Figure 2.
Figure 2. Accelerated integrin inactivation in Pfn1fl/fl-Pf4Creplatelets. (A) β3-integrin (Itgb3) localization and recruitment of talin-1 (Tln1) to β-integrin tails was assessed by immunostaining and confocal microscopy (Leica TCS SP5, 100×/1.4 oil STED White objective, Leica Microsystems) of fibrinogen-spread platelets (n = 6 vs 6). Scale bars, 3 μm. (B) Assessment of the Tln1 and β3-integrin distribution pattern in at least 70 platelets of 6 animals per group. Values are mean ± SD (n = 6 vs 6). Representative heat maps (C) and histograms (D) of the cellular stiffness measured by atomic force microscopy on fibrinogen-spread platelets of 5 animals per group. Washed platelets were stimulated for 5 or 30 minutes with thrombin (Thr, T) and collagen-related peptide (CRP, C) (E-F) or CRP alone (G). Activation of αIIbβ3-integrins and phosphatidylserine exposure on the outer leaflet of the platelet membrane were determined by adding JON/A-PE antibody and Anxa5-FITC protein 5 minutes prior to the end of the incubation time. Analysis was performed by flow cytometry. Flow cytometry plots are representative of at least 6 animals per group. (F-G) Percentage of platelets per quadrant (Q); Q1, JON/A+ Anxa5− (upper left); Q2, JON/A+ Anxa5+ (upper right); Q3, JON/A− Anxa5+ (lower right); Q4, JON/A− Anxa5− (lower left). Values are mean ± SD of 6 vs 7 animals. Resting (H-I) or 2.5 μM latrunculin A-pretreated (J-K) platelets were left untreated or preincubated for 10 minutes in the presence of the calpain inhibitor MDL-28170 (200 μM). Subsequently, samples were stimulated with the calcium ionophore A23187 (10 μM) or thrombin (0.1 U/mL) and CRP (10 μg/mL), lysed, and processed for immunoblotting. Full-length (Ab762) and calpain-cleaved (AbΔ747) β3-integrin were probed with the respective antibodies and analyzed by densitometry. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) served as control. Values are mean ± SD of 6 vs 7 animals. (H,J) Immunoblots are representative of at least 6 animals per group. Platelets were either pretreated with 5 μM of the actin polymerization inhibiting drug cytochalasin D (L) or 200 μM of the calpain inhibitor MDL-28170 (M-N). Subsequently, the activation and localization of β3-integrin and Tln1 was assessed by flow cytometry (L,N) or immunostaining and confocal microscopy (Leica TCS SP5, 100×/1.4 oil STED White objective, Leica Microsystems) on fibrinogen-spread platelets (M). Values are mean ± SD of 4 vs 4 animals. Unpaired Student t test was used to assess statistical differences between the groups: ***P < .001; **P < .01; *P < .05.

Accelerated integrin inactivation in Pfn1fl/fl-Pf4Creplatelets. (A) β3-integrin (Itgb3) localization and recruitment of talin-1 (Tln1) to β-integrin tails was assessed by immunostaining and confocal microscopy (Leica TCS SP5, 100×/1.4 oil STED White objective, Leica Microsystems) of fibrinogen-spread platelets (n = 6 vs 6). Scale bars, 3 μm. (B) Assessment of the Tln1 and β3-integrin distribution pattern in at least 70 platelets of 6 animals per group. Values are mean ± SD (n = 6 vs 6). Representative heat maps (C) and histograms (D) of the cellular stiffness measured by atomic force microscopy on fibrinogen-spread platelets of 5 animals per group. Washed platelets were stimulated for 5 or 30 minutes with thrombin (Thr, T) and collagen-related peptide (CRP, C) (E-F) or CRP alone (G). Activation of αIIbβ3-integrins and phosphatidylserine exposure on the outer leaflet of the platelet membrane were determined by adding JON/A-PE antibody and Anxa5-FITC protein 5 minutes prior to the end of the incubation time. Analysis was performed by flow cytometry. Flow cytometry plots are representative of at least 6 animals per group. (F-G) Percentage of platelets per quadrant (Q); Q1, JON/A+ Anxa5− (upper left); Q2, JON/A+ Anxa5+ (upper right); Q3, JON/A− Anxa5+ (lower right); Q4, JON/A− Anxa5− (lower left). Values are mean ± SD of 6 vs 7 animals. Resting (H-I) or 2.5 μM latrunculin A-pretreated (J-K) platelets were left untreated or preincubated for 10 minutes in the presence of the calpain inhibitor MDL-28170 (200 μM). Subsequently, samples were stimulated with the calcium ionophore A23187 (10 μM) or thrombin (0.1 U/mL) and CRP (10 μg/mL), lysed, and processed for immunoblotting. Full-length (Ab762) and calpain-cleaved (AbΔ747) β3-integrin were probed with the respective antibodies and analyzed by densitometry. Glyceraldehyde-3-phosphate dehydrogenase (Gapdh) served as control. Values are mean ± SD of 6 vs 7 animals. (H,J) Immunoblots are representative of at least 6 animals per group. Platelets were either pretreated with 5 μM of the actin polymerization inhibiting drug cytochalasin D (L) or 200 μM of the calpain inhibitor MDL-28170 (M-N). Subsequently, the activation and localization of β3-integrin and Tln1 was assessed by flow cytometry (L,N) or immunostaining and confocal microscopy (Leica TCS SP5, 100×/1.4 oil STED White objective, Leica Microsystems) on fibrinogen-spread platelets (M). Values are mean ± SD of 4 vs 4 animals. Unpaired Student t test was used to assess statistical differences between the groups: ***P < .001; **P < .01; *P < .05.

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