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Our previous studies revealed that fibrin interacts with the VLDL receptor (VLDLR) through a pair of its βN-domains and this interaction promotes transendothelial migration of leukocytes and, thereby, inflammation. In agreement, the NDSK-II fragment representing the central part of the fibrin molecule and containing these domains stimulates leukocyte transmigration. However, the recombinant (β15-66) 2 fragment corresponding to a pair of the βN-domains inhibits NDSK-II-stimulated leukocyte transmigration. To explain this paradox, we hypothesized that fibrin βN-domains have dual function in fibrin-dependent inflammation, namely, their C-terminal regions containing the VLDLR-binding sites promote leukocyte transmigration while their N-terminal regions are responsible for inhibition of this process. To test this hypothesis and to further clarify the molecular mechanisms underlying fibrin-induced VLDLR-dependent pathway of leukocyte transmigration and its inhibition, we prepared the dimeric (β15-44) 2 and (β40-66) 2 fragments corresponding to the N- and C-terminal regions of the βN-domains and studied their effect on endothelial permeability and transendothelial migration of leukocytes. The results obtained revealed that (β40-66) 2 bound to the VLDLR with high affinity and promoted endothelial permeability and leukocyte transmigration while (β15-44) 2 did not interact with this receptor and had no effect on leukocyte transmigration, in agreement with our hypothesis. We also found that the first three N-terminal residues of the βN-domains play a critical role in the inhibitory properties of these domains. Further, the inhibitory properties of the βN-domains were expressed only upon their isolation from the fibrin molecule. The question of whether their inhibitory function may play a role in fibrin remains to be addressed., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

According to the current view, binding of fibrin degradation product E 1 fragment to endothelial VE-cadherin promotes transendothelial migration of leukocytes and thereby inflammation, and fibrin-derived β15-42 peptide reduces leukocyte transmigration by competing with E 1 for binding to VE-cadherin and, in addition, by signaling through Src kinase Fyn. However, the very low affinity of β15-42 to VE-cadherin raised a question about its ability to inhibit E 1 -VE-cadherin interaction. Further, our previous study revealed that fibrin promotes leukocyte transmigration through the very-low-density lipoprotein (VLDL) receptor (VLDLR)-dependent pathway and suggested a possible link between the inhibitory properties of β15-42 and this pathway. To test such a link and the proposed inhibitory mechanisms for β15-42, we performed in vitro experiments using surface plasmon resonance, enzyme-linked immunosorbent assay, and leukocyte transendothelial migration assay, and in vivo studies with wild-type and VLDLR-deficient mice using mouse model of peritonitis. The experiments revealed that β15-42 cannot inhibit E 1 -VE-cadherin interaction at the concentrations used in the previous in vivo studies leaving the proposed Fyn-dependent signaling mechanism as a viable explanation for the inhibitory effect of β15-42. While testing this mechanism, we confirmed that Fyn plays a critical role in controlling fibrin-induced transendothelial migration of leukocytes and found that signaling through the VLDLR-dependent pathway results in inhibition of Fyn, thereby increasing leukocyte transmigration. Furthermore, our in vivo experiments revealed that β15-42 inhibits this pathway, thereby preventing inhibition of Fyn and reducing leukocyte transmigration. Thus, this study clarifies the molecular mechanism underlying the VLDLR-dependent pathway of leukocyte transmigration and reveals that this pathway is a target for β15-42., Competing Interests: None declared., (Georg Thieme Verlag KG Stuttgart · New York.)

In spite of numerous studies on the involvement of fibrinogen in transendothelial migration of leukocytes and thereby inflammation, there is still no clear understanding of which fibrin(ogen) species can stimulate leukocyte transmigration. Although we have previously proposed that interaction of fibrin with the VLDL receptor (VLDLR) promotes leukocyte transmigration, there is no direct experimental evidence for the involvement of fibrin in this process. To address these questions, we performed systematic studies of interaction of VLDLR with fibrinogen, fibrin, and their isolated recombinant BβN- and βN-domains, respectively, and the effect of various fibrin(ogen) species on transendothelial migration of leukocytes. The results obtained revealed that freshly purified fibrinogen does not interact with VLDLR in solution and has practically no effect on leukocyte transmigration. They also indicate that the VLDLR-binding site is cryptic in fibrinogen and becomes accessible upon its adsorption onto a surface or upon its conversion into fibrin. We also found that the D-D:E 1 complex and higher molecular mass fibrin degradation products, as well as soluble fibrin and fibrin polymers (clots) anchored to the endothelial monolayer, promote leukocyte transmigration mainly through the VLDL receptor-dependent pathway. Thus, the results of the present study suggest that fibrin degradation products and soluble fibrin that may be present in the circulation in vivo, as well as fibrin clots that may be deposited on the surface of inflamed endothelium, promote leukocyte transmigration. These findings further clarify the molecular mechanisms underlying the fibrin-VLDLR-dependent pathway of leukocyte transmigration and provide an explanation for a possible (patho)physiological role of this pathway., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Our previous studies revealed that the interaction of fibrin with the very low density lipoprotein receptor (VLDLR) promotes transendothelial migration of leukocytes and thereby inflammation, and localised the fibrin-binding site to CR-domains 2-4 of this receptor. In the present study, we tested interaction of three anti-VLDLR monoclonal antibodies, mAb 1H10, 1H5, and 5F3, with recombinant fragments of VLDLR containing various combinations of its CR-domains and found that the epitopes for mAb 1H10 and mAb 1H5 overlap with the fibrin-binding site of VLDLR. Based on these findings, we hypothesised that mAb 1H10 and mAb 1H5 should inhibit fibrin-VLDLR interaction and modulate leukocyte transmigration. To test this hypothesis, we first demonstrated that these monoclonal antibodies both have high affinity to the fibrin-binding fragments of the VLDL receptor and efficiently inhibit interaction between the VLDLR-binding fragment of fibrin and the fibrin-binding fragments of VLDLR. Next, in the in vitro experiments using leukocyte transendothelial migration assay we found that both monoclonal antibodies efficiently inhibit leukocyte transmigration induced by fibrin mimetic NDSK-II. Finally, in vivo experiments using mouse model of peritonitis revealed that mAb 1H10 and mAb 1H5 both significantly reduce infiltration of leukocytes into the peritoneum. Furthermore, our experiments using mouse model of myocardial ischemia-reperfusion injury revealed that both monoclonal antibodies significantly reduce myocardial injury induced by ischaemia-reperfusion. Thus, the results obtained indicate that monoclonal antibodies 1H10 and 1H5 are novel specific inhibitors of fibrin-VLDLR-dependent leukocyte transmigration pathway. They may represent potential therapeutics for treatment of fibrin-dependent inflammation including myocardial ischaemia-reperfusion injury., Competing Interests: Conflicts of Interest: None declared.

While testing the effect of the (β15-66)(2) fragment, which mimics a pair of fibrin βN-domains, on the morphology of endothelial cells, we found that this fragment induces redistribution of vascular endothelial-cadherin in a process that is inhibited by the receptor-associated protein (RAP). Based on this finding, we hypothesized that fibrin may interact with members of RAP-dependent low-density lipoprotein (LDL) receptor family. To test this hypothesis, we examined the interaction of (β15-66)(2), fibrin, and several fibrin-derived fragments with 2 members of this family by ELISA and surface plasmon resonance. The experiments showed that very LDL (VLDL) receptor (VLDLR) interacts with high affinity with fibrin through its βN-domains, and this interaction is inhibited by RAP and (β15-66)(2). Furthermore, RAP inhibited transendothelial migration of neutrophils induced by fibrin-derived NDSK-II fragment containing βN-domains, suggesting the involvement of VLDLR in fibrin-dependent leukocyte transmigration. Our experiments with VLDLR-deficient mice confirmed this suggestion by showing that, in contrast to wild-type mice, fibrin-dependent leukocyte transmigration does not occur in such mice. Altogether, the present study identified VLDLR as a novel endothelial cell receptor for fibrin that promotes fibrin-dependent leukocyte transmigration and thereby inflammation. Establishing the molecular mechanism underlying this interaction may result in the development of novel inhibitors of fibrin-dependent inflammation.