Chemokine CXCL7 Heterodimers: Structural Insights, CXCR2 Receptor Function, and Glycosaminoglycan Interactions.

Chemokines mediate diverse fundamental biological processes, including combating infection. Multiple chemokines are expressed at the site of infection; thus chemokine synergy by heterodimer formation may play a role in determining function. Chemokine function involves interactions with G-protein-coupled receptors and sulfated glycosaminoglycans (GAG). However, very little is known regarding heterodimer structural features and receptor and GAG interactions. Solution nuclear magnetic resonance (NMR) and molecular dynamics characterization of platelet-derived chemokine CXCL7 heterodimerization with chemokines CXCL1, CXCL4, and CXCL8 indicated that packing interactions promote CXCL7-CXCL1 and CXCL7-CXCL4 heterodimers, and electrostatic repulsive interactions disfavor the CXCL7-CXCL8 heterodimer. As characterizing the native heterodimer is challenging due to interference from monomers and homodimers, we engineered a "trapped" disulfide-linked CXCL7-CXCL1 heterodimer. NMR and modeling studies indicated that GAG heparin binding to the heterodimer is distinctly different from the CXCL7 monomer and that the GAG-bound heterodimer is unlikely to bind the receptor. Interestingly, the trapped heterodimer was highly active in a Ca2+ release assay. These data collectively suggest that GAG interactions play a prominent role in determining heterodimer function in vivo. Further, this study provides proof-of-concept that the disulfide trapping strategy can serve as a valuable tool for characterizing the structural and functional features of a chemokine heterodimer.

Structural Basis of Native CXCL7 Monomer Binding to CXCR2 Receptor N-Domain and Glycosaminoglycan Heparin.

CXCL7, a chemokine highly expressed in platelets, orchestrates neutrophil recruitment during thrombosis and related pathophysiological processes by interacting with CXCR2 receptor and sulfated glycosaminoglycans (GAG). CXCL7 exists as monomers and dimers, and dimerization (~50 µM) and CXCR2 binding (~10 nM) constants indicate that CXCL7 is a potent agonist as a monomer. Currently, nothing is known regarding the structural basis by which receptor and GAG interactions mediate CXCL7 function. Using solution nuclear magnetic resonance (NMR) spectroscopy, we characterized the binding of CXCL7 monomer to the CXCR2 N-terminal domain (CXCR2Nd) that constitutes a critical docking site and to GAG heparin. We found that CXCR2Nd binds a hydrophobic groove and that ionic interactions also play a role in mediating binding. Heparin binds a set of contiguous basic residues indicating a prominent role for ionic interactions. Modeling studies reveal that the binding interface is dynamic and that GAG adopts different binding geometries. Most importantly, several residues involved in GAG binding are also involved in receptor interactions, suggesting that GAG-bound monomer cannot activate the receptor. Further, this is the first study that describes the structural basis of receptor and GAG interactions of a native monomer of the neutrophil-activating chemokine family.

Dynamics and thermodynamic properties of CXCL7 chemokine.

Chemokines form a family of signaling proteins mainly responsible for directing the traffic of leukocytes, where their biological activity can be modulated by their oligomerization state. We characterize the dynamics and thermodynamic stability of monomer and homodimer structures of CXCL7, one of the most abundant platelet chemokines, using experimental methods that include circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy, and computational methods that include the anisotropic network model (ANM), molecular dynamics (MD) simulations and the distance constraint model (DCM). A consistent picture emerges for the effects of dimerization and Cys5-Cys31 and Cys7-Cys47 disulfide bonds formation. The presence of disulfide bonds is not critical for maintaining structural stability in the monomer or dimer, but the monomer is destabilized more than the dimer upon removal of disulfide bonds. Disulfide bonds play a key role in shaping the characteristics of native state dynamics. The combined analysis shows that upon dimerization flexibly correlated motions are induced between the 30s and 50s loop within each monomer and across the dimer interface. Interestingly, the greatest gain in flexibility upon dimerization occurs when both disulfide bonds are present, and the homodimer is least stable relative to its two monomers. These results suggest that the highly conserved disulfide bonds in chemokines facilitate a structural mechanism that is tuned to optimally distinguish functional characteristics between monomer and dimer.

A peptide inhibitor of cytomegalovirus infection from human hemofiltrate.

Naturally occurring substances with antimicrobial activity can serve as a starting point for the rational design of new drugs to treat infectious diseases. Here, we screened a library of peptides derived from human hemofiltrate for inhibitory effects on human cytomegalovirus (CMV) infection. We isolated a previously unknown derivative of the neutrophil-activating peptide 2, which we termed CYVIP, for CMV-inhibiting peptide. The peptide blocked infection with human and mouse CMV as well as with herpes simplex virus type 1 in different cell types. We found that CYVIP interferes with virus attachment to the cell surface, and structure-activity relationship studies revealed that positively charged lysine and arginine residues of CYVIP are essential for its inhibitory activity. The N-terminal 29 amino acids of the peptide were sufficient for inhibition, and substitution with an acidic residue further improved its activity. The target structure of CYVIP on the cell surface seems to be the sulfate residues of heparan sulfate proteoglycans, which are known to serve as herpesvirus attachment receptors. Our data suggest that O-sulfation of heparan sulfate is required for binding of CYVIP, and furthermore, that the initial interaction of CMV particles with cells takes place preferentially via 6-O-linked sulfate groups. These findings about CYVIP's mode of action lay the basis for further development of antivirals interfering with attachment of CMV to cells, a crucial step of the infection cycle.

Exploring platelet chemokine antimicrobial activity: nuclear magnetic resonance backbone dynamics of NAP-2 and TC-1.

The platelet chemokines neutrophil-activating peptide-2 (NAP-2) and thrombocidin-1 (TC-1) differ by only two amino acids at their carboxy-terminal ends. Nevertheless, they display a significant difference in their direct antimicrobial activities, with the longer NAP-2 being inactive and TC-1 being active. In an attempt to rationalize this difference in activity, we studied the structure and the dynamics of both proteins by nuclear magnetic resonance (NMR) spectroscopy. Using 15N isotope-labeled protein, we confirmed that the two monomeric proteins essentially have the same overall structure in aqueous solution. However, NMR relaxation measurements provided evidence that the negatively charged carboxy-terminal residues of NAP-2 experience a restricted motion, whereas the carboxy-terminal end of TC-1 moves in an unrestricted manner. The same behavior was also seen in molecular dynamic simulations of both proteins. Detailed analysis of the protein motions through model-free analysis, as well as a determination of their overall correlation times, provided evidence for the existence of a monomer-dimer equilibrium in solution, which seemed to be more prevalent for TC-1. This finding was supported by diffusion NMR experiments. Dimerization generates a larger cationic surface area that would increase the antimicrobial activities of these chemokines. Moreover, these data also show that the negatively charged carboxy-terminal end of NAP-2 (which is absent in TC-1) folds back over part of the positively charged helical region of the protein and, in doing so, interferes with the direct antimicrobial activity.

Influence of nanoporesize on platelet adhesion and activation.

In this study we have evaluated the influence of biomaterial nano-topography on platelet adhesion and activation. Nano-porous alumina membranes with pore diameters of 20 and 200 nm were incubated with whole blood and platelet rich plasma. Platelet number, adhesion and activation were determined by using a coulter hematology analyzer, scanning electron microscopy, immunocytochemical staining in combination with light microscopy and by enzyme immunoassay. Special attention was paid to cell morphology, microparticle generation, P-selectin expression and beta-TG production. Very few platelets were found on the 200 nm alumina as compared to the 20 nm membrane. The platelets found on the 20 nm membrane showed signs of activation such as spread morphology and protruding filipodia as well as P-selectin expression. However no microparticles were detected on this surface. Despite the fact that very few platelets were found on the 200 nm alumina in contrast to the 20 nm membrane many microparticles were detected on this surface. Interestingly, all microparticles were found inside circular shaped areas of approximately 3 mum in diameter. Since this is the approximate size of a platelet we speculate that this is evidence of transient, non-adherent platelet contact with the surface, which has triggered platelet microparticle generation. To the authors knowledge, this is the first study that demonstrates how nanotexture can influence platelet microparticle generation. The study highlights the importance of understanding molecular and cellular events on nano-level when designing new biomaterials.

The human CXC chemokine granulocyte chemotactic protein 2 (GCP-2)/CXCL6 possesses membrane-disrupting properties and is antibacterial.

Granulocyte chemotactic protein 2 (GCP-2)/CXCL6 is a CXC chemokine expressed by macrophages and epithelial and mesenchymal cells during inflammation. Through binding and activation of its receptors (CXCR1 and CXCR2), it exerts neutrophil-activating and angiogenic activities. Here we show that GCP-2/CXCL6 itself is antibacterial. Antibacterial activity against gram-positive and gram-negative pathogenic bacteria of relevance to mucosal infections was seen at submicromolar concentrations (minimal bactericidal concentration at which 50% of strains tested were killed, 0.063 +/- 0.01 to 0.37 +/- 0.03 muM). In killed bacteria, GCP-2/CXCL6 associated with bacterial surfaces, which showed membrane disruption and leakage. A structural prediction indicated the presence of three antiparallel NH(2)-terminal beta-sheets and a short amphipathic COOH-terminal alpha-helix; the latter feature is typical of antimicrobial peptides. However, when the synthetic derivatives corresponding to the NH(2)-terminal (50 amino acids) and COOH-terminal (19 amino acids, corresponding to the putative alpha-helix) regions were compared, higher antibacterial activity was observed for the NH(2)-terminus-derived peptide, indicating that the holopeptide is necessary for full antibacterial activity. An artificial model of bacterial membranes confirmed these findings. The helical content of GCP-2/CXCL6 in the presence or absence of lipopolysaccharide or negatively charged membranes was studied by circular dichroism. As with many antibacterial peptides, membrane disruption by GCP-2/CXCL6 was dose-dependently reduced in the presence of NaCl, which, we here demonstrate, inhibited the binding of the peptide to the bacterial surface. Compared with CXC chemokines ENA-78/CXCL5 and NAP-2/CXCL7, GCP-2/CXCL6 showed a 90-fold-higher antibacterial activity. Taken together, GCP/CXCL6, in addition to its chemotactic and angiogenic properties, is likely to contribute to direct antibacterial activity during localized infection.

A synthetic congener modeled on a microbicidal domain of thrombin- induced platelet microbicidal protein 1 recapitulates staphylocidal mechanisms of the native molecule.

Thrombin-induced platelet microbicidal protein 1 (tPMP-1) is a staphylocidal peptide released by activated platelets. This peptide initiates its microbicidal activity by membrane permeabilization, with ensuing inhibition of intracellular macromolecular synthesis. RP-1 is a synthetic congener modeled on the C-terminal microbicidal alpha-helix of tPMP-1. This study compared the staphylocidal mechanisms of RP-1 with those of tPMP-1, focusing on isogenic tPMP-1-susceptible (ISP479C) and -resistant (ISP479R) Staphylococcus aureus strains for the following quantitative evaluations: staphylocidal efficacy; comparative MIC; membrane permeabilization (MP) and depolarization; and DNA, RNA, and protein synthesis. Although the proteins had similar MICs, RP-1 caused significant killing of ISP479C (<50% survival), correlating with extensive MP (>95%) and inhibition of DNA and RNA synthesis (>90%), versus substantially reduced killing of ISP479R (>80% survival), with less MP (55%) and less inhibition of DNA or RNA synthesis (70 to 80%). Interestingly, RP-1-induced protein synthesis inhibition was equivalent in both strains. RP-1 did not depolarize the cell membrane and caused a relatively short postexposure growth inhibition. These data closely parallel those previously reported for tPMP-1 against this strain set and exemplify how synthetic molecules can be engineered to reflect structure-activity relationships of functional domains in native host defense effector molecules.

Quality assessment of heparin coatings by their binding capacities of coagulation and complement enzymes.

In vitro testing of blood contacting materials before clinical application is generally advisable. Four heparin coatings from different manufacturers were tested for adsorbed proteins and soluble activation markers. The surface with the highest antithrombin, thrombin, high-molecular-weight-kininogen (HMWK) and the lowest fibrinogen binding capacity (Carmeda, Medtronic) showed significantly lower levels of granulocytes and platelet activation (beta-TG, PMN-elastase release). No statistically significant differences in soluble markers of the coagulation system could be detected (F1 + 2, TAT). Interestingly, complement activation (TCC) was significantly reduced within the group of the lowest adsorption of the complement factor C3. Our data demonstrate that there is a relation between the binding affinity of proteins (C1-inhibitor, C3-complement) and the consecutive changes in complement activation (TCC). Therefore, measuring adsorbed proteins on artificial surfaces is a suitable, sensitive and very reproducible method for assessing the thrombogenicity of biomaterials.

Novel C-terminally truncated isoforms of the CXC chemokine beta-thromboglobulin and their impact on neutrophil functions.

The neutrophil agonist neutrophil-activating peptide-2 (NAP-2) arises through proteolytic processing of platelet-derived N-terminally extended inactive precursors, the most abundant one being connective tissue-activating peptide-III (CTAP-III). Apart from N-terminal processing, C-terminal processing also appears to participate in the functional regulation of NAP-2, as indicated by our recent identification of an isoform missing four C-terminal amino acids, NAP-2 (1-66), which was about threefold more potent than full-size NAP-2. In the present study, we report on the discovery and identification of natural NAP-2 (1-63), an isoform truncated by seven C-terminal residues. Functional and receptor-binding analyses demonstrated that NAP-2 (1-63) represents the most active isoform, being about fivefold more potent than full-size NAP-2. Analyses of rNAP-2 isoforms successively truncated at the C terminus by up to eight residues suggest functionally important roles for acidic residues and for the leucine at position 63, a residue highly conserved within CXC chemokines. Finally, we report on a novel C-terminally truncated isoform of CTAP-III (CTAP-III (1-81)) representing the potential precursor of NAP-2 (1-66). We show that C-terminal truncation in CTAP-III enhances its potency to desensitize chemokine-induced neutrophil activation, indicating that C-terminal processing might not only serve to enhance neutrophil activation, but might as well participate in the down-regulation of an inflammatory response.

Fibronectin, platelet factor 4 and beta-thromboglobulin in endemic hepatosplenic schistosomiasis: relation to acute hematemesis.

Some platelet alpha-granule contents were assessed in parallel with other markers of hemostatic imbalance in 50 patients with hepatosplenic schistosomiasis (15 patients with compensated hepatosplenomegaly, 15 patients with advanced hepatic fibrosis and ascites and 20 patients during an acute attack of hematemesis from ruptured esophageal varices). Platelet factor 4 (PF4), beta-thromboglobulin (beta-TG), fibronectin (FN), prothrombin fragment 1 + 2, thrombin-antithrombin (TAT) complexes, fibrin degradation products (FbDP) and D-dimer were assessed in schistosomal patients compared to controls (15 healthy subjects). A significant increase in both thrombin (high TAT and prothrombin fragment 1 + 2 levels) and plasmin (high FbDP and D-dimer levels) generation was detected in decompensated patients establishing the presence of a steady state of low-grade disseminated intravascular coagulation, with and without overt bleeding, in these patients. A decrease in plasma FN concentration was found in diseased groups compared to controls. The reduction in plasma levels of FN paralleled the defective liver function and matched the relative decrease in tissue FN in liver specimens of decompensated patients suggesting that FN levels can be used to evaluate the pathological staging of the disease. A significant increase in beta-TG and PF4 levels was noted in decompensated patients with ascites and/or acute hematemesis compared both to controls and compensated patients reflecting platelet alpha-granule release and consequently increased in vivo platelet activation which may initiate and/or perpetuate the pathophysiological mechanisms of the hemostatic imbalance underlying the hemorrhagic diathesis in hepatosplenic schistosomiasis.

Pre-adsorption of a cellulose ether onto polymer surfaces: adsorption of adhesins and platelet activation.

The effect of pre-adsorbed cellulose ethers upon the adsorption of plasma proteins at polymer surfaces was measured by an enzyme-linked immunosorbent assay. The resulting adsorption of plasma proteins was correlated with the sticking and activation of platelets at the blood-material interface, measured by scanning electron microscopy and release of beta-thromboglobulin. Retained Ca2+ activity was made possible by hirudin, a specific thrombin inhibitor, from medicinal leeches as an anticoagulant drug, thereby keeping the initial coagulation cascade and calcium-dependent cell-protein interactions intact during the experiments. The results show that pre-adsorption of a hydrophobic cellulose ether with a flocculation temperature < 37 degrees C, on polyurethane and polytetrafluoroethylene, decreased the adsorption of fibrinogen, fibronectin and vitronectin. A corresponding decrease was found in the number of cells sticking to the surface and in the release of beta-thromboglobulin from platelets.

[Nonparametric normal range for thrombocyte parameters in childhood]. / Nichtparametrischer Normalbereich für thrombozytäre Parameter im Kindesalter.

In order to establish an univariate nonparametric pediatric tolerance region platelet function has been investigated in 105 healthy children and adolescents. In comparison to adult normal values, the bleeding time is shortened, spontaneous platelet aggregation is enhanced as well as collagen-induced platelet aggregation. 30% of the children showed an increased disaggregation in ADP-induced aggregation. A slight delay was found in the spreading of thrombocytes. Platelet volume shifted to the left. Values of beta-thromboglobulin were raised. Compared to adult values no alterations could be found in platelet shape-change. Changes of platelet functions were more apparent in the younger children.

Effect of ritanserin, a 5-hydroxytryptamine2-receptor antagonist, on platelet function and thrombin generation at the site of plug formation in vivo.

To investigate the role of serotonin in platelet plug formation we studied, in eight healthy volunteers, the effect of ritanserin (a 5-hydroxytryptamine2-receptor antagonist) on the platelet release reaction (represented by beta-thromboglobulin release) platelet prostaglandin metabolism (represented by thromboxane B2 formation), and thrombin generation (represented by fibrinopeptide A formation) in the microvasculature. After administration of ritanserin lower amounts of thromboxane B1 were generated in the initial stages of plug formation, suggesting an inhibitory effect on the platelet prostaglandin metabolism. Similar amounts of beta-thromboglobulin were released after the administration of ritanserin compared with placebo, indicating a minor effect of ritanserin on the release reaction. Reduction of thrombin formation by ritanserin in the later stages of hemostasis suggested an inhibitory effect of this substance on the procoagulatory activity of platelets or endothelial cells. This could be attributable to interference with the formation or function of coagulation factor complexes on cell surfaces, or it could be the consequence of a reduction of the platelet activity.