Platelet calcium signaling responses
While strong platelet activation in patients with KHE was mostly
unimpaired, we decided to analyze the responses to rhodocytin23 and fucoidan 34 in comparison
with the conventional platelet activation (Fig. 3). The key
intracellular signal that triggers platelet functional responses is
cytosolic calcium concentration 35, while platelet
shape change and reversible fibrinogen binding are the most sensitive
platelet responses 27,36.
Cytosolic calcium concentration in quiescent state was significantly
lower in KHE samples (Fig. 3A). Calcium mobilization in response to 2 µM
ADP was also decreased in comparison to healthy controls (Fig. 3B).
Fibrinogen-binding responses (Fig. 3C) were diminished in some of the
patients. The most severe platelet dysfunction was observed again in P1.
Impaired platelet responses to stimulation in KHE could possibly be
explained by general dysfunction of platelet signaling associated with
CLEC-2 malfunctioning. For patients with KHE we observed unaltered
calcium mobilization upon stimulation with rhodocytin or fucoidan (Fig.
3A). However, in the presence of CLEC-2 inhibitors 2CP (2 μM) and HB125
(2 μM), calcium responses to rhodocytin (Fig. 3A) were significantly
reduced in KHE samples; while the impact of these inhibitors was less
pronounced for samples from healthy donors. Interestingly, being
administered at 2 μM, 2CP and HB125 themselves induced calcium
mobilization in platelets comparably to rhodocytin (200 nM) in KHE
samples and higher than rhodocytin (200 nM) in healthy controls (Fig.
3A). This might be related to partial agonistic properties of these
CLEC-2 inhibitors which upon binding to the podoplanin/rhodocytin
binding site on CLEC-2 could trigger its activation to some extent,
especially, when administered at a high dose.
Platelet di-aggregation
In order to understand the functional significance of the observed
decrease in platelet signaling responses in KHE (Fig. 3), aggregometry
studies with weak platelet stimulation were performed. While
thrombocytopenia is commonly observed in KHE/KMP, LaSca aggregometry was
performed, as this method requires low platelet concentrations in probe
(Fig. 4).
Typical plots of overall aggregation of platelets in KHE upon
stimulation with 800 nM ADP (black curves) and 20 μg/mL CRP (red curves)
are shown in Figure 4A. In most of the patients, diminished initial
velocity of aggregation upon stimulation with ADP (Fig. 4B) was
observed, while maximal aggregation was mostly unimpaired (Fig. 4C). On
the other hand, all the parameters of collagen-induced platelet
aggregation were decreased in patients with KHE (Fig. 4D,E). These data
are in line with previous findings on the reduced platelet
responsiveness to stimulation with collagen (Fig. 1) as well as the
impaired weak activation in KHE/KMP (Fig. 3). LaSca assays demonstrated
that both 2CP and HB125 could cause decrease in initial velocity of
aggregation (Fig. 4F) upon weak stimulation with 10 nM rhodocytin.
However, the inhibitors had no effect on maximum aggregation in patients
with KHE (Fig. 4G).