Figure 4.
Patients with KHE/KMP have decreased levels of CLEC-2 and GPVI on the platelet surface. (A) Scheme of the computational model of CLEC-2 induced signaling in platelets: CLEC-2 activation results in the receptor clustering and phosphorylation by Syk kinases. In resting platelets, small amount of active Syk is maintained by active SFK kinases, which, in turn, are maintained active by CD148 phosphatases. Nonactive Syk bind to phosphorylated and clustered CLEC-2 receptors and also become active. Active Syk phosphorylate LAT and TULA-2. TULA-2 is the negative regulator of platelet Syk activation.40 PLCγ2 and PI3K bind to phosphorylated LAT, and PI3K becomes active. PI3K produces PIP3 from PIP2, which is bound by PH-domain of Btk. Hereby, Btk becomes active and activates PLCγ2, which hydrolyzes PIP2 and produces IP3, which initiates cytosolic calcium signaling. (B) Dependance of the maximally achievable cytosolic calcium concentration from CLEC-2 number on the resting platelets, predicted by the computational model. Physiological number of CLEC-2 per platelet35 is highlighted in red. (C) Typical results of the microscopic immunofluorescence analysis of the CLEC-2 expression on the platelet surface of the patients with KHE/KMP and healthy donors. (D-E) Fluorescence intensities of the anti–CLEC-2 antibodies (D) and anti-NMII antibodies (E) of the patients with KHE/KMP and healthy donors. Blurred corresponds to unique platelet measurements, and bright dots correspond to patients. Each color corresponds to unique patients. Seven healthy donors and 6 patients with KHE/KMP were analyzed. (F) Typical results of the microscopic immunofluorescence analysis of the GPVI expression on the platelet surface (GPVI; NMII) of the patients with KHE/KMP and healthy donors. (G-H) Fluorescence intensities of the anti-GPVI antibodies (G) and anti-NMII antibodies (H) of the KHE/KMP patients and healthy donors. Blurred corresponds to unique platelet measurements, and bright dots correspond to patients. Each color corresponds to unique patients. Seven healthy donors and 6 patients with KHE/KMP were analyzed. Statistical significance was calculated using Mann-Whitney U test. ∗P < .05; ∗∗P < .01. CLEC-2, anti–CLEC-2 antibodies; GPVI, anti-GPVI antibodies; NMII, nonmuscular myozin II (used for platelet identification).

Patients with KHE/KMP have decreased levels of CLEC-2 and GPVI on the platelet surface. (A) Scheme of the computational model of CLEC-2 induced signaling in platelets: CLEC-2 activation results in the receptor clustering and phosphorylation by Syk kinases. In resting platelets, small amount of active Syk is maintained by active SFK kinases, which, in turn, are maintained active by CD148 phosphatases. Nonactive Syk bind to phosphorylated and clustered CLEC-2 receptors and also become active. Active Syk phosphorylate LAT and TULA-2. TULA-2 is the negative regulator of platelet Syk activation.40 PLCγ2 and PI3K bind to phosphorylated LAT, and PI3K becomes active. PI3K produces PIP3 from PIP2, which is bound by PH-domain of Btk. Hereby, Btk becomes active and activates PLCγ2, which hydrolyzes PIP2 and produces IP3, which initiates cytosolic calcium signaling. (B) Dependance of the maximally achievable cytosolic calcium concentration from CLEC-2 number on the resting platelets, predicted by the computational model. Physiological number of CLEC-2 per platelet35 is highlighted in red. (C) Typical results of the microscopic immunofluorescence analysis of the CLEC-2 expression on the platelet surface of the patients with KHE/KMP and healthy donors. (D-E) Fluorescence intensities of the anti–CLEC-2 antibodies (D) and anti-NMII antibodies (E) of the patients with KHE/KMP and healthy donors. Blurred corresponds to unique platelet measurements, and bright dots correspond to patients. Each color corresponds to unique patients. Seven healthy donors and 6 patients with KHE/KMP were analyzed. (F) Typical results of the microscopic immunofluorescence analysis of the GPVI expression on the platelet surface (GPVI; NMII) of the patients with KHE/KMP and healthy donors. (G-H) Fluorescence intensities of the anti-GPVI antibodies (G) and anti-NMII antibodies (H) of the KHE/KMP patients and healthy donors. Blurred corresponds to unique platelet measurements, and bright dots correspond to patients. Each color corresponds to unique patients. Seven healthy donors and 6 patients with KHE/KMP were analyzed. Statistical significance was calculated using Mann-Whitney U test. ∗P < .05; ∗∗P < .01. CLEC-2, anti–CLEC-2 antibodies; GPVI, anti-GPVI antibodies; NMII, nonmuscular myozin II (used for platelet identification).

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