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).