Platelet extravasation across inflamed venules depends on CD18, mast cells and neutrophils

Platelets and neutrophils are often recruited at sites of microvascular inflammation. While neutrophil extravasation in inflammation is well characterized, platelet extravasation in inflammation is poorly understood and has only been documented in a small number of studies. We showed previously that mice expressing low levels of the leukocyte integrin CD18 (CD18hypo) had reduced total recruitment of platelets (intravascular and extravascular) in the corneal limbus following corneal epithelial wound injury. In this study we sought to determine the role of CD18 in platelet extravasation. Following corneal wound injury, wild‐type (WT) mice had evidence of platelet extravasation across limbal venules; however, CD18hypo mice had markedly reduced platelet extravasation. In view of isolated reports that platelets express CD18, we compared blood platelet counts and ex vivo platelet function under shear stress between CD18hypo and WT mice. Blood platelet counts were measured with a Scil Vet ABC hematology analyzer (Scil animal care company). Flow chamber assays were conducted using a microfluidic BioFlux System (Fluxion Biosciences) with plates coated with type I collagen (25 μg/ml; Helena) and blocked with 5% Bovine serum albumin. The labeled blood was perfused over the collagen‐coated surfaces at two values of fixed shear stress (16 dyne/cm 2 and 30 dyne/cm 2 ). Platelet counts and platelet adhesion to collagen at both levels of shear stress were comparable between CD18hypo and WT mice. Since a variety of leukocytes express CD18, including mast cells and neutrophils, we performed experiments to assess the role of these leukocytes in platelet extravasation. We found that CD18hypo mice had a small but statistically significant decrease in mast cell number and a marked decrease in degranulated mast cells as compared to WT mice following wound injury. Further, corneal epithelial wound injury in mice deficient in mast cells (Kit W‐sh/W‐sh ) resulted in a marked decrease in platelet extravasation as compared to WT mice. Finally, antibody‐induced depletion of circulating neutrophils resulted in a marked decrease in platelet extravasation relative to control mice. Overall, these studies demonstrate that platelet extravasation in a corneal wound injury model of inflammation depends on CD18, mast cells, and neutrophils. Support or Funding Information Supported by: NIH EY018239, EY17120, HL079368 and VA BLR&D Merit Review Award I01 BX002551. M.A.B. was supported by NHLBI T32 HL139425 Extravascular platelets (green) are evident surrounding venules 24 hours postwounding in wild type mice. Figure 1 Platelets from CD18 hypomorphic mice had similar adhesion under shear as those from Wild‐type mice; blood platelet counts were similar in both strains Figure 2