Discussion

In this study, platelet responsiveness to conventional and CLEC-2-induced activation for patients with a podoplanin-expressing vascular tumor (KHE/KMP) was characterized. We found that platelet functionality and platelet aggregation tends to be impaired in patients with KHE and is significantly impaired in the patient with KMP (Fig. 1). Platelet integrin activation measured both by PAC-1 binding, fibrinogen binding and aggregation appears to be the most affected parameter (Fig. 1B, Fig. 2, Fig. 3B, Fig. 4B,D). Surprisingly, platelet calcium mobilization in response to CLEC-2 stimulation was unaltered in the patients’ samples and we found that two novel low molecular weight CLEC-2 inhibitors successfully block platelet activation by rhodocytin (Fig. 3A, Fig. 4).
While podoplanin is considered the key platelet activator in KHE/KMP, we analyzed platelet responses to CLEC-2 agonists rhodocytin and fucoidan. Platelet cytosolic calcium increased normally upon stimulation with rhodocytin or fucoidan in KHE/KMP (Fig. 3A). Interestingly, either of the inhibitors of CLEC-2-podoplanin interaction, 2CP or HB125, reduced noticeably calcium mobilization in response to rhodocytin, with more pronounced effect for KHE/KMP platelets. Moreover, aggregometry assay demonstrated that both 2CP and HB125 decrease initial velocity of aggregation in patients with KHE upon stimulation with rhodocytin. These findings suggest that CLEC-2 receptor and the components of CLEC-2:podoplanin downstream signaling pathway could be a promising target for pathogenetic therapy of KMP and related potentially life-threatening hematologic complications. To confirm this, furtherin vitro and in vivo studies are required.
Platelet CLEC-2 interaction with podoplanin is considered the key mechanism of platelet activation in KHE, but it may not be the only one involved in pathogenesis of KMP. Undoubtedly, KHE are tumors with extremely rapid growth and progression, and contain regions with impaired endothelial integrity, with exposure to tissue factor, VWF and collagen 6, that can also lead to platelet activation, coagulation and thrombi formation. These additional mechanisms have been proven by positive tumor staining for VWF in 80% cases37. Thus, further investigations are required to establish the biochemical basis of this phenomenon.
This study has several limitations. First, the study was conducted in a small cohort of patients. Despite the small sample size due to extreme rarity of KHE/KMP, the results obtained are still very valuable. The second limitation of this study is that the included patients are not treatment-naïve and most of them obtained hematologic response prior to enrollment. Thus, proposed hypotheses of this study require confirmation in a cohort of patients with newly diagnosed KHE/KMP.