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S. Wichaiyo et al.
injury (Figure 8A and B), in association with enhanced vascular leakage (Figure 8A and C) and increased fibrin deposition (Figure 8D and E). Moreover, decreased wound neutrophils (Figure 8F and G) and macrophages (Figure 8H and I), and higher wound monocytes (Online Supplementary Figure S8A and B) but unaltered level of TNF-α (Online Supplementary Figure S8C and D) were observed in anti-podoplanin-treated Gp6-/- mice. The re- epithelialization (Online Supplementary Figure S8E and F), granulation tissue formation (Online Supplementary Figure S8E and H), and angiogenesis (Online Supplementary Figure S8I and J) were also increased in anti-podoplanin-treated Gp6-/- mice. However, no significant change was seen in wound contraction (Online Supplementary Figure S8E and G). Similar to DKO mice, extravasation of platelets was detected together with the presence of anti-podoplanin antibody on pericytes and other perivascular cells in anti- podoplanin-treated Gp6-/- mice at this time (Online Supplementary Figure S9).
These data indicate that the accelerated wound healing in DKO mice is not due to developmental defects or thrombocytopenia, but due to a combined loss of the interaction of platelet CLEC-2 and GPVI with their respective ligands.
Discussion
In this study, we show that combined deletion of platelet CLEC-2 and GPVI promotes healing of a full- thickness skin wound in mice compared to WT or single- knockout mice. The accelerated wound closure is accom- panied by impairment of vascular integrity, rapid re- epithelialization and an increase in granulation tissue for- mation, resulting in a smaller wound scar. This is also associated with elevated levels of fibrinogen and fibrin in the tissue, reduced infiltration of leukocytes, and enhanced angiogenesis. A proposed model for the multi- factorial regulation of accelerated wound healing in the absence of the two ITAM receptors is shown in Online Supplementary Figure S10.
Wound healing is a multistep process involving coagu- lation, vascular permeability changes, inflammation, cell proliferation, and cell migration.1,2,21 In our study, we show that deletion of CLEC-2 and GPVI in platelets leads to increased bleeding into the wound, in association with accelerated wound repair. These effects are not due to blood/lymphatic vessel mixing or the reduction in platelet count in the DKO mice as similar results were observed in anti-podoplanin-antibody treated Gp6-/- mice, which do not have these defects. Acceleration of wound healing is also observed in mice treated with histamine23 or serum fraction of the natural latex from rubber tree,21 which enhances vascular permeability, a milder form of vascular leakage. Therefore, impaired vascular integrity as seen in the DKO platelets or increased permeability leads to extravasation of growth factors, cytokines, and plasma proteins to the tissue, and promotion of wound healing.
The role of blood coagulation in wound repair has been studied in mice expressing low TF5 or lacking FIX (hemo- philia B mice).3,5 In these two models, the deficiency in clotting factors leads to a persistent hematoma formation (due to a prolonged subcutaneous bleeding) and a reduc- tion in fibrin generation, both of which contribute to a delay in wound repair. Chronic hemorrhage also con-
tributes to the characteristics of non-healing wounds in cancers, which promote tumor growth.24-26 In contrast, we show that in the presence of an intact coagulation cas- cade, the transient and self-limited bleeding into the wound (which is rich in TF5,22) caused by the impairment of vascular integrity at an initial stage, is associated with accelerated wound repair. This is most likely due to increased entry of platelets, clotting factors, and plasma proteins, leading to fibrinogen accumulation and fibrin generation. Thus, the mechanism of bleeding determines whether it is beneficial or detrimental to wound repair.
Fibrinogen and fibrin are not only crucial for clot forma- tion, but also act as a natural suture/sealant, providing a matrix for cell migration as well as a reservoir for growth factors and cytokines.2 For example, fibrin exposes plas- minogen to migrating keratinocytes, which in turn con- vert plasminogen into plasmin to mediate fibrinolysis, allowing the cells to move along the fibrin(ogen) matrix.27- 29 Similarly, endothelial cells proliferate and migrate over fibrin(ogen), forming a capillary tube that contributes to angiogenesis during wound healing.30-32 In DKO mice, we therefore propose that fibrin(ogen) accumulation pro- motes re-epithelialization and angiogenesis, and acceler- ates wound repair.
In contrast, fibrinogen33 and a high concentration of fib- rin34 inhibit neutrophil migration as previously reported and in line with our in vitro data. These data may explain our observations that in DKO mice, wound neutrophils at day 3 did not increase over the amount seen at day 1 post injury. This is in contrast to wound neutrophil accumula- tion in WT, which was higher at day 3 than at day 1 post injury. Increased fibrin content in the wound was also observed in DKO mice compared to WT at day 3 post injury. Therefore, the physical obstruction and anti-migra- tory properties of the fibrin clot is likely to inhibit neu- trophil wound entry observed in DKO mice. Moreover, given that, at this early phase, there was no significant dif- ference in the level of blood neutrophils between WT and DKO mice, altered neutrophil turnover/apoptosis in DKO mice is unlikely to make a significant contribution to the reduction in wound neutrophils observed in DKO com- pared to WT at day 3 post injury.
Consistent with our data, several lines of evidence demonstrate improved wound healing in neutrophil- depleted conditions.35-38 Indeed, neutrophil infiltration mediates damage through a production of proteases,35 oxidative radicals, elastase,39 and neutrophil extracellular traps,37 all of which can delay the healing process. A sim- ilar effect of fibrin(ogen) might also apply to monocyte/macrophage recruitment since fibrinogen has an anti-adhesive effect against monocytes.40 A recent ster- ile wound model has demonstrated that Ly6C+ mono- cytes are present throughout wound healing and differen- tiate into M1 macrophages during inflammatory phase and M2 macrophages during the reparative phase.41 In addition, it has been shown that TNF-α secretion is increased during monocyte-to-M1-macrophage differen- tiation.41,42 The reduction in TNF-α during the inflamma- tory phase is associated with the increase in wound monocytes but not macrophages. This suggests an inhibi- tion in monocyte-to-M1-macrophage transition in DKO mice, resulting in a significant change in immune cell infiltration in the wound.
Depletion of macrophages in the first five days has pre- viously been shown to delay the later stages of wound
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haematologica | 2019; 104(8)