Antibodies to MHC class I antigens have been shown to activate endothelial cells to release von Willebrand factor (vWf) and P-selectin from storage granules. These adhesion molecules induce platelet activation and aggregation that results in release of pro-inflammatory cytokines. Recently, hyaluronidase 2 (HYAL2) was reported to be contained in platelets and externalized on their surfaces upon activation. HYAL2 is one of the four enzymes currently known to cleave hyaluronan, a major component of extracellular matrix. Hyaluronan normally exists as a high molecular weight polymer, which suppresses immunogenicity and cell proliferation. HYAL2 expressed by platelets has been shown to cleave hyaluronan into fragments with proinflammatory properties in vitro. Since increased amounts of hyaluronan were observed in renal biopsies with acute or chronic rejection, platelet mediated fragmentation may enhance inflammation and graft injury. Therefore, in this study, we investigated the relationship between platelet activation by alloantibodies and generation of hyaluronan fragments in renal transplants.

MHC mismatched B10.A (H-2a) kidneys were transplanted on to immune deficient B6 Rag1-/- recipients. A mixture of IgG1, 2a, and 2b monoclonal antibodies to H-2a was transferred intraperitoneally. One hour after alloantibody transfer, complement activation was detected by C4d staining. Immunohistochemistry also demonstrated widespread intravascular aggregates of vWf and P-selectin coated platelets. Platelet activation correlated with increased circulating hyaluronan relative to control mice that received isoantibody as measured by ELISA. To test the role of platelets in alloantibody induced hyaluronan fragmentation, platelets were depleted with anti-CD42b antibody 1 hour before alloantibody transfer. Depletion of platelets decreased levels of hyaluronan in both sera and graft homogenates.

In conclusion, our data indicate that alloantibodies can alter extracellular matrix by activating platelets, and hyaluronan may be a potential target through which platelets regulate immune responses to organ transplants. In addition, increased level of circulating hyaluronan might serve as an indicator of platelet activation and graft injury.

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