Figure 5. The DUB inhibitor degrasyn... | American Society of Hematology

Figure 5.

The DUB inhibitor degrasyn does not significantly affect Ca2+ mobilization, or thrombin-, T6 -, or VWF-induced platelet aggregation, but significantly affects collagen-induced platelet aggregation and platelet adhesion to fibrinogen, VWF, and collagen. (A) Washed platelets loaded with Calbryte 520 AM dye were untreated or treated with the indicated dose of degrasyn or milrinone for 20 minutes. Then, samples were treated with 25 μM of the PAR-1 receptor–activating peptide (SFLLRN; T6), and Ca2+ mobilization visualized for 5 minutes in a Hamamatsu FDSS/μCell fluorescence plate reader. (B) Washed platelets (3 × 108/mL) were treated with 2.5 μM or 5 μM degrasyn for 20 minutes at room temperature and then activated with either 10 μM of PAR-1 activating peptide (T6), 0.2 U/mL thrombin (Thr), 10 μg/mL collagen, or 3.76 μg/mL VWF + 1.8 mg/mL ristocetin in an aggregometer cuvette. Changes in light transmission were measured at 37°C with stirring. Representative aggregation tracing. (C) Quantitation and statistical analysis of 4 independent aggregation experiments. The data are reported as the mean ± SD of the maximal aggregation (MA). Student t test results: ∗∗P = .0062. (D) Washed platelets were either treated or untreated with 5 μM degrasyn for 20 minutes and then added to wells precoated with fibrinogen (10 μg/mL), VWF (5 μg/mL), or collagen (10 μg/mL). After 1 hour, the wells were washed, and platelet adhesion was assessed by measuring residual alkaline phosphatase activity. Results of 3 independent experiments, reported as mean ± SD adhesion units (optical density). Student t test results: ∗∗∗∗P < .0001; ∗∗P = .0018. (E) Washed platelets (1 × 106/mL) were either untreated or treated with 5 μM degrasyn for 20 minutes and then allowed to adhere to cover slips coated with 100 μg/mL fibrinogen for 45 minutes, fixed, and labeled with antiubiquitin antibody P4D1 in combination with Alexa-594 secondary antibody. Images were acquired using an Abberior Facility Line confocal microscope using a 100× original magnification 1.45NA oil lens. Images were processed using FIJI/ImageJ for contrast and scale bar (10 μm). ns, not significant; RFU, relative fluorescence unit.

The DUB inhibitor degrasyn does not significantly affect Ca²⁺ mobilization, or thrombin-, T6 -, or VWF-induced platelet aggregation, but significantly affects collagen-induced platelet aggregation and platelet adhesion to fibrinogen, VWF, and collagen. (A) Washed platelets loaded with Calbryte 520 AM dye were untreated or treated with the indicated dose of degrasyn or milrinone for 20 minutes. Then, samples were treated with 25 μM of the PAR-1 receptor–activating peptide (SFLLRN; T6), and Ca²⁺ mobilization visualized for 5 minutes in a Hamamatsu FDSS/μCell fluorescence plate reader. (B) Washed platelets (3 × 10⁸/mL) were treated with 2.5 μM or 5 μM degrasyn for 20 minutes at room temperature and then activated with either 10 μM of PAR-1 activating peptide (T6), 0.2 U/mL thrombin (Thr), 10 μg/mL collagen, or 3.76 μg/mL VWF + 1.8 mg/mL ristocetin in an aggregometer cuvette. Changes in light transmission were measured at 37°C with stirring. Representative aggregation tracing. (C) Quantitation and statistical analysis of 4 independent aggregation experiments. The data are reported as the mean ± SD of the maximal aggregation (MA). Student t test results: ∗∗P = .0062. (D) Washed platelets were either treated or untreated with 5 μM degrasyn for 20 minutes and then added to wells precoated with fibrinogen (10 μg/mL), VWF (5 μg/mL), or collagen (10 μg/mL). After 1 hour, the wells were washed, and platelet adhesion was assessed by measuring residual alkaline phosphatase activity. Results of 3 independent experiments, reported as mean ± SD adhesion units (optical density). Student t test results: ∗∗∗∗P < .0001; ∗∗P = .0018. (E) Washed platelets (1 × 10⁶/mL) were either untreated or treated with 5 μM degrasyn for 20 minutes and then allowed to adhere to cover slips coated with 100 μg/mL fibrinogen for 45 minutes, fixed, and labeled with antiubiquitin antibody P4D1 in combination with Alexa-594 secondary antibody. Images were acquired using an Abberior Facility Line confocal microscope using a 100× original magnification 1.45NA oil lens. Images were processed using FIJI/ImageJ for contrast and scale bar (10 μm). ns, not significant; RFU, relative fluorescence unit.

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