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.