αMI-domain of integrin Mac-1 binds the cytokine pleiotrophin using multiple mechanisms.

The integrin Mac-1 (αMß2, CD11b/CD18, CR3) is an adhesion receptor expressed on macrophages and neutrophils. Mac-1 is also a promiscuous integrin that binds a diverse set of ligands through its αMI-domain. However, the binding mechanism of most ligands remains unclear. We have characterized the interaction of αMI-domain with the cytokine pleiotrophin (PTN), a protein known to bind αMI-domain and induce Mac-1-mediated cell adhesion and migration. Our data show that PTN's N-terminal domain binds a unique site near the N- and C-termini of the αMI-domain using a metal-independent mechanism. However, a stronger interaction is achieved when an acidic amino acid in a zwitterionic motif in PTN's C-terminal domain chelates the divalent cation in the metal ion-dependent adhesion site of active αMI-domain. These results indicate that αMI-domain can bind ligands using multiple mechanisms and that the active αMI-domain has a preference for motifs containing both positively and negatively charged amino acids.

αMI-domain of Integrin Mac-1 Binds the Cytokine Pleiotrophin Using Multiple Mechanisms.

The integrin Mac-1 (αMß2, CD11b/CD18, CR3) is an important adhesion receptor expressed on macrophages and neutrophils. Mac-1 is also the most promiscuous member of the integrin family that binds a diverse set of ligands through its αMI-domain. However, the binding mechanism of most ligands is not clear. We have determined the interaction of αMI-domain with the cytokine pleiotrophin (PTN), a cationic protein known to bind αMI-domain and induce Mac-1-mediated cell adhesion and migration. Our data show that PTN's N-terminal domain binds a unique site near the N- and C-termini of the αMI-domain using a metal-independent mechanism. However, stronger interaction is achieved when an acidic amino acid in a zwitterionic motif in PTN's C-terminal domain chelates the divalent cation in the metal ion-dependent adhesion site of the active αMI-domain. These results indicate that αMI-domain can bind ligands using multiple mechanisms, and suggest that active αMI-domain prefers acidic amino acids in zwitterionic motifs.

Platelet factor 4 improves survival in a murine model of antibiotic-susceptible and methicillin-resistant Staphylococcus aureus peritonitis.

The complement receptor CR3, also known as integrin Mac-1 (CD11b/CD18), is one of the major phagocytic receptors on the surface of neutrophils and macrophages. We previously demonstrated that in its protein ligands, Mac-1 binds sequences enriched in basic and hydrophobic residues and strongly disfavors negatively charged sequences. The avoidance by Mac-1 of negatively charged surfaces suggests that the bacterial wall and bacterial capsule possessing net negative electrostatic charge may repel Mac-1 and that the cationic Mac-1 ligands can overcome this evasion by acting as opsonins. Indeed, we previously showed that opsonization of Gram-negative Escherichia coli with several cationic peptides, including PF4 (Platelet Factor 4), strongly augmented phagocytosis by macrophages. Here, we investigated the effect of recombinant PF4 (rPF4) on phagocytosis of Gram-positive Staphylococcus aureus in vitro and examined its impact in a mouse model of S. aureus peritonitis. Characterization of the interaction of rPF4 with nonencapsulated and encapsulated S. aureus showed that rPF4 localizes on the bacterial surface, thus making it available for Mac-1. Furthermore, rPF4 did not have direct bactericidal and bacteriostatic activity and was not toxic to host cells. rPF4 enhanced phagocytosis of S. aureus bioparticles by various primary and cultured Mac-1-expressing leukocytes by several folds. It also increased phagocytosis of live nonencapsulated and encapsulated bacteria. Notably, the augmentation of phagocytosis by rPF4 did not compromise the intracellular killing of S. aureus by macrophages. Using a murine S. aureus peritonitis model, we showed that treatment of infected mice with rPF4 caused a significant increase in the clearance of antibiotic-susceptible S. aureus and its methicillin-resistant (MRSA) variant and markedly improved survival. These findings indicate that rPF4 binding to the bacterial surface circumvents its antiphagocytic properties, improving host defense against antibiotic-susceptible and antibiotic-resistant bacteria.

PLATELET FACTOR 4 (PF4) IMPROVES SURVIVAL IN A MURINE MODEL OF ANTIBIOTIC-SUSCEPTIBLE AND METHICILLIN-RESISTANT STAPHYLOCOCCUS AUREUS PERITONITIS.

The complement receptor CR3, also known as integrin Mac-1 (CD11b/CD18), is one of the major phagocytic receptors on the surface of neutrophils and macrophages. We previously demonstrated that in its protein ligands, Mac-1 binds sequences enriched in basic and hydrophobic residues and strongly disfavors negatively charged sequences. The avoidance by Mac-1 of negatively charged surfaces suggests that the bacterial wall and bacterial capsule possessing net negative electrostatic charge may repel Mac-1 and that the cationic Mac-1 ligands can overcome this evasion by acting as opsonins. Indeed, we previously showed that opsonization of Gram-negative Escherichia coli with several cationic peptides, including PF4 (Platelet Factor 4), strongly augmented phagocytosis by macrophages. Here, we investigated the effect of recombinant PF4 (rPF4) on phagocytosis of Gram-positive Staphylococcus aureus in vitro and examined its impact in a mouse model of S. aureus peritonitis. Characterization of the interaction of rPF4 with nonencapsulated and encapsulated S. aureus showed that rPF4 localizes on the bacterial surface, thus making it available for Mac-1. Furthermore, rPF4 did not have direct bactericidal and bacteriostatic activity and was not toxic to host cells. rPF4 enhanced phagocytosis of S. aureus bioparticles by various primary and cultured Mac-1-expressing leukocytes by several folds. It also increased phagocytosis of live nonencapsulated and encapsulated bacteria. Notably, the augmentation of phagocytosis by rPF4 did not compromise the intracellular killing of S. aureus by macrophages. Using a murine S. aureus peritonitis model, we showed that treatment of infected mice with rPF4 caused a significant increase in the clearance of antibiotic-susceptible S. aureus and its methicillin-resistant (MRSA) variant and markedly improved survival. These findings indicate that rPF4 binding to the bacterial surface circumvents its antiphagocytic properties, improving host defense against antibiotic-susceptible and antibiotic-resistant bacteria.

The CIS association of CD47 with integrin Mac-1 regulates macrophage responses by stabilizing the extended integrin conformation.

CD47 is a ubiquitously expressed cell surface integrin-associated protein. Recently, we have demonstrated that integrin Mac-1 (αMß2, CD11b/CD18, CR3), the major adhesion receptor on the surface of myeloid cells, can be coprecipitated with CD47. However, the molecular basis for the CD47-Mac-1 interaction and its functional consequences remain unclear. Here, we demonstrated that CD47 regulates macrophage functions directly interacting with Mac-1. In particular, adhesion, spreading, migration, phagocytosis, and fusion of CD47-deficient macrophages were significantly decreased. We validated the functional link between CD47 and Mac-1 by coimmunoprecipitation analysis using various Mac-1-expressing cells. In HEK293 cells expressing individual αM and ß2 integrin subunits, CD47 was found to bind both subunits. Interestingly, a higher amount of CD47 was recovered with the free ß2 subunit than in the complex with the whole integrin. Furthermore, activating Mac-1-expressing HEK293 cells with phorbol 12-myristate 13-acetate (PMA), Mn2+, and activating antibody MEM48 increased the amount of CD47 in complex with Mac-1, suggesting CD47 has a greater affinity for the extended integrin conformation. Notably, on the surface of cells lacking CD47, fewer Mac-1 molecules could convert into an extended conformation in response to activation. Additionally, we identified the binding site in CD47 for Mac-1 in its constituent IgV domain. The complementary binding sites for CD47 in Mac-1 were localized in integrin epidermal growth factor-like domains 3 and 4 of the ß2 and calf-1 and calf-2 domains of the αM subunits. These results indicate that Mac-1 forms a lateral complex with CD47, which regulates essential macrophage functions by stabilizing the extended integrin conformation.

Hypofibrinogenemia with preserved hemostasis and protection from thrombosis in mice with an Fga truncation mutation.

Genetic variants within the fibrinogen Aα chain encoding the αC-region commonly result in hypodysfibrinogenemia in patients. However, the (patho)physiological consequences and underlying mechanisms of such mutations remain undefined. Here, we generated Fga270 mice carrying a premature termination codon within the Fga gene at residue 271. The Fga270 mutation was compatible with Mendelian inheritance for offspring of heterozygous crosses. Adult Fga270/270 mice were hypofibrinogenemic with ∼10% plasma fibrinogen levels relative to FgaWT/WT mice, linked to 90% reduction in hepatic Fga messenger RNA (mRNA) because of nonsense-mediated decay of the mutant mRNA. Fga270/270 mice had preserved hemostatic potential in vitro and in vivo in models of tail bleeding and laser-induced saphenous vein injury, whereas Fga-/- mice had continuous bleeding. Platelets from FgaWT/WT and Fga270/270 mice displayed comparable initial aggregation following adenosine 5'-diphosphate stimulation, but Fga270/270 platelets quickly disaggregated. Despite ∼10% plasma fibrinogen, the fibrinogen level in Fga270/270 platelets was ∼30% of FgaWT/WT platelets with a compensatory increase in fibronectin. Notably, Fga270/270 mice showed complete protection from thrombosis in the inferior vena cava stasis model. In a model of Staphylococcus aureus peritonitis, Fga270/270 mice supported local, fibrinogen-mediated bacterial clearance and host survival comparable to FgaWT/WT, unlike Fga-/- mice. Decreasing the normal fibrinogen levels to ∼10% with small interfering RNA in mice also provided significant protection from venous thrombosis without compromising hemostatic potential and antimicrobial function. These findings both reveal novel molecular mechanisms underpinning fibrinogen αC-region truncation mutations and highlight the concept that selective fibrinogen reduction may be efficacious for limiting thrombosis while preserving hemostatic and immune protective functions.

Fibrin polymer on the surface of biomaterial implants drives the foreign body reaction.

Implantation of biomaterials and medical devices in the body triggers the foreign body reaction (FBR) which is characterized by macrophage fusion at the implant surface leading to the formation of foreign body giant cells and the development of the fibrous capsule enveloping the implant. While adhesion of macrophages to the surface is an essential step in macrophage fusion and implanted biomaterials are known to rapidly acquire a layer of host proteins, a biological substrate that is responsible for this process in vivo is unknown. Here we show that mice with genetically imposed fibrinogen deficiency display a dramatic reduction of macrophage fusion on biomaterials implanted intraperitoneally and subcutaneously and are protected from the formation of the fibrin-containing fibrous capsule. Furthermore, macrophage fusion on biomaterials implanted in FibAEK mice that express a mutated form of fibrinogen incapable of thrombin-mediated polymerization was strongly reduced. Despite the lack of fibrin, the capsule was formed in FibAEK mice, although it had a different composition and distinct mechanical properties than that in wild-type mice. Specifically, while mononuclear α-SMA-expressing macrophages embedded in the capsule of both strains of mice secreted collagen, the amount of collagen and its density in the tissue of FibAEK mice was reduced. These data identify fibrin polymer as a key biological substrate driving the development of the FBR.

Transition of podosomes into zipper-like structures in macrophage-derived multinucleated giant cells.

Macrophage fusion resulting in the formation of multinucleated giant cells (MGCs) is a multistage process that requires many adhesion-dependent steps and involves the rearrangement of the actin cytoskeleton. The diversity of actin-based structures and their role in macrophage fusion is poorly understood. In this study, we revealed hitherto unrecognized actin-based zipper-like structures (ZLSs) that arise between MGCs formed on the surface of implanted biomaterials. We established an in vitro model for the induction of these structures in mouse macrophages undergoing IL-4-mediated fusion. Using this model, we show that over time MGCs develop cell-cell contacts containing ZLSs. Live-cell imaging using macrophages isolated from mRFP- or eGFP-LifeAct mice demonstrated that ZLSs are dynamic formations undergoing continuous assembly and disassembly and that podosomes are precursors of these structures. Immunostaining experiments showed that vinculin, talin, integrin αMß2, and other components of podosomes are present in ZLSs. Macrophages deficient in WASp or Cdc42, two key molecules involved in actin core organization in podosomes, as well as cells treated with the inhibitors of the Arp2/3 complex, failed to form ZLSs. Furthermore, E-cadherin and nectin-2 were found between adjoining membranes, suggesting that the transition of podosomes into ZLSs is induced by bridging plasma membranes by junctional proteins.

An actin-based protrusion originating from a podosome-enriched region initiates macrophage fusion.

Macrophage fusion resulting in the formation of multinucleated giant cells occurs in a variety of chronic inflammatory diseases, yet the mechanism responsible for initiating this process is unknown. Here, we used live cell imaging to show that actin-based protrusions at the leading edge initiate macrophage fusion. Phase-contrast video microscopy demonstrated that in the majority of events, short protrusions (∼3 µm) between two closely apposed cells initiated fusion, but occasionally we observed long protrusions (∼12 µm). Using macrophages isolated from LifeAct mice and imaging with lattice light sheet microscopy, we further found that fusion-competent protrusions formed at sites enriched in podosomes. Inducing fusion in mixed populations of GFP- and mRFP-LifeAct macrophages showed rapid spatial overlap between GFP and RFP signal at the site of fusion. Cytochalasin B strongly reduced fusion and when rare fusion events occurred, protrusions were not observed. Fusion of macrophages deficient in Wiskott-Aldrich syndrome protein and Cdc42, key molecules involved in the formation of actin-based protrusions and podosomes, was also impaired both in vitro and in vivo. Finally, inhibiting the activity of the Arp2/3 complex decreased fusion and podosome formation. Together these data suggest that an actin-based protrusion formed at the leading edge initiates macrophage fusion.

Interaction between the integrin Mac-1 and signal regulatory protein α (SIRPα) mediates fusion in heterologous cells.

Macrophage fusion leading to the formation of multinucleated giant cells is a hallmark of chronic inflammation. Several membrane proteins have been implicated in mediating cell-cell attachment during fusion, but their binding partners remain unknown. Recently, we demonstrated that interleukin-4 (IL-4)-induced fusion of mouse macrophages depends on the integrin macrophage antigen 1 (Mac-1). Surprisingly, the genetic deficiency of intercellular adhesion molecule 1 (ICAM-1), an established ligand of Mac-1, did not impair macrophage fusion, suggesting the involvement of other counter-receptors. Here, using various approaches, including signal regulatory protein α (SIRPα) knockdown, recombinant proteins, adhesion and fusion assays, biolayer interferometry, and peptide libraries, we show that SIRPα, which, similar to ICAM-1, belongs to the Ig superfamily and has previously been implicated in cell fusion, interacts with Mac-1. The following results support the conclusion that SIRPα is a ligand of Mac-1: (a) recombinant ectodomain of SIRPα supports adhesion of Mac-1-expressing cells; (b) Mac-1-SIRPα interaction is mediated through the ligand-binding αMI-domain of Mac-1; (c) recognition of SIRPα by the αMI-domain conforms to general principles governing binding of Mac-1 to many of its ligands; (d) SIRPα reportedly binds CD47; however, anti-CD47 function-blocking mAb produced only a limited inhibition of macrophage adhesion to SIRPα; and (e) co-culturing of SIRPα- and Mac-1-expressing HEK293 cells resulted in the formation of multinucleated cells. Taken together, these results identify SIRPα as a counter-receptor for Mac-1 and suggest that the Mac-1-SIRPα interaction may be involved in macrophage fusion.

Fabricating Optical-quality Glass Surfaces to Study Macrophage Fusion.

Visualizing the formation of multinucleated giant cells (MGCs) from living specimens has been challenging due to the fact that most live imaging techniques require propagation of light through glass, but on glass macrophage fusion is a rare event. This protocol presents the fabrication of several optical-quality glass surfaces where adsorption of compounds containing long-chain hydrocarbons transforms glass into a fusogenic surface. First, preparation of clean glass surfaces as starting material for surface modification is described. Second, a method is provided for the adsorption of compounds containing long-chain hydrocarbons to convert non-fusogenic glass into a fusogenic substrate. Third, this protocol describes fabrication of surface micropatterns that promote a high degree of spatiotemporal control over MGC formation. Finally, fabricating glass bottom dishes is described. Examples of use of this in vitro cell system as a model to study macrophage fusion and MGC formation are shown.

Leukocyte integrin Mac-1 (CD11b/CD18, αMß2, CR3) acts as a functional receptor for platelet factor 4.

Platelet factor 4 (PF4) is one of the most abundant cationic proteins secreted from α-granules of activated platelets. Based on its structure, PF4 was assigned to the CXC family of chemokines and has been shown to have numerous effects on myeloid leukocytes. However, the receptor for PF4 remains unknown. Here, we demonstrate that PF4 induces leukocyte responses through the integrin Mac-1 (αMß2, CD11b/CD18). Human neutrophils, monocytes, U937 monocytic and HEK293 cells expressing Mac-1 strongly adhered to immobilized PF4 in a concentration-dependent manner. The cell adhesion was partially blocked by anti-Mac-1 mAb and inhibition was enhanced when anti-Mac-1 antibodies were combined with glycosaminoglycans, suggesting that cell-surface proteoglycans act cooperatively with Mac-1. PF4 also induced Mac-1-dependent migration of human neutrophils and murine WT, but not Mac-1-deficient macrophages. Coating of Escherichia coli bacteria or latex beads with PF4 enhanced their phagocytosis by macrophages by ∼4-fold, and this process was blocked by different Mac-1 antagonists. Furthermore, PF4 potentiated phagocytosis by WT, but not Mac-1-deficient macrophages. As determined by biolayer interferometry, PF4 directly bound the αMI-domain, the major ligand-binding region of Mac-1, and this interaction was governed by a Kd of 1.3 ± 0.2 µm Using the PF4-derived peptide library, synthetic peptides duplicating the αMI-domain recognition sequences and recombinant mutant PF4 fragments, the binding sites for αMI-domain were identified in the PF4 segments Cys12-Ser26 and Ala57-Ser70 These results identify PF4 as a ligand for the integrin Mac-1 and suggest that many immune-modulating effects previously ascribed to PF4 are mediated through its interaction with Mac-1.

Pleiotrophin, a multifunctional cytokine and growth factor, induces leukocyte responses through the integrin Mac-1.

Pleiotrophin (PTN) is a multifunctional, cationic, glycosaminoglycan-binding cytokine and growth factor involved in numerous physiological and pathological processes, including tissue repair and inflammation-related diseases. PTN has been shown to promote leukocyte responses by inducing their migration and expression of inflammatory cytokines. However, the mechanisms through which PTN mediates these responses remain unclear. Here, we identified the integrin Mac-1 (αMß2, CD11b/CD18) as the receptor mediating macrophage adhesion and migration to PTN. We also found that expression of Mac-1 on the surface of human embryonic kidney (HEK) 293 cells induced their adhesion and migration to PTN. Accordingly, PTN promoted Mac-1-dependent cell spreading and initiated intracellular signaling manifested in phosphorylation of Erk1/2. While binding to PTN, Mac-1 on Mac-1-expressing HEK293 cells appears to cooperate with cell-surface proteoglycans because both anti-Mac-1 function-blocking mAb and heparin were required to block adhesion. Moreover, biolayer interferometry and NMR indicated a direct interaction between the αMI domain, the major ligand-binding region of Mac-1, and PTN. Using peptide libraries, we found that in PTN the αMI domain bound sequences enriched in basic and hydrophobic residues, indicating that PTN conforms to the general principle of ligand-recognition specificity of the αMI domain toward cationic proteins/peptides. Finally, using recombinant PTN-derived fragments, we show that PTN contains two distinct Mac-1-binding sites in each of its constitutive domains. Collectively, these results identify PTN as a ligand for the integrin Mac-1 on the surface of leukocytes and suggest that this interaction may play a role in inflammatory responses.

Development of fusogenic glass surfaces that impart spatiotemporal control over macrophage fusion: Direct visualization of multinucleated giant cell formation.

Implantation of synthetic material, including vascular grafts, pacemakers, etc. results in the foreign body reaction and the formation of multinucleated giant cells (MGCs) at the exterior surface of the implant. Despite the long-standing premise that fusion of mononucleated macrophages results in the formation of MGCs, to date, no published study has shown fusion in context with living specimens. This is due to the fact that optical-quality glass, which is required for the majority of live imaging techniques, does not promote macrophage fusion. Consequently, the morphological changes that macrophages undergo during fusion as well as the mechanisms that govern this process remain ill-defined. In this study, we serendipitously identified a highly fusogenic glass surface and discovered that the capacity to promote fusion was due to oleamide contamination. When adsorbed on glass, oleamide and other molecules that contain long-chain hydrocarbons promoted high levels of macrophage fusion. Adhesion, an essential step for macrophage fusion, was apparently mediated by Mac-1 integrin (CD11b/CD18, αMß2) as determined by single cell force spectroscopy and adhesion assays. Micropatterned glass further increased fusion and enabled a remarkable degree of spatiotemporal control over MGC formation. Using these surfaces, we reveal the kinetics that govern MGC formation in vitro. We anticipate that the spatiotemporal control afforded by these surfaces will expedite studies designed to identify the mechanism(s) of macrophage fusion and MGC formation with implication for the design of novel biomaterials.

Identification of Human Cathelicidin Peptide LL-37 as a Ligand for Macrophage Integrin αMß2 (Mac-1, CD11b/CD18) that Promotes Phagocytosis by Opsonizing Bacteria.

LL-37, a cationic antimicrobial peptide, has numerous immune-modulating effects. However, the identity of a receptor(s) mediating the responses in immune cells remains uncertain. We have recently demonstrated that LL-37 interacts with the αMI-domain of integrin αMß2 (Mac-1), a major receptor on the surface of myeloid cells, and induces a migratory response in Mac-1-expressing monocyte/macrophages as well as activation of Mac-1 on neutrophils. Here, we show that LL-37 and its C-terminal derivative supported strong adhesion of various Mac-1-expressing cells, including HEK293 cells stably transfected with Mac-1, human U937 monocytic cells and murine IC-21 macrophages. The cell adhesion to LL-37 was partially inhibited by specific Mac-1 antagonists, including mAb against the αM integrin subunit and neutrophil inhibitory factor, and completely blocked when anti-Mac-1 antibodies were combined with heparin, suggesting that cell surface heparan sulfate proteoglycans act cooperatively with integrin Mac-1. Coating both Gram-negative and Gram-positive bacteria with LL-37 significantly potentiated their phagocytosis by macrophages, and this process was blocked by a combination of anti-Mac-1 mAb and heparin. Furthermore, phagocytosis by wild-type murine peritoneal macrophages of LL-37-coated latex beads, a model of foreign surfaces, was several fold higher than that of untreated beads. By contrast, LL-37 failed to augment phagocytosis of beads by Mac-1-deficient macrophages. These results identify LL-37 as a novel ligand for integrin Mac-1 and demonstrate that the interaction between Mac-1 on macrophages and bacteria-bound LL-37 promotes phagocytosis.

The Role of Integrins αMß2 (Mac-1, CD11b/CD18) and αDß2 (CD11d/CD18) in Macrophage Fusion.

The subfamily of ß2 integrins is implicated in macrophage fusion, a hallmark of chronic inflammation. Among ß2 family members, integrin Mac-1 (αMß2, CD11b/CD18) is abundantly expressed on monocyte/macrophages and mediates critical adhesive reactions of these cells. However, the role of Mac-1 in macrophage fusion leading to the formation of multinucleated giant cells remains unclear. Moreover, the role of integrin αDß2 (CD11d/CD18), a receptor with recognition specificity overlapping that of Mac-1, is unknown. We found that multinucleated giant cells are formed in the inflamed mouse peritoneum during the resolution phase of inflammation, and their numbers were approximately twofold higher in wild-type mice than in Mac-1(-/-) mice. Analyses of isolated inflammatory peritoneal macrophages showed that IL-4-induced fusion of Mac-1-deficient cells was strongly reduced compared with wild-type counterparts. The examination of adhesive reactions known to be required for fusion showed that spreading, but not adhesion and migration, was reduced in Mac-1-deficient macrophages. Fusion of αDß2-deficient macrophages was also significantly decreased, albeit to a smaller degree. Deficiency of intercellular adhesion molecule 1, a counter-receptor for Mac-1 and αDß2, did not alter the fusion rate. The results indicate that both Mac-1 and αDß2 support macrophage fusion with Mac-1 playing a dominant role and suggest that Mac-1 may mediate cell-cell interactions with a previously unrecognized counter-receptor(s).

Deposition of fibrinogen on the surface of in vitro thrombi prevents platelet adhesion.

The initial accumulation of platelets after vessel injury is followed by thrombin-mediated generation of fibrin which is deposited around the plug. While numerous in vitro studies have shown that fibrin is highly adhesive for platelets, the surface of experimental thrombi in vivo contains very few platelets suggesting the existence of natural anti-adhesive mechanisms protecting stabilized thrombi from platelet accumulation and continuous thrombus propagation. We previously showed that adsorption of fibrinogen on pure fibrin clots results in the formation of a nonadhesive matrix, highlighting a possible role of this process in surface-mediated control of thrombus growth. However, the deposition of fibrinogen on the surface of blood clots has not been examined. In this study, we investigated the presence of intact fibrinogen on the surface of fibrin-rich thrombi generated from flowing blood and determined whether deposited fibrinogen is nonadhesive for platelets. Stabilized fibrin-rich thrombi were generated using a flow chamber and the time that platelets spend on the surface of thrombi was determined by video recording. The presence of fibrinogen and fibrin on the surface of thrombi was analyzed by confocal microscopy using specific antibodies. Examination of the spatial distribution of two proteins revealed the presence of intact fibrinogen on the surface of stabilized thrombi. By manipulating the surface of thrombi to display either fibrin or intact fibrinogen, we found that platelets adhere to fibrin- but not to fibrinogen-coated thrombi. These results indicate that the fibrinogen matrix assembled on the outer layer of stabilized in vitro thrombi protects them from platelet adhesion.

Fibrinogen matrix deposited on the surface of biomaterials acts as a natural anti-adhesive coating.

Adsorption of fibrinogen on the luminal surface of biomaterials is a critical early event during the interaction of blood with implanted vascular graft prostheses which determines their thrombogenicity. We have recently identified a nanoscale process by which fibrinogen modifies the adhesive properties of various surfaces for platelets and leukocytes. In particular, adsorption of fibrinogen at low density promotes cell adhesion while its adsorption at high density results in the formation of an extensible multilayer matrix, which dramatically reduces cell adhesion. It remains unknown whether deposition of fibrinogen on the surface of vascular graft materials produces this anti-adhesive effect. Using atomic force spectroscopy, single cell force spectroscopy, and standard adhesion assays with platelets and leukocytes, we have characterized the adhesive and physical properties of the contemporary biomaterials, before and after coating with fibrinogen. We found that uncoated PET, PTFE and ePTFE exhibited high adhesion forces developed between the AFM tip or cells and the surfaces. Adsorption of fibrinogen at the increasing concentrations progressively reduced adhesion forces, and at ≥2 µg/ml all surfaces were virtually nonadhesive. Standard adhesion assays performed with platelets and leukocytes confirmed this dependence. These results provide a better understanding of the molecular events underlying thrombogenicity of vascular grafts.

The opioid peptide dynorphin A induces leukocyte responses via integrin Mac-1 (αMß2, CD11b/CD18).

BACKGROUND: Opioid peptides, including dynorphin A, besides their analgesic action in the nervous system, exert a broad spectrum of effects on cells of the immune system, including leukocyte migration, degranulation and cytokine production. The mechanisms whereby opioid peptides induce leukocyte responses are poorly understood. The integrin Mac-1 (αMß2, CD11b/CD18) is a multiligand receptor which mediates numerous reactions of neutrophils and monocyte/macrophages during the immune-inflammatory response. Our recent elucidation of the ligand recognition specificity of Mac-1 suggested that dynorphin A and dynorphin B contain Mac-1 recognition motifs and can potentially interact with this receptor. RESULTS: In this study, we have synthesized the peptide library spanning the sequence of dynorphin AB, containing dynorphin A and B, and showed that the peptides bound recombinant αMI-domain, the ligand binding region of Mac-1. In addition, immobilized dynorphins A and B supported adhesion of the Mac-1-expressing cells. In binding to dynorphins A and B, Mac-1 cooperated with cell surface proteoglycans since both anti-Mac-1 function-blocking reagents and heparin were required to block adhesion. Further focusing on dynorphin A, we showed that its interaction with the αMI-domain was activation independent as both the α7 helix-truncated (active conformation) and helix-extended (nonactive conformation) αMI-domains efficiently bound dynorphin A. Dynorphin A induced a potent migratory response of Mac-1-expressing, but not Mac-1-deficient leukocytes, and enhanced Mac-1-mediated phagocytosis of latex beads by murine IC-21 macrophages. CONCLUSIONS: Together, the results identify dynorphins A and B as novel ligands for Mac-1 and suggest a role for the Dynorphin A-Mac-1 interactions in the induction of nonopiod receptor-dependent effects in leukocytes.

Ligand recognition specificity of leukocyte integrin αMß2 (Mac-1, CD11b/CD18) and its functional consequences.

The broad recognition specificity exhibited by integrin α(M)ß2 (Mac-1, CD11b/CD18) has allowed this adhesion receptor to play innumerable roles in leukocyte biology, yet we know little about how and why α(M)ß2 binds its multiple ligands. Within α(M)ß2, the α(M)I-domain is responsible for integrin's multiligand binding properties. To identify its recognition motif, we screened peptide libraries spanning sequences of many known protein ligands for α(M)I-domain binding and also selected the α(M)I-domain recognition sequences by phage display. Analyses of >1400 binding and nonbinding peptides derived from peptide libraries showed that a key feature of the α(M)I-domain recognition motif is a small core consisting of basic amino acids flanked by hydrophobic residues. Furthermore, the peptides selected by phage display conformed to a similar pattern. Identification of the recognition motif allowed the construction of an algorithm that reliably predicts the α(M)I-domain binding sites in the α(M)ß2 ligands. The recognition specificity of the α(M)I-domain resembles that of some chaperones, which allows it to bind segments exposed in unfolded proteins. The disclosure of the α(M)ß2 binding preferences allowed the prediction that cationic host defense peptides, which are strikingly enriched in the α(M)I-domain recognition motifs, represent a new class of α(M)ß2 ligands. This prediction has been tested by examining the interaction of α(M)ß2 with the human cathelicidin peptide LL-37. LL-37 induced a potent α(M)ß2-dependent cell migratory response and caused activation of α(M)ß2 on neutrophils. The newly revealed recognition specificity of α(M)ß2 toward unfolded protein segments and cationic proteins and peptides suggests that α(M)ß2 may serve as a previously proposed "alarmin" receptor with important roles in innate host defense.