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There is increasing evidence that platelets participate in multiple pathophysiological processes other than thrombosis and hemostasis, such as immunity, inflammation, embryonic development, and cancer progression. A recent study revealed that heme (hemin)-activated platelets induce macrophage extracellular traps (METs) and exacerbate rhabdomyolysis-induced acute kidney injury (RAKI); however, how hemin activates platelets remains unclear. Here, we report that both C-type lectin-like receptor-2 (CLEC-2) and glycoprotein VI (GPVI) are platelet hemin receptors and are involved in the exacerbation of RAKI. We investigated hemin-induced platelet aggregation in humans and mice, binding of hemin to CLEC-2 and GPVI, the RAKI-associated phenotype in a mouse model, and in vitro MET formation. Using western blotting and surface plasmon resonance, we showed that hemin activates human platelets by stimulating the phosphorylation of SYK and PLCγ2 and directly binding to both CLEC-2 and GPVI. Furthermore, hemin-induced murine platelet aggregation was partially reduced in CLEC-2–depleted and FcRγ-deficient (equivalent to GPVI-deficient) platelets and almost completely inhibited in CLEC-2–depleted FcRγ-deficient (double-knockout) platelets. In addition, hemin-induced murine platelet aggregation was inhibited by the CLEC-2 inhibitor cobalt hematoporphyrin or GPVI antibody (JAQ-1). Renal dysfunction, tubular injury, and MET formation were attenuated in double-knockout RAKI mice. Furthermore, in vitro MET formation assay showed that the downstream signaling pathway of CLEC-2 and GPVI is involved in MET formation. We propose that both CLEC-2 and GPVI in platelets play an important role in RAKI development.

The overall prognosis of patients with sarcoma-based cancers has changed little in the last 20 years. There is an urgent need to investigate the metastatic potential of these tumors and to develop anti-metastatic drugs. It is becoming increasingly clear that platelets play an important role in the establishment of metastasis of carcinoma cells and could be a useful therapeutic target for patients with carcinoma. However, little is known about the role of platelets in sarcoma progression. Here, we investigated how osteosarcoma progression relates to platelet function to explore the possibility of anti-platelet therapy. We found that, similar to carcinoma cells, podoplanin (also known as Aggrus)-positive osteosarcoma cells induce platelet aggregation and activation. Administration of anti-glycoprotein Ibα (GPIbα, also known as CD42b) antibody reduced the lung metastasis of osteosarcoma. The supernatant from platelets cocultured with osteosarcoma cells contained several growth factors and promoted proliferation, invasiveness, and sphere formation of osteosarcoma cells in vitro. In addition, the development of lung metastasis was highly dependent on direct interaction between osteosarcoma cells and platelets. To explore the therapeutic target, we focused on the interactions between podoplanin on osteosarcoma and C-type lectin-like receptor (CLEC)-2 on platelets. The administration of a depleting antibody against CLEC-2 efficiently suppressed osteosarcoma metastasis into the lung. We also analyzed clinical data from patient samples at primary and metastatic sites. Although GPIbα expression was similar between the two sites, there was a significant increase in podoplanin at the metastatic site compared to that in the primary site, and the level of podoplanin expression in the primary site correlated with patient prognosis. These findings suggest that blockade of interactions between platelets CLEC-2 and osteosarcoma podoplanin represent the most promising therapeutic strategy for preventing the lung metastasis of osteosarcoma. © 2020 American Society for Bone and Mineral Research.

In humans, platelet count within the normal range is required for physiological hemostasis, but, adversely, platelets also support pathological thrombosis. Moreover, by releasing growth factors, they may enhance neoplastic proliferation. We hypothesize that platelet count correlates with platelet-dependent pathologies, even within the range of hemostatic competence. Because platelet production is promoted by thrombopoietin signaling through the myeloproliferative leukemia virus oncogene (cMPL), a receptor expressed on megakaryocytes, we evaluated the feasibility of selective targeting of hepatic thrombopoietin production to test this hypothesis. We synthesized murine- and primate-specific antisense oligonucleotides (THPO-ASO) that silence hepatic thrombopoietin gene (THPO) expression without blocking extrahepatic THPO. Repeated doses of THPO-ASO were administered to mice and a baboon, causing a sustained 50% decline in plasma thrombopoietin levels and platelet count within 4 weeks in both species. To investigate whether reducing platelet count within the translationally relevant hemostatic range could alter a neoplastic process, we administered THPO-ASO to 6-week-old transgenic MMTV-PyMT mice that develop early ductal atypia that progresses into cMPL-negative fatal metastatic breast cancer within 2 to 3 months. THPO-ASO treatment increased the average time to euthanasia (primary humane endpoint) at 2 cm(3) combined palpable tumor volume. Our results show that THPO-ASO reduced blood platelet count, plasma platelet factor 4, vascular endothelial growth factor, thrombopoietin levels, bone marrow megakaryocyte density, tumor growth rate, proliferation index, vascularization, platelet and macrophage content, and pulmonary metastases vs untreated controls. These findings confirm that sustained and moderate pharmacological platelet count reduction is feasible with THPO-ASO administration and can delay progression of certain platelet-dependent pathological processes within a safe hemostatic platelet count range.

Platelets participate in not only thrombosis and hemostasis but also other pathophysiological processes, including tumor metastasis and inflammation. However, the putative role of platelets in the development of solid organs has not yet been described. Here, we report that platelets regulate lung development through the interaction between the platelet-activation receptor, C-type lectin-like receptor-2 (Clec-2; encoded by Clec1b), and its ligand, podoplanin, a membrane protein. Clec-2 deletion in mouse platelets led to lung malformation, which caused respiratory failure and neonatal lethality. In these embryos, α-smooth muscle actin-positive alveolar duct myofibroblasts (adMYFs) were almost absent in the primary alveolar septa, which resulted in loss of alveolar elastic fibers and lung malformation. Our data suggest that the lack of adMYFs is caused by abnormal differentiation of lung mesothelial cells (luMCs), the major progenitor of adMYFs. In the developing lung, podoplanin expression is detected in alveolar epithelial cells (AECs), luMCs, and lymphatic endothelial cells (LECs). LEC-specific podoplanin knockout mice showed neonatal lethality and Clec1b-/--like lung developmental abnormalities. Notably, these Clec1b-/--like lung abnormalities were also observed after thrombocytopenia or transforming growth factor-β depletion in fetuses. We propose that the interaction between Clec-2 on platelets and podoplanin on LECs stimulates adMYF differentiation of luMCs through transforming growth factor-β signaling, thus regulating normal lung development.

Tumor metastasis is the major cause of deaths in cancer patients and is modulated by intertwined stress-responsive signaling cascades. Here we demonstrate that deletion of stress-responsive apoptosis signal-regulating kinase 1 (Ask1) in platelets results in unstable hemostasis and drastic attenuation of tumor lung metastasis, both of which are attributable to platelet dysfunction. Platelet-specific deletion of Ask1 in mice leads to defects in ADP-dependent platelet aggregation, unstable hemostasis and subsequent attenuation of tumor metastasis. We also revealed that activating phosphorylation of Akt is attenuated in Ask1-deficient platelets, contrary to the previous reports suggesting that Akt is negatively regulated by ASK1. Mechanistically, ASK1-JNK/p38 axis phosphorylates an ADP receptor P2Y12 at Thr345, which is required for the ADP-dependent sustained Akt activity that is vital to normal platelet functions. Our findings offer insight into positive regulation of Akt signaling through P2Y12 phosphorylation as well as MAPK signaling in platelets by ASK1 and suggest that ASK1-JNK/p38 axis provides a new therapeutic opportunity for tumor metastasis.

Megakaryopoiesis is the hierarchical differentiation of hematopoietic stem cells into megakaryocytes. Differentiating megakaryocytes undergo maturation characterized by endomitosis and produce numerous platelets through proplatelet formation. C-type lectin-like receptor 2 (CLEC-2) is a podoplanin (PDPN) receptor mainly expressed on platelets and megakaryocytes. Deletion of platelet/megakaryocyte CLEC-2 causes thrombocytopenia in mice; however, its contribution to megakaryopoiesis remains unknown. Here, we show that megakaryopoiesis is promoted through the CLEC-2/PDPN interaction in the vicinity of arterioles in the bone marrow (BM). We have also identified PDPN-expressing BM arteriolar stromal cells, tentatively termed as BM fibroblastic reticular cell (FRC)-like cells. Platelet/megakaryocyte-specific CLEC-2 conditional knockout (cKO) mice showed a decrease in the number of immature megakaryocytes. CLEC-2 wild-type megakaryocyte expansion was augmented in vitro by the addition of recombinant PDPN, but not cKO megakaryocytes. Moreover, megakaryocyte colonies were colocalized with periarteriolar BM FRC-like cells in the BM. Coculture of megakaryocytes with BM FRC-like cells augmented megakaryocyte expansion, which was dependent upon the CLEC-2/PDPN interaction. Furthermore, we found that the CLEC-2/PDPN interaction induces BM FRC-like cells to secrete chemokine (C-C motif) ligand 5 (CCL5) to facilitate proplatelet formation. These observations indicate that a reciprocal interaction between CLEC-2 on megakaryocytes and PDPN on BM FRC-like cells contributes to the periarteriolar megakaryopoietic microenvironment in mouse BM.

Cancer-associated thrombosis is a common first presenting sign of malignancy and is currently the second leading cause of death in cancer patients after their malignancy. However, the molecular mechanisms underlying cancer-associated thrombosis remain undefined. In this study, we aimed to develop a better understanding of how cancer cells affect the coagulation cascade and platelet activation to induce a prothrombotic phenotype. Our results show that colon cancer cells trigger platelet activation in a manner dependent on cancer cell tissue factor (TF) expression, thrombin generation, activation of the protease-activated receptor 4 (PAR4) on platelets and consequent release of ADP and thromboxane A2. Platelet-colon cancer cell interactions potentiated the release of platelet-derived extracellular vesicles (EVs) rather than cancer cell-derived EVs. Our data show that single colon cancer cells were capable of recruiting and activating platelets and generating fibrin in plasma under shear flow. Finally, in a retrospective analysis of colon cancer patients, we found that the number of venous thromboembolism events was 4.5 times higher in colon cancer patients than in a control population. In conclusion, our data suggest that platelet-cancer cell interactions and perhaps platelet procoagulant EVs may contribute to the prothrombotic phenotype of colon cancer patients. Our work may provide rationale for targeting platelet-cancer cell interactions with PAR4 antagonists together with aspirin and/or ADP receptor antagonists as a potential intervention to limit cancer-associated thrombosis, balancing safety with efficacy.

The third-generation tyrosine kinase inhibitor (TKI) ponatinib has been associated with high rates of acute ischemic events. The pathophysiology responsible for these events is unknown. We hypothesized that ponatinib produces an endothelial angiopathy involving excessive endothelial-associated von Willebrand factor (VWF) and secondary platelet adhesion. In wild-type mice and ApoE−/− mice on a Western diet, ultrasound molecular imaging of the thoracic aorta for VWF A1-domain and glycoprotein-Ibα was performed to quantify endothelial-associated VWF and platelet adhesion. After treatment of wild-type mice for 7 days, aortic molecular signal for endothelial-associated VWF and platelet adhesion were five- to sixfold higher in ponatinib vs sham therapy (P < .001), whereas dasatinib had no effect. In ApoE−/− mice, aortic VWF and platelet signals were two- to fourfold higher for ponatinib-treated compared with sham-treated mice (P < .05) and were significantly higher than in treated wild-type mice (P < .05). Platelet and VWF signals in ponatinib-treated mice were significantly reduced by N-acetylcysteine and completely eliminated by recombinant ADAMTS13. Ponatinib produced segmental left ventricular wall motion abnormalities in 33% of wild-type and 45% of ApoE−/− mice and corresponding patchy perfusion defects, yet coronary arteries were normal on angiography. Instead, a global microvascular angiopathy was detected by immunohistochemistry and by intravital microscopy observation of platelet aggregates and nets associated with endothelial cells and leukocytes. Our findings reveal a new form of vascular toxicity for the TKI ponatinib that involves VWF-mediated platelet adhesion and a secondary microvascular angiopathy that produces ischemic wall motion abnormalities. These processes can be mitigated by interventions known to reduce VWF multimer size.

The platelet activation receptor C-type lectin-like receptor 2 (CLEC-2) interacts with podoplanin on the surface of certain types of tumor cells, and this interaction facilitates tumor metastasis. CLEC-2 is also involved in thrombus formation and its stabilization. Because CLEC-2–depleted mice are protected from experimental lung metastasis and thrombus formation and do not show increased bleeding time, CLEC-2 may serve as a good target for antimetastatic or antithrombotic drugs. We screened 6770 compounds for their capability to inhibit CLEC-2–podoplanin binding using an enzyme-linked immunosorbent assay. In the first screening round, 63 compounds were identified and further evaluated by flow cytometry using CLEC-2–expressing cells. We identified protoporphyrin IX (H2-PP) as the most potent inhibitor and modified its hematoporphyrin moiety to be complexed with cobalt (cobalt hematoporphyrin [Co-HP]), which resulted in an inhibitory potency much stronger than that of H2-PP. Surface plasmon resonance analysis and molecular docking study showed that Co-HP binds directly to CLEC-2 at N120, N210, and K211, previously unknown podoplanin-binding sites; this binding was confirmed by analysis of CLEC-2 mutants with alterations in N120 and/or K211. Co-HP at a concentration of 1.53 μM inhibited platelet aggregation mediated through CLEC-2, but not that mediated through other receptors. IV administration of Co-HP to mice significantly inhibited hematogenous metastasis of podoplanin-expressing B16F10 cells to the lung as well as in vivo arterial and venous thrombosis, without a significant increase in tail-bleeding time. Thus, Co-HP may be a promising molecule for antimetastatic and antiplatelet treatment that does not cause bleeding tendency.

Platelet recruitment to sites of vascular injury is mediated by von Willebrand factor (VWF). The shear-induced unraveling of ultra-large VWF multimers causes the formation of a “stringlike” conformation, which rapidly recruits platelets from the bloodstream. A disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13) regulates this process by cleaving VWF to prevent aberrant platelet adhesion; it is unclear whether the activity of ADAMTS13 itself is regulated. The serine proteases α-thrombin and plasmin have been shown to cleave ADAMTS13. Based on sequence homology, we hypothesized that activated coagulation factor XI (FXIa) would likewise cleave ADAMTS13. Our results show that FXIa cleaves ADAMTS13 at the C-terminal domains, generating a truncated ADAMTS13 with a deletion of part of the thrombospondin type-1 domain and the CUB1-2 domains, while α-thrombin cleaves ADAMTS13 near the CUB1-2 domains and plasmin cleaves ADAMTS13 at the metalloprotease domain and at the C-terminal domain. Using a cell surface immunoassay, we observed that FXIa induced the deletion of the CUB1-2 domains from ADAMTS13 on the surface of endothelial cells. Removal of the C-terminal domain of ADAMTS13 by FXIa or α-thrombin caused an increase in ADAMTS13 activity as measured by a fluorogenic substrate (FRETS) and blocked the ability of ADAMTS13 to cleave VWF on the endothelial cell surface, resulting in persistence of VWF strands and causing an increase in platelet adhesion under flow conditions. We have demonstrated a novel mechanism for coagulation proteinases including FXIa in regulating ADAMTS13 activity and function. This may represent an additional hemostatic function by which FXIa promotes local platelet deposition at sites of vessel injury.

C-type lectin-like receptor 2 (CLEC-2) has been identified on the surface of platelets as a receptor for a platelet activating snake venom, rhodocytin/aggretin. CLEC-2 belongs to a C-type lectin superfamily and binds to a sialoglycoprotein, podoplanin, in vivo. Platelets play a crucial role in hemostasis and thrombosis, but recent studies have uncovered multiple roles of platelets beyond hemostasis in physiology and pathology. The interaction between platelet CLEC-2 and podoplanin is the key to several roles of platelets beyond hemostasis. The spatial and temporal expression patterns of podoplanin regulate vascular/lymphatic development, maintenance of vascular integrity, tissue regeneration, and some pathological processes including tumor metastasis and thromboinflammation. CLEC-2 facilitates blood/lymphatic vessel separation during embryonic development by binding to podoplanin on lymphatic endothelial cells. The leakage of platelets from hyperpermeable vessels for maintaining vascular integrity during inflammation depends on CLEC-2. During wound healing, the expression of podoplanin in keratinocytes is upregulated, which helps in the process. Podoplanin is expressed on the surface of tumor cells and facilitates hematogenous metastasis by inducing platelet aggregation through CLEC-2. During thrombotic processes, such as development of deep vein thrombosis, podoplanin is upregulated on unknown cells in the vessel wall in the area of inflammation, facilitates thrombus formation, and promotes further inflammation by binding to CLEC-2. In this article, the roles of platelets beyond hemostasis are comprehensively reviewed.

Essentials We generated recombinant rhodocytin that could aggregate platelets via CLEC-2. Recombinant wild-type rhodocytin formed heterooctamer with four α- and β-subunits. Asp 4 in α-subunit of rhodocytin was required for binding to CLEC-2. Inhibitory mutant of rhodocytin blocked podoplanin-dependent hematogenous metastasis. Summary Background Rhodocytin, a disulfide-linked heterodimeric C-type lectin from Calloselasma rhodostoma consisting of α-subunits and β-subunits, induces platelet aggregation through C-type lectin-like receptor 2 (CLEC-2). CLEC-2 is a physiological binding partner of podoplanin (PDPN), which is expressed on some tumor cell types, and is involved in tumor cell-induced platelet aggregation and tumor metastasis. Thus, modified rhodocytin may be a possible source of anti-CLEC-2 drugs for both antiplatelet and antimetastasis therapy. However, its molecular function has not been well characterized, because of the lack of recombinant rhodocytin that induces platelet aggregation. Objective To produce recombinant rhodocytin, in order to verify its function with mutagenesis, and to develop an anti-CLEC-2 drug based on the findings. Methods We used Chinese hamster ovary cells to express recombinant rhodocytin (wild-type [WT] and mutant), which was analyzed for induction/inhibition of platelet aggregation with light transmission aggregometry, the formation of multimers with blue native PAGE, and binding to CLEC-2 with flow cytometry. Finally, we investigated whether mutant rhodocytin could suppress PDPN-induced metastasis in an experimental lung metastasis mouse model. Results Functional WT] rhodocytin (αWTβWT) was obtained by coexpression of both subunits. Asp4 in α-subunits of rhodocytin was required for CLEC-2 binding. αWTβWT formed a heterooctamer similarly to native rhodocytin. Moreover, an inhibitory mutant of rhodocytin (αWTβK53A/R56A), forming a heterotetramer, bound to CLEC-2 without inducing platelet aggregation, and blocked CLEC-2-PDPN interaction-dependent platelet aggregation and experimental lung metastasis. Conclusion These findings provide molecular characterization information on rhodocytin, and suggest that mutant rhodocytin could be used as a therapeutic agent to target CLEC-2.

During the developmental stages, when the primary lymph sac arises from the cardinal vein, podoplanin in lymphatic endothelial cells (LECs) activates platelets by binding to CLEC-2, which facilitates blood/lymphatic vessel separation. One of the mechanisms for this process is that granule contents from activated platelets inhibit the migration and proliferation of LECs, thereby facilitating separation of the vessels. Angiogenesis consists of several steps, including activation of endothelial cells (ECs), migration to the site of the vessel branch, proliferation, and formation of tube-like structures. Granule contents released from activated platelets, including VEGF and FGF, are required for EC proliferation. On the other hand, direct contact between platelets and ECs via platelet membrane proteins, including GPIbα and αIIbβ3, prompts angiogenesis by stabilizing developing vessels. Platelets also play a crucial role in maintaining the vascular integrity of high endothelial venules in lymph nodes and inflammatory vessels via the podoplanin receptor CLEC-2 and the collagen receptor glycoprotein VI. Platelets also regulate cerebrovascular patterning and integrity during development via CLEC-2 and podoplanin in neuroepithelial cells. Thus, platelets play a role, not only in thrombosis and hemostasis, but also in lymphangiogenesis, angiogenesis, and maintenance of vascular integrity.

Essentials The role of C-type lectin-like receptor-2 (CLEC-2) in cancer progression is unclear. CLEC-2-depleted mouse model is generated by using a rat anti-mouse CLEC-2 monoclonal antibody. CLEC-2 depletion inhibits hematogenous tumor metastasis of podoplanin-expressing B16F10 cells. CLEC-2 depletion prolongs cancer survival by suppressing thrombosis and inflammation. SummaryBackground C-type lectin-like receptor 2 (CLEC-2) is a platelet activation receptor of sialoglycoprotein podoplanin, which is expressed on the surface of certain types of tumor cells. CLEC-2–podoplanin interactions facilitate hematogenous tumor metastasis. However, direct evidence of the role of CLEC-2 in hematogenous metastasis and cancer progression is lacking. Objective and methods We generated immunological CLEC-2-depleted mice by using anti-mouse CLEC-2 monoclonal antibody 2A2B10 and investigated whether CLEC-2 promoted hematogenous tumor metastasis and tumor growth and exacerbated the prognosis of mice bearing podoplanin-expressing B16F10 melanoma cells. Results Our results showed that hematogenous metastasis was significantly inhibited in CLEC-2-depleted mice. B16F10 cells co-cultured with wild-type platelets, but not with CLEC-2-deficient platelets, showed increased proliferation. However, B16F10 cell proliferation was not inhibited in CLEC-2-depleted mice. Histological analysis showed that thrombus formation in tumor vessels was significantly inhibited and functional vessel density was significantly increased in CLEC-2-depleted mice. These data suggest that CLEC-2 deficiency may inhibit thrombus formation in tumor vessels and increase the density of functional vessels, thus improving oxygen and nutrient supply to tumors, indirectly promoting tumor proliferation. Furthermore, the overall survival of CLEC-2-depleted mice was significantly prolonged, which may be due to the suppression of thrombus formation in the lungs and subsequent inhibition of systemic inflammation and cachexia. Conclusions These data provide a rationale for the targeted inhibition of CLEC-2 as a new strategy for preventing hematogenous tumor metastasis and for inhibiting cancer-related thromboembolism.

Essentials Regeneration role of C-type lectin receptor-2 (CLEC-2) after 70% hepatectomy (HPx) was investigated. Wild-type or CLEC-2 deleted from platelets of chimeric mice (flKO) underwent HPx. The liver/body weight ratio was significantly lower in the flKO than in the wild-type. CLEC-2 plays an essential role in liver regeneration after HPx. SummaryBackground and aim The aim of the present study was to investigate the role of C-type lectin receptor (CLEC)-2 in liver regeneration following partial liver resection in mice. Materials and methods Irradiated chimeric mice transplanted with fetal liver cells from wild-type (WT) mice, CLEC-2-deleted (KO) mice or mice with CLEC-2 deleted specifically from platelets (flKO) were generated. Mice underwent 70% partial hepatectomy (PH). Immunohistochemical staining was performed to investigate the expression of the endogenous ligand for CLEC-2, podoplanin. The accumulation of platelets in the liver was also quantified. The hepatic expression of the IL-6/gp130 and STAT3, Akt and ERK1/2 was also examined. Results The liver/body weight ratio and expression of all cell proliferation markers were significantly lower in the flKO group than in the WT group. The expression of phosphorylated (p) Akt and pERK1/2 was similar in the WT and flKO groups. On the other hand, the expression of pSTAT3 and IL-6 was significantly stronger in the WT group than in the flKO group. The expression of podoplanin was detected in the hepatic sinusoids of both groups. However, the extent to which platelets accumulated in hepatic sinusoids was significantly less in the flKO group than in the WT group. Conclusion CLEC-2 was involved in hepatic regeneration after liver resection and CLEC-2-related liver regeneration was attributed to the interaction between platelets and sinusoidal endothelial cells.