble agonists, such as thrombin, TxA2
, and ADP, is dispensable for the contribution of these cells to the maintenance of vascular integrity. This is surprising, as all 3 agonists play key roles in the ability of platelets to form thrombi at sites of vascular injury (1
). Furthermore, ADP and TxA2
provide critical feedback signaling for platelet activation via ITAM receptors (1
). As shown recently, however, platelets contribute to vascular integrity in inflammation by a mechanism that is independent of platelet aggregation and hemostasis (26
), but likely depends on the release of vasoactive mediators stored in platelet granules. Activation by ITAM receptors may trigger a specific release reaction different from that induced by GPCRs. Such agonist-specific differences in the released granule content have been described recently, but these studies were focused on the different agonists for GPCRs (43
Finally, our work highlighted similarities between the molecular mechanisms regulating vascular integrity in inflammation and those facilitating the separation of blood and lymphatic vessels during development. As shown by various groups, lymphatic vascular development during embryogenesis is dependent on platelet activation via the CLEC2/SLP-76 axis (11
). Blood-filled lymphatics were also observed in adult mice transplanted with bone marrow from Clec2–/–
). In contrast, no such defects have been described for mice lacking components of the GPCR signaling machinery or for mice deficient in key receptors for platelet adhesion and aggregation. It is important to note, however, that defects in lymphatic vascular development have not been described for mice lacking GPVI, which suggests that the molecular mechanisms regulating vascular development and vascular integrity in the adult differ, at least with regard to triggering platelet activation.
Critical to our studies was the development of an adoptive transfer system for platelets into TP mice. Here, selective depletion of circulating platelets was achieved in Tg mice with antibodies that recognize hIL-4R, a heterologous antigen expressed on circulating platelets in these mice. With the use of this technique, we were able to rapidly deplete virtually all circulating platelets in hIL-4Rα/GPIbα–Tg mice and replace them with platelets defective in all major GPCR or ITAM receptors. Importantly, this antibody-mediated depletion of platelets did not affect neutrophils or other blood cells (data not shown). In comparison, peripheral platelet counts in mice lacking the thrombopoietin receptor c-Mpl, a key regulator of megakaryocytopoiesis, are only reduced by approximately 90% compared with controls (44
). The remaining platelets are fully functional, and c-mpl–/–
mice do not bleed. Furthermore, c-Mpl deficiency also affects other hematopoietic progenitor cells and thus does not represent a platelet-specific model. Mice lacking the transcription factor p47 NF-E2 (45
) have near-complete TP due to a defect on thrombopoiesis, which leads to excessive hemorrhage and perinatal lethality. In addition, p47 NF-E2 knockout mice show several red blood cell defects. Finally, TP induced by chemotherapeutic agents, such as 1,4-butanediol dimethanesulfonate (Busulfan; ref. 47
), is accompanied by leukopenia, and thus cannot be used for studying inflammation in mice.
In summary, we here described the comprehensive analysis of the platelet signaling response required to maintain vascular integrity in inflammation. By generating mice with multiple platelet-specific signaling defects, we demonstrated that vascular integrity in inflammation depended on both ITAM receptors, GPVI and CLEC2, as well as the adapter protein SLP-76. In contrast, signaling via the major GPCRs was dispensable for this response. Our studies highlight potential complications associated with novel antiplatelet drugs targeting the ITAM signaling pathway, which may lead to blood-lymph mixing or to bleeding at sites of inflammation.