Marian Brennan

Integrins as therapeutic targets: lessons and opportunities

The integrins are a large family of cell adhesion molecules that are essential for the regulation of cell growth and function. The identification of key roles for integrins in a diverse range of diseases, including cancer, infection, thrombosis and autoimmune disorders, has revealed their substantial potential as therapeutic targets. However, so far, pharmacological inhibitors for only three integrins have received marketing approval. This article discusses the structure and function of integrins, their roles in disease and the chequered history of the approved integrin antagonists. Recent advances in the understanding of integrin function, ligand interaction and signalling pathways suggest novel strategies for inhibiting integrin function that could help harness their full potential as therapeutic targets.

Fibronectin-binding proteins of Staphylococcus aureus mediate activation of human platelets via fibrinogen and fibronectin bridges to integrin GPIIb/IIIa and IgG binding to the FcgammaRIIa receptor.

Staphylococcus aureus is a leading cause of infective endocarditis (IE). Platelet activation promoted by S. aureus resulting in aggregation and thrombus formation is an important step in the pathogenesis of IE. Here, we report that the fibrinogen/fibronectin‐binding proteins FnBPA and FnBPB are major platelet‐activating factors on the surface of S. aureus from the exponential phase of growth. Truncated derivatives of FnBPA, presenting either the fibrinogen‐binding A domain or the fibronectin‐binding BCD region, each promoted platelet activation when expressed on the surface of S. aureus or Lactococcus lactis, indicating two distinct mechanisms of activation. FnBPA‐promoted platelet activation is mediated by fibrinogen and fibronectin bridges between the A domain and the BCD domains, respectively, to the low affinity form of the integrin GPIIb/IIIa on resting platelets. Antibodies recognizing the FnBPA A domain or the complex between the FnBPA BCD domains and fibronectin were essential for activation promoted by bacteria expressing the A domain or the BCD domain respectively. Activation was inhibited by a monoclonal antibody (IV‐3) specific for the FcγRIIa IgG receptor on platelets. We propose that the activation of quiescent platelets by bacteria expressing FnBPs involves the formation of a bridge between the bacterial cell and the platelet surface by (i) fibronectin and fibrinogen interacting with the low affinity form of GPIIb/IIIa and (ii) by antibodies specific to FnBPs that engage the platelet Fc receptor FcγRIIa. Platelet activation by S. aureus clinical IE isolates from both the exponential and stationary phases of growth was completely inhibited by monoclonal antibody IV‐3 suggesting that the IgG–FcγRIIa interaction is of fundamental importance for platelet activation mediated by this organism. This suggests new avenues for development of therapeutics against vascular infections.

Roles for fibrinogen, immunoglobulin and complement in platelet activation promoted by Staphylococcus aureus clumping factor A

Staphylococcus aureus is an important cause of infective endocarditis (IE) in patients without a history of prior heart valve damage. The ability to stimulate the activation of resting platelets and their subsequent aggregation is regarded as an important virulence factor of bacteria that cause IE. Clumping factor A is the dominant surface protein responsible for platelet activation by S. aureus cells in the stationary phase of growth. This study used Lactococcus lactis as a surrogate host to study the mechanism of ClfA‐promoted platelet activation. Expression of ClfA from a nisin‐inducible promoter demonstrated that a minimum level of surface‐expressed ClfA was required. Using platelets that were purified from plasma, the requirement for both bound fibrinogen and immunoglobulin was demonstrated. The immunoglobulin G (IgG) requirement is consistent with the potent inhibition of platelet activation by a monoclonal antibody specific for the platelet FcγRIIa receptor. Furthermore the IgG must contain antibodies specific for the ClfA A domain. A model is proposed whereby bacterial cells armed with a sufficient number of surface‐bound fibrinogen molecules can engage resting platelet glycoprotein GPIIb/IIIa, aided by bound IgG molecules, which encourages the clustering of FcγRIIa receptors. This can trigger activation of signal transduction leading to activation of GPIIb/IIIa and aggregation of platelets. In addition, analysis of a mutant of ClfA totally lacking the ability to bind fibrinogen revealed a second, although less efficient, mechanism of platelet activation. The fibrinogen‐independent pathway required IgG and complement deposition to trigger platelet aggregation

Fibrinogen and elastin bind to the same region within the A domain of fibronectin binding protein A, an MSCRAMM of Staphylococcus aureus.

The fibronectin binding protein, FnBPA, is a multifunctional microbial surface component recognizing adhesive matrix molecule (MSCRAMM) that promotes bacterial adherence to immobilized fibrinogen and elastin via the N‐terminal A domain. The binding site for fibrinogen and elastin was localized to subdomains N2N3. A three‐dimensional structural model of FnBPA was created based on the known crystal structure of the domains N2N3 of clumping factor A (ClfA). The role of individual residues in the putative ligand binding trench was examined by testing the affinity of mutants for fibrinogen and elastin. Two residues (N304 and F306) were crucial for binding both ligands and are in the equivalent positions to residues known to be important for fibrinogen binding by ClfA. A peptide comprising the C‐terminus of the γ‐chain of fibrinogen and a monoclonal anti‐rAFnBPA antibody were potent inhibitors of the FnBPA–elastin interaction. This suggests that FnBPA binds to fibrinogen and elastin in a similar manner. Amino acid sequence divergence of 26.5% occurred between the A domains of FnBPA from strains 8325–4 and P1. Most variant residues were predicted to be located on the surface of domains N2N3 while few occurred in the putative ligand binding trench and the latching peptide explaining limited immunocross reactivity while ligand binding activity is conserved.

Both complement- and fibrinogen-dependent mechanisms contribute to platelet aggregation mediated by Staphylococcus aureus clumping factor B.

ABSTRACT Staphylococcus aureus can stimulate activation and aggregation of platelets, which are thought to be factors in the development of infective endocarditis. Previous studies have identified clumping factor A (ClfA) and fibronectin binding proteins A and B (FnBPA and FnBPB) as potent platelet aggregators. These proteins are able to stimulate rapid platelet aggregation by either a fibrinogen- or a fibronectin-dependent process which also requires antibodies specific to each protein. Slower aggregation has been seen in other systems where specific fibrinogen binding ligands are absent and platelet aggregation is mediated by complement and specific antibodies. Bacteria expressing ClfB aggregate platelets with a longer lag time than ClfA or FnBPA and FnBPB. In order to investigate whether ClfB causes platelet aggregation in a complement- or fibrinogen-dependent manner, a non-fibrinogen-binding mutant of ClfB (ClfB Q235A) was constructed. Lactococcus lactis expressing ClfB Q235A was able to stimulate platelet aggregation in platelet-rich plasma without a significant increase in lag time. The requirements for platelet aggregation were investigated using gel-filtered platelets. Fibrinogen and specific anti-ClfB antibodies were found to be sufficient to allow platelet aggregation mediated by the wild-type ClfB protein. It seems that ClfB causes platelet aggregation by a fibrinogen-dependent mechanism. The non-fibrinogen-binding ClfB mutant was unable to stimulate platelet aggregation under these conditions. However, bacteria expressing ClfB Q235A caused platelet aggregation in a complement-dependent manner which required specific anti-ClfB antibodies.

Elucidating the role of Staphylococcus epidermidis serine–aspartate repeat protein G in platelet activation

Summary.  Background: Staphylococcus epidermidis is a commensal of the human skin that has been implicated in infective endocarditis and infections involving implanted medical devices. S. epidermidis induces platelet aggregation by an unknown mechanism. The fibrinogen‐binding protein serine–aspartate repeat protein G (SdrG) is present in 67–91% of clinical strains. Objectives: To determine whether SdrG plays a role in platelet activation, and if so to investigate the role of fibrinogen in this mechanism. Methods: SdrG was expressed in a surrogate host, Lactococcus lactis, in order to investigate its role in the absence of other staphylococcal components. Platelet adhesion and platelet aggregation assays were employed. Results:  L. lactis expressing SdrG stimulated platelet aggregation (lag time: 2.9 ± 0.5 min), whereas the L. lactis control did not. L. lactis SdrG‐induced aggregation was inhibited by αIIbβ3 antagonists and aspirin. Aggregation was dependent on both fibrinogen and IgG, and the platelet IgG receptor FcγRIIa. Preincubation of the bacteria with Bβ‐chain fibrinopeptide inhibited aggregation (delaying the lag time six‐fold), suggesting that fibrinogen acts as a bridging molecule. Platelets adhered to L. lactis SdrG in the absence of fibrinogen. Adhesion was inhibited by αIIbβ3 antagonists, suggesting that this direct interaction involves αIIbβ3. Investigation using purified fragments of SdrG revealed a direct interaction with the B‐domains. Adhesion to the A‐domain involved both a fibrinogen and an IgG bridge. Conclusion: SdrG alone is sufficient to support platelet adhesion and aggregation through both direct and indirect mechanisms.

Molecular interactions and inhibition of the staphylococcal biofilm-forming protein SdrC

Significance The bacterial pathogen Staphylococcus aureus shows a remarkable ability to aggregate, thereby contributing to the formation of cellular communities that are difficult to eradicate. In this study, we dissect the homophilic interactions at play during S. aureus cell–cell adhesion, focusing on the key surface protein SdrC. We discover that SdrC is engaged in low-affinity homophilic bonds that promote intercellular adhesion, and that it also favors strong hydrophobic interactions with surfaces, emphasizing that this protein is a multifunctional adhesin. We also show that SdrC-dependent cell-surface attachment, cell–cell adhesion, and biofilm formation can be efficiently blocked by a peptide, thus suggesting this approach could be used for antibiofilm therapy. Staphylococcus aureus forms biofilms on indwelling medical devices using a variety of cell-surface proteins. There is growing evidence that specific homophilic interactions between these proteins represent an important mechanism of cell accumulation during biofilm formation, but the underlying molecular mechanisms are still not well-understood. Here we report the direct measurement of homophilic binding forces by the serine-aspartate repeat protein SdrC and their inhibition by a peptide. Using single-cell and single-molecule force measurements, we find that SdrC is engaged in low-affinity homophilic bonds that promote cell–cell adhesion. Low-affinity intercellular adhesion may play a role in favoring biofilm dynamics. We show that SdrC also mediates strong cellular interactions with hydrophobic surfaces, which are likely to be involved in the initial attachment to biomaterials, the first stage of biofilm formation. Furthermore, we demonstrate that a peptide derived from β-neurexin is a powerful competitive inhibitor capable of efficiently blocking surface attachment, homophilic adhesion, and biofilm accumulation. Molecular modeling suggests that this blocking activity may originate from binding of the peptide to a sequence of SdrC involved in homophilic interactions. Our study opens up avenues for understanding the role of homophilic interactions in staphylococcal adhesion, and for the design of new molecules to prevent biofilm formation during infection.

In Vitro Activities of Synthetic Host Defense Propeptides Processed by Neutrophil Elastase against Cystic Fibrosis Pathogens

ABSTRACT The antimicrobial and hemolytic activities of a host defense peptide can be controlled by its modification as a propeptide of reduced net charge, which can then be processed by neutrophil elastase, a serine protease involved in chronic airway inflammation and infections associated with cystic fibrosis.

A novel platinum complex of the histone deacetylase inhibitor belinostat: Rational design, development and in vitro cytotoxicity

The successful design and synthesis of a novel Pt complex of the histone deacteylase inhibitor belinostat are reported. Molecular modelling assisted in the identification of a suitable malonate derivative of belinostat (mal-p-Bel) for complexation to platinum. Reaction of [Pt(NH3)2(H2O)2](NO3)2 with the disodium salt of mal-p-Bel gave cis-[Pt(NH3)2(mal-p-Bel-2H)] (where -2H indicates that mal-p-Bel is doubly deprotonated) in excellent yield. An in vitro cytotoxicity study revealed that cis-[Pt(NH3)2(mal-p-Bel-2H)] possesses (i) considerable cytotoxicity against reported ovarian cancer cell lines, (ii) enhanced cytotoxicity relative to the previously reported Pt histone deacetylase inhibitor conjugate, cis-[Pt(II)(NH3)2(malSAHA-2H)] and (iii) favourable cyto-selective properties as compared to cisplatin and belinostat.

C-reactive protein binds to αIIbβ3

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