Interaction of fibrin with VE-cadherin and anti-inflammatory effect of fibrin-derived fragments

Background: The interaction of the fibrin beta N-domain with VE-cadherin on endothelial cells is implicated in transendothelial migration of leukocytes, and the beta 15-42 fragment representing part of this domain has been shown to inhibit this process. However, our previous study revealed that only a dimeric (beta 15-66)(2) fragment, corresponding to the full-length beta N-domain and mimicking its dimeric arrangement in fibrin, bound to VE-cadherin. Objective: To test our hypothesis that dimerization of beta 15-42-containing fragments increases their affinity for VE-cadherin and ability to inhibit transendothelial migration of leukocytes. Methods: Interaction of beta 15-42-containing fragments with VE-cadherin was characterized by ELISA and surface plasmon resonance. The inhibitory effect of such fragments was tested in vitro with a leukocyte transendothelial migration assay and in vivo with mouse models of peritonitis and myocardial ischemia-reperfusion injury. Results: First, we prepared the monomeric beta 15-42 and beta 15-64 fragments and their dimeric forms, (beta 15-44)(2) and (beta 15-66)(2), and studied their interaction with the fibrin-binding domain of VE-cadherin, VE-cad(3). The experiments revealed that both dimeric fragments bound to VE-cad(3) with high affinity, whereas the affinities of beta 15-42 and beta 15-64 were significantly lower. Next, we tested the ability of these fragments to inhibit leukocyte transmigration in vitro and infiltration into the inflamed peritoneum in vivo, and found that the inhibitory effects of the dimers on these processes were also superior. Furthermore, (beta 15-44)(2) significantly reduced myocardial injury induced by ischemia-reperfusion. Conclusion: The results confirm our hypotheses and indicate that (beta 15-66)(2) and (beta 15-44)(2), which exhibited much higher affinity for VE-cadherin, are highly effective in suppressing inflammation by inhibiting leukocyte transmigration.