Antimicrobial peptides derived from the cartilage.-specific C-type Lectin Domain Family 3 Member A (CLEC3A) - potential in the prevention and treatment of septic arthritis.

OBJECTIVE: To investigate the antimicrobial activity of peptides derived from C-type Lectin Domain Family 3 Member A (CLEC3A), shed light on the mechanism of antimicrobial activity and assess their potential application in prevention and treatment of septic arthritis. DESIGN: We performed immunoblot to detect CLEC3A peptides in human cartilage extracts. To investigate their antimicrobial activity, we designed peptides and recombinantly expressed CLEC3A domains and used them to perform viable count assays using E.coli, P.aeruginosa and S.aureus. We investigated the mechanism of their antimicrobial activity by fluorescence and scanning electron microscopy, performed ELISA-style immunoassays and transmission electron microscopy to test for lipopolysaccharide binding and surface plasmon resonance to test for lipoteichoic acid (LTA) binding. We coated CLEC3A peptides on titanium, a commonly used prosthetic material, and performed fluorescence microscopy to quantify bacterial adhesion. Moreover, we assessed the peptides' cytotoxicity against primary human chondrocytes using MTT cell viability assays. RESULTS: CLEC3A fragments were detected in human cartilage extracts. Moreover, bacterial supernatants lead to fragmentation of recombinant and cartilage-derived CLEC3A. CLEC3A-derived peptides killed E.coli, P.aeruginosa and S.aureus, permeabilized bacterial membranes and bound lipopolysaccharide and LTA. Coating CLEC3A antimicrobial peptides (AMPs) on titanium lead to significantly reduced bacterial adhesion to the material. In addition, microbicidal concentrations of CLEC3A peptides in vitro displayed no direct cytotoxicity against primary human chondrocytes. CONCLUSIONS: We identify cartilage-specific AMPs originating from CLEC3A, resolve the mechanism of their antimicrobial activity and point to a novel approach in the prevention and treatment of septic arthritis using potent, non-toxic, AMPs.

Antibacterial and antibiofilm effects of sodium hypochlorite against Staphylococcus aureus isolates derived from patients with atopic dermatitis.

BACKGROUND: Atopic dermatitis (AD) is characterized by an increased susceptibility to skin infections. Staphylococcus aureus is reported to dominate in AD lesions and reports have revealed the presence of staphylococcal biofilms. These infections contribute to aggravation of the eczema. Sodium hypochlorite is known to reduce bacterial load of skin lesions, as well as disease severity, in patients with AD, but the effect on biofilms is unknown. OBJECTIVES: To investigate the antimicrobial and antibiofilm effects of sodium hypochlorite against S. aureus isolates derived from patients with AD. METHODS: Skin biopsies derived from patients with infected AD were examined by scanning electron microscopy (SEM). Using radial diffusion assays, biofilm assays and confocal laser scanning microscopy, we assessed the effect of sodium hypochlorite on S. aureus isolates derived from lesional skin of patients with AD. RESULTS: SEM revealed clusters of coccoid bacteria embedded in fibrin and extracellular substances at the skin of a patient with infected AD. At concentrations of 0·01-0·08%, sodium hypochlorite showed antibacterial effects against planktonic cells. Eradication of S. aureus biofilms in vitro was observed in concentrations ranging from 0·01% to 0·16%. Confocal laser scanning microscopy confirmed these results. Finally, when human AD skin was subjected to sodium hypochlorite in an ex vivo model, a dose of 0·04% reduced the bacteria derived from AD skin. CONCLUSIONS: Sodium hypochlorite has antimicrobial and antibiofilm effects against clinical S. aureus isolates. Our findings suggest usage of a higher concentration than currently used in bleach baths of patients with skin-infected AD.

Acetic acid as a decontamination method for sink drains in a nosocomial outbreak of metallo-ß-lactamase-producing Pseudomonas aeruginosa.

BACKGROUND: Pseudomonas aeruginosa may colonize water systems via biofilm formation. In hospital environments, contaminated sinks have been associated with nosocomial transmission. Here we describe a prolonged outbreak of a metallo-ß-lactamase-producing P. aeruginosa (Pae-MBL) associated with sink drains, and propose a previously unreported decontamination method with acetic acid. AIM: To describe a nosocomial outbreak of Pae-MBL associated with hospital sink drains and to evaluate acetic acid as a decontamination method. METHODS: The outbreak was investigated by searching the microbiology database, microbiological sampling and strain typing. Antibacterial and antibiofilm properties of acetic acid were evaluated in vitro. Pae-MBL-positive sinks were treated with 24% acetic acid once weekly and monitored with repeated cultures. FINDINGS: Fourteen patients with positive cultures for Pae-MBL were identified from 2008 to 2014. The patients had been admitted to three wards, where screening discovered Pae-MBL in 12 sink drains located in the patient bathrooms. Typing of clinical and sink drain isolates revealed identical or closely related strains. Pae-MBL biofilm was highly sensitive to acetic acid with a minimum biofilm eradication concentration of 0.75% (range: 0.19-1.5). Weekly treatment of colonized sink drains with acetic acid resulted in negative cultures and terminated transmission. CONCLUSION: Acetic acid is highly effective against Pae-MBL biofilms, and may be used as a simple method to decontaminate sink drains and to prevent nosocomial transmission.

Osteopontin binds and modulates functions of eosinophil-recruiting chemokines.

BACKGROUND: Allergic asthma is characterized by eosinophilic inflammation and airway obstruction. There is also an increased risk of pulmonary infection caused by Streptococcus pneumoniae, in particular during severe asthma where high levels of the glycoprotein, osteopontin (OPN), are present in the airways. Eosinophils can be recruited by chemokines activating the receptor CCR3 including eotaxin-1/CCL11, eotaxin-2/CCL24, eotaxin-3/CCL26, RANTES/CCL5, and MEC/CCL28. In addition to inducing chemotaxis, several of these molecules have defensin-like antibacterial properties. This study set out to elucidate the functional consequences of OPN binding to eosinophil-recruiting chemokines. METHODS: Antibacterial activities of the chemokines were investigated using viable count assays and electron microscopy. Binding studies were performed by means of surface plasmon resonance. The potential interference of OPN with antibacterial, receptor-activating, and lipopolysaccharide-neutralizing abilities of these chemokines was investigated. RESULTS: We found that OPN bound all eosinophil-recruiting chemokines with high affinity except for CCL5. The eosinophil-recruiting chemokines all displayed bactericidal activity against S. pneumoniae, but only CCL26 and CCL28 retained high antibacterial activity in the presence of sodium chloride at physiologic concentrations. Preincubation of the chemokines with OPN strongly inhibited their antibacterial activity against S. pneumoniae but did not affect their ability to activate CCR3. All chemokines investigated showed LPS-neutralizing activity that was impaired by OPN only in the case of CCL24. CONCLUSIONS: The data suggest that OPN may impair host defense activities of the chemokines without affecting their eosinophil-recruiting properties. This could be one mechanism explaining the increased vulnerability to acquire pneumococcal infection in parallel with sustained allergic inflammation in asthma.

The neutrophil-recruiting chemokine GCP-2/CXCL6 is expressed in cystic fibrosis airways and retains its functional properties after binding to extracellular DNA.

Infections in cystic fibrosis (CF), often involving Pseudomonas aeruginosa, result from a dysregulated airway immunity where one hallmark is the accumulation of necrotic and apoptotic immune cells, in particular neutrophils. In addition, neutrophils actively release DNA, forming neutrophil extracellular traps (NETs) that contain antimicrobial proteins. Altogether, free DNA in complex with actin accumulates in the airway lumen, resulting in highly viscous sputum that provides an anionic matrix, binding cationic antimicrobial proteins. In this study, granulocyte chemotactic protein 2 (GCP-2)/CXCL6, a neutrophil-activating chemokine with bactericidal properties, was detected in the airway epithelium of CF patients and was also present in azurophilic and specific granules of neutrophils. Elastase of neutrophils, but not of P. aeruginosa, completely degraded CXCL6 (chemokine (C-X-C motif) ligand 6). In addition, CXCL6 colocalized with extracellular DNA in both CF sputa and in in vitro-formed NETs. In vitro, CXCL6 bound DNA with a KD of 2,500 nM. Interestingly, both the bactericidal and the receptor-activating properties of CXCL6 (against neutrophils) remained largely unaffected in the presence of DNA. However, the chemotactic properties of CXCL6 were reduced by the presence of DNA. Taken together, CXCL6 is expressed in CF, retaining its functional properties even after binding to the anionic scaffold that extracellular DNA provides in CF.

Modulation of Hemostatic and Inflammatory Responses by Leptospira Spp

Leptospirosis is a worldwide spread zoonotic and neglected infectious disease of human and veterinary concern that is caused by pathogenic Leptospira species. In severe infections, hemostatic impairments such as coagulation/fibrinolysis dysfunction are frequently observed. These complications often occur when the host response is controlled and/or modulated by the bacterial pathogen. In the present investigation, we aimed to analyze the modulation of the hemostatic and inflammatory host responses by the bacterial pathogen Leptospira. The effects of leptospires and their secreted products on stimulation of human intrinsic and extrinsic pathways of coagulation were investigated by means of altered clotting times, assembly and activation of contact system and induction of tissue factor. We show that both extrinsic and intrinsic coagulation cascades are modulated in response to Leptospira or leptospiral secreted proteins. We further find that the pro-inflammatory mediator bradykinin is released following contact activation at the bacterial surface and that procoagulant microvesicles are shed from monocytes in response to infection. Also, we show that human leptospirosis patients present higher levels of circulating pro-coagulant microvesicles than healthy individuals. Here we show that both pathways of the coagulation system are modulated by leptospires, possibly leading to altered hemostatic and inflammatory responses during the disease. Our results contribute to the understanding of the leptospirosis pathophysiological mechanisms and may open new routes for the discovery of novel treatments for the severe manifestations of the disease

Eotaxin-3 (CCL26) exerts innate host defense activities that are modulated by mast cell proteases.

BACKGROUND: During bacterial infections of the airways, a Th1-profiled inflammation promotes the production of several host defense proteins and peptides with antibacterial activities including ß-defensins, ELR-negative CXC chemokines, and the cathelicidin LL-37. These are downregulated by Th2 cytokines of the allergic response. Instead, the eosinophil-recruiting chemokines eotaxin-1/CCL11, eotaxin-2/CCL24, and eotaxin-3/CCL26 are expressed. This study set out to investigate whether these chemokines could serve as innate host defense molecules during allergic inflammation. METHODS: Antibacterial activities of the eotaxins were investigated using viable count assays, electron microscopy, and methods assessing bacterial permeabilization. Fragments generated by mast cell proteases were characterized, and their potential antibacterial, receptor-activating, and lipopolysaccharide-neutralizing activities were investigated. RESULTS: CCL11, CCL24, and CCL26 all showed potent bactericidal activity, mediated through membrane disruption, against the airway pathogens Streptococcus pneumoniae, Staphylococcus aureus, Nontypeable Haemophilus influenzae, and Pseudomonas aeruginosa. CCL26 retained bactericidal activity in the presence of salt at physiologic concentrations, and the region holding the highest bactericidal activity was the cationic and amphipathic COOH-terminus. Proteolysis of CCL26 by chymase and tryptase, respectively, released distinct fragments of the COOH- and NH2 -terminal regions. The COOH-terminal fragment retained antibacterial activity while the NH2 -terminal had potent LPS-neutralizing properties in the order of CCL26 full-length protein. An identical fragment to NH2 -terminal fragment generated by tryptase was obtained after incubation with supernatants from activated mast cells. None of the fragments activated the CCR3-receptor. CONCLUSIONS: Taken together, the findings show that the eotaxins can contribute to host defense against common airway pathogens and that their activities are modulated by mast cell proteases.

Targeted mass spectrometry analysis of neutrophil-derived proteins released during sepsis progression.

Early diagnosis of severe infectious diseases is essential for timely implementation of lifesaving therapies. In a search for novel biomarkers in sepsis diagnosis we focused on polymorphonuclear neutrophils (PMNs). Notably, PMNs have their protein cargo readily stored in granules and following systemic stimulation, an immediate increase of neutrophil-borne proteins can be observed into the circulation of sepsis patients. We applied a combination of mass spectrometry (MS) based approaches, LC-MS/MS and selected reaction monitoring (SRM), to characterise and quantify the neutrophil proteome in healthy or disease conditions. With this approach we identified a neutrophil-derived protein abundance pattern in blood plasma consisting of 20 proteins that can be used as a protein signature for severe infectious diseases. Our results also show that SRM is highly sensitive, specific, and reproducible and, thus, a promising technology to study a complex, dynamic and multifactorial disease such as sepsis.

Antimicrobial activity of fibrinogen and fibrinogen-derived peptides--a novel link between coagulation and innate immunity.

Fibrinogen is a key player in the blood coagulation system, and is upon activation with thrombin converted into fibrin that subsequently forms a fibrin clot. In the present study, we investigated the role of fibrinogen in the early innate immune response. Here we show that the viability of fibrinogen-binding bacteria is affected in human plasma activated with thrombin. Moreover, we found that the peptide fragment GHR28 released from the ß-chain of fibrinogen has antimicrobial activity against bacteria that bind fibrinogen to their surface, whereas non-binding strains are unaffected. Notably, bacterial killing was detected in Group A Streptococcus bacteria entrapped in a fibrin clot, suggesting that fibrinogen and coagulation is involved in the early innate immune system to quickly wall off and neutralise invading pathogens.

Induction of anti-ß2 -glycoprotein I autoantibodies in mice by protein H of Streptococcus pyogenes.

BACKGROUND: The antiphospholipid syndrome (APS) is characterized by the persistent presence of anti-ß(2) -glycoprotein I (ß(2) -GPI) autoantibodies. ß(2) -GPI can exist in two conformations. In plasma it is a circular protein, whereas it adopts a fish-hook conformation after binding to phospholipids. Only the latter conformation is recognized by patient antibodies. ß(2) -GPI has been shown to interact with Streptococcus pyogenes. OBJECTIVE: To evaluate the potential of S. pyogenes-derived proteins to induce anti-ß(2) -GPI autoantibodies. METHODS AND RESULTS: Four S. pyogenes surface proteins (M1 protein, protein H, streptococcal collagen-like protein A [SclA], and streptococcal collagen-like protein B [SclB]) were found to interact with ß(2) -GPI. Only binding to protein H induces a conformational change in ß(2) -GPI, thereby exposing a cryptic epitope for APS-related autoantibodies. Mice were injected with the four proteins. Only mice injected with protein H developed antibodies against the patient antibody-related epitope in domain I of ß(2) -GPI. Patients with pharyngotonsillitis caused by S. pyogenes who developed anti-protein H antibodies also generated anti-ß(2) -GPI antibodies. CONCLUSIONS: Our study has demonstrated that a bacterial protein can induce a conformational change in ß(2) -GPI, resulting in the formation of antiß(2) -GPI autoantibodies. This constitutes a novel mechanism for the formation of anti-ß(2) -GPI autoantibodies.

Perfusion of human placenta with hemoglobin introduces preeclampsia-like injuries that are prevented by α1-microglobulin.

BACKGROUND: Preeclamptic women have increased plasma levels of free fetal hemoglobin (HbF), increased gene expression of placental HbF and accumulation of free HbF in the placental vascular lumen. Free hemoglobin (Hb) is pro-inflammatory, and causes oxidative stress and tissue damage. METHODOLOGY: To show the impact of free Hb in PE, we used the dual ex vivo placental perfusion model. Placentas were perfused with Hb and investigated for physical parameters, Hb leakage, gene expression and morphology. The protective effects of α(1)-microglobulin (A1M), a heme- and radical-scavenger and antioxidant, was investigated. RESULTS: Hb-addition into the fetal circulation led to a significant increase of the perfusion pressure and the feto-maternal leakage of free Hb. Morphological damages similar to the PE placentas were observed. Gene array showed up-regulation of genes related to immune response, apoptosis, and oxidative stress. Simultaneous addition of A1M to the maternal circulation inhibited the Hb leakage, morphological damage and gene up-regulation. Furthermore, perfusion with Hb and A1M induced a significant up-regulation of extracellular matrix genes. SIGNIFICANCE: The ex vivo Hb-perfusion of human placenta resulted in physiological and morphological changes and a gene expression profile similar to what is observed in PE placentas. These results underline the potentially important role of free Hb in PE etiology. The damaging effects were counteracted by A1M, suggesting a role of this protein as a new potential PE therapeutic agent.

Biofilm formation by Propionibacterium acnes is a characteristic of invasive isolates.

Propionibacterium acnes is a common and probably underestimated cause of delayed joint prosthesis infection. Bacterial biofilm formation is central in the pathogenesis of infections related to foreign material, and P. acnes has been shown to form biofilm both in vitro and in vivo. Here, biofilm formation by 93 P. acnes isolates, either from invasive infections (n = 45) or from the skin of healthy people (n = 48), was analysed. The majority of isolates from deep infections produced biofilm in a microtitre model of biofilm formation, whereas the skin isolates were poor biofilm producers (p <0.001 for a difference). This indicates a role for biofilm formation in P. acnes virulence. The type distribution, as determined by sequencing of recA, was similar among isolates isolated from skin and from deep infections, demonstrating that P. acnes isolates with different genetic backgrounds have pathogenic potential. The biofilm formed on plastic and on bone cement was analysed by scanning electron microscopy (EM) and by transmission EM. The biofilm was seen as a 10-mum-thick layer covering the bacteria and was composed of filamentous as well as more amorphous structures. Interestingly, the presence of human plasma in solution or at the plastic surface inhibits biofilm formation, which could explain why P. acnes primarily infect plasma-poor environments of, for example, joint prostheses and cerebrospinal shunts. This work underlines the importance of biofilm formation in P. acnes pathogenesis, and shows that biofilm formation should be considered in the diagnosis and treatment of invasive P. acnes infections.

The activation peptide of thrombin-activatable fibrinolysis inhibitor: a role in activity and stability of the enzyme?

BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a 56-kDa procarboxypeptidase. Proteolytic enzymes activate TAFI into TAFIa, an inhibitor of fibrinolysis, by cleaving off the N-terminal activation peptide (amino acids 1-92), from the enzyme moiety. Activated TAFI is unstable, with a half-life of approximately 10 min at 37 degrees C. So far, it is unknown whether the activation peptide is released or remains attached to the catalytic domain, and whether it influences TAFIa's properties. The current study was performed to clarify these issues. METHODS: TAFI was activated, and the activity and half-life of the enzyme were determined in the presence and absence of the activation peptide. RESULTS: TAFIa was active both before and after removal of the activation peptide, and the half-life of TAFIa was identical in the two preparations. Furthermore, we observed that intrinsically inactivated TAFIa (TAFIai) aggregated into large, insoluble complexes that could be removed by centrifugation. CONCLUSIONS: The data presented in this article show that the activation peptide of TAFI is not required for TAFIa activity and that the activation peptide has no effect on the stability of the enzyme. These results are in favour of a model in which the activation peptide solely stabilizes the structure of the proenzyme. After activation of TAFI and subsequent breakage of interactions between the activation peptide and the catalytic domain, the activation peptide is no longer capable of performing this stabilizing task, and the integrity of the catalytic domain is lost rapidly. The resulting TAFIai is more prone to proteolysis and aggregation.

Neutrophil degranulation mediates severe lung damage triggered by streptococcal M1 protein.

Streptococcus pyogenes of the M1 serotype can cause streptococcal toxic shock syndrome commonly associated with acute lung injury. The aim of the present study was to investigate the role of neutrophils and their secretion products in M1 protein-induced lung damage. The degranulation of neutrophils by M1 protein was studied in whole blood using marker analysis for individual granule subsets. In mice, M1 protein was injected intravenously and the lung damage was assessed by histology, electron microscopy, cell count in bronchoalveolar lavage fluid and analysis of lung vascular permeability. Comparisons were made in mice with intact white blood count, neutropenic mice and neutropenic mice injected with the secretion of activated neutrophils. In whole blood, M1 protein forms complexes with fibrinogen that bind to beta(2)-integrins on the neutrophil surface, resulting in degranulation of all four subsets of neutrophil granules. Intravenous injection of M1 protein into mice induced neutrophil accumulation in the lung, increase in vascular permeability and acute lung damage. Depletion of neutrophils from the circulation completely abrogated lung injury and vascular leakage. Interestingly, the lung damage was restored by injecting neutrophil secretion. The present data suggest that neutrophil granule proteins are directly responsible for lung damage induced by the streptococcal M1 protein.

Pseudomonas aeruginosa-induced infection and degradation of human wound fluid and skin proteins ex vivo are eradicated by a synthetic cationic polymer.

OBJECTIVES: Antimicrobial peptides are important effectors of innate immunity. Bacteria display multiple defence mechanisms against these peptides. For example, Pseudomonas aeruginosa releases potent proteinases that inactivate the human cathelicidin LL-37. Hence, in conditions characterized by persistent bacterial colonization, such as in P. aeruginosa-infected skin wounds, there is a need for efficient means of reducing bacterial load. Here, the effect of the cationic molecule polyhexamethylenebiguanide (PHMB) was evaluated. METHODS: Infection models in human wound fluid and human skin were established. Radial diffusion methods, bacterial growth and bactericidal assays were used for determination of effects of PHMB on bacteria in the presence of plasma, wound fluid or human skin. At the protein and tissue levels, SDS-PAGE, light microscopy and scanning electron microscopy were used to study the effects of P. aeruginosa infection before and after addition of PHMB. RESULTS: PHMB killed common ulcer-derived bacteria in the presence of human wound fluid. Furthermore, elastase-expressing P. aeruginosa completely degraded wound fluid proteins as well as human skin during infection ex vivo. The infection, and consequent protein degradation, was reversed by PHMB. CONCLUSIONS: The ex vivo infection models presented here should be helpful in the screening of novel antimicrobials and constitute a prerequisite for future clinical studies.

Interactions between surface proteins of Streptococcus pyogenes and coagulation factors modulate clotting of human plasma.

Invasive and toxic infections caused by Streptococcus pyogenes are connected with high morbidity and mortality. Typical symptoms of these infections are hypotension, edema formation, tissue necrosis, and bleeding disorders. Here we report that components of the coagulation system including fibrinogen, factors V, XI, and XII, and H-kininogen, are assembled at the surface of S. pyogenes through specific interactions with bacterial surface proteins. In plasma environment, absorption of fibrinogen by S. pyogenes causes a hypocoagulatory state resulting in prolonged clotting times and impaired fibrin network formation. Moreover, the binding of coagulation factors and the subsequent activation of the coagulation system at the bacterial surface lead to the formation of a fibrin network covering S. pyogenes bacteria adhering to epithelial cells. The results suggest that interactions between S. pyogenes and components of the coagulation system contribute to some of the symptoms seen in severe infections caused by this important human pathogen.

Association of chondroadherin with collagen type II.

Chondroadherin is a cell binding, leucine-rich repeat protein found in the territorial matrix of articular cartilage. Several members of the leucine-rich repeat protein family present in the extracellular matrix of e.g. cartilage have been shown to interact with collagen and influence collagen fibrillogenesis. We show that complexes of monomeric collagen type II and chondroadherin can be released under non-denaturing conditions from articular cartilage treated with p-aminophenylmercuric acetate to activate resident matrix metalloproteinases. Purified complexes as well as complexes formed in vitro between recombinant chondroadherin and collagen type II were studied by electron microscopy. Chondroadherin was shown to bind to two sites on collagen type II. The interaction was characterized by surface plasmon resonance analysis showing K(D) values in the nanomolar range. Both chondroadherin and collagen interact with chondrocytes, partly via the same receptor, but give rise to different cellular responses. By also interacting with each other, a complex system is created which may be of functional importance for the communication between the cells and its surrounding matrix and/or in the regulation of collagen fibril assembly.

Biglycan and decorin bind close to the n-terminal region of the collagen VI triple helix.

The binding of native biglycan and decorin to pepsin-extracted collagen VI from human placenta was examined by solid phase assay and by measurement of surface plasmon resonance in the BIAcore(TM)2000 system. Both proteoglycans exhibited a strong affinity for collagen VI with dissociation constants (K(D)) of approximately 30 nm. Removal of the glycosaminoglycan chains by chondroitinase ABC digestion did not significantly affect binding. In coprecipitation experiments, biglycan and decorin bound to collagen VI and equally competed with the other, suggesting that biglycan and decorin bind to the same binding site on collagen VI. This was confirmed by electron microscopy after negative staining of complexes between gold-labeled proteoglycans and collagen VI, demonstrating that both biglycan and decorin bound exclusively to a domain close to the interface between the N terminus of the triple helical region and the following globular domain. In solid phase assay using recombinant collagen VI fragments, it was shown that the alpha2(VI) chain probably plays a role in the interaction.

Zinc-dependent conformational changes in domain D5 of high molecular mass kininogen modulate contact activation.

Human high molecular mass kininogen (HK) participates as nonenzymatic cofactor in the contact system. Here, we show that recombinant domain D5 of HK (rD5) prolongs the clotting time of the intrinsic pathway of coagulation and attenuates the generation of bradykinin. Further studies indicate that a correct fold of domain D5 within HK is required for the activation of the contact system. The folding of rD5 seems to be modulated by the metal ions Zn2+, Ni2+, and Cu2+ as a specific antibody directed against the zinc-binding site in HK binds to HK and rD5 in a metal ion concentration dependent manner. The finding that these three metal ions specifically affect contact activation suggests that they regulate the accessibility of rD5 for negatively charged surfaces. Support for the assumption that the observed phenomena are due to conformational changes was obtained by fluorescence spectroscopy of rD5, demonstrating that its fluorescence spectrum was changed in the presence of ZnCl2. Moreover, negative staining electron microscopy experiments suggest that the zinc-induced changes in D5 also affect the conformation of the entire HK protein. The present data emphasize the role of zinc and other metal ions in the regulation of contact activation.

The N-terminal N5 subdomain of the alpha 3(VI) chain is important for collagen VI microfibril formation.

Collagen VI assembly is unique within the collagen superfamily in that the alpha 1(VI), alpha 2(VI), and alpha 3(VI) chains associate intracellularly to form triple helical monomers, and then dimers and tetramers, which are secreted from the cell. Secreted tetramers associate end-to-end to form the distinctive extracellular microfibrils that are found in virtually all connective tissues. Although the precise protein interactions involved in this process are unknown, the N-terminal globular regions, which are composed of multiple copies of von Willebrand factor type A-like domains, are likely to play a critical role in microfibril formation, because they are exposed at both ends of the tetramers. To explore the role of these subdomains in collagen VI intracellular and extracellular assembly, alpha 3(VI) cDNA expression constructs with sequential N-terminal deletions were stably transfected into SaOS-2 cells, producing cell lines that express alpha 3(VI) chains with N-terminal globular domains containing modules N9-N1, N6-N1, N5-N1, N4-N1, N3-N1, or N1, as well as the complete triple helix and C-terminal globular domain (C1-C5). All of these transfected alpha 3(VI) chains were able to associate with endogenous alpha 1(VI) and alpha 2(VI) to form collagen VI monomers, dimers, and tetramers, which were secreted. Importantly, cells that expressed alpha 3(VI) chains containing the N5 subdomain, alpha 3(VI) N9-C5, N6-C5, and N5-C5, formed microfibrils and deposited a collagen VI matrix. In contrast, cells that expressed the shorter alpha 3(VI) chains, N4-C5, N3-C5, and N1-C5, were severely compromised in their ability to form end-to-end tetramer assemblies and failed to deposit a collagen VI matrix. These data demonstrate that the alpha 3(VI) N5 module is critical for microfibril formation, thus identifying a functional role for a specific type A subdomain in collagen VI assembly.