Podoplanin-Fc burns out platelets

In this issue of Blood, Cueni and colleagues have demonstrated that podoplanin-Fc not only displays an inhibitory effect on lymphatic vessel formation, but also induces platelet aggregation and disseminated intravascular coagulation (DIC) in transgenic mice.

Podoplanin is a small glycosylated transmembrane protein that is regulated by the lymphatic-specific homeobox gene Prox-1. It is known to be mainly expressed in the lymphatic endothelial cells and is therefore considered as one of the most widely applicable lymphatic markers along with Prox-1, LYVE-1, and VEGFR3. The exact role of podoplanin is not clearly elucidated; however, podoplanin is important for the proper formation and function of the lymphatic vasculature as demonstrated in podoplanin-deficient mice which develop dilated and dysfunctional lymphatic vessels, as well as lymphedema.¹  It has been reported that podoplanin induces platelet aggregation in vitro^2,3  via the C-type lectin-like receptor-2 on the surface of platelets³  (reviewed by Tammela and Alitalo⁴ ).

Recombinant soluble fusion proteins that link the extracellular domain of a membrane protein to the Fc region of immunoglobulin G (IgG) are increasingly being developed and applied, both for experimental purposes, and also for therapeutic indications. Usually, the fusion protein is designed to compete with the native membrane-bound receptor (an analog of fusion protein) for its ligand, hence, sequestering the ligand away from its native receptor, suppressing the endogenous signal transduction.

In this issue, Cueni et al produced a recombinant soluble fusion protein targeted for podoplanin which consists of the extracellular portion of human podoplanin linked to the Fc region of human IgG₁ (podoplanin-Fc) to alter the action of endogenous, membrane-bound podoplanin and subsequently examine its effects on the growth and/or function of lymphatic vessels.⁵  The authors report that podoplanin-Fc caused an inhibitory effect on lymphatic vessel formation in vitro and in vivo, which was somewhat predicted. However, unexpectedly, podoplanin-Fc was also found to bind and activate platelets inducing their aggregation. In transgenic mice designed to produce podoplanin-Fc under the control of the keratin 14 promoter (K14 podoplanin-Fc transgenic mice), the synthesis of podoplanin-Fc was solely confined to the skin. However, the fusion protein produced in the epidermis readily diffused and entered the systemic circulation, causing diffuse platelet aggregation. Unpredictable sudden deaths occurred in 15% of these K14 podoplanin-Fc–transgenic animals without any apparent preceding events: in these cases, postmortem examination revealed signs of gastrointestinal hemorrhage and anemia. The seemingly healthy transgenic mice also developed intravascular thrombi which were podoplanin-positive in multiple vital organs. Thrombocytopenia and impaired coagulation were also observed. These findings perfectly match the clinical manifestation of human DIC.

This study provides direct in vivo evidence identifying podoplanin as a molecule that interacts with platelets to induce platelet aggregation. The results are in agreement with a recent report⁶  that described the capacity of podoplanin to bind, activate, and aggregate platelets in vivo during the process of developmental separation of the lymphatic system from the blood vessels. Podoplanin^−/− embryos fail to accomplish lymphatic separation because the necessary platelet aggregation cannot occur at the junction of the lymph sac and cardinal vein, which are podoplanin⁺ structures in the wild type. The precise podoplanin receptor and downstream signaling pathway need to be further dissected, but meanwhile it seems reasonable to suggest the C-type lectin-like receptor-2 as a potential candidate, which is known to transduce its signals via the tyrosine kinase Syk and the adaptor protein SLP-76.^4,5,7 

Importantly, this study proposed a novel genetic animal model that could be a platform for DIC research. According to Cueni and colleagues, the phenotype of K14-podoplanin-Fc mice closely resembles the profile of human DIC, in which microthrombi occurred within the systemic circulation, along with thrombocytopenia and consumption coagulopathy. Hopefully, this model will boost our understanding of DIC, which would no doubt be applicable in oncology, trauma, infectious disease, and so forth (reviewed by Levi and Ten Cate⁸ ).

At the same time, these findings warn us of the theoretical risk of DIC developing during therapeutic approaches attempting to modulate podoplanin level and/or activity. It seems reasonable to appreciate the possibility of secondary alterations of platelet function in therapeutic strategies that are aimed at adjusting the lymphatic system itself.