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.