Nataly P. Podolnikova

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

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

Control of integrin αIIbβ3 outside-in signaling and platelet adhesion by sensing the physical properties of fibrin(ogen) substrates

The physical properties of substrates are known to control cell adhesion via integrin-mediated signaling. Fibrin and fibrinogen, the principal components of hemostatic and pathological thrombi, may represent biologically relevant substrates whose variable physical properties control adhesion of leukocytes and platelets. In our previous work, we have shown that binding of fibrinogen to the surface of fibrin clot prevents cell adhesion by creating an antiadhesive fibrinogen layer. Furthermore, fibrinogen immobilized on various surfaces at high density supports weak cell adhesion whereas at low density it is highly adhesive. To explore the mechanism underlying differential cell adhesion, we examined the structural and physical properties of surfaces prepared by deposition of various concentrations of fibrinogen using atomic force microscopy and force spectroscopy. Fibrinogen deposition at high density resulted in an aggregated multilayered material characterized by low adhesion forces. In contrast, immobilization of fibrinogen at low density produced a single layer in which molecules were directly attached to the solid surface, resulting in higher adhesion forces. Consistent with their distinct physical properties, low- but not high-density fibrinogen induced strong alpha(IIb)beta(3)-mediated outside-in signaling in platelets, resulting in their spreading. Moreover, while intact fibrin gels induced strong signaling in platelets, deposition of fibrinogen on the surface of fibrin resulted in diminished cell signaling. The data suggest that deposition of a multilayered fibrinogen matrix prevents stable cell adhesion by modifying the physical properties of surfaces, which results in reduced force generation and insufficient signaling. The mechanism whereby circulating fibrinogen alters adhesive properties of fibrin clots may have important implications for control of thrombus formation and thrombogenicity of biomaterials.

The assembly of nonadhesive fibrinogen matrices depends on the αC regions of the fibrinogen molecule.

Background: Surface-induced aggregation of fibrinogen results in the assembly of an extensible multilayered matrix, which prevents integrin-mediated cell adhesion. Results: Without the αC regions, the fibrinogen molecules assemble a defective, poorly extensible matrix supporting sustained cell adhesion. Conclusion: The assembly of nonadhesive fibrinogen multilayer requires the αC regions of the molecule. Significance: The molecular mechanism for the assembly of the fibrinogen multilayer is identified. Adsorption of fibrinogen on fibrin clots and other surfaces strongly reduces integrin-mediated adhesion of platelets and leukocytes with implications for the surface-mediated control of thrombus growth and blood compatibility of biomaterials. The underlying mechanism of this process is surface-induced aggregation of fibrinogen, resulting in the assembly of a nanoscale multilayered matrix. The matrix is extensible, which makes it incapable of transducing strong mechanical forces via cellular integrins, resulting in insufficient intracellular signaling and weak cell adhesion. To determine the mechanism of the multilayer formation, the physical and adhesive properties of fibrinogen matrices prepared from human plasma fibrinogen (hFg), recombinant normal (rFg), and fibrinogen with the truncated αC regions (FgAα251) were compared. Using atomic force microscopy and force spectroscopy, we show that whereas hFg and rFg generated the matrices with a thickness of ∼8 nm consisting of 7–8 molecular layers, the deposition of FgAα251 was terminated at two layers, indicating that the αC regions are essential for the multilayer formation. The extensibility of the matrix prepared from FgAα251 was 2-fold lower than that formed from hFg and rFg. In agreement with previous findings that cell adhesion inversely correlates with the extensibility of the fibrinogen matrix, the less extensible FgAα251 matrix and matrices generated from human fibrinogen variants lacking the αC regions supported sustained adhesion of leukocytes and platelets. The persistent adhesiveness of matrices formed from fibrinogen derivatives without the αC regions may have implications for conditions in which elevated levels of these molecules are found, including vascular pathologies, diabetes, thrombolytic therapy, and dysfibrinogenemia.

The Role of Integrins αMβ2 (Mac-1, CD11b/CD18) and αDβ2 (CD11d/CD18) in Macrophage Fusion

The subfamily of β2 integrins is implicated in macrophage fusion, a hallmark of chronic inflammation. Among β2 family members, integrin Mac-1 (αMβ2, 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 αDβ2 (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 αDβ2-deficient macrophages was also significantly decreased, albeit to a smaller degree. Deficiency of intercellular adhesion molecule 1, a counter-receptor for Mac-1 and αDβ2, did not alter the fusion rate. The results indicate that both Mac-1 and αDβ2 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).

The Opioid Peptide Dynorphin a Induces Leukocyte Responses via Integrin Mac-1 (αMβ2, CD11b/CD18)

BackgroundOpioid 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 (αMβ2, 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.ResultsIn 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.ConclusionsTogether, 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.

Identification of Human Cathelicidin Peptide LL-37 as a Ligand for Macrophage Integrin αMβ2 (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 αMβ2 (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.

Leukocyte Integrin Mac-1 (CD11b/CD18, αMβ2, 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 (αMβ2, 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 (αMβ2, 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.

Selective Photonic Disinfection of Cell Culture Using a Visible Ultrashort Pulsed Laser

Microbial contamination of cell culture is a major problem encountered both in academic labs and in the biotechnology/pharmaceutical industries. A broad spectrum of microbes, including mycoplasma, bacteria, fungi, and viruses are the causative agents of cell-culture contamination. Unfortunately, the existing disinfection techniques lack selectivity and/or lead to the development of drug-resistance, and more importantly there is no universal method to address all the microbes. Here, we report a novel, chemical-free visible ultrashort pulsed laser method for cell-culture disinfection. The ultrashort pulsed laser technology inactivates pathogens with mechanical means, a paradigm shift from the traditional pharmaceutical and chemical approaches. We demonstrate that ultrashort pulsed laser treatment can efficiently inactivate mycoplasma, bacteria, yeast, and viruses with good preservation of mammalian cell viability. Our results indicate that this ultrashort pulsed laser technology has the potential to serve as a universal method for the disinfection of cell culture.

Skelemin association with αIIbβ3 integrin: a structural model.

Over the last two decades, our knowledge concerning intracellular events that regulate integrin’s affinity to their soluble ligands has significantly improved. However, the mechanism of adhesion-induced integrin clustering and development of focal complexes, which could further mature to form focal adhesions, still remains under-investigated. Here we present a structural model of tandem IgC2 domains of skelemin in complex with the cytoplasmic tails of integrin αIIbβ3. The model of tertiary assembly is generated based upon NMR data and illuminates a potential link between the essential cell adhesion receptors and myosin filaments. This connection may serve as a basis for generating the mechanical forces necessary for cell migration and remodeling.