Identification of Neural (N)-Cadherin as a Novel Endothelial Cell Receptor for Fibrin and Localization of the Complementary Binding Sites.

Based on the high structural homology between vascular endothelial (VE)-cadherin and neural (N)-cadherin, we hypothesized that fibrin, which is known to interact with VE-cadherin and promote angiogenesis through this interaction, may also interact with N-cadherin. To test this hypothesis, we prepared fibrin and its plasmin-produced and recombinant fragments covering practically all parts of the fibrin molecule. We also prepared the soluble extracellular portion of N-cadherin (sN-cadherin), which includes all five extracellular N-cadherin domains, and studied its interaction with fibrinogen, fibrin, and the aforementioned fibrin fragments using two independent methods, ELISA and SPR. The experiments confirmed our hypothesis, revealing that fibrin interacts with sN-cadherin with high affinity. Furthermore, the experiments localized the N-cadherin binding site within the fibrin ßN-domains. Notably, the recombinant dimeric (ß15-66)2 fragment, corresponding to these domains and mimicking their dimeric arrangement in fibrin, preserved the N-cadherin-binding properties of fibrin. To localize the fibrin binding site within N-cadherin, we performed ELISA and SPR experiments with (ß15-66)2 and recombinant N-cadherin fragments representing its individual extracellular domains and combinations thereof. The results obtained indicate that the interaction of fibrin with N-cadherin occurs through the third and fifth extracellular domains of the latter. This is in contrast to our previous study, which revealed that fibrin interacts only with the third extracellular domain of VE-cadherin. In conclusion, our study identified N-cadherin as a novel receptor for fibrin and localized complementary binding sites within both fibrin and N-cadherin. The pathophysiological role of this interaction remains to be established.

Current View on the Molecular Mechanisms Underlying Fibrin(ogen)-Dependent Inflammation.

Numerous studies have revealed the involvement of fibrinogen in the inflammatory response. To explain the molecular mechanisms underlying fibrinogen-dependent inflammation, two bridging mechanisms have been proposed in which fibrin(ogen) bridges leukocytes to endothelial cells. The first mechanism suggests that bridging occurs via the interaction of fibrinogen with the leukocyte receptor Mac-1 and the endothelial receptor ICAM-1 (intercellular adhesion molecule-1), which promotes leukocyte transmigration and enhances inflammation. The second mechanism includes bridging of leukocytes to the endothelium by fibrin degradation product E1 fragment through its interaction with leukocyte receptor CD11c and endothelial VE-cadherin to promote leukocyte transmigration. The role of E1 in promoting inflammation is inhibited by the fibrin-derived ß15-42 fragment, and this has been suggested to result from its ability to compete for the E1-VE-cadherin interaction and to trigger signaling pathways through the src kinase Fyn. Our recent study revealed that the ß15-42 fragment is ineffective in inhibiting the E1- or fibrin-VE-cadherin interaction, leaving the proposed signaling mechanism as the only viable explanation for the inhibitory function of ß15-42. We have discovered that fibrin interacts with the very-low-density lipoprotein (VLDL) receptor, and this interaction triggers a signaling pathway that promotes leukocyte transmigration through inhibition of the src kinase Fyn. This pathway is inhibited by another pathway induced by the interaction of ß15-42 with a putative endothelial receptor. In this review, we briefly describe the previously proposed molecular mechanisms underlying fibrin-dependent inflammation and their advantages/disadvantages and summarize our recent studies of the novel VLDL receptor-dependent pathway of leukocyte transmigration which plays an important role in fibrin-dependent inflammation.

Dual functions of the fibrin ßN-domains in the VLDL receptor-dependent pathway of transendothelial migration of leukocytes.

Our previous studies revealed that fibrin interacts with the VLDL receptor (VLDLR) through a pair of its ßN-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 (ß15-66)2 fragment corresponding to a pair of the ßN-domains inhibits NDSK-II-stimulated leukocyte transmigration. To explain this paradox, we hypothesized that fibrin ßN-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 (ß15-44)2 and (ß40-66)2 fragments corresponding to the N- and C-terminal regions of the ßN-domains and studied their effect on endothelial permeability and transendothelial migration of leukocytes. The results obtained revealed that (ß40-66)2 bound to the VLDLR with high affinity and promoted endothelial permeability and leukocyte transmigration while (ß15-44)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 ßN-domains play a critical role in the inhibitory properties of these domains. Further, the inhibitory properties of the ßN-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.

The Story of the Fibrin(ogen) αC-Domains: Evolution of Our View on Their Structure and Interactions.

Although much has been established concerning the overall structure and function of fibrinogen, much less has been known about its two αC regions, each consisting of an αC-connector and an αC-domain, but new information has been accumulating. This review summarizes the state of our current knowledge of the structure and interactions of fibrinogen's αC regions. A series of studies with isolated αC regions and their fragments demonstrated that the αC-domain forms compact ordered structures consisting of N- and C-terminal subdomains including ß sheets and suggested that the αC-connector has a poly(L-proline) type II structure. Functionally, the αC-domains interact intramolecularly with each other and with the central region of the molecule, first demonstrated by electron microscopy and then quantified by optical trap force spectroscopy. Upon conversion of fibrinogen into fibrin, the αC-domains switch from intra- to intermolecular interactions to form ordered αC polymers. The formation of αC polymers occurs mainly through the homophilic interaction between the N-terminal subdomains; interaction between the C-terminal subdomains and the αC-connectors also contributes to this process. Considerable evidence supports the idea that the αC-regions accelerate fibrin polymerization and affect the final structure of fibrin clots. The interactions between αC-regions are important for the mechanical properties of clots, increasing their stiffness and extensibility. Conversion of fibrinogen into fibrin results in exposure of multiple binding sites in its αC regions, providing interaction of fibrin with different proteins and cell types during hemostasis and wound healing. This heretofore mysterious part of the fibrinogen molecule is finally giving up its secrets.

Structural Basis for the Interaction of Fibrin with the Very Low-Density Lipoprotein Receptor Revealed by NMR and Site-Directed Mutagenesis.

Interaction of fibrin with the very low-density lipoprotein receptor (VLDLR) promotes transendothelial migration of leukocytes and thereby inflammation. To establish the structural basis for this interaction, we have previously localized the VLDLR-binding site to fibrin ßN-domains including fibrin ß chain sequence 15-64 and determined the NMR solution structure of the VLDLR(2-4) fragment containing fibrin-binding CR domains 2-4 of VLDLR. In this study, we identified amino acid residues in VLDLR and the ßN-domains that are involved in the interaction using NMR and site-directed mutagenesis. The results obtained revealed that Lys47 and Lys53 of the second and third positively charged clusters of the ßN-domain, respectively, interact with Trp20 and Asp25 of the CR2 domain and Trp63 and Glu68 of the CR3 domain, respectively. This finding indicates that Lys residues of the ßN-domain interact with the Lys-binding site of the CR domains in a manner proposed earlier for the interaction of other members of the LDL receptor family with their ligands. In addition, Gly15 of the ßN-domain and its first positively charged cluster contribute to the high-affinity interaction with VLDLR. Molecular modeling based on the results obtained and analysis of the previously published structures of such domains complexed with RAP and HRV2 allowed us to propose a model of interaction of fibrin ßN-domains with the fibrin-binding CR domains of the VLDL receptor.

Illustrated State-of-the-Art Capsules of the ISTH 2020 Congress.

This year's Congress of the International Society of Thrombosis and Haemostasis (ISTH) was hosted virtually from Philadelphia July 17-21, 2021. The conference, now held annually, highlighted cutting-edge advances in basic, population and clinical sciences of relevance to the Society. Despite being held virtually, the 2021 congress was of the same scope and quality as an annual meeting held in person. An added feature of the program is that talks streamed at the designated times will then be available on-line for asynchronous viewing. The program included 77 State of the Art (SOA) talks, thematically grouped in 28 sessions, given by internationally recognized leaders in the field. The SOA speakers were invited to prepare brief illustrated reviews of their talks that were peer reviewed and are included in this article. The topics, across the main scientific themes of the congress, include Arterial Thromboembolism, Coagulation and Natural Anticoagulants, COVID-19 and Coagulation, Diagnostics and Omics, Fibrinogen, Fibrinolysis and Proteolysis, Hemophilia and Rare Bleeding Disorders, Hemostasis in Cancer, Inflammation and Immunity, Pediatrics, Platelet Disorders, von Willebrand Disease and Thrombotic Angiopathies, Platelets and Megakaryocytes, Vascular Biology, Venous Thromboembolism and Women's Health. These illustrated capsules highlight the major scientific advances with potential to impact clinical practice. Readers are invited to take advantage of the excellent educational resource provided by these illustrated capsules. They are also encouraged to use the image in social media to draw attention to the high quality and impact of the science presented at the congress.

Structure and function of fibrinogen BßN-domains.

Two BßN-domains of fibrinogen are formed by the N-terminal portions of its two Bß chains including amino acid residues Bß1-65. Although their folding status is not well understood and the recombinant disulfide-linked (Bß1-66)2 fragment corresponding to a pair of these domains was found to be unfolded, some data suggest that these domains may be folded in the parent molecule. In contrast, their major functional properties are well established. Removal of fibrinopeptides B (amino acid residues Bß1-14) from these domains upon fibrinogen to fibrin conversion results in the exposure of multiple binding sites in fibrin ßN-domains (residues ß15-65). These sites provide interactions of the ßN-domains with different proteins and cells and their participation in various physiological and pathological processes including fibrin assembly, fibrin-dependent angiogenesis, and fibrin-dependent leukocyte transmigration and thereby inflammation. The major goal of the present review is to summarize current view on the structure and function of these domains in fibrinogen and fibrin and their role in the above-mentioned processes.

Fibrin-VLDL Receptor-Dependent Pathway Promotes Leukocyte Transmigration by Inhibiting Src Kinase Fyn and is a Target for Fibrin ß15-42 Peptide.

According to the current view, binding of fibrin degradation product E1 fragment to endothelial VE-cadherin promotes transendothelial migration of leukocytes and thereby inflammation, and fibrin-derived ß15-42 peptide reduces leukocyte transmigration by competing with E1 for binding to VE-cadherin and, in addition, by signaling through Src kinase Fyn. However, the very low affinity of ß15-42 to VE-cadherin raised a question about its ability to inhibit E1-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 ß15-42 and this pathway. To test such a link and the proposed inhibitory mechanisms for ß15-42, 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 ß15-42 cannot inhibit E1-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 ß15-42. 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 ß15-42 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 ß15-42.

Nuclear Magnetic Resonance Solution Structure of the Recombinant Fragment Containing Three Fibrin-Binding Cysteine-Rich Domains of the Very Low Density Lipoprotein Receptor.

Our previous studies revealed that interaction of fibrin with the very low density lipoprotein (VLDL) receptor plays a prominent role in transendothelial migration of leukocytes and thereby inflammation. The major goal of our subsequent studies is to establish the structural basis for this interaction. As the first step toward this goal, we localized the fibrin-binding sites within cysteine-rich (CR) domains 2-4 of the VLDL receptor. In this study, we have made a next step toward this goal by establishing the nuclear magnetic resonance solution structure of the recombinant VLDLR(2-4) fragment containing all three fibrin-binding CR domains of this receptor. The structure revealed that all three CR domains have a similar general fold. Each domain contains a calcium-binding loop, and the loop in the CR3 domain has a unique conformation relative to the other two. Domains CR2 and CR3 interact with each other, while CR4 is flexible relative to the other two domains. In addition, analysis of the electrostatic potential surface of VLDLR(2-4) revealed extended negatively charged regions in each of its CR domains. The presence of these regions suggests that they may interact with three positively charged clusters of the fibrin ßN domain whose involvement in interaction with the VLDL receptor was demonstrated earlier. Altogether, these findings provide a solid background for our next step toward establishing the structural basis for fibrin-VLDL receptor interaction.

Effect of fibrinogen, fibrin, and fibrin degradation products on transendothelial migration of leukocytes.

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 BßN- and ßN-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:E1 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.

Interaction of Fibrin with the Very Low-Density Lipoprotein (VLDL) Receptor: Further Characterization and Localization of the VLDL Receptor-Binding Site in Fibrin ßN-Domains.

Our recent study revealed that fibrin and the very low-density lipoprotein receptor (VLDLR) interact with each other through a pair of fibrin ßN-domains and CR domains of the receptor and this interaction promotes transendothelial migration of leukocytes and thereby inflammation. The major objectives of this study were to further clarify the molecular mechanism of fibrin-VLDLR interaction and to identify amino acid residues in the ßN-domains involved in this interaction. Our binding experiments with the (ß15-66)2 fragment, which corresponds to a pair of fibrin ßN-domains, and the VLDLR(1-8) fragment, consisting of eight CR domains of VLDLR, revealed that interaction between them strongly depends on ionic strength and chemical modification of all Lys or Arg residues in (ß15-66)2 results in abrogation of this interaction. To identify which of these residues are involved in the interaction, we mutated all Lys or Arg residues in each of the three positively charged Lys/Arg clusters of the (ß15-66)2 fragment, as well as single Arg17 and Arg30, and tested the affinity of the mutants obtained for VLDLR(1-8) by an enzyme-linked immunosorbent assay and surface plasmon resonance. The experiments revealed that the second and third Lys/Arg clusters make the major contribution to this interaction while the contribution of the first cluster is moderate. The results obtained suggest that interaction between fibrin and the VLDL receptor employs the "double-Lys/Arg" recognition mode previously proposed for the interaction of the LDL receptor family members with their ligands. They also provide valuable information for the development of highly specific peptide-based inhibitors of fibrin-VLDLR interaction.

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.

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 ßN 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 (ß15-66)2 fragment corresponding to a pair of VLDLR-binding ßN 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 (ß15-66)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 (ß15-66)2 was detected by fluorescence spectroscopy. In addition, formation of a complex between VLDLR(2-4)HT and (ß15-66)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.

On the mechanism of αC polymer formation in fibrin.

Our previous studies revealed that the fibrinogen αC-domains undergo conformational changes and adopt a physiologically active conformation upon their self-association into αC polymers in fibrin. In the present study, we analyzed the mechanism of αC polymer formation and tested our hypothesis that self-association of the αC-domains occurs through the interaction between their N-terminal subdomains and may include ß-hairpin swapping. Our binding experiments performed by size-exclusion chromatography and optical trap-based force spectroscopy revealed that the αC-domains self-associate exclusively through their N-terminal subdomains, while their C-terminal subdomains were found to interact with the αC-connectors that tether the αC-domains to the bulk of the molecule. This interaction should reinforce the structure of αC polymers and provide the proper orientation of their reactive residues for efficient cross-linking by factor XIIIa. Molecular modeling of self-association of the N-terminal subdomains confirmed that the hypothesized ß-hairpin swapping does not impose any steric hindrance. To "freeze" the conformation of the N-terminal subdomain and prevent the hypothesized ß-hairpin swapping, we introduced by site-directed mutagenesis an extra disulfide bond between two ß-hairpins of the bovine Aα406-483 fragment corresponding to this subdomain. The experiments performed by circular dichroism revealed that Aα406-483 mutant containing Lys429Cys/Thr463Cys mutations preserved its ß-sheet structure. However, in contrast to wild-type Aα406-483, this mutant had lower tendency for oligomerization, and its structure was not stabilized upon oligomerization, in agreement with the above hypothesis. On the basis of the results obtained and our previous findings, we propose a model of fibrin αC polymer structure and molecular mechanism of assembly.

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 (ß15-66)(2) fragment, which mimics a pair of fibrin ßN-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 (ß15-66)(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 ßN-domains, and this interaction is inhibited by RAP and (ß15-66)(2). Furthermore, RAP inhibited transendothelial migration of neutrophils induced by fibrin-derived NDSK-II fragment containing ßN-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.

Structure, stability, and interaction of fibrin αC-domain polymers.

Our previous studies revealed that in fibrinogen the αC-domains are not reactive with their ligands, suggesting that their binding sites are cryptic and become exposed upon its conversion to fibrin, in which these domains form αC polymers. On the basis of this finding, we hypothesized that polymerization of the αC-domains in fibrin results in the exposure of their binding sites and that these domains adopt the physiologically active conformation only in αC-domain polymers. To test this hypothesis, we prepared a recombinant αC region (residues Aα221-610) including the αC-domain (Aα392-610), demonstrated that it forms soluble oligomers in a concentration-dependent and reversible manner, and stabilized such oligomers by covalently cross-linking them with factor XIIIa. Cross-linked Aα221-610 oligomers were stable in solution and appeared as ordered linear, branching filaments when analyzed by electron microscopy. Spectral studies revealed that the αC-domains in such oligomers were folded into compact structures of high thermal stability with a significant amount of ß-sheets. These findings indicate that cross-linked Aα221-610 oligomers are highly ordered and mimic the structure of fibrin αC polymers. The oligomers also exhibited functional properties of polymeric fibrin because, in contrast to the monomeric αC-domain, they bound tPA and plasminogen and stimulated activation of the latter by the former. Altogether, the results obtained with cross-linked Aα221-610 oligomers clarify the structure of the αC-domains in fibrin αC polymers and confirm our hypothesis that their binding sites are exposed upon polymerization. Such oligomers represent a stable, soluble model of fibrin αC polymers that can be used for further structure-function studies of fibrin αC-domains.

Fibrinopeptides A and B release in the process of surface fibrin formation.

Fibrinogen adsorption on a surface results in the modification of its functional characteristics. Our previous studies revealed that fibrinogen adsorbs onto surfaces essentially in 2 different orientations depending on its concentration in the solution: "side-on" at low concentrations and "end-on" at high concentrations. In the present study, we analyzed the thrombin-mediated release of fibrinopeptides A and B (FpA and FpB) from fibrinogen adsorbed in these orientations, as well as from surface-bound fibrinogen-fibrin complexes prepared by converting fibrinogen adsorbed in either orientation into fibrin and subsequently adding fibrinogen. The release of fibrinopeptides from surface-adsorbed fibrinogen and from surface-bound fibrinogen-fibrin complexes differed significantly compared with that from fibrinogen in solution. The release of FpB occurred without the delay (lag phase) characteristic of its release from fibrinogen in solution. The amount of FpB released from end-on adsorbed fibrinogen and from adsorbed fibrinogen-fibrin complexes was much higher than that of FpA. FpB is known as a potent chemoattractant, so its preferential release suggests a physiological purpose in the attraction of cells to the site of injury. The N-terminal portions of fibrin ß chains including residues Bß15-42, which are exposed after cleavage of FpB, have been implicated in many processes, including angiogenesis and inflammation.

Noncovalent interaction of alpha(2)-antiplasmin with fibrin(ogen): localization of alpha(2)-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.

Structure, stability, and interaction of the fibrin(ogen) alphaC-domains.

Our recent study established the NMR structure of the recombinant bAalpha406-483 fragment corresponding to the NH(2)-terminal half of the bovine fibrinogen alphaC-domain and revealed that at increasing concentrations this fragment forms oligomers (self-associates). The major goals of the study presented here were to determine the structure and self-association of the full-length human fibrinogen alphaC-domains. To accomplish these goals, we prepared a recombinant human fragment, hAalpha425-503, homologous to bovine bAalpha406-483, and demonstrated using NMR, CD, and size-exclusion chromatography that its overall fold and ability to form oligomers are similar to those of bAalpha406-483. We also prepared recombinant hAalpha392-610 and bAalpha374-568 fragments corresponding to the full-length human and bovine alphaC-domains, respectively, and tested their structure, stability, and ability to self-associate. Size-exclusion chromatography revealed that both fragments form reversible oligomers in a concentration-dependent manner. Their oligomerization was confirmed in sedimentation equilibrium experiments, which also established the self-association affinities of these fragments and revealed that the addition of each monomer to assembling alphaC-oligomers substantially increases the stabilizing free energy. In agreement, unfolding experiments monitored by CD established that self-association of both fragments results in a significant increase in their thermal stability. Analysis of CD spectra of both fragments revealed that alphaC self-association results in an increase in the level of regular structure, implying that the COOH-terminal half of the alphaC-domain adopts an ordered conformation in alphaC-oligomers and that this domain contains two independently folded subdomains. Altogether, these data further clarify the structure of the human and bovine alphaC-domains and the molecular mechanism of their self-association into alphaC-polymers in fibrin.

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.