Critical Role for Cyclophilin D and the Mitochondrial Permeability Transition Pore (MPTP) in Platelet Activation.

Dual stimulation of platelets with thrombin and collagen results in the formation of a unique subpopulation of highly activated platelets. Characteristics of the highly activated platelet subpopulation include

1. increased surface retention of procoagulant alpha granule proteins,

2. high-level phosphatidylserine (PS) externalization, and

3. modulation of the fibrinogen receptor αIIbβ3 as evidenced by their decreased recognition by antibodies to activated αIIbβ3 such as PAC-1 and JON/A.

Formation of the highly activated platelet subpopulation is closely correlated with a rapid loss of mitochondrial transmembrane potential (ΔΨ_m), a marker of MPTP formation. To test whether formation of the MPTP might regulate the development of the highly activated platelet subpopulation, platelet activation responses were examined in the presence of inhibitors and activators of MPTP formation. Cyclosporine, an inhibitor of MPTP formation, inhibited both PS externalization and αIIbβ3 modulation following dual stimulation with thrombin and the glycoprotein VI agonist convulxin (58 ± 4% vs. 9 ± 3%, p<0.01). Conversely, thrombin stimulation of platelets in the presence of H₂O₂ (100μM), an MPTP activator, increased PS externalization and αIIbβ3 modulation relative to platelets stimulated with thrombin alone (11 ± 3% vs. 48 ± 6%, p<0.05).

Platelet activation responses were examined in cyclophilin D null (CypD −/−) mice, which have marked impairment of MPTP formation. Following dual agonist stimulation with thrombin and convulxin, both αIIbβ3 modulation and platelet PS externalization were significantly abrogated in CypD −/− platelets relative to wild type (7 ± 1% vs. 69 ± 1%, p<0.01). Alpha granule release, however, was unaffected in the absence of CypD. In vitro tests of platelet function similarly demonstrated that CypD −/− platelets had marked impairment of platelet prothrombinase activity relative to wild-type platelets after stimulation with thrombin and convulxin, but normal platelet aggregation responses.

We then tested the hypothesis that CypD −/− mice would have an altered thrombotic response to arterial injury. Following photochemical injury of the carotid artery endothelium, a stable occlusive thrombus formed more rapidly in CypD −/− than in wild-type mice (16 ± 2 vs. 32 ± 7 min, p<0.05). Tail-bleeding time was unaffected. These results strongly implicate cyclophilin D and the MPTP as critical regulators of the subset of platelet activation responses occurring in the highly activated platelet subpopulation and suggest that activation of this novel platelet mitochondrial signaling pathway might play an important role in the regulation of the thrombotic response in vivo.