Background: Lymphoma is
commonly associated with platelet dysfunction, usually evidenced by the
occurrence of thrombosis in lymphoma patients. However, active bleeding is
also a frequent occurrence. Bleeding is typically attributed to
thrombocytopenia which occurs due to impaired megakaryocyte production in the
bone marrow. There is a growing body of evidence demonstrating that platelets
can actively sequester growth factors, inflammatory proteins and even miRNAs
from their microenvironment
and this can affect platelet function. The secretome of lymphoma cells, which
contains lymphoma-derived growth factors and cytokines could therefore
contribute to bleeding symptoms in lymphoma patients via thrombocytopathy in
the presence of adequate megakaryocyte production.
Aims: The study
aimed to determine the effect of the secretome from L-1236 Hodgkin’s lymphoma
cells on the activation and function of platelets from healthy subjects.
Hodgkin’s lymphoma cells were cultured at specific cell concentrations
(3_105–5_106) for specific times (24, 48, 72 and 96 hours) in RPMI growth
media under normal growth conditions (37 _C, 5% CO2 in air). Conditioned
media was collected at the specified times. Platelet rich plasma (PRP) was
purified from citrated whole blood samples from healthy volunteer donors. PRP
was incubated with L-1236 conditioned media for 1 hour at 37 _C. Platelet
aggregometry was performed following stimulation with 5 mM ADP. Platelet activation was assessed by analysis of
CD62P and PAC-1 by flow cytometry.
conditioned media significantly inhibited the expression of CD62P and PAC-1
on the surface of platelets at all cell concentrations and all culture times
tested. The inhibition appeared to be dose-dependent, with conditioned media
collected from 5_106 cells after 96 hours causing maximal inhibition. This dose
response effect was also true for the aggregometry results, with inhibition
of ADP-stimulated aggregation.
activation and function is significantly affected by the secretome of L-1236
Hodgkin lymphoma cells.
provide further evidence of a link between lymphoma and platelet function.
Further work aims to characterise the specific proteins responsible for this
moderation of platelet function.
Background: Since the late
19th century, platelet abnormalities have been observed in cancer patients
(Sabrkhany et al., 2011). However, it is known that incidences of thrombosis
are increased in patients with malignant lymphoma (Goldschmidt et al., 2003),
while in some patients, the occurrence of thrombocytopenia is unexplained
(Bal et al., 2010). Although primarily involved in haemostasis, platelets
have been shown to play an important role in the progression of many types of
cancer as regulators of angiogenesis. Platelets have been shown to activate
in response to various tissue factors and cytokines which are released by
tumour cells (Jurasz et al., 2004). Lymphoma secretome that contains molecules
which involved in the crosstalk of the microenvironment are thought to aid in
the growth and survival of lymphoma cells. Lymphoma cells are restricted to a
specific microenvironment in lymphatic tissues.
However, these soluble molecules in the secretome from the lymphoma cells,
may leave the lymph nodes and move into the peripheral blood stream, where
the molecules are exposed to new microenvironmental components, such as platelets.
The relationship between lymphoma cells and platelets is not well known (Bal
et al., 2010). Therefore, this novel study aims to determine the effect of
the secretome from L-1236 Hodgkin’s lymphoma cells on the activation and
function of platelets from healthy subjects. This data highlights the
importance of platelets in relation to lymphoma.
Methods: The L-1236
(5×105-5×106 cells/ml) secretome, which were cultured in (Roswell Park
Memorial Institute) RPMI culture medium at normal condition (37°C, 5% CO2 in
air), were collected at (24, 48, 72 and 96 hours). Platelet-rich plasma (PRP)
was purified from citrated whole blood from six healthy donors. PRP was incubated
with L-1236 secretome for 1-hour at 37°C. Platelet stimulated aggregation
with 5μM adenosine diphosphate (ADP) was performed. P-selectin (CD62P) and
alpha 2b-beta3 complex (PAC-1) expression on platelets were analysed by flow
cytometry. Total protein concentration in platelet lysates was analysed by
bicinchoninic acid protein assay.
secretome significantly inhibited the expression of CD62P and
PAC-1(p<0.05) and this was associated with inhibition of ADP-stimulated
aggregation. Total protein concentration in platelet lysates was increased
following incubation with the L-1236 secretome.
activation and function are significantly affected by the L-1236 secretome.
The results provide further evidence of a link between lymphoma and platelet function.