Platelets are well recognized as a key player in primary hemostasis and thrombosis, but their role in the subsequent initiation of tissue repair is less well characterized. Within the hemostatic thrombus, ballooned platelets reminiscent of the procoagulant or necrotic platelet are first observed in the periphery (extravascular and in contact with collagen) and subsequently throughout the platelet plug. The key role of cyclophilin D (CypD) in the regulation of platelet mitochondrial permeability transition pore (mPTP) and procoagulant platelet formation is well established. Furthermore, the cyclophilin inhibitor cyclosporine is known to both block procoagulant platelet formation in vitroand to result in delayed wound healing in patients. While the consequences of CypD's absence have been studied in vivo in models of venous and arterial thrombosis, the effects on wound healing and tissue repair have not been investigated.

Here we investigated the role of CypD-dependent events in the setting of cutaneous wound healing. Punch biopsies (6 mm) were placed and wound diameter (days 0, 1,2, 5,7,10,14) was examined. In mice with a global absence of CypD and in mice with platelet/megakaryocyte-specific CypD deficiency, wound healing was substantially and similarly delayed (n=9 -11 for each group). This demonstrates that platelet CypD-mediated processes play a key role in the initiation of hemostatic thrombus resolution and subsequent wound healing. The initial resolution of wound healing was delayed by ~ 2-3 days. This resulted in a significant difference at days 4 and 7 post-injury in the size of the wounds of these mice (p<0.05). To elaborate possible mechanisms by which platelet CypD-deficiency resulted in delayed wound healing, skin from the area of the wound was harvested at key early time points (day 0,1,2,5) and histological examination was performed. Early in the initiation of wound resolution, polymorphonuclear cells (PMNs) successively infiltrate the platelet plug. In mice lacking CypD, either globally or platelet-specific, polymorphonuclear cell (PMN) infiltration of the resolving thrombus on days 1 and 2 was markedly impaired. However, in all mice, PMNs amassed at the edges of the wound at 2 days post-injury. Furthermore, subsequent wound resolution was dysregulated in the absence of platelet CypD. Wounds from these mice (day 5) demonstrated marked blebbing and structural abnormalities. Remarkably, these delays in wound healing and PMN succession occurred in the absence of either gross or microscopic bleeding consistent with the absence of a defect in initial hemostasis previously reported in platelet-CypD deficient mice.

These results indicate that platelets, independent of their hemostatic role, play a key role in the initiation of wound resolution. And that, subsequent to hemostasis, a platelet CypD-mediated process, presumably the procoagulant platelet transition, is required for the timely and orchestrated initiation of wound resolution and successive wound PMN infiltration. These results are consistent with the established in vitro role of CypD in mediating the procoagulant platelet morphologic transition and the procoagulant platelet's known role in mediating neutrophil activation. Finally, our results identify a key platelet-dependent mechanism by which therapy with the immunosuppressant cyclosporine can result in delayed wound healing.

Jobe: CSL: Consultancy; Shire: Consultancy; Octapharma: Consultancy.