Platelet senescence and phosphatidylserine exposure

Abstract

Background: The exposure of phosphatidylserine occurs during platelet (PLT) activation and during in vitro storage. Phosphatidylserine exposure also occurs during apoptosis after the release of mitochondrial cytochrome c. We have examined the role of cytochrome c release, mitochondrial membrane potential (ΔΨm), and cyclophilin D (CypD) in phosphatidylserine exposure due to activation and storage.

Study design and methods: The exposure of phosphatidylserine and the loss of ΔΨm were determined in a flow cytometer using fluorescein isothiocyanate-lactadherin and JC-1, a lipophilic cationic reporter dye. The role of CypD was determined with cyclosporin A and CypD-deficient murine PLTs. Cytochrome c-induced caspase-3 and Rho-associated kinase I (ROCK1) activation were determined by immunoblotting and using their inhibitors.

Results: Collagen- and thrombin-induced exposure of phosphatidylserine was accompanied by a decrease in ΔΨm. Cyclosporin A inhibited the phosphatidylserine exposure and the loss of ΔΨm. CypD(-/-) mice had decreased loss of ΔΨm and impaired phosphatidylserine exposure. Collagen and thrombin did not induce the release of cytochrome c nor the activation of caspase-3 and ROCK1. In contrast, in PLTs stored for more than 5 days, the phosphatidylserine exposure was associated with cytochrome c-induced caspase-3 and ROCK1 activation. ABT737, a BH3 mimetic that induces mitochondrial pathway of apoptosis, induced cytochrome c release and activation of caspase-3 and ROCK1 and phosphatidylserine exposure independent of CypD.

Conclusion: These results show that in stored PLTs cytochrome c release and the subsequent activation of caspase-3 and ROCK1 mediate phosphatidylserine exposure and it is distinct from activation-induced phosphatidylserine exposure.

Figure 1. Effect of cyclosporine A on platelet phosphatidylserine expression

Washed human platelets (2 × 10⁸/ml) were incubated with cyclosporine A (2 μM) or buffer alone and stimulated with a combination of collagen (5 μg/ml) and thrombin (1 unit/ml) in Panel A or ABT737 (10 μM) in Panel B. The phosphatidylserine expression was measured by FITC-lactadherin. Panel C. The percentages of platelets with fluorescence above background levels (as defined in the gate G in panels A and B) were plotted with the means and standard deviations of a representative triplicate measurements.

Figure 2. Effect of cyclophilin D deficiency on phosphatidylserine expression in murine platelets

Washed platelets from control or CypD^-/- mice were treated with a combination of collagen and thrombin (Panel A) or ABT737 (Panel B) as in figure 1. The phosphatidylserine expression was measured by FITC-lactadherin. Panel C. The percentages of platelets with fluorescence above background levels (as defined in the gate G in panels A and B) were plotted with the means and standard deviations of a representative triplicate measurements.

Figure 3. Effect of cyclosporine A on ΔΨm

Washed human platelets (2 × 10⁸/ml) were incubated with buffer alone (Panels A and B) or cyclosporine A (2 μM) (Panels C and D) for 20 minutes at room temperature followed by activation with a combination of collagen and thrombin (Panels A and B) or ABT737 (Panels B and D). The samples were labeled with JC-1 and the loss of ΔΨm was measured as a decrease in the red fluorescence. The gate P shows the platelet population with loss of ΔΨm. Panel E. The percentage of platelets with loss of ΔΨm in gate P were plotted with means and standard deviations of a representative triplicate measurements.

Figure 4. Effect of cyclophilin D deficiency on ΔΨm in murine platelets

Washed platelets (2 × 10⁸/ml), from CypD^+/+ (control mice) in Panels A and C or CypD^-/- mice in Panels B and D, were stimulated a combination of collagen and thrombin (Panels A and B) or ABT737 (Panels C and D) and then labeled with JC-1. The loss of ΔΨm was determined as in Figure 3. Panel E shows the means and standard deviations of a representative triplicate measurements of platelets with loss of ΔΨm in gate P.

Figure 5. Release of cytochrome c

Panel A. Washed Jurkat cells (5×10⁵) were incubated with buffer alone or etoposide(10 μM) for 5 h and release of cytochrome c to the cytosol was determined by SDS-PAGE of the extract followed by electrophoretic transfer to PVDF membranes and immunoblotting with a polyclonal antibody to cytochrome c. Panels B and C. Washed platelets (2 × 10⁸/ml) were incubated with a combination of collagen and thrombin (Panel B) or ABT737 (Panel C). Release of cytochrome c to the cytosol was determined as in Panel A.

Figure 6. Phosphatidylserine exposure and cleavage of caspase-3 and ROCK1

Panel A. Washed platelets (2 × 10⁸/ml) were treated with a combination of collagen and thrombin or ABT737 and the activation of caspase-3 were determined by immunoblotting following SDS-PAGE with a polyclonal antibody to caspase-3. Activation of caspase-3 results in the cleavage of intact molecule (35 kDa) into a 17 kDa fragment. Panel B. Washed platelets were incubated with caspase 3 inhibitor zDEVD fmk (20 μM) for 20 minutes and stimulated with combination of collagen and thrombin or with ABT737 and the phosphatidylserine exposure was measured with fluoresein-lactadherin. Shown are the means and standard deviations of a triplicate measurement. Panel C. Washed platelets were incubated with ROCK1 inhibitor Y-27632 (20 μM) for 20 minutes, stimulated with combination of collagen and thrombin or with ABT737 and the phosphatidylserine exposure was measured with fluorescein-lactadherin. Shown are the means and standard deviations of a triplicate measurement. Panel D. Washed platelets were treated with a combination of collagen and thrombin or ABT737. Platelet extracts (20 μg/well) were subjected to SDS-PAGE and immunoblotted with an antibody specific to the carboxy-terminal fragment of ROCK1.

Figure 7. Phosphatidylserine exposure and caspase-3 and ROCK1 cleavage in stored human platelets

Panel A. Phosphatidylserine expression on fresh and 6-day old stored platelets was determined with FITC-lactadherin as in figure 1. Panel B. The percentages of platelets with fluorescence above background levels as defined in the gate G in figure 1. Panel C and D. Platelet extracts (20 μg/well) from fresh and 6 day old stored platelets were subjected SDS-PAGE, transferred to PVDF membranes and blotted with a polyclonal antibody to caspase-3 (Panel C) or monoclonal antibody to activation fragment 1 (Panel D). Lane 1, fresh platelets treated with ABT737; Lane 2, fresh platelets and Lanes 3-5, six-day old platelets from three different individuals.