Results

Patient characteristics

7 patients were enrolled to the study. Most of them (71%) were male, and the median age at enrollment was 11 months. The most common tumor site was soft tissues of the upper body, and the disease predominantly manifested since newborn with a deep thrombocytopenia and an associated coagulopathy (KMP). Biopsy demonstrated that 6/7 patients had KHE, and one patient (Patient 4) had kaposiform lymphangiomatosis according to histological examination. In 5/7 cases, immunohistochemistry was performed and revealed positive staining for podoplanin, CD31, and CD34.
None of the subjects was treatment-naïve. Six patients had a history of prior KMP at disease manifestation, but had adequate blood cell counts at enrollment. One patient (Patient 1) presented with KMP (thrombocytopenia, severe coagulopathy) at the time of enrollment. Patients 1, 2, 4, and 5 received cyclophosphamide and vinblastine with/without liposomal doxorubicin (LD), Patient 7 received LD and vincristine, Patient 6 received LD and sirolimus, Patient 3 received sirolimus and hydrocortisone. Two patients also received β-blockers: propranolol (Patient 2), or atenolol (Patient 7). All clinical, laboratory, and treatment data are summarized in Table S1.

Platelet activation in response to strong stimulation

Based on the fact that KHE/KMP pathogenesis is partially based on platelet-tumor interactions, we expected altered platelet functionality in KHE. Therefore, we studied platelet functional responses (shape change, GPIb shedding, granule release, integrin activation and phosphatidylserine exposure) in response to a 10 min-stimulation with either combination of CRP (GPVI agonist) and SFLLRN (PAR1 agonist), or combination of AYGPKF (PAR4 agonist), SFLLRN and ADP, by endpoint flow cytometry (Fig. 1).
Altogether, platelet responses to strong activation were within normal ranges (Fig. 1). For P1 and P4 an increased FSC MFI was observed (Fig. 1A). For P1, this could be explained by inflammation related increased platelet consumption, while for P4 this could be an effect of tubulin ring disruption by vinblastine, also observed in microscopy (Fig. S2). Platelet integrin activation in response to thrombin and purinergic receptors stimulation (“2TR+ADP” in Figure 1) was significantly diminished in KHE patients (Fig. 1B), while other platelet responses to activation were on the lower boundary of those of healthy donors. P1 alone demonstrated significantly altered platelet responses, which is consistent with DIC-like phenotype of this patient.
Immunofluorescence microscopy of peripheral blood smears was performed in 5/7 patients (Patients 3-7) and revealed normal platelet staining for GPIb, GPIIb, VWF, P-selectin, LAMP-1, -2, -3, and NMMIIa in all the patients (data not shown). In patients 4, 5, and 7 abnormal diffuse distribution of platelet β1-tubulin was observed (Fig. S2). This phenomenon could be explained by treatment with tubulin-targeted agents, vincristine or vinblastine.