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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 β 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.

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. ispartof: RESEARCH AND PRACTICE IN THROMBOSIS AND HAEMOSTASIS vol:5 issue:5 ispartof: location:United States status: published

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 β15–42 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 β15–42 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 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 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 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.

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 βN 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.

SummaryOur 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.

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...

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: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.

Our recent study revealed that fibrin and the VLDL receptor (VLDLR) interact with each other through a pair of fibrin βN-domains and CR domains of the receptor and this interaction promotes transendothelial migration of leukocytes and thereby inflammation. The major objectives of the present study were to further clarify the molecular mechanism of fibrin-VLDLR interaction and to identify amino acid residues in the βN-domains involved in this interaction. Our binding experiments with the (β15–66)(2) fragment, which corresponds to a pair of fibrin βN-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 (β15–66)(2) results in abrogation of this interaction. To identify which of these residues are involved in the interaction, we mutated all Lys or Arg in each of the three positively charged Lys/Arg clusters of the (β15–66)(2) fragment, as well as single Arg17 and Arg30, and tested the affinity of the mutants obtained to VLDLR(1–8) by ELISA and SPR. 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.

SummaryUpon conversion of fibrinogen into fibrin, fibrinogen αC-domains containing the RGD recognition motif form ordered αC polymers. Our previous study revealed that polymerisation of these domains promotes integrin-dependent adhesion and spreading of endothelial cells, as well as integrin-mediated activation of the FAK and ERK1/2 signalling pathways. The major goal of this study was to test the impact of αC-domain polymerisation on endothelial cell migration and proliferation during wound healing, and to clarify the mechanism underlying superior activity of αC polymers toward endothelial cells. In an in vitro wound healing assay, confluent endothelial cell monolayers on tissue culture plates coated with the αC monomer or αC polymers were wounded by scratching and wound closure was monitored by timelapse videomicroscopy. Although the plates were coated with equal amounts of αC species, as confirmed by ELISA, wound closure by the cells occurred much faster on αC polymers, indicating that αC-domain polymerisation promotes cell migration and proliferation. In agreement, endothelial cell proliferation was also more efficient on αC polymers, as revealed by cell proliferation assay. Wound closure on both types of substrates was equally inhibited by the integrin-blocking GRGDSP peptide and a specific antagonist of the ERK1/2 signalling pathway. In contrast, blocking the FAK signaling pathway by a specific antagonist decreased wound closure only on αC polymers. These results indicate that polymerisation of the αC-domains enhances integrin-dependent endothelial cell migration and proliferation mainly through the FAK signalling pathway. Furthermore, clustering of integrin-binding RGD motifs in αC polymers is the major mechanism triggering these events.

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.

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 β-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.

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.

A number of new as well as previously described fragments derived from the D region of bovine fibrinogen by limited proteolysis have been characterized by sequence analysis, differential scanning calorimetry and circular dichroism. Determination of the extremities of the polypeptide chains forming individual fragments allowed the scheme of proteolysis and the borders between domains in the D region of fibrinogen to be established. It was also found that the most thermostable region of the D fragment (TSD) can be substantially reduced in size without loss of its compact structure. The alpha-helical content of the newly prepared 21-kDa TSD2 and 16-kDa TSD3 fragments were 82% and 75%, respectively, strongly supporting a coiled-coil structure for this region of the fibrinogen molecule. The DX and DZ fragments, prepared from a chymotryptic digest of the DLA fragment, were found to be similar to the DL and DY fragments, respectively, except for an internal cleavage at K393-T394 in their beta chains. This cleavage leads to destabilization of all thermolabile domains, indicating interaction between them. The DL and DY fragments, containing only one polymerization site in their beta chains, were able to inhibit fibrin polymerization at high concentration. However, these same fragments failed to bind to fibrin-Sepharose under conditions where their structural analogues, DX and DZ, were tightly bound, indicating that cleavage after K393 substantially increases the affinity of this site.

The carboxyl-terminal regions of the fibrinogen Aalpha chains (alphaC regions) form compact alphaC-domains tethered to the bulk of the molecule with flexible alphaC-connectors. It was hypothesized that in fibrinogen two alphaC-domains interact intramolecularly with each other and with the central E region preferentially through its N-termini of Bbeta chains and that removal of fibrinopeptides A and B upon fibrin assembly results in dissociation of the alphaC regions and their switch to intermolecular interactions. To test this hypothesis, we studied the interactions of the recombinant alphaC region (Aalpha221-610 fragment) and its subfragments, alphaC-connector (Aalpha221-391) and alphaC-domain (Aalpha392-610), between each other and with the recombinant (Bbeta1-66)2 and (beta15-66)2 fragments and NDSK corresponding to the fibrin(ogen) central E region, using laser tweezers-based force spectroscopy. The alphaC-domain, but not the alphaC-connector, bound to NDSK, which contains fibrinopeptides A and B, and less frequently to desA-NDSK and (Bbeta1-66)2 containing only fibrinopeptides B; it was poorly reactive with desAB-NDSK and (beta15-66)2 both lacking fibrinopeptide B. The interactions of the alphaC-domains with each other and with the alphaC-connector were also observed, although they were weaker and heterogeneous in strength. These results provide the first direct evidence for the interaction between the alphaC-domains and the central E region through fibrinopeptide B, in agreement with the hypothesis given above, and indicate that fibrinopeptide A is also involved. They also confirm the hypothesized homomeric interactions between the alphaC-domains and display their interaction with the alphaC-connectors, which may contribute to covalent cross-linking of alpha polymers in fibrin.

According to the existing hypothesis, in fibrinogen, the COOH-terminal portions of two Aalpha chains are folded into compact alphaC-domains that interact intramolecularly with each other and with the central region of the molecule; in fibrin, the alphaC-domains switch to an intermolecular interaction resulting in alphaC-polymers. In agreement, our recent NMR study identified within the bovine fibrinogen Aalpha374-538 alphaC-domain fragment an ordered compact structure including a beta-hairpin restricted at the base by a 423-453 disulfide linkage. To establish the complete structure of the alphaC-domain and to further test the hypothesis, we expressed a shorter alphaC-fragment, Aalpha406-483, and performed detailed analysis of its structure, stability, and interactions. NMR experiments on the Aalpha406-483 fragment identified a second loose beta-hairpin formed by residues 459-476, yielding a structure consisting of an intrinsically unstable mixed parallel/antiparallel beta-sheet. Size-exclusion chromatography and sedimentation velocity experiments revealed that the Aalpha406-483 fragment forms soluble oligomers whose fraction increases with an increase in concentration. This was confirmed by sedimentation equilibrium analysis, which also revealed that the addition of each monomer to an assembling alphaC-oligomer substantially increases its stabilizing free energy. In agreement, unfolding experiments monitored by CD established that oligomerization of Aalpha406-483 results in increased thermal stability. Altogether, these experiments establish the complete NMR solution structure of the Aalpha406-483 alphaC-domain fragment, provide direct evidence for the intra- and intermolecular interactions between the alphaC-domains, and confirm that these interactions are thermodynamically driven.

The polymerization of fibrin occurs primarily through interactions between N-terminal A- and B-knobs, which are exposed by the cleavage of fibrinopeptides A and B, respectively, and between corresponding a- and b-holes in the γ- and β-modules. Of the potential knob-hole interactions—A:a, B:b, A:b, and B:a—the first has been shown to be critical for fibrin formation, but the roles of the others have remained elusive. Using laser tweezers–based force spectroscopy, we observed and quantified individual B:b and A:b interactions. Both desA-fibrin with exposed A-knobs and desB-fibrin bearing B-knobs interacted with fragment D from the γD364H fibrinogen containing b-holes but no functional a-holes. The strength of single B:b interactions was found to be 15 to 20 pN, approximately 6-fold weaker than A:a interactions. B:b binding was abrogated by B-knob mimetic peptide, the (β15-66)2 fragment containing 2 B-knobs, and a monoclonal antibody against the β15-21 sequence. The interaction of desB-fibrin with fragment D containing a- and b-holes produced the same forces that were insensitive to A-knob mimetic peptide, suggesting that B:a interactions were absent. These results directly demonstrate for the first time B:b binding mediated by natural B-knobs exposed in a fibrin monomer.

Nonsubstrate interaction of thrombin with fibrinogen promotes sequential cleavage of fibrinopeptides A and B (fpA and fpB, respectively) from the latter, resulting in its conversion into fibrin. The recently established crystal structure of human thrombin in complex with the central part of human fibrin clarified the mechanism of this interaction. Here, we reveal new details of the structure and present the results of molecular modeling of the fpA- and fpB-containing portions of the Aalpha and Bbeta chains, not identified in the complex, in both fibrinogen and protofibrils. The analysis of the results reveals that in fibrinogen the fpA-containing portions are in a more favorable position to bind in the active site cleft of bound thrombin. Surface plasmon resonance experiments establish that the fpB-containing portions interact with the fibrin-derived dimeric D-D fragment, suggesting that in protofibrils they bind to the newly formed DD regions bringing fpB into the vicinity of bound thrombin. These findings provide a coherent rationale for the preferential removal of fpA from fibrinogen at the first stage of fibrin assembly and the accelerated cleavage of fpB from protofibrils and/or fibrils at the second stage.

Interactions of endothelial cells with fibrin(ogen) are implicated in inflammation, angiogenesis, and wound healing. Cross-linking of the fibrinogen αC domains with factor XIIIa generates ordered αC oligomers mimicking polymeric arrangement of the αC domains in fibrin. These oligomers and those prepared with tissue transglutaminase were used to establish a mechanism of the αC domain–mediated interaction of fibrin with endothelial cells. Cell adhesion and chemical cross-linking experiments revealed that oligomerization of the αC domains by both transglutaminases significantly increases their RGD (arginyl–glycyl–aspartate)–dependent interaction with endothelial αVβ3 and to a lesser extent with αVβ5 and α5β1 integrins. The oligomerization promotes integrin clustering, thereby increasing cell adhesion, spreading, formation of prominent peripheral focal contacts, and integrin-mediated activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK) signaling pathways. The enhanced integrin clustering is likely caused by ordered juxtaposition of RGD-containing integrin-binding sites upon oligomerization of the αC domains and increased affinity of these domains for integrins. Our findings provide new insights into the mechanism of the αC domain–mediated interaction of endothelial cells with fibrin and imply its potential involvement in cell migration. They also suggest a new role for transglutaminases in regulation of integrin-mediated adhesion and signaling via covalent modification of integrin ligands.

The leukocyte integrin αMβ2 (Mac-1) is a multiligand receptor that mediates a range of adhesive reactions of leukocytes during the inflammatory response. This integrin binds the coagulation protein fibrinogen providing a key link between thrombosis and inflammation. However, the mechanism by which αMβ2 binds fibrinogen remains unknown. Previous studies indicated that a model in which two fibrinogen γC domain sequences, P1 (γ190–202) and P2 (γ377–395), serve as the αMβ2 binding sites cannot fully account for recognition of fibrinogen by integrin. Here, using surface plasmon resonance, we examined the interaction of the ligand binding αMI-domain of αMβ2 with the D fragment of fibrinogen and showed that this ligand is capable of associating with several αMI-domain molecules. To localize the alternative αMI-domain binding sites, we screened peptide libraries covering the complete sequences of the γC and βC domains, comprising the majority of the D fragment structure, for αMI-domain binding. In addition to the P2 and P1 peptides, the αMI-domain bound to many other sequences in the γC and βC scans. Similar to P1 and P2, synthetic peptides derived from γC and βC were efficient inhibitors of αMβ2-mediated cell adhesion and were able to directly support adhesion suggesting that they contain identical recognition information. Analyses of recognition specificity using substitutional peptide libraries demonstrated that the αMI-domain binding depends on basic and hydrophobic residues. These findings establish a new model of αMβ2 binding in which the αMI-domain interacts with multiple sites in fibrinogen and has the potential to recognize numerous sequences. This paradigm may have implications for mechanisms of promiscuity in ligand binding exhibited by integrin αMβ2.

Nonsubstrate interactions of thrombin with fibrin play an important role in modulating its procoagulant activity. To establish the structural basis for these interactions, we crystallized d -Phe-Pro-Arg-chloromethyl ketone-inhibited human thrombin in complex with a fragment, E ht , corresponding to the central region of human fibrin, and solved its structure at 3.65-Å resolution. The structure revealed that the complex consists of two thrombin molecules bound to opposite sides of the central part of E ht in a way that seems to provide proper orientation of their catalytic triads for cleavage of fibrinogen fibrinopeptides. As expected, binding occurs through thrombin's anion-binding exosite I. However, only part of it is involved in forming an interface with the complementary negatively charged surface of E ht . Among residues constituting the interface, Phe-34, Ser-36A, Leu-65, Tyr-76, Arg-77A, Ile-82, and Lys-110 of thrombin and the Aα chain Trp-33, Phe-35, Asp-38, Glu-39, the Bβ chain Ala-68 and Asp-69, and the γ chain Asp-27 and Ser-30 of E ht form a net of polar contacts surrounding a well defined hydrophobic interior. Thus, despite the highly charged nature of the interacting surfaces, hydrophobic contacts make a substantial contribution to the interaction.

Accumulation of lipoprotein(a) (Lp(a)) in atherosclerotic plaques is mediated through interaction of fibrin-(ogen) deposits with the apolipoprotein(a) (apo(a)) moiety of Lp(a). It was suggested that because apo(a) competes with plasminogen for binding to fibrin, causing inhibition of fibrinolysis, it could also promote atherothrombosis. Because the fibrin(ogen) alphaC-domains bind plasminogen and tissue-type plasminogen activator with high affinity in a Lys-dependent manner, we hypothesized that they could also bind apo(a). To test this hypothesis, we studied the interaction between the recombinant apo(a) A10 isoform and the recombinant alphaC-fragment (Aalpha-(221-610)) corresponding to the alphaC-domain by enzyme-linked immunosorbent assay and surface plasmon resonance. Both methods revealed a high affinity interaction (Kd = 19-21 nm) between the immobilized alphaC-fragment and apo(a), indicating that the former contains an apo(a)-binding site. This affinity was comparable to that of apo(a) for fibrin. At the same time, no interaction was observed between soluble fibrinogen and immobilized apo(a), suggesting that, in the former, this and other apo(a)-binding sites are cryptic. Further experiments with truncated recombinant variants of the alphaC-fragment allowed localization of the apo(a)-binding site to the Aalpha-(392-610) region. The presence of epsilon-aminocaproic acid only slightly inhibited binding of apo(a) to the alphaC-fragment, indicating the Lys-independent nature of their interaction. In agreement, the influence of plasminogen or tissue-type plasminogen activator on binding of apo(a) to the alphaC-fragment was minimal. These results indicate that the alphaC-domains contain novel high affinity apo(a)-binding sites that may provide a Lys-independent mechanism for bringing Lp(a) to places of fibrin deposition such as injured vessels or atherosclerotic lesions.

The 39-kDa receptor-associated protein (RAP) is an endoplasmic reticulum resident protein that binds to the low density lipoprotein receptor-related protein (LRP) as well as certain members of the low density lipoprotein receptor superfamily and antagonizes ligand binding. In order to identify important functional regions of RAP, studies were performed to define the domain organization and domain boundaries of this molecule. Differential scanning calorimetry (DSC) experiments revealed that the process of thermal denaturation of RAP is highly reversible and occurs in a broad temperature range with two well resolved heat absorption peaks. A good fit of the endotherm was obtained with four two-state transitions suggesting these many cooperative domains in the molecule. A number of recombinant fragments of RAP were expressed in bacteria, and their domain composition and stability were characterized by DSC, circular dichroism, and fluorescence spectroscopy. The results confirmed that RAP is composed of four independently folded domains, D1, D2, D3, and D4, that encompass residues 1-92, 93-163, 164-216, and 217-323, respectively. The first and the fourth domains preserved their structure and stability when isolated, whereas the compact structure of the fragment corresponding to D2 seems to be altered when isolated from the parent molecule. Isolated D3 was partially degraded during isolation from bacterial lysates. The isolated D4 was capable of binding with high affinity to LRP whereas neither D1 nor D2 bound. At the same time a fragment containing both D1 and D2 exhibited high affinity binding to LRP. These facts combined with the thermodynamic analysis of the melting process of the fragments containing D1 and D2 indicate that these two domains interact with each other and that the proper folding of the second domain into a native-like active conformation requires presence of the first domain.

The interaction of leukocyte integrin alphaMbeta2 (CD11b/CD18, Mac-1) with fibrinogen has been implicated in the inflammatory response by contributing to leukocyte adhesion to the endothelium and subsequent transmigration. Previously, it has been demonstrated that a peptide, P1, corresponding to residues 190-202 in the gamma-chain of fibrinogen, binds to alphaM beta2 and blocks the interaction of fibrinogen with the receptor and that Asp199 within P1 is important to activity. We have demonstrated, however, that a double mutation of Asp199-Gly200 to Gly-Ala in the recombinant gamma-module of fibrinogen, spanning region 148-411, did not abrogate alphaM beta2 recognition and considered that other binding sites in the gamma-module may participate in the receptor recognition. We have found that synthetic peptide P2, duplicating gamma377-395, inhibited adhesion of alphaM beta2-transfected cells to immobilized D100 fragment of fibrinogen in a dose-dependent manner. In addition, immobilized P2 directly supported efficient adhesion of the alphaM beta2-expressing cells, including activated and non-activated monocytoid cells. The I domain of alphaM beta2 was implicated in recognition of P2, as the biotinylated recombinant alphaMI domain specifically bound to both P2 and P1 peptides. Analysis of overlapping peptides spanning P2 demonstrated that it may contain two functional sequences: gamma377-386 (P2-N) and gamma383-395 (P2-C), with the latter sequence being more active. In the three-dimensional structure of the gamma-module, gamma190-202 and gamma377-395 reside in close proximity, forming two antiparallel beta strands. The juxtapositioning of these two sequences may form an unique and complex binding site for alphaM beta2.

The structural organization of streptokinase was established through detailed study of its denaturation by differential scanning calorimetry. Streptokinase exhibited a complex endotherm whose shape was sensitive to changing pH. In all cases the endotherms were easily described by four two-state transitions indicating unambiguously the presence of four independently folded domains in the molecule. Two of them were slightly destabilized by lowering pH from 7.0 to 3.8 while the other two were stabilized in this pH range. Two proteolytic fragments of streptokinase were examined, a 37-kDa fragment beginning at Ile1 with a cleavage following Phe62, and a 17-kDa fragment beginning at Lys 147. At pH 8.5, three two-state transitions were observed in the former and two in the latter indicating this many domains in each and suggesting that the fragments are formed by a step-wise removal of individual domains from the parent molecule. Comparison of the melting of these fragments with that of streptokinase allowed the first two transitions in the parent protein to be assigned to the melting of two NH2-terminal domains and the two higher-temperature transitions to the melting of the two COOH-terminal domains. The latter two domains strongly interact with each other since the absence of the most stable extreme COOH-terminal domain in both fragments resulted in a strong destabilization of its neighbor whose melting occurred with a midpoint near room temperature. The two NH2-terminal domains seem to be more independent. One of them melts similarly in the parent protein and both fragments while the other, formed by the 1-146 region, is less stable in the 37-kDa fragment. This destabilization is most probably due to the cleavage after Phe62 which, based on the sequence similarity of streptokinase with serine proteases, may be part of a surface-oriented loop.

Thermal denaturation of platelet glycoprotein IIbIIIa (integrin alpha IIb beta 3) was investigated by spectrofluorimetry and differential scanning calorimetry (DSC). Two forms of the protein were compared: active IIbIIIa, i.e., that fraction that binds to RGD-Sepharose, and inactive IIbIIIa, the non-binding fraction. At pH 8.5 in the presence of octyl glucoside and Ca2+ both forms exhibited a broad complex endotherm consisting of a well expressed low-temperature heat-absorption peak in the range of 40-65 degrees C followed by a broad peak stretching over 65-110 degrees C. Each endotherm could be deconvoluted into at least eight transitions reflecting the melting of at least this many independently folded domains. The first two transitions in the region of the low-temperature peak had similar positions in both forms while at least some of the other transitions occurred at higher temperature in the active protein suggesting that some of the domains are more stable in the latter. When both fractions of IIbIIIa were heated in the fluorometer a sigmoidal transition was observed in the region of the first endothermic peak where the two thermolabile domains melt. This transition was destabilized by 15 degrees C in the presence of EDTA, suggesting that these domains are formed by the 243-468 region of the IIb subunit which contains four Ca(2+)-binding motifs. It was further stabilized by 3 degrees C upon addition of the GRGDSPK peptide in the presence of Ca2+ while in EDTA the peptide had no effect. This is consistent with the involvement of Ca(2+)-binding region in the formation of the ligand-binding site. A 66-kDa chymotryptic fragment, containing the 17-kDa NH2-terminal portion of the IIIa subunit disulfide-linked to its 50-kDa COOH-terminal portion including the cysteine-rich core, exhibited a fluorescence-detected Ca(2+)-independent transition in the region where the higher temperature DSC-detected transitions occur suggesting that some of the latter may be connected with the melting of the corresponding portions of IIbIIIa.

The fibulins are an emerging family of extracellular matrix and blood proteins presently having two members designated fibulin-1 and −2. Fibulin-1 is the predominant fibulin in blood, present at a concentration of 30-40 μg/ml (~1000-fold higher than fibulin-2). During the course of isolating fibulin-1 from plasma by immunoaffinity chromatography, a 340-kDa polypeptide was consistently found to co-purify. This protein was identified as fibrinogen (Fg) based on its electrophoretic behavior and reactivity with Fg monoclonal antibodies. Radioiodinated fibulin-1 was shown to bind to Fg transferred onto nitrocellulose filters after SDS-polyacrylamide gel electrophoresis. In enzyme-linked immunosorbent assay, fibulin-1 bound to Fg (and fibrin) adsorbed onto microtiter well plastic, and conversely, Fg bound to fibulin-1-coated wells. The binding of Fg to fibulin-1 was also observed in surface plasmon resonance assays, and a dissociation constant (Kd) of 2.9 ± 1.6 μM was derived. In addition, fluorescence anisotropy experiments demonstrated that the interaction was also able to occur in fluid phase, which suggests that complexes of fibulin-1 and Fg could exist in the blood. To localize the portion of Fg that is responsible for interacting with fibulin-1, proteolytic fragments of Fg were evaluated for their ability to promote fibulin-1 binding. Fragments containing the carboxyl-terminal region of the Bβ chain (residues 216-468) were able to bind to fibulin-1. In addition, it was found that fibulin-1 was able to incorporate into fibrin clots formed in vitro and was immunologically detected within newly formed fibrin-containing thrombi associated with human atherectomy specimens. The interaction between fibulin-1 and Fg highlights potential new roles for fibulin-1 in hemostasis as well as thrombosis.

Scanning microcalorimetry and spectrofluorimetry were applied to a study of the thermal stability and interaction of the modules within natural human protein C (PC) and recombinant protein C (rPC), a potential therapeutic anticoagulant expressed in transgenic pigs. Upon heating in the presence of 2 mM EDTA, pH 8.5, each protein exhibited a similar heat absorption peak with a Tm of approximately 62 degrees C corresponding to the melting of the serine protease (SP) module. Deconvolution of this peak indicated that the SP module consists of two domains that unfold independently. At pH below 3.8, a second peak appeared at extremely high temperature corresponding to the unfolding of the two interacting epidermal growth factor-like (EGF) domains. This second peak occurred at a temperature about 20 degrees C lower in rPC than in PC indicating that the EGF domains in the recombinant protein are less stable. The isolated 6-kDa gamma-carboxyglutamic acid-rich (Gla) fragment as well as a 25-kDa Gla-(EGF)2 fragment both exhibited a sigmoidal fluorescence-detected denaturation transition in the same temperature region as the SP domains, but only in the presence of Ca2+. In 2 mM Ca2+, the first heat absorption peak in both intact proteins became biphasic, indicating Ca(2+)-induced structural changes. By contrast, Ca2+ had very little effect on the melting of Gla-domain-less protein C. This indicates that not Ca2+ itself but the Ca(2+)-loaded Gla domain is responsible for conformational changes in the SP domain of the parent protein. Detailed analysis of the shape of the endotherms obtained in Ca2+ and EDTA suggests that Ca2+ induces compact structure in the Gla domain which appears to interact strongly with the SP domain(s) of protein C.

Differential scanning calorimetry was used to study the domain structure and intramolecular interactions of tPA/uPA chimeras. A high temperature transition centered near 90 degrees C was observed upon melting of the tPA/uPA chimera (amino acids 1-274 of tPA and 138-411 of uPA) and its variant lacking the finger and epidermal growth factor-like modules (residues 1-3 and 87-274 of tPA and 138-411 of uPA). Since neither of the two parent plasminogen activators display such a stable structure, one may suggest that a new stabilizing intramolecular interaction occurs in the chimeras. We found that occupation of the lysine binding site of tPA by a lysine or arginine side chain from the urokinase moiety is responsible for the high temperature transition as well as for the failure of the chimeras to exhibit the expected fibrin binding properties. All uPA species, single- and two-chain high molecular weight uPA (Pro-Uk and HMW-Uk) and two-chain low molecular weight uPA (LMW-Uk), interact intermolecularly with tPA and its kringle-containing derivatives. This intermolecular interaction was strongly inhibited by epsilon-aminocaproic acid indicating that the lysine binding site of tPA is involved. The binding of uPA with the fluorescein-labeled A-chain of tPA, registered by changes in fluorescence anisotropy, was estimated to have a Kd range of 1-7 microM. The interaction of tPA with uPA determined by solid-phase assays appeared to be tighter, with a Kd range of 50-300 nM. Two synthetic peptides, with and without carboxyl-terminal lysine, corresponding to urokinase residues 144-158 and 144-157, were approximately 100-fold more potent than epsilon-aminocaproic acid with respect to inhibition of the tPA-uPA interaction, indicating that the tPA binding site on urokinase is located within this sequence, close to the activation site Lys158-Ile159. The discovered intermolecular interaction may be related to the reported synergistic effect of simultaneous administration of these two plasminogen activators.

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 β-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 β-hairpin swapping does not impose any steric hindrance. To "freeze" the conformation of the N-terminal subdomain and prevent the hypothesized β-hairpin swapping, we introduced by site-directed mutagenesis an extra disulfide bond between two β-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 β-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.

The NH2-terminal 29-kDa Fib-1 fragment consisting of the first five finger modules of fibronectin (F1-5) binds reversibly to fibrin and facilitates cross-linking by Factor XIII. To narrow down the fibrin-binding site within this region, we have used recombinant technology to express a number of individual fingers, rF1, rF2, rF3, rF4, and rF5, and their pairs, rF1-2 rF2-3, and rF4-5, as fusion proteins in Escherichia coli. These recombinant fragments were separated from the carrier maltose-binding protein by digestion with human factor Xa or other proteases, and their structural integrity was confirmed by spectroscopic and calorimetric methods. The recombinant F1 and F4-5 exhibited fluorescence-detected melting transitions of the same magnitude and with the same midpoint (Tm) as their natural analogues prepared from Fib-1 by proteolysis. Differential scanning calorimetry experiments further demonstrated that these fragments are properly folded and have compact structures identical to the natural ones. Isolated rF4 melts at a much lower temperature than rF5 or the bimodular fragment rF4-5, indicating the loss of a stabilizing interaction between fingers 4 and 5. Comparison of fluorescence spectra of individual rF4 and rF5 with that of rF4-5 was also consistent with an interaction that affects the environment of Trp residue(s). rF2 also melts at a lower temperature than rF3 or rF2-3, suggesting a stabilizing interaction between the second and third fingers as well. When tested on fibrin-Sepharose, only the bimodular fragment rF4-5 was able to bind. All other fragments, including individual fingers 4 and 5, failed to bind. Thus, fibrin binding is not a common property of all fingers. The results indicate that a recognition site for fibrin is located within fingers 4 and 5. The interaction between these neighboring domains may play an important role in proper orientation of the residues forming this site.

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.

The melting of several serine proteases that had been reacted with different peptidylchloromethylketone (cmk) inhibitors was studied by fluorescence spectroscopy and calorimetry. These inhibitors, which cross-link the two domains of the proteases, invariably increased the melting temperature by as much as 28.5 degrees C. The magnitude of the effect was dependent on the size and composition of the peptide moieties. The delta G of unfolding of tosyl-Phe-cmk-chymotrypsin was 13.5 kcal/mol compared to only 8.3 kcal/mol for chymotrypsin. Binding of cmk inhibitors also protected the two interacting domains of urokinase from acid-induced decooperation and caused them to merge into a highly cooperative structure upon refolding at low pH. Fluorescence-detected melting curves of Glu-Gly-Arg-cmk-urokinase indicated that unfolding/refolding at pH 4.5 is characterized by dramatic hysteresis; the cooling curves fell close to those obtained upon heating or cooling of the uninhibited enzyme. Upon second heating, the melting curves were similar to those of the original. The hysteresis effects are interpreted as follows. The tethered tripeptide binds to the active site, causing the protein to melt at much higher temperature in a single cooperative step, as if the two domains are merged into one cooperative unit. Upon cooling, the unfolded protein, with the inhibitor still attached, refolds at the same temperature as the underivatized protein. Only after the native structure is formed does the peptide moiety again bind and stabilize toward a second heating. At lower pH, second heating produced biphasic or triphasic melting curves that were attributed to differential protonation of acid-titratable groups on the enzyme and/or inhibitor at the time of refolding. Similar effects were observed with other trypsin-like proteases, indicating that the hysteresis and bi- and triphasic refolding at low pH are rather general for this class of enzyme.

The locations of the carboxyl-terminal two thirds of the A alpha chains, or the alpha C domains, were determined for fibrinogen and some of its derivatives by electron microscopy of rotary-shadowed preparations. A monoclonal antibody, G8, to the carboxyl-terminal 150 amino acids of the A alpha chain, binds near the central region of fibrinogen, indicating that the alpha C domains of most molecules are not normally visible because they are on or near the amino-terminal disulfide knot. At pH 3.5, fibrinogen and fibrin monomers appear to be similar, with a projection terminating in a small globular domain from each end of most molecules. In contrast, fragment X monomers, produced by cleavage of the alpha C domains from fibrinogen with plasmin, show no such projections. When fibrin monomer is brought to neutral pH under conditions where polymerization is delayed, individual molecules are still visible showing the alpha C domains as a single additional nodule near the central region. Moreover, analysis of clusters of molecules reveals some intermolecular associations via the alpha C domains. A 40-kDa fragment comprising the alpha C domain has been isolated from a plasmin digest of fibrinogen and characterized by SDS-polyacrylamide gel electrophoresis and determination of amino-terminal amino acid sequences. Electron microscopy of alpha C fragments reveals individual globular structures, as well as oligomeric aggregates. The addition of alpha C fragments to fibrin monomer followed by dilution to neutral pH to initiate polymerization results in lower turbidity, longer lag period, and slower maximum rate of turbidity increase. Also, electron microscopy reveals complexes of alpha C fragments with fibrin monomer at neutral pH. It appears that the free alpha C fragments can bind to the alpha C domains of fibrin, competing with the normal alpha C domain interactions involved in polymerization.

Dansyl-labeled tetrapeptide Gly-His-Arg-Pro which mimics the central fibrin polymerization site was used to investigate its binding to a number of fibrinogen fragments containing different numbers of domains. The tetrapeptide was found to bind to fragments DH(95 kDa), DL(82 kDa) and DY(63 kDa) but not to the TSD(28 kDa) fragment. The DY fragment differs from the TSD by the presence of beta and beta C domains. Therefore these domains, which are formed by the C-terminal part of the beta chain, possess a polymerization site complementary to the Gly-His-Arg containing counterpart.

Coagulation factor IX has the modular composition Gla-(EGF)2-SP, where Gla, EGF, and SP represent the gamma-carboxy-Glu-rich, epidermal growth factor-like, and serine protease modules, respectively. The protein melts in two distinct temperature regions. The SP module melts at lower temperature between 42 and 55 degrees C, depending on the pH, with irreversible loss of esterolytic activity. The endotherm corresponding to this transition is readily described by a two-state transition indicating the melting of a single cooperative unit. A thrombin-generated 12-kDa fragment representing the COOH-terminal half of the SP module and a 45-kDa fragment containing the NH2-terminal half of the SP module and the rest of the molecule can be separated by exclusion chromatography in 3 M urea and recombined in its absence. Both fragments retain a compact structure as evidenced by melting transitions near 60 degrees C at neutral pH. This indicates that the SP module contains two independently folded domains that strongly interact with each other and seem to merge into one cooperative unit in the intact protein. At low pH at high temperature a second peak appears which is also observed in a 19-kDa fragment containing the EGF modules. Thermodynamic analysis of this second peak showed that the two EGF modules are independently folded and provided evidence for a weak interaction between them. Fluorescence and CD measurements indicated that the secondary structure of the isolated 6-kDa Gla fragment is substantially increased in the presence of Ca2+. The Ca2+-loaded Gla fragment undergoes a sigmoidal unfolding transition as revealed by fluorescence and CD measurements. This same transition in a 25-kDa Gla-(EGF)2 fragment was stabilized by more than 10 degrees C, indicating a strong interaction between the Ca(2+)-loaded Gla and EGF domains. Thus, factor IX consists of five independently folded interacting domains.

Background: Exposure of cryptic, functional sites on fibrinogen upon its adsorption to hydrophobic surfaces of biomaterials has been linked to an inflammatory response and fibrosis. Such adsorption also induces ordered fibrinogen aggregation which is poorly understood. Objective: To investigate hydrophobic surface-induced fibrinogen aggregation. Methods: Contact and lateral force scanning probe microscopy, yielding topography, image dimensions and fiber elastic modulus measurements were used along with transmission and scanning electron microscopy. Fibrinogen aggregation was induced under non-enzymatic conditions by adsorption on a trioctyl-surface monolayer (trioctylmethylamine) grafted onto silica clay plates. Results: A more than one molecule thick coating was generated by adsorption on the plate from 100 to 200 {mu}g mL{sup -1} fibrinogen solutions, and three-dimensional networks formed from 4 mg mL{sup -1} fibrinogen incubated with uncoated or fibrinogen-coated plates. Fibrils appeared laterally assembled into branching and overlapping fibers whose heights from the surface ranged from approximately 3 to 740 nm. The elastic modulus of fibrinogen fibers was 1.55 MPa. No fibrils formed when fibrinogen lacking {alpha}C-domains was used as a coating or was incubated with intact fibrinogen-coated plates, or when the latter plates were sequentially incubated with anti-A{alpha}529-539 mAb and intact fibrinogen. When an anti-A{alpha}241-476 mAb was used instead, fine, longmore » fibers formed. Similarly, sequential incubations of fibrinogen-coated plates with recombinant {alpha}C-domain (A{alpha}392-610 fragment) or {alpha}C-connector (A{alpha}221-372 fragment) and fibrinogen resulted in distinctly fine fiber networks. Conclusions: Adsorption-induced fibrinogen self-assembly is initiated by a more than one molecule-thick surface layer and eventuates in three-dimensional networks whose formation requires fibrinogen with intact {alpha}C-domains.« less

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.

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 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.

The melting of recombinant tissue plasminogen activator (rtPA) has been investigated by differential scanning calorimetry and fluorescence spectroscopy. At neutral pH, rtPA melts with only partial reversibility in a single sharp peak that can be deconvoluted into four transitions. By contrast, at acidic pH the melting process is spread over a broad range of temperature and is highly reversible. Under these conditions five transitions are resolved by deconvolution analysis. Additional measurements in 6 M guanidinium chloride reveal a sixth transition representing an extremely stable domain. Comparison of the melting curves of several fragments with those of the parent protein allowed all of the transitions to be assigned. The results indicate that rtPA is comprised of six independently folded domains. Two of these domains correspond to the two kringle modules whose thermodynamic properties are similar to those of the kringles in plasminogen. Two additional domains are formed by the epidermal growth factor (EGF)-like and finger modules, the latter of which is extremely stable, requiring the presence of a chemical denaturant for its melting to be observed. The serine protease module contains two more domains which at neutral pH melt cooperatively in a single transition but at low pH melt independently, accounting for the greater number of transitions observed there. Measurements with a 50-kDa fragment lacking the C-terminal half of the serine protease module and with a variant lacking the finger and EGF domains indicate that the serine protease domains interact strongly with and are stabilized by the finger and/or EGF domains in the intact protein. This interaction between domains located at opposite ends of the rtPA molecule produces a more compact structure. A better understanding of such interactions may enhance efforts to engineer plasminogen activators with improved thrombolytic properties.

Many different terms related to fibrinogen and fibrin have come into general use in the fibrin(ogen) field, but there is much confusion and controversy over some of the terminology. The existing terminology related to fibrinogen structure and fibrin polymerization and recommendations on the standardization of commonly used nomenclature have been discussed at the 52nd and 53rd annual meetings of the ISTH Scientific and Standardization Committee (SSC), Subcommittee on Fibrinogen and Factor XIII. The recommendations on nomenclature of fibrinogen and fibrin presented here are based on numerous comments and suggestions made by the Subcommittee members and leading scientists from the fibrin(ogen) field. They have been approved by the Subcommittee at the 54th ISTH SSC meeting in Vienna in 2008.

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.

Incorporation of fibronectin into fibrin clots is important for the formation of a provisional matrix that promotes cell adhesion and migration during wound healing. Previous studies revealed that this incorporation occurs through noncovalent interaction between two NH2-terminal Fib-1 regions of fibronectin (one on each chain) and the alphaC-regions of fibrin, and is further reinforced by factor XIIIa-mediated covalent cross-linking of fibronectin to the fibrin matrix. To clarify the role of another pair of fibrin-binding regions, Fib-2, located at the disulfide-linked COOH-terminal ends of fibronectin, we prepared by limited proteolysis a dimeric 140 kDa (Fib-2)2 fragment containing both Fib-2 regions and tested its interaction with recombinant fragments corresponding to the alphaC regions of fibrin(ogen). In both ELISA and surface plasmon resonance (SPR) experiments 140 kDa (Fib-2)2 bound to the immobilized Aalpha221-610 alphaC-fragment. However, the affinity of binding was substantially lower than that for Fib-1. Ligand blotting and ELISA established that the Fib-2 binding site is located in the connector part of the alphaC region including residues Aalpha221-391. Analysis of the SPR-detected binding of fibronectin to the immobilized Aalpha221-610 alphaC-fragment revealed two types of fibronectin-binding sites, one with high affinity and another one with much lower affinity. Competition experiments revealed about 30% inhibition of the Fib-2 mediated binding by increasing concentrations of Fib-1 fragment suggesting partial overlap of the two sets of binding sites. Based on these results and our previous studies we propose a mechanism of interaction of fibronectin with fibrin in which both Fib-1 and Fib-2 play a role.

Human fibrinogen gamma chain variants, termed gamma' chains, contain a unique 20-residue sequence after gamma chain residue 407 that ends at gamma'427, and is designated gamma'(427L). Full-length (FL) gamma'(427L) chains are constituents of a fibrin-dependent thrombin inhibitory system known as antithrombin I, whereas a gamma' chain processed in vivo, termed gamma'(423P), lacks the C-terminal tetrapeptide EDDL, and does not bind thrombin. Together, the gamma'(423P) and gamma'(427L) chains comprise the total plasma fibrinogen gamma' chain content.Lowered plasma gamma' chain content (i.e. gamma' chain-containing fibrinogen/total fibrinogen ratio) has been shown to correlate with susceptibility to venous thrombosis, thus prompting this study on the total and FL gamma' chain content in 45 subjects with thrombotic microangiopathy (TMA), a disorder characterized by microvascular thrombosis.We measured by enzyme-linked immunosorbent assay the total gamma' chain-containing fibrinogen/total fibrinogen (Total gamma'-fgn/Total fgn) ratio and the FL gamma' chain-containing fibrinogen/total fibrinogen (FL gamma'-fgn/Total fgn) ratio in these plasmas and in healthy subjects (n = 87).In healthy subjects, the mean Total gamma'-fgn/Total fgn ratio was 0.127, whereas the FL gamma'-fgn/Total fgn ratio was somewhat lower at 0.099 (P0.0001), a difference reflecting the presence of gamma'(423P) chains. In TMA plasmas, both the Total gamma'-fgn and FL gamma'-fgn/Total fgn ratios (0.099 and 0.084, respectively) were lower than those of their healthy subject counterparts (P0.0001).These findings in TMA suggest that reductions in the gamma' chain content indicate reduced antithrombin I activity that may contribute to microvascular thrombosis in TMA.

This study tested the hypothesis that high-affinity binding of macromolecular ligands to the alphaIIbbeta3 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 alphaIIbbeta3 and two model ligands-echistatin, a 6-kDa recombinant protein with an RGD integrin-targeting sequence, and fibrinogen's gamma-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 alphaIIb/beta3 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 degrees C with the ligand-mimetic peptide cHarGD--promotes complex formation with fibrinogen's gamma-module. We also observed that the gamma-module's flexible carboxy terminus was not required for alphaIIbbeta3 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. Echistatin's binding energy is sufficient to rearrange the subunit interface, but regulated ligands like fibrinogen must rely on priming to overcome conformational barriers.

Fibrinogen is a 340-kDa plasma protein that plays a prominent role in hemostasis, thrombosis, wound healing, inflammation, angiogenesis, and tumorigenesis. This multitude of fibrinogen functions is connected with its complex multidomain structure that provides its homophilic interaction upon fibrin assembly and its interactions with numerous proteins and cell types in other processes. Fibrinogen consists of two identical disulfide-linked subunits each of which is composed of three non-identical polypeptide chains, Aα, Bβ, and γ (1; 2). These chains assemble to form at least 20 distinct domains grouped into several major structural regions (3–6). The disulfide-linked NH2-terminal portions of all six chains form the central E region, the COOH-terminal portions of the Bβ and γ chains and the middle portion of the Aα chain form two distal D regions, and the remaining COOH-terminal two-thirds of two Aα chains form two αC regions. The D-E-D regions account for three nodules observed in numerous electron microscopy studies (7–10), while the fourth nodule observed in some molecules near the central nodule E corresponds to the two interacting αC regions, often referred to as αC-domains (9; 11; 12) (Figure 1A). Figure 1 Schematic representation of the interacting αC-domains in fibrinogen and their dissociation upon its conversion to fibrin. Panel A shows fibrinogen molecule consisting of the D-E-D nodules linked by coiled coil connectors and two αC-domains ... Activation of the coagulation cascade leads to the generation of thrombin which converts fibrinogen into fibrin by cleavage of fibrinopeptides A and B (FPA and FPB1, respectively) from its central region. This cleavage results in spontaneous polymerization of individual fibrin molecules into double-stranded protofibrils (Figure 1B) which then aggregate laterally to produce thicker fibers composing fibrin clots (1; 10). According to our current view, in fibrinogen the αC-domains interact intramolecularly with each other and with the central E region, most probably via its FPB, while upon fibrin assembly they dissociate and switch from intra- to intermolecular interaction thus promoting lateral aggregation of protofibrils (12–16). The functional role of the αC-domains is not restricted to their participation in fibrin assembly. They are involved in controlling activation of plasma transglutaminase factor XIII (17), which subsequently cross-links fibrin clots to increase their mechanical stability. The human fibrinogen αC-domains promote cell adhesion via their Aα572-574 RGD sequence2 and via bound fibronectin (18–21), and may contribute to the development of atherothrombosis (22). They are also involved in regulation of fibrinolysis via binding of plasminogen, its activator tPA, its inhibitor α2-antiplasmin, and via modulating the structure of fibrin clots (16; 23–25). The fibrinogen D and E regions can be separated by limited proteolysis resulting in the D and E fragments, respectively, while the αC regions are vulnerable to proteolytic enzymes, which degrade them into smaller pieces (1; 2). The success in crystallization of these fragments resulted in determination of the three-dimensional structure of individual fibrinogen regions and promoted X-ray studies of the entire molecule. Particularly, the X-ray structures have been established for the human fibrinogen D fragment (26) and for the bovine and human fibrinogen E fragments (6; 27). Further, a low resolution structure of most of the fibrinogen molecule was obtained from a crystallographic study of a proteolytically truncated bovine fibrinogen (28), followed by a higher resolution structure of intact chicken fibrinogen (29). Altogether, these studies have established the three-dimensional structure of more than two-thirds of the molecule including the complete D regions and most of the E region; that of the αC-domains remains to be determined. In the absence of the three-dimensional structure of the αC regions, the current view on their structural organization is based on a number of studies performed in different laboratories mainly by electron microscopy, differential scanning calorimetry (DSC), and sequence analysis (3; 4; 9; 11; 15). These studies revealed that in fibrin(ogen) each αC region forms a compact globular entity, αC-domain, attached to the bulk of the molecule with a flexible connector (15). The full-length αC region and its halves corresponding to αC-connector and the αC-domain have been prepared by recombinant technique (24; 30). A detailed analysis of their folding status by several methods has confirmed that the αC-domain contain a compact cooperative structure while the connector is flexible (30). At the same time, there is also an alternative view suggesting that the αC-domains represent “free-swimming appendages” devoid of any ordered structure (1; 8). This view was reinforced by an X-ray study of intact chicken fibrinogen, in which the αC-domains were not observed in electron density maps (29). Thus, the question whether these domains contain compact ordered structure is still debated (31; 32). In this study, we report the NMR solution structure of the recombinant bovine fibrinogen αC-domain fragment including residues Aα374-5383, which reveals a type I′ β-hairpin and a collapsed hydrophobic cluster, and discuss a possible role of such a structure in the previously observed intra- and intermolecular interactions.

The leukocyte integrin alphaMbeta2 (Mac-1) is a multiligand receptor that mediates a range of adhesive reactions of leukocytes during the inflammatory response. This integrin binds the coagulation protein fibrinogen providing a key link between thrombosis and inflammation. However, the mechanism by which alphaMbeta2 binds fibrinogen remains unknown. Previous studies indicated that a model in which two fibrinogen gammaC domain sequences, P1 (gamma190-202) and P2 (gamma377-395), serve as the alphaMbeta2 binding sites cannot fully account for recognition of fibrinogen by integrin. Here, using surface plasmon resonance, we examined the interaction of the ligand binding alphaMI-domain of alphaMbeta2 with the D fragment of fibrinogen and showed that this ligand is capable of associating with several alphaMI-domain molecules. To localize the alternative alphaMI-domain binding sites, we screened peptide libraries covering the complete sequences of the gammaC and betaC domains, comprising the majority of the D fragment structure, for alphaMI-domain binding. In addition to the P2 and P1 peptides, the alphaMI-domain bound to many other sequences in the gammaC and betaC scans. Similar to P1 and P2, synthetic peptides derived from gammaC and betaC were efficient inhibitors of alphaMbeta2-mediated cell adhesion and were able to directly support adhesion suggesting that they contain identical recognition information. Analyses of recognition specificity using substitutional peptide libraries demonstrated that the alphaMI-domain binding depends on basic and hydrophobic residues. These findings establish a new model of alphaMbeta2 binding in which the alphaMI-domain interacts with multiple sites in fibrinogen and has the potential to recognize numerous sequences. This paradigm may have implications for mechanisms of promiscuity in ligand binding exhibited by integrin alphaMbeta2.