Sergiy Yakovlev

Biomolecular Characterization of CD44-Fibrin(ogen) Binding DISTINCT MOLECULAR REQUIREMENTS MEDIATE BINDING OF STANDARD AND VARIANT ISOFORMS OF CD44 TO IMMOBILIZED FIBRIN(OGEN)

CD44 and fibrin(ogen) play critical roles in the hematogenous dissemination of tumor cells, including colon carcinomas. We recently reported that CD44 is the primary fibrin, but not fibrinogen, receptor on LS174T colon carcinomas. However, the biochemical nature of this interaction and the roles of CD44 standard (CD44s) versus CD44 variant (CD44v) isoforms in fibrin(ogen) recognition have yet to be delineated. Microspheres, coated with CD44 immunopurified from LS174T or T84 colon carcinoma cells, which express primarily CD44v, effectively bind to immobilized fibrin, but not fibrinogen, in shear flow. In contrast, CD44s from HL-60 cells binds to both immobilized fibrin and fibrinogen under flow. Use of highly specific enzymes and metabolic inhibitors reveals that LS174T CD44 binding to fibrin is dependent on O-glycosylation of CD44, whereas CD44s-fibrin(ogen) interaction has an absolute requirement for N-, but not O-, linked glycans. The presence of chondroitin and dermatan sulfate on CD44 standard and variant isoforms facilitates fibrin recognition. Use of the anti-CD44 function-blocking monoclonal antibody Hermes-1 nearly abolishes binding of LS174T CD44 to fibrin, although it has no effect on CD44s-fibrin(ogen) interaction. The CD44-binding site is localized within the N-terminal portion of the fibrin β chains, including amino acid residues (β15-66). Surface plasmon resonance experiments revealed high affinity binding of immobilized CD44 with solubilized fibrin but not fibrinogen. Collectively, these data suggest that immobilization of fibrinogen exposes a cryptic site that mediates binding to CD44s but not CD44v. Our findings may provide a rational basis for designing novel therapeutic strategies to combat metastasis.

The Interaction of Integrin αIIbβ3 with Fibrin Occurs through Multiple Binding Sites in the αIIb β-Propeller Domain

Background: During thrombus formation, platelet integrin αIIbβ3 binds fibrin; however, the mechanism of this interaction is unclear. Results: Mutations of discontinuous negatively charged and aromatic residues in the αIIb β-propeller domain impair fibrin clot retraction and cell adhesion. Conclusion: Integrin αIIbβ3 has multiple binding sites for fibrin. Significance: Uncovered recognition specificity of αIIbβ3 for fibrin may be used to select inhibitors of this interaction. The currently available antithrombotic agents target the interaction of platelet integrin αIIbβ3 (GPIIb-IIIa) with fibrinogen during platelet aggregation. Platelets also bind fibrin formed early during thrombus growth. It was proposed that inhibition of platelet-fibrin interactions may be a necessary and important property of αIIbβ3 antagonists; however, the mechanisms by which αIIbβ3 binds fibrin are uncertain. We have previously identified the γ370–381 sequence (P3) in the γC domain of fibrinogen as the fibrin-specific binding site for αIIbβ3 involved in platelet adhesion and platelet-mediated fibrin clot retraction. In the present study, we have demonstrated that P3 can bind to several discontinuous segments within the αIIb β-propeller domain of αIIbβ3 enriched with negatively charged and aromatic residues. By screening peptide libraries spanning the sequence of the αIIb β-propeller, several sequences were identified as candidate contact sites for P3. Synthetic peptides duplicating these segments inhibited platelet adhesion and clot retraction but not platelet aggregation, supporting the role of these regions in fibrin recognition. Mutant αIIbβ3 receptors in which residues identified as critical for P3 binding were substituted for homologous residues in the I-less integrin αMβ2 exhibited reduced cell adhesion and clot retraction. These residues are different from those that are involved in the coordination of the fibrinogen γ404–411 sequence and from auxiliary sites implicated in binding of soluble fibrinogen. These results map the binding of fibrin to multiple sites in the αIIb β-propeller and further indicate that recognition specificity of αIIbβ3 for fibrin differs from that for soluble fibrinogen.

Noncovalent Interaction of α2-Antiplasmin with Fibrin(ogen): Localization of α2-Antiplasmin-Binding Sites

Covalent incorporation (cross-linking) of plasmin inhibitor alpha(2)-antiplasmin (alpha(2)-AP) into fibrin clots increases their resistance to fibrinolysis. We hypothesized that alpha(2)-AP may also interact noncovalently with fibrin prior to its covalent cross-linking. To test this hypothesis, we studied binding of alpha(2)-AP to fibrin(ogen) and its fragments by an enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance. The experiments revealed that alpha(2)-AP binds to polymeric fibrin and surface-adsorbed fibrin(ogen), while no binding was observed with fibrinogen in solution. To localize the alpha(2)-AP-binding sites, we studied the interaction of alpha(2)-AP with the fibrin(ogen)-derived D(1), D-D, and E(3) fragments, and the recombinant alphaC region and its constituents, alphaC connector and alphaC domain and its subdomains, which together encompass practically the whole fibrin(ogen) molecule. In the ELISA, alpha(2)-AP bound to immobilized D(1), D-D, alphaC region, alphaC domain, and its C-terminal subdomain. The binding was Lys-independent and was not inhibited by plasminogen or tPA. Furthermore, the affinity of alpha(2)-AP for D-D was significantly increased in the presence of plasminogen, while that to the alphaC domain remained unaffected. Altogether, these results indicate that the fibrin(ogen) D region and the C-terminal subdomain of the alphaC domain contain high-affinity alpha(2)-AP-binding sites that are cryptic in fibrinogen and exposed in fibrin or adsorbed fibrinogen, and the presence of plasminogen facilitates interaction of alpha(2)-AP with the D regions. The discovered noncovalent interaction of alpha(2)-AP with fibrin may contribute to regulation of the initial stage of fibrinolysis and provide proper orientation of the cross-linking sites to facilitate covalent cross-linking of alpha(2)-AP to the fibrin clot.

Integrin αIIbβ3:ligand interactions are linked to binding-site remodeling

This study tested the hypothesis that high‐affinity binding of macromolecular ligands to the αIIbβ3 integrin is tightly coupled to binding‐site remodeling, an induced‐fit process that shifts a conformational equilibrium from a resting toward an open receptor. Interactions between αIIbβ3 and two model ligands—echistatin, a 6‐kDa recombinant protein with an RGD integrin‐targeting sequence, and fibrinogens γ‐module, a 30‐kDa recombinant protein with a KQAGDV integrin binding site—were measured by sedimentation velocity, fluorescence anisotropy, and a solid‐phase binding assay, and modeled by molecular graphics. Studying echistatin variants (R24A, R24K, D26A, D26E, D27W, D27F), we found that electrostatic contacts with charged residues at the αIIb/β3 interface, rather than nonpolar contacts, perturb the conformation of the resting integrin. Aspartate 26, which interacts with the nearby MIDAS cation, was essential for binding, as D26A and D26E were inactive. In contrast, R24K was fully and R24A partly active, indicating that the positively charged arginine 24 contributes to, but is not required for, integrin recognition. Moreover, we demonstrated that priming—i.e., ectodomain conformational changes and oligomerization induced by incubation at 35°C with the ligand‐mimetic peptide cHarGD—promotes complex formation with fibrinogens γ‐module. We also observed that the γ‐modules flexible carboxy terminus was not required for αIIbβ3 integrin binding. Our studies differentiate priming ligands, which bind to the resting receptor and perturb its conformation, from regulated ligands, where binding‐site remodeling must first occur. Echistatins binding energy is sufficient to rearrange the subunit interface, but regulated ligands like fibrinogen must rely on priming to overcome conformational barriers.

Interaction of fibrin(ogen) with the endothelial cell receptor VE-cadherin: localization of the fibrin-binding site within the third extracellular VE-cadherin domain.

Interaction of fibrin with endothelial cells through their receptor VE-cadherin has been implicated in modulation of angiogenesis and inflammation. Previous studies identified the VE-cadherin-binding site in the fibrin betaN-domains formed by the NH(2)-terminal regions of fibrin beta chains and revealed that the recombinant dimeric (beta15-66)(2) fragment mimicking these domains preserves the VE-cadherin-binding properties of fibrin. To test if the other fibrin(ogen) regions/domains are involved in this interaction and localize the complementary fibrin-binding site in VE-cadherin, we prepared several recombinant fragments containing individual extracellular domains of VE-cadherin or combinations thereof, as well as several fragments corresponding to various fibrin(ogen) regions, and tested the interactions between them by ELISA and surface plasmon resonance. The experiments revealed that the betaN-domains are the only fibrin(ogen) regions involved in the interaction with VE-cadherin. They also localized the fibrin-binding site to the third extracellular domain of VE-cadherin and established that the fibrin-binding properties of this domain are not influenced by the presence or absence of the neighboring domains. In addition, the experiments confirmed that calcium ions, which are required to maintain proper conformation and adhesive properties of VE-cadherin, do not influence the fibrin-binding properties of the latter.

Aciculin interacts with filamin C and Xin and is essential for myofibril assembly, remodeling and maintenance

ABSTRACT Filamin C (FLNc) and Xin actin-binding repeat-containing proteins (XIRPs) are multi-adaptor proteins that are mainly expressed in cardiac and skeletal muscles and which play important roles in the assembly and repair of myofibrils and their attachment to the membrane. We identified the dystrophin-binding protein aciculin (also known as phosphoglucomutase-like protein 5, PGM5) as a new interaction partner of FLNc and Xin. All three proteins colocalized at intercalated discs of cardiac muscle and myotendinous junctions of skeletal muscle, whereas FLNc and aciculin also colocalized in mature Z-discs. Bimolecular fluorescence complementation experiments in developing cultured mammalian skeletal muscle cells demonstrated that Xin and aciculin also interact in FLNc-containing immature myofibrils and areas of myofibrillar remodeling and repair induced by electrical pulse stimulation (EPS). Fluorescence recovery after photobleaching (FRAP) experiments showed that aciculin is a highly dynamic and mobile protein. Aciculin knockdown in myotubes led to failure in myofibril assembly, alignment and membrane attachment, and a massive reduction in myofibril number. A highly similar phenotype was found upon depletion of aciculin in zebrafish embryos. Our results point to a thus far unappreciated, but essential, function of aciculin in myofibril formation, maintenance and remodeling.

Identification of VLDLR as a novel endothelial cell receptor for fibrin that modulates fibrin-dependent transendothelial migration of leukocytes

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.

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

Summary.  Background: The interaction of the fibrin βN‐domain with VE‐cadherin on endothelial cells is implicated in transendothelial migration of leukocytes, and the β15–42 fragment representing part of this domain has been shown to inhibit this process. However, our previous study revealed that only a dimeric (β15–66)2 fragment, corresponding to the full‐length βN‐domain and mimicking its dimeric arrangement in fibrin, bound to VE‐cadherin. Objective: To test our hypothesis that dimerization of β15–42‐containing fragments increases their affinity for VE‐cadherin and ability to inhibit transendothelial migration of leukocytes. Methods: Interaction of β15–42‐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 β15–42 and β15–64 fragments and their dimeric forms, (β15–44)2 and (β15–66)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 β15–42 and β15–64 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, (β15–44)2 significantly reduced myocardial injury induced by ischemia–reperfusion. Conclusion: The results confirm our hypotheses and indicate that (β15‐66)2 and (β15‐44)2, which exhibited much higher affinity for VE‐cadherin, are highly effective in suppressing inflammation by inhibiting leukocyte transmigration.

Interaction of Fibrin with the Very Low Density Lipoprotein Receptor: Further Characterization and Localization of the Fibrin-Binding Site

Our recent study revealed that fibrin interacts with the very low density lipoprotein receptor (VLDLR) on endothelial cells through its βN domains, and this interaction promotes transendothelial migration of leukocytes and thereby inflammation. The major aims of this study were to further characterize this interaction and localize the fibrin-binding site in the VLDLR. To localize the fibrin-binding site, we expressed a soluble extracellular portion of this receptor, sVLDLRHT, its N- and C-terminal regions, VLDLR(1-8)HT and des(1-8)VLDLRHT, respectively, and a number of VLDLR fragments containing various combinations of CR domains and confirmed their proper folding by fluorescence spectroscopy. Interaction of these fragments with the (β15-66)2 fragment corresponding to a pair of VLDLR-binding βN domains of fibrin was tested by different methods. Our experiments performed by an enzyme-linked immunosorbent assay and surface plasmon resonance revealed that the VLDLR(1-8)HT fragment containing eight CR domains of VLDLR and its subfragments, VLDLR(1-4)HT and VLDLR(2-4)HT, interact with (β15-66)2 with practically the same affinity as sVLDLRHT while the affinity of VLDLR(2-3)HT was ∼2-fold lower. In contrast, des(1-8)VLDLRHT exhibited no binding. Formation of the complex in solution between the fibrin-binding fragments of VLDLR and (β15-66)2 was detected by fluorescence spectroscopy. In addition, formation of a complex between VLDLR(2-4)HT and (β15-66)2 in solution was confirmed by size-exclusion chromatography. Thus, the results obtained indicate that minimal fibrin-binding structures are located within the second and third CR domains of the VLDL receptor and the presence of the fourth CR domain is required for high-affinity binding. They also indicate that tryptophan residues of CR domains are involved in this binding.

Anti-VLDL receptor monoclonal antibodies inhibit fibrin-VLDL receptor interaction and reduce fibrin-dependent leukocyte transmigration

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.