Platelet Basic Protein Research Articles

Down-regulation of neutrophil functions by the ELR+CXC chemokine platelet basic protein

The platelet-derived neutrophil-activating peptide 2 (NAP-2, 70 amino acids) belongs to the ELR+ CXC subfamily of chemokines. Similar to other members of this group, such as IL-8, NAP-2 activates chemotaxis and degranulation in neutrophils (polymorphonuclear [PMN]) through chemokine receptors CXCR-1 and CXCR-2. However, platelets do not secrete NAP-2 as an active chemokine but as the C-terminal part of several precursors that lack PMN-stimulating capacity. As we have previously shown, PMN themselves may liberate NAP-2 from the precursor connective tissue-activating peptide III (CTAP-III, 85 amino acids) by proteolysis. Instead of inducing cell activation, continuous accumulation of the chemokine in the surroundings of the processing cells results in the down-regulation of specific surface-expressed NAP-2 binding sites and in the desensitization of chemokine-induced PMN degranulation. Thus, NAP-2 precursors may be regarded as indirect mediators of functional desensitization in neutrophils. In the current study we investigated the biologic impact of another major NAP-2 precursor, the platelet basic protein (PBP, 94 amino acids). We show that PBP is considerably more potent than CTAP-III to desensitize degranulation and chemotaxis in neutrophils. We present data suggesting that the high desensitizing capacity of PBP is based on its enhanced proteolytic cleavage into NAP-2 by neutrophil-expressed cathepsin G and that it involves efficient down-regulation of surface-expressed CXCR-2 while CXCR-1 is hardly affected. Correspondingly, we found PBP and, less potently, CTAP-III to inhibit CXCR-2– but not CXCR-1– dependent chemotaxis of neutrophils toward NAP-2. Altogether our findings demonstrate that the anti-inflammatory capacity of NAP-2 is governed by the species of its precursors.

Megakaryocyte maturation is associated with expression of the cxc chemokine connective tissue activating peptide ctap III

Abstract Connective tissue activating peptide III (CTAP III) is a CXC chemokine, derived from platelet basic protein (PBP), which has been identified in platelets, activated macrophages, neutrophils and in T lymphocytes. CTAP III can support stem cell derived hematopoiesis yet clearly inhibits the proliferation of committed megakaryocytes (MK) progenitors. This investigation was aimed at characterizing CTAP III in human MK and determining it association with proliferation and differentiation. We report high expression of CTAP III in mature MK from normal human bone marrow (BM) and from BM of patients with immune thrombocytopenic purpura (ITP) or chronic myeloid leukemia (CML) and in ex-vivo expanded MK. Immunostaining with anti-CTAPIII antibodies demonstrated its prominent presence in platelet producing like zones within the cytoplasm and intense staining around the periphery of the large mature MK. In logarithmically growing cell lines DAMI, CHRF-288 or MEG01 megakaryoblasts only 15% of the cells, which were primarily large cells (>30 μM), expressed CTAPIII. The addition of phorbol ester PMA, which causes proliferation arrest and differentiation induction, stimulated high levels of CTAPIII expression after 24 hours in more than 75% of the cells, being expressed in both the small cells and the large cells. RT-PCR reveled upregulation of CTAPIII mRNA after one hour of exposure to PMA which was sustained for 24 hours. While the biological function of CTAPIII in MK is not known, these studies demonstrate that molecular upregulation of CTAP III in MK is associated with maturation and like other chemokines may be involved in proliferation arrest and cellular interactions with extracellular matrix.

Activation of the Megakaryocyte-specific Gene Platelet Basic Protein (PBP) by the Ets Family Factor PU.1

Platelet basic protein (PBP) is a chemokine family member that is only found in platelets and their precursors megakaryocytes. The PBP gene is physically linked to the gene for another platelet-specific chemokine, platelet factor 4. While the biological basis of platelet factor 4 expression has been pursued by others, the regulatory features controlling the platelet-specific expression of PBP have not been investigated. In this article, we examined the molecular basis by which this megakaryocyte-specific gene is regulated. Transient expression studies of truncated reporter constructs containing from 4.5 to 0.1 kilobases of the functional PBP gene 5'-flanking region, demonstrated that the proximal 0.1 kilobases of the promoter was sufficient for high levels of expression in human erythroleukemia and CHRF-288 cells, two megakaryocytic cell lines. However, none of these constructs was expressed above background levels in HeLa and 293 cells, two non-megakaryocytic cell lines. Further truncation of this promoter suggested that there was an important regulatory element(s) within a pyrimidine-rich tract. Mobility shift analysis of the pyrimidine-rich tract defined a region between -85 and -64 which bound to a nuclear factor(s). This region contains sequences matching the consensus Ets-binding site from -78 to -75 base pairs. In particular, we noted that this site matched a PU.1 consensus sequence known as a PU box. Mobility shift and supershift studies with nuclear extracts as well as recombinant PU.1 protein and anti-PU.1 antibody further confirmed that PU.1 was the specific Ets family factor that bound to this site. Transient expression assays using reporter constructs which contained point mutations that abrogated PU.1 binding also significantly reduced PBP promoter activity in human erythroleukemia and CHRF cells. In addition, while all reporter gene constructs containing PBP promoters were completely inactive in HeLa cells, transactivation experiments using a PU.1 expression construct demonstrated that exogenous expression of PU.1 could increase reporter gene expression up to 8-fold in these cells. Finally, the role of PU.1 in PBP gene expression was compared between wild-type and PU.1-null embryonic stem (ES) cells that were differentiated in vitro into cells that resembled megakaryocytes both morphologically and immunologically. We found that PBP gene expression in the differentiated PU.1(-/-) null ES cells (as determined by semi-quantitative reverse transcriptase-polymerase chain reaction) was more than four times lower than that in the wild-type ES cells, while other platelet-specific genes were expressed equally or similarly in the two ES cell lines. Previous reports have shown that PU.1 is expressed in several hematopoietic lineages, including megakaryocytes. However, the functional role of PU.1 has only been previously demonstrated in the myeloid and lymphoid lineages. Therefore, our studies are the first to show the biological importance of this nuclear factor in the regulated expression of a megakaryocyte-specific gene.

The amino-terminal residues in the crystal structure of connective tissue activating peptide-III (des10) block the ELR chemotactic sequence

α-Chemokines comprise a family of cytokines that are chemotactic for neutrophils and have a structure similar to platelet factor 4 (PF4), in which the first two cysteine residues are separated by one residue (Cys-X-Cys). The two α-chemokines, connective tissue activating peptide-III (CTAP-III) and neutrophil activating peptide-2 (NAP-2), are carboxyl-terminal fragments of platelet basic protein (PBP) that are generated by monocyte-derived proteases. NAP-2 strongly stimulates neutrophils that are present during inflammation whereas its precursors, PBP and CTAP-III, are inactive, although they also possess the highly conserved, amino-terminal sequence, Glu-Leu-Arg (ELR), that is critical for receptor binding. To resolve this conundrum, we have determined the crystal structure of recombinant Asp-CTAP, which has ten fewer amino-terminal residues than CTAP-III but five more than NAP-2. The space group is P 21, with unit cell dimensions a=43.8 Å, b=76.8 Å, c=43.8 Å, and β =97.0°, and a tetramer in the asymmetric unit. The molecular replacement method, with the NAP-2 tetramer as a starting model, was used to determine the initial phase information. The final R -factor is 0.196 (Rfree=0.251) for 2σ data from 7.0 to 1.75 Å resolution. This high-resolution model of Asp-CTAP is the longest defined structure of an α-chemokine to date. The electron density map shows an over-all structure for Asp-CTAP that is very similar to that of NAP-2, but with the additional five amino-terminal residues folding back through a type-II turn, thereby stabilizing the oligomeric “inactive” state, and masking the critical ELR receptor binding region that is exposed in the structure of NAP-2.

Regulation of neutrophil activation by proteolytic processing of platelet-derived alpha-chemokines.

In recent years evidence has been accumulated that platelets besides their function in coagulation play an important role in inflammation and wound repair. Upon activation platelets release a variety of mediators, among which members of the α-chemokine subfamily of proinflammatory cytokines have been identified. These platelet-derived polypeptides do not only comprise members of the so-called ß-thromboglobulin family, such as platelet basic protein (PBP), connective tissue-activating peptide III (CTAP-III), and neutrophil-activating peptide 2 (NAP-2)1, but also platelet factor 4 (PF4)2,3 and the ß-chemokine RANTES (Regulated upon activation normal T cell expressed and probably secreted)4. While ß-chemokines have been shown to activate monocytes, T lymphocytes and eosinophils, α-chemokines such as IL-8, NAP-2 and melanoma growth-stimulating activity (MGSA/gro-α) appear to represent rather selective activators of polymorphonuclear leukocytes (PMN)5. Importantly, their biological activity, such as chemotaxis and degranulation-inducing capacity, has been demonstrated to be closely connected with the presence of an N-terminal glutamic acid-leucine-arginine (ELR) motif. Only recently, attention has been paid to the regulatory properties of α-chemokines. In the present article we will focus on two aspects of regulation of PMN functions, namely 1. the proteolytic processing of platelet-derived α-chemokines as a regulatory event in the induction and modulation of PMN activation, and 2. the phenotypic and functional consequences for the PMN under the constraints of such regulatory events.KeywordsProteolytic ProcessingNeutrophil ActivationPlatelet FactorInactive PrecursorElastase ReleaseThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Structure and bioactivity of recombinant human CTAP-III and NAP-2.

Connective tissue-activating peptide III (CTAP-III) and neutrophil-activating peptide-2 (NAP-2) are both derived from a common precursor, platelet basic protein (PBP), which is stored in the alpha-granules of platelets and released upon their activation. CTAP-III is an 85-residue peptide which is converted to NAP-2 by enzymic removal of the 15 amino-terminal residues. Both peptides play a role in the early stages of wound healing and inflammation through different activities. We have cloned the cDNA for PBP and expressed constructs coding for the CTAP-III and NAP-2 polypeptides in Escherichia coli. We have purified and renatured these recombinant proteins. The integrity of the recombinant proteins has been ascertained by in vitro bioassays. CTAP-III causes 51% histamine release from the basophilic cell lin KU812 at 10(-7) M, whereas NAP-2 only causes 28% release at the same concentration. In assays on human neutrophils, NAP-2 had an EC50 of 2 x 10(-8) M in chemotaxis, an EC50 of 3 x 10(-8) M for shape change, and could displace IL-8 from neutrophils with a Kd of 7.5 x 10(-9) M. CTAP-III had no activity in these assays. The disulfide bonds have been identified by peptide mapping and sequence analysis, and are in the positions predicted by homology to interleukin-8 and platelet factor 4. Measurement of the molecular mass at physiologic concentrations by gel permeation chromatography has shown that CTAP-III forms predominantly tetramers and dimers, whereas NAP-2 is only dimetric. SDS/PAGE analysis of samples cross-linked with disuccinimidyl suberate support these topologies. We postulate a mechanism for tetramer formation based on the interaction of the amino-terminal extension in CTAP-III involving a helix-helix interaction that could stabilize the association of two CTAP-III dimers.

Isolation and purification of neutrophil-activating peptide-4: A chemokine missing two cysteines

Publisher Summary This chapter describes the isolation of a chemokine that is lacking the motif Cys-X-Cys, which is a conserved structural element of the C-X-C branch of chemokines. Chemokines identified in platelets include the C-X-C chemokines platelet factor 4 (PF4) and platelet basic protein, which is the precursor of connective tissue activating peptide-III (CTAP-III), β-thromboglobulin, and neutrophil-activating peptide-2 (NAP-2), as well as the C-C chemokine RANTES (regulated and normal T-lymphocyte expressed and presumably secreted). Apart from the release of chemokines from platelets, a number of different chemokine mRNAs could be identified to be expressed in platelets. During purification of neutrophil chemotactic activity seen in platelet lysates the chapter identifies a novel chemokine with close similarity to PF4. Because this activity was identified as a neutrophil attractant, this chemokine was termed NAP-4.

Leukocyte-derived growth factor links the PDGF and CXC chemokine families of peptides.

Leukocytes produce many biological mediators that orchestrate the subsequent cellular events during wound healing. We have identified a novel cytokine, leukocyte-derived growth factor (LDGF), which is mitogenic for connective tissue cells. Sequence analysis of the LDGF peptide revealed that it is a precursor of other known peptides including platelet basic protein (PBP), connective tissue activating peptide III (CTAP-III), and neutrophil activating peptide 2 (NAP-2). None of these shorter peptides are active as mitogens for fibroblasts. LDGF appears to stimulate fibroblast growth by stimulation of tyrosine kinase activity of the PDGF receptors. One of the truncated products of LDGF, NAP-2, is a potent neutrophil chemoattractant. Peptides larger than NAP-2, such as PBP and CTAP-III, are not active as neutrophil chemoattractants. Collectively, these findings demonstrate that the LDGF peptide must remain intact in order to retain its fibroblast mitogenic activity. If the LDGF peptide is processed to release the carboxyl terminal half to generate NAP-2, a peptide with proinflammatory activity is generated. These results indicate that the multiple peptides produced from the LDGF-PBP gene posses divergent biological activities that could regulate different phases of the repair process.

Presence of Autoantibodies to Interleukin-8 or Neutrophil-Activating Peptide-2 in Patients With Heparin-Associated Thrombocytopenia

Eighty-seven patients with heparin-associated thrombocytopenia (HAT) showed either a positive heparin platelet aggregometry test result and/or the presence of antibodies to heparin-platelet factor 4 (H-PF4) complexes by enzyme-linked immunosorbent assay (ELISA). Fifteen of these patients lacked antibodies to H-PF4, and plasma from these patients was analyzed for the presence of antibodies to PF4-related chemokines, Neutrophil-activating peptide-2 (NAP-2) and interleukin-8 (IL-8). Of these 15 patients, 6 showed antibodies to IL-8 and 3 to the platelet basic protein (PBP)-derived protein, NAP-2. Antibodies to IL-8 and NAP-2 were not observed in control patients (n = 38), patients with HAT and H-PF4 autoantibodies (n = 72), patients with autoimmune diseases (n = 21), or patients with non-HAT thrombocytopenia (n = 30). Five of these nine patients with anti-IL-8 or anti-NAP-2 developed thrombosis during heparin treatment, which is not statistically different from the patients with H-PF4 antibodies. The existence of autoantibodies to IL-8 and NAP-2 in HAT patients highlights the significance of chemokines in the pathogenesis of HAT. The contribution of heparin in vitro was minimal in patients with anti-IL-8 and anti-NAP-2 antibodies, suggesting a biologic difference from the majority of patients with HAT and anti-PF4 antibodies. It may be that antibodies to IL-8 and NAP-2 have weaker affinity for heparin and that the ELISA system may not reflect in vivo heparin-chemokine complex formation. Alternatively, antichemokine autoantibodies may predate heparin exposure, and the role of heparin in initiating HAT may be to mobilize the chemokines and to target them to platelets, neutrophils, or endothelial cells. Subsequent chemokine-binding autoantibodies then lead to cell activation resulting in thrombocytopenia and thrombosis.

Thrombin-activated Human Platelets Release two NAP-2 Variants that Stimulate Polymorphonuclear Leukocytes

Thrombin-activated human platelets release substance(s) of a proteic nature which induce an increase in the intracellular calcium concentration in polymorphonuclear leukocytes (PMN). Aim of this study was to characterize the platelet released product(s) responsible for PMN stimulation. PMN-stimulating activity was isolated from platelet supernatant by FPLC and HPLC. The N-terminal sequence analysis revealed that the purified fractions consisted in 90% of a peptide of 73 amino acids and in 10% of a peptide of 74 amino acids; both are truncated forms of the connective tissue-activating peptide III (CTAP-III), a platelet alpha-granule product, and have 3 and 4 additional amino acids at the N-terminus compared with the neutrophil-activating peptide 2 (NAP-2): Asp-Leu-Tyr and Ser-Asp-Leu-Tyr, respectively. Treatment of platelet supernatant (previously depleted of PMN-activating nucleotides) with Affi-gel heparin resulted in the disappearance of PMN-stimulating effects, suggesting that NAP-2 variants, which are heparin-binding proteins, account for ATP-independent PMN-stimulating activity of the supernatant. Cross-desensitization between rNAP-2 and the platelet supernatant and inhibition by the anti-NAP-2 antibody are in agreement with this conclusion. Although NAP-2 and its variants are reportedly generated from the inactive precursors, CTAP-III and platelet basic protein, through a proteolytic cleavage, NAP-2 variants were not generated in our system by proteases deriving from platelets or contaminating leukocytes. Indeed, treatment of intact platelet suspensions with different protease inhibitors failed to modify the calcium stimulating activity of the resulting supernatants. In conclusion, thrombin-activated platelets release NAP-2 variants which are not generated outside the platelets by proteolytic processing but are released in an active form. This finding enhances our understanding of platelet-PMN interaction in thrombosis and inflammation.

The Crystal Structure of Recombinant Human Neutrophil-activating Peptide-2 (M6L) at 1.9-Å Resolution

Neutrophil-activating peptide-2 (NAP-2) is a 70-residue carboxyl-terminal fragment of platelet basic protein, which is found in the alpha-granules of human platelets. NAP-2, which belongs to the CXC family of chemokines that includes interleukin-8 and platelet factor 4, binds to the interleukin-8 type II receptor and induces a rise in cytosolic calcium, chemotaxis of neutrophils, and exocytosis. Crystals of recombinant NAP-2 in which the single methionine at position 6 was replaced by leucine to facilitate expression belong to space group P1 (unit cell parameters a = 40.8, b = 43.8, and c = 44.7 A and alpha = 98.4 degrees, beta = 120.3 degrees, and gamma = 92.8 degrees), with 4 molecules of NAP-2 (Mr = 7600) in the asymmetric unit. The molecular replacement solution calculated with bovine platelet factor 4 as the starting model was refined using rigid body refinement, manual fitting in solvent-leveled electron density maps, simulated annealing, and restrained least squares to an R-factor of 0.188 for 2 sigma data between 7.0- and 1.9-A resolution. The final refined crystal structure includes 265 solvent molecules. The overall tertiary structure, which is similar to that of platelet factor 4 and interleukin-8, includes an extended amino-terminal loop, three strands of antiparallel beta-sheet arranged in a Greek key fold, and one alpha-helix at the carboxyl terminus. The Glu-Leu-Arg sequence that is critical for receptor binding is fully defined by electron density and exhibits multiple conformations.

CXC Chemokines Connective Tissue Activating Peptide-III and Neutrophil Activating Peptide-2 are Heparin/Heparan Sulfate-degrading Enzymes

Heparan sulfate proteoglycans at cell surfaces or in extracellular matrices bind diverse molecules, including growth factors and cytokines, and it is believed that the activities of these molecules may be regulated by the metabolism of heparan sulfate. In this study, purification of a heparan sulfate-degrading enzyme from human platelets led to the discovery that the enzymatic activity residues in at least two members of the platelet basic protein (PBP) family known as connective tissue activating peptide-III (CTAP-III) and neutrophil activating peptide-2. PBP and its N-truncated derivatives, CTAP-III and neutrophil activating peptide-2, are CXC chemokines, a group of molecules involved in inflammation and wound healing. SDS-polyacrylamide gel electrophoresis analysis of the purified heparanase resulted in a single broad band at 8-10 kDa, the known molecular weight of PBP and its truncated derivatives. Gel filtration chromatography of heparanase resulted in peaks of activity corresponding to monomers, dimers, and tetramers; these higher order aggregates are known to form among the chemokines. N-terminal sequence analysis of the same preparation indicated that only PBP and truncated derivatives were present, and commercial CTAP-III from three suppliers had heparanase activity. Antisera produced in animals immunized with a C-terminal synthetic peptide of PBP inhibited heparanase activity by 95%, compared with activity of the purified enzyme in the presence of the preimmune sera. The synthetic peptide also inhibited heparanase by 95% at 250 microM, compared to the 33% inhibition of heparanase activity by two other peptides. The enzyme was determined to be an endoglucosaminidase, and it degraded both heparin and heparan sulfate with optimal activity at pH 5.8. Chromatofocusing of the purified heparanase resulted in two protein peaks: an inactive peak at pI7.3, and an active peak at pI 4.8-5.1. Sequence analysis showed that the two peaks contained identical protein, suggesting that a post-translational modification activates the enzyme.

Subunit association and structural analysis of platelet basic protein and related proteins investigated by 1H NMR spectroscopy and circular dichroism.

Platelet basic protein (PBP) (94 residues) is naturally processed via N-terminal cleavage to yield connective tissue activating peptide-III (85 residues), beta-thromboglobulin (81 residues), and neutrophil activating peptide-2 (70 residues). Chemical cross-linking and gel filtration data indicate that each homolog can form dimers and tetramers. Subunit association equilibria for dimer (KD) and tetramer (KT) formation have been derived for each species from 1H NMR (600 MHz) spectral analysis of slowly exchanging (NMR time scale) monomer- dimer-tetramer aggregation state populations. In general, raising the pH from about pH 3.5 to pH 6 increases KD by two to three orders in magnitude and decreases KT by some 50-fold. Ionic strength effects also suggest that intersubunit electrostatic interactions are critical to subunit association. Subunit stabilization can be ranked proportional to N-terminal chain length: platelet basic protein > connective tissue activating peptide-III > beta-thromboglobulin > neutrophil activating peptide-2. Under more physiologic conditions, PBP family monomers are favored at normal cytokine protein concentrations and may form the biologically active state. CD and NMR data indicate conservation of alpha-helix and anti-parallel beta-sheet structure among PBP-related species and support the idea that the extended N terminus folds over and masks the neutrophil activation domain and is part of the intersubunit binding domain.

Molecular cloning and characterisation of a neutrophil chemotactic protein from porcine platelets.

In our search for novel chemoattractant factors, we have purified a heparin-binding protein from porcine platelets which is a potent chemoattractant for human neutrophils. The protein has 80 amino acids and a molecular mass of 8597.5Da as measured by electrospray mass spectrometry. It has been characterised by amino acid sequencing and shown to have highest identity to members of the human platelet basic-protein-family. Its N-terminal sequence is intermediate in length between the human connective-tissue-activating polypeptide III (CTAP-III) and neutrophil-activating polypeptide-2 (NAP-2). The porcine NAP-2/CTAP-III shows the classic CXC cysteine spacing found towards the N-terminus in the chemokine alpha family and contains the ELR motif which has been shown to be essential for neutrophil chemotaxis. We have isolated mRNA from porcine platelets and constructed a cDNA library containing 1.0 x 10(6) independent clones. Using probes based on the protein sequence we have isolated a full length-clone for this gene, with an open reading frame containing 119 amino acids. Despite overall similarity between the human and porcine proteins, the N-terminal region is almost completely different between the two species, with only two identical amino acids. The proteolytic cleavage sites required for processing of human platelet basic protein are completely missing in the porcine homologue, implying a different processing pathway or mechanism. The porcine protein is capable of agonizing certain effects of both NAP-2 and CTAP-III when incubated with human cells indicating that the same porcine protein may be involved in both processes.

Cell type specific expression of members of the IL-8/NAP-1 gene family

The expression and regulation of seven human members of a family of related cytokines, which play a role as effectors of inflammation, were analysed in hemopoietic cells and in fibroblasts. In T lymphocytes all genes: platelet basic protein (PBP); platelet factor 4 (PF-4); IL-8/NAP-1; IP-10; GRO; pAT464 and pAT744 were induced by stimulation with phytohemagglutinin and phorbol 12-myristate 13-acetate (PHA/PMA). In contrast to T cells, only some of the genes were induced upon terminal differentiation of pro-monocytic cells and upon serum stimulation of resting fibroblasts. This distinct expression indicates functional differences of the individual proteins. The expression of inflammatory mediators in fibroblasts suggests the involvement of these cells in inflammatory reactions.

Basophil activation by members of the chemokine superfamily.

The human chemokine superfamily represents a new class of structurally homologous cell-derived peptides with multiple functions within the immune system (1-7). According to the position of the first two cysteins in the primary sequence, the members of the PF-4 superfamily can be divided into two branches: the “C-X-C branch” (α chemokines) including interleukin 8 (IL-8)/neutrophil-activating peptide 1 (NAP-1), platelet basic protein (PBP) and the N-terminally processed forms connective tissue activating protein III (CTAPIII) and NAP-2, platelet factor 4 (PF-4), ENA-78 and the gro peptides α, β, γ; and the “CC branch” (β chemokines) comprising monocyte chemotactic protein 1 (MCP-1), MCP2/HC14, MCP-3, RANTES, MIP-1α, MIP-1β and I-309. Some of the these peptides are well characterised with regards to their biological activities, but only little information is available for others who’s existence is sometimes based solely on gene expression (6,8,9). Recent studies showed that particular chemokines are strongly up-regulated in inflammatory disorders such as atherosclerosis, rheumatoid arthritis, and wound healing emphasising their clinical potential (6,10-12). Furthermore, several in vitro findings indicate that the division of this superfamily into two branches based on structural characteristics reflects also some fundamental differences in the range of biological activities of the α and the β chemokines. α chemokines such as IL-8/NAP-1, NAP-2, ENA-78 or MGSA exert their proinflammatory activity mainly through their neutrophil-activating capacity (1-4), whereas β chemokines preferentially attract different mononuclear cell types (4-7). This observation suggests that α chemokines play a central role in acute inflammatory events characterised by a polynuclear infiltrate, whereas β chemokines seem to be important in regulating chronic inflammatory processes in which monocytes and lymphocytes predominate.KeywordsHistamine ReleaseAllergic InflammationMediator ReleaseHematopoietic Growth FactorBasophil ActivationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Isolation, characterization, and immunological detection of neutrophil-activating peptide 2: a proteolytic degradation product of platelet basic protein.

Neutrophil-activating peptide 2 (NAP-2), corresponding to platelet basic protein fragment 25-94, was prepared by chymotryptic digestion of its precursors, low affinity platelet factor 4 or beta-thromboglobulin, followed by purification by high performance liquid chromatography. NAP-2 (0.1-1.5 microns) caused the release of human granulocyte elastase from cytochalasin B-treated neutrophils in a dose-dependent manner. In the same system, beta-thromboglobulin, human platelet factor 4, S-pyridylethyl NAP-2, and platelet basic protein C-terminal fragment (77-94) were inactive, whereas platelet basic protein fragment 22-89 had low, but significant, activity. Sensitive immunological identification of NAP-2 based on nonequilibrium isoelectric focusing and immunoblotting is described.

Platelet Factor 4: Production, Structure, and Physiologic and Immunologic Action

Platelet factor 4 (PF4) is a protein found in megakaryocytes and platelet α-granules (1, 2). Immunocytochemical studies show that it is present as well in mast cell granules (3) and on the endothelium of human umbilical veins, but not arteries (4). Early work on PF4 has been reviewed elsewhere (5–7). Human platelets contain about 18 ± 4 μg of PF4/109 (8).Thrombospondin, platelet-derived growth factor, and compounds derived from platelet basic protein such as β-thromboglobulin (β-TG) are found in platelet α-granules in addition to PF4. All are secreted when platelets are appropriately stimulated; for example, since thrombin is a strong stimulus, these compounds are present in much higher concentrations in serum than in plasma (e.g., 5334 vs 1.8 ng/ml for PF4 [9]). They are also secreted after contact of platelets with collagen in damaged blood vessels, for example.Production and StructurePF4 is synthesized by megakaryocytes (10), an ability that correlates with cytoplasmic maturity (2, 11). The PF4 is firs...

Induction of members of the IL-8/NAP-1 gene family in human T lymphocytes is suppressed by cyclosporin A

Human members of a family of structurally related cytokines, which play a role as effectors of inflammation, were analyzed for their expression and regulation in T lymphocytes. Members of this gene family include Platelet Basic Protein (PBP); Platelet Factor 4 (PF-4); IL-8/NAP-1; IP-10, a gamma interferon induced protein; GRO; pAT 464 and pAT 744. In resting T lymphocytes the RNAs of the individual genes could not be detected, but all genes were induced upon stimulation with PHA or with PHA/PMA. The induction of five genes was blocked by the immunosuppresive drug cyclosporin A (CSA), which appears to affect initial events in T cell activation. This expression in T lymphocytes, especially the sensitivity to CSA, indicates a common immunmodulatory role of these structural related proteins.

Formation of neutrophil-activating peptide 2 from platelet-derived connective-tissue-activating peptide III by different tissue proteinases

Neutrophil-activating peptide 2 (NAP-2) is generated by cleavage of two inactive precursors, connective-tissue-activating peptide III (CTAP-III) and platelet basic protein (PBP), which are stored in the alpha-granules of blood platelets. Using highly purified CTAP-III as the substrate we studied the generation of NAP-2 by several neutral tissue proteinases. CTAP-III was rapidly cleaved by chymotrypsin, cathepsin G and trypsin, yielding products with neutrophil-stimulating activity. This activity remained unchanged for 24 h in the presence of chymotrypsin, decreased only slowly in the presence of cathepsin G, but was rapidly destroyed by trypsin. CTAP-III was also degraded by human neutrophil elastase and porcine pancreatic elastase, but no active fragments were obtained. By contrast, no degradation of CTAP-III was observed with thrombin, plasmin or 'granzymes' from cytolytic T-lymphocyte granules. Two active fragments of CTAP-III, generated by chymotrypsin or cathepsin G, were purified and partially sequenced, and were found to have the same N-terminal sequence as NAP-2. These results indicate that both proteinases cleave preferentially the bond between amino acids 15 (Tyr) and 16 (Ala) of CTAP-III. We conclude that chymotrypsin-like proteolytic activity in the vicinity of activated platelets may generate NAP-2 intravascularly. Due to its presence in the primary granules of neutrophils and monocytes cathepsin G is likely to be involved in this process.