Volume 14 Supplement 1
New horizon in platelet function: with special reference to a recently-found molecule, CLEC-2
© The Author(s). 2016
Published: 4 October 2016
Platelets play a key role in the pathophysiological processes of hemostasis and thrombus formation. However, platelet functions beyond thrombosis and hemostasis have been increasingly identified in recent years. A large body of evidence now exists which suggests that platelets also play a key role in inflammation, immunity, malignancy, and furthermore in organ development and regeneration, such as the liver.
We have recently identified CLEC-2 on the platelet membrane, which induces intracellular activation signals upon interaction of a snake venom, rhodocytin. Later we discovered that podoplanin, present in renal podocytes and lymphatic endothelial cells, both of which are not accessible to platelets in blood stream, is an endogenous ligand for CLEC-2. In accord with our expectation, platelet-specific CLEC-2 knockout mice have a phenotype of edema, lymphatic vessel dilatation, and the presence of blood cells in lymphatic vessels. It is suggested that lymphatic/blood vessel separation during the developmental stage is governed by cytokines released from platelets activated by the interaction between platelet CLEC-2 and podoplanin present on lymphatic endothelial cells.
Recombinant CLEC-2 bound to early atherosclerotic lesions and normal arterial walls, co-localizing with vascular smooth muscle cells (VSMCs). Flow cytometry and immunocytochemistry showed that recombinant CLEC-2, but not an anti-podoplanin antibody, bound to VSMCs, suggesting that CLEC-2 ligands other than podoplanin are present in VSMCs. Protein arrays and Biacore analysis were used to identify S100A13 as a CLEC-2 ligand in VSMCs. S100A13 was released upon oxidative stress, and expressed in the luminal area of atherosclerotic lesions.
Megakaryopoiesis is promoted through the CLEC-2/podoplanin interaction in the vicinity of arterioles, not sinusoids or lymphatic vessels. There exist podoplanin-expressing bone-marrow (BM) arteriolar stromal cells, tentatively termed as BM fibroblastic reticular cell (FRC)-like cells, and megakaryocyte colonies were co-localized with periarteriolar BM FRC-like cells in the BM. CLEC-2/podoplanin interaction induces BM FRC-like cells to secrete CCL5 to facilitate proplatelet formation. These observations indicate that a reciprocal interaction with between CLEC-2 on megakaryocytes and podoplanin on BM FRC-like cells contributes to the periarteriolar megakaryopoietic microenvironment in mouse BM.
KeywordsPlatelets Thrombosis Beyond hemostasis Immunity CLEC-2 Lymphangiogeneis Smooth muscle cells Megakaryopoiesis
Platelets play a key role in the pathophysiological processes of hemostasis and thrombus formation. However, platelet functions beyond thrombosis and hemostasis have been increasingly identified in recent years. This review reports on the newly identified platelet functions beyond hemostasis, especially with the reference to CLEC-2.
In primitive organisms such as horseshoes crabs, except for red blood cells, there is only one additional type of blood cells, hemocytes, which is involved in bactericidal function, inflammation, immunity, and hemostasis. In more highly evolved creatures, there are five types of white blood cells and platelets which respectively take care of specific functions, and platelets have long been considered to be an expert in thrombosis and hemostasis but nothing else. However, as described above, platelets are now increasingly considered to play as liaison interactive effects between different cell types and tissues.
Platelets are the second most abundant cell type in the circulation after red blood cells. Platelets contain three types of cytoplasmic granules, including α-granules, dense granules and lysosome, which contain a large number of autocrine and paracrine substances. Recent reports demonstrate that intra-granular proteins are differentially released upon different stimulation, suggesting that platelets can provide specific substances to specific tissues in appropriate conditions. In addition to its vast number in circulation, its small size and its ability to form microparticles which trespass various tissues, allows the detection of small breaches in any space as well as in the circulation. Microparticles are small fragments of membranes shed by mechanisms often involving metalloproteases. While leukocytes and endothelial cells can also produce microparticles, the majority (70 to 90 %) of microparticles in the circulation is considered to come from activated platelets. Platelet-derived microparticles express adhesive molecules and they may also contain proinfmmatory molecules such as IL-1β which may communicate proinflammatory signals to extravascular tissues. Furthermore, platelet-derived microparticles are also known to contain microRNA, which, upon release from microparticles, may be transferred to other cell types, where they modulate inflammation and immunity, as well as in developmental biology and angiogenesis. Thus, platelets and their microparticles can be figuratively described as the best drug-delivery system in living things .
Discovery of CLEC-2
Rhodocytin, a snake venom obtained from the Malayan pit viper, Calloselasma rhodostoma activates platelets with a manner similar to collagen. Rhodocytin affinity chromatography and TOF-MASS spectrometry were utilized, and we identified a new class of platelet activation receptor, c-type lectin-like receptor 2 (CLEC-2) .
While CLEC-2 is expressed to a limited degree in liver sinusoidal endothelial cells, and liver Kupffer cells, CLEC-2 is abundantly and specifically expressed on platelets and megakaryocytes in humans . In mice, in addition to platelets, neutrophils and macrophages also express CLEC-2, and it appears to mediate phagocytosis and proinflammatory cytokine expression.
Thrombosis and hemostasis
Previously, CLEC-2 was considered to play only a minor role in hemostasis, because CLEC-2 knockout resulted in no or only a moderate increase in bleeding time. However, a recent report on severely defective hemostasis in GPVI and CLEC-2 double knockout mice suggests that GPVI and CLEC-2 compensate for each other, preventing severe blood loss, that they serve together to play a key role in hemostasis . In the closely related context, it is of note that GPVI and CLEC-2 with the shared signal transduction pathways such as Syk, SLP-76, and PLCγ2 play a critical role in maintaining vascular integrity during development and inflammation .
Role of platelets in liver regeneration
There is an increasing body evidence to suggest that platelets are involved in various stages of liver regeneration. They are not only involved in the early phase of liver generation, but platelet transfusion and thrombocytosis can enhance hepatocyte regeneration after liver injury. Recent studies have reported that platelets are recruited to the sinusoidal and Disse’s space and probably due to direct contact with certain cell types such as stellate cells and sinusoidal endothelial cells, they release bioactive compounds which stimulate hepatocyte proliferation . However, up to date, the molecules on the platelet membranes which are involved in this process have not been identified.
We have recently found that hepatocyte proliferation is attenuated by clopidogrel, which inhibits platelet activation, and that liver regeneration is impaired with CLEC-2-knockout mice, suggesting that CLEC-2 on the platelet membrane is the key molecule which links with platelets and hepatocyte proliferation (manuscript being submitted).
Megakaryopoiesis and thrombopoiesis
There is an increasing body of evidence to suggest that platelets participate in various patho-physiological processes beyond those related to thrombosis and hemostasis, and that CLEC-2 on the platelet membrane by interacting with its ligands, podoplanin and others, contributes to a number of these processes.
C-type lectin-like receptor 2
Lymphatic endothelial cell
Vascular smooth muscle cell
Human umbilical vein-derived endothelial cell
Neutrophil extracellular trap
Fibroblastic reticular cell
Publication fees for this article have been funded by APSTH 2016.
This article has been published as part of Thrombosis Journal Volume 14 Supplement 1, 2016. The full contents of the supplement are available at https://thrombosisjournal.biomedcentral.com/articles/supplements/volume-14-supplement-1.
Availability of data and materials
ST performed the experiments related to megakaryopoiesis. KS analyzed and interpreted the entire data related to CLEC-2. YO gave advice to the project, and was a major contributor in writing this manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
Consent for publication
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