Purification and properties of M protein extracted from group A streptococci with pepsin: covalent structure of the amino terminal region of type 24 M antigen.

M protein was extracted from type 24, group A streptococci with pepsin at pH 5.8 and was further purified by ammonium sulfate precipitation, ribonuclease digestion, ion-exchange chromatography, and isoelectric focusing. The purified pepsin extract of M (pep M) protein was shown to be free of nontype-specific immunoreactivity in (a) complement fixation tests with heterologous M antiserum, (b) skin tests in normal adult guinea pigs, and (c) passive hemagglutination tests for the presence of lipoteichoic acid sensitizing or antigenic activity. The pep M24 was highly immunogenic; two of three rabbits developed opsonic antibody titers of 1:256 and the third a titer of 1:32 6 wk after a single injection of 100-pg doses of pep M24 emulsified in complete Freund's adjuvant. The antisera lacked nontype-specific antibodies and produced single precipitin lines in agar gel diffusion tests against crude HC1 extracts of the homologous M protein. Thus, the type-specific antigenic determinant(s) of type 24 M protein appears to be separable from immunotoxic, cross-reactive antigens without loss of immunogenicity in rabbits. The mobility of pep M24 upon electrophoresis in 10 percent sodium dodecyl sulfate pelyacrylamide gel was consistent with an average mol wt of 33,500 daltons. Amino acid analysis demonstrated a predominance of alanine, followed by glutamic acid, lysine, leucine, and aspartic acid. Pep M24 contained an estimated six to seven methionine residues and approximately ten phenylalanine residues per molecule. No other aromatic amino acids were detected. Automatic Edman degradation of pep M24 yielded the sequence of the first 29 amino acids (the amino terminal amino acid being valine) of the amino terminal region of the molecule. The detection of only one new amino acid at each step of Edman degradation confirmed the homogeneity of the purified pep M24.

A synthetic peptide derived from the sequence of a type I collagen receptor inhibits type I collagen-mediated platelet aggregation.

A synthetic peptide-1, an 18 amino acid residue peptide derived from a hydrophilic domain of a cloned platelet type I collagen receptor, was used to study the role of the receptor on types I and III collagen-induced platelet aggregation and the release of ATP. The peptide inhibits the type I, but not the type III, collagen-induced platelet aggregation and the release of ATP in a dose-dependent manner. The [125I]peptide-1 specifically binds to type I collagen-coated microtiter wells in a dose-dependent manner (with Kd = 10 nM). The binding of [125I]peptide-1 can be inhibited by an excess of unlabeled peptide-1 suggesting that the binding is specific. The labeled peptide-1 does not bind to type III collagen-coated microtiter wells. Results from an enzyme-linked immunosorbent assay show that the peptide reacts with the poly- and monoclonal antibodies raised against the purified platelet type I collagen receptor (Mr 65 kD). The peptide also inhibits the adhesion of platelets on type I collagen matrix and rabbit aortic segments in a dose-dependent manner. These results suggest that the reactive site of the platelet receptor for type I collagen resides in this portion of the molecule.

Binding of collagen alpha1 chains to human platelets.

We previously reported that purified alpha1 chains of type 1 chick skin collagen induce platelet aggregation. We now describe immunological and biochemical evidence that the peptide binds to intact platelets as an early event in the induction of platelet aggregation and the release reaction. Antibody against alpha1 (I) was obtained by immunizing rabbits with complete Freund's adjuvant mixed with purified alpha1. Immunofluorescence studies showed that alpha1(I)-treated platelets exhibited strong immunofluorescence. The intensity of fluorescence was markedly decreased by the pretreatment of platelets with alpha1-CB5 and glucosylgalactosylhydroxylysine. Dose-response curves of platelet aggregation induced by alpha1 and the binding of alpha1 by washed intact platelets are correlated. The biochemical studies showed that the binding of the alpha1 chain to washed intact platelets was platelet concentration and temperature dependent, and that it reached a maximum in 10 min. The process was reversible and specific, with an association constant of 1.7 muM. The inhibitor of alpha1-induced platelet aggregation, glucosylgalactosyl hydroxylysine, inhibited the alpha1 binding. These results suggest that alpha1(I) chains bind to specific receptor site(s) on platelet membranes to trigger aggregation and the release reaction.

Cloning, characterization, and functional studies of a nonintegrin platelet receptor for type I collagen.

A cDNA (1.6 kb) encoding a platelet protein receptor that binds type I collagen has been isolated from a human bone marrow cDNA library by using a degenerate oligonucleotide probe derived from the amino acid sequence of a CNBr fragment of the purified receptor. Computer search revealed that this cDNA represents the coding sequence of a unique protein. Using the prokaryotic expression system pKK 223-3-65 cDNA, a 54-kD recombinant protein was obtained and purified to apparent homogeneity. In an eukaryotic expression vector (pcDNA3-65 cDNA), a 65-kD protein was identified that was recognized by monoclonal anti-65 kD antibody (anti-65m). The recombinant protein binds to type I, but not to type III collagen by affinity column chromatography. The binding of the recombinant protein to type I collagen-coated Petri dishes is inhibited by anti-65m in a dose-dependent manner. The pcDNA3-65 cDNA-transfected nonadherent T cells express the protein, allowing them to attach to a type I collagen matrix, and are inhibited by anti-65m in a dose-dependent manner. Like the receptor protein purified from platelet membranes, the recombinant protein inhibits type I collagen-induced platelet aggregation and the adhesion of [14C]serotonin-labeled platelets to type I collagen in a dose-dependent manner. The recombinant protein neither binds to type III collagen-coated Petri dishes nor inhibits type III collagen and ADP-induced platelet aggregation, indicating specificity for type I collagen.

Binding of chemotactic collagen-derived peptides to fibroblasts. The relationship to fibroblast chemotaxis.

We previously showed that collagen, alpha-chains, and collagen-derived peptide fragments induce chemotactic migration of human fibroblasts in vitro. We now describe biochemical and immunological evidence showing there are binding sites for collagen peptides on fibroblast membranes.By the use of (14)C-labeled alpha1(I) chain, binding to intact fibroblasts was demonstrated. The process was reversible, and time- and fibroblast concentration-dependent. Scatchard plot analyses of the data obtained for the binding of alpha1(I) suggested that there are congruent with 16 x 10(6) binding sites per fibroblast with an association constant of 1.1 x 10(7)/M for alpha1(I). Dissociation of the bound radioactivity and subsequent chromatographic analysis on agarose A-1.5 m revealed that the alpha1 was unaltered. The binding of (14)C-labeled alpha1 was inhibited by each of the CNBr peptides derived from alpha1 chain of chick skin collagen and CNBr peptide mixtures of various genetic types of collagen chains. Immunofluorescence studies with anti-alpha1 antibody showed that alpha1-treated fibroblasts exhibited strong immunofluorescence. The intensity of fluorescence was markedly diminished by prior absorption of the antibody with alpha1. The alpha1-treated cells stained with preimmune sera did not show significant fluorescence.Dose-response curves of fibroblast chemotaxis induced by alpha1 and the binding of alpha1 by fibroblasts correlate closely. Furthermore, the potency of alpha1-CNBr peptides as chemotactic agents correlates with their ability to inhibit the binding of labeled alpha1(I). These data suggest the hypothesis that collagenderived peptides cause fibroblast chemotactic migration by acting on fibroblast membranes.

Activation of phospholipase D in human platelets by collagen and thrombin and its relationship to platelet aggregation.

Stimulation of phospholipase D after activation of cell surface receptors has been reported in many cell types. We have investigated the mechanism of activation of this enzyme by collagen in the human platelet by assaying the release of [3H]methylcholine from [3H]methylphosphatidylcholine. Results from these studies suggest that phospholipase D activity is regulated by reversible phosphorylation. Phospholipase D activity was stimulated when platelet-rich plasma was preincubated with collagen and was not inhibited by aspirin. Among various aggregating agents tested, collagen and thrombin but not ADP activated phospholipase D activity (2- to 3-fold). The addition of sphingosine inhibited phospholipase D activity. Preincubation of platelet-rich plasma with sphingosine inhibited collagen- and thrombin-induced platelet aggregation and the release of ATP. The inhibitory effect of sphingosine on collagen- and thrombin- induced platelet aggregation and release of ATP was dose-dependent. The functional significance of phospholipase D activation was also tested by examining the effect of the product, phosphatidic acid, on collagen-induced platelet aggregation and release of ATP. Platelet shape change and the reversibility of platelet aggregation resulted by the addition of phosphatidic acid to platelet-rich plasma. Furthermore, the simultaneous addition of phosphatidic acid and collagen shortened the latency period but had no effect on platelet aggregation. Two platelet proteins (47 kDa and 22 kDa) increased in phosphorylation after the addition of 1 microM phosphatidic acid which did not cause platelet aggregation. These results suggest that collagen stimulates phospholipase D activity which plays a secondary role in platelet aggregation and the release reaction.

Recent development of peptides from glycoproteins IIb (alphaIIb) and IIIa (beta3) that inhibit platelet fibrinogen binding.

The glycoprotein (GP) IIb/IIIa (alphaIIbbeta3) found on platelets binds fibrinogen when platelets are activated, thereby mediating the platelet aggregation process. Blockading of alphaIIbbeta3 has been proposed to prevent platelet aggregation independent of the substance(s) responsible for activating the platelets. This inhibition of platelet aggregation is thought to be an effective therapeutic approach to various thromboembolic syndromes. The development of various forms of alphalambdapietaalpha;IIbbeta3 inhibitors has resulted in the inhibition of platelet aggregation, although studies of alphaIIbbeta3 receptor function and various alphaIIbbeta3 inhibitors have demonstrated the potential for these agents to produce effects on other aspects of platelet function as well as having non-platelet effects. This review describes the newly derived peptides from 1) glycoprotein IIb (alphaIIb) that interferes with platelet aggregation by inhibiting the binding of fibrinogen to alphaIIbbeta3 and from 2) GP IIIa (beta3) by blocking the alphaIIbbeta3 complex formation. These peptides may become effective agents to block the interaction of ADP, type I collagen, and type III collagen (type I collagen and type III collagen are present in abundant amounts in blood vessel walls) with platelets.

The effect of somatostatin on platelet aggregation.

Somatostatin was infused intravenously over two hours to rabbits in a dose of 1.5 mug/kg min with an initial loading dose of 50 mug/kg. This dose inhibited platelet aggregation in response to both ADP and collagen. Lower doses of SRIF had no effect. The addition of SRIF in vitro to either human or rabbit platelet rich plasma also had no effect on collagen or ADP-induced platelet aggregation.

Collagen-platelet interaction: platelet non-integrin receptors.

Platelet-collagen interaction is a complex event that involves ligand-receptor interaction. There are many adhesive non-integrin receptors for platelets to interact with various types of collagens. These non-integrin receptors also serve as signal transducers both from the outside of platelets to the inside and possibly vice versa. The present review covers basic aspects of non-integrin receptor function and various signal transduction pathways.

Phosphorylation-dephosphorylation states at different sites affect phosphoprotein phosphatase 1 activity.

We have previously reported that the binding of type I collagen to its receptor initiates platelet aggregation involving phosphoprotein phosphatase 1 (PP 1), which coprecipitates with the 65-kDa platelet type I collagen receptor. Phosphorylation of the anti-PP1 precipitation PP1 decreases its enzyme activity. In the present investigation, the mechanism of the decreased enzyme activity was studied by examining the phosphorylation of PP 1 on serine/threonine or tyrosine residues. Phosphoamino acid analysis of the PP 1 indicates that serine, threonine, and tyrosine can all be phosphorylated. We find that the activity of PP 1 decreases with serine/threonine phosphorylation but that phosphorylation of tyrosine residue activates enzyme activity. These results indicate that the activity of platelet phosphoprotein phosphatase 1 is controlled by phosphorylation and dephosphorylation states at multiple, different site(s).

Effect of monoclonal antibody on the adhesion of platelets to aortic segments.

A purified monoclonal antibody raised against a purified platelet 65 kDa protein that binds to type I collagen receptor was used to see if the antibody can block the adhesion of platelets to damaged aortic segments. The purified antibody binds to the surface of human platelets by flow cytometry. The adhesion of platelets to the damaged aortic segments was significantly reduced by the pre-incubation of the platelet-rich plasma with the antibody, suggesting that the antibody binds to platelets and blocks the adhesion of platelets to the aortic segments.

Collagen-induced platelet aggregation and release reaction: role of ras protein.

The relationship between the platelet collagen receptor and ras protein was examined by immunoprecipitation of control and collagen stimulated platelets with both antibodies. Both ras protein and receptor are coprecipitated by both antibodies. The coprecipitated samples contained GTP and GDP which were separated by thin layer chromatography. The effect of guanine nucleotide binding protein (G-protein, 21ras) on platelet aggregation was examined by using a guanine triphosphate analogue (GTP-gamma-S). Results of streptolysin O permeabilized experiments show that the addition of the analogue to permeabilized platelet-rich plasma causes platelet aggregation and release of ATP in a dose-dependent fashion. The aggregation induced by GTP-gamma-S could not be obtained with GDP, GDP-beta-S, or 5'-GMP, suggesting that the effect of GTP-gamma-S is specific. The platelet aggregation induced by the analogue was inhibited in a dose-dependent manner by phenylmethanesulfonyl fluoride (PMSF) but not apyrase. GTP-gamma-S induced thromboxane B2 formation was also decreased by PMSF. Formation of thromboxane B2 was also blocked by the addition of PMSF and ethanol suggesting that GTP-gamma-S increases arachidonic acid release from permeabilized platelets. These results suggest that both GTP-gamma-S and GTP enhance phospholipase A2 activity which releases arachidonic acid in permeabilized platelets and subsequently causes platelets to aggregate.

The effect of low dose of 12-O-tetradecanoyl-phorbol-13-acetate on collagen platelet interactions.

We and others have reported that phorbol ester doses ranging from 0.25 to 25 micrograms/ml induce human platelets to aggregate and release serotonin. In this paper, we report the effect of low doses of phorbol (0.5 to 50 ng/ml) on subthreshold amounts of collagen induced platelet aggregation. The platelet aggregation induced by the addition of subthreshold amounts of collagen can be enhanced by low doses of phorbol. The combined low doses of phorbol and subthreshold amounts of collagen induced platelet aggregation can be inhibited by the addition of aspirin and imidazole. The increase in platelet aggregation induced by the combined low doses of phorbol and subthreshold amounts of collagen is probably mediated by the collagen fibril formation.

Okadaic acid and vanadate inhibit collagen-induced platelet aggregation; the functional relation of phosphatases on platelet aggregation.

The different specific inhibitors for phosphoserine/threonine and phosphotyrosine protein phosphatases were used to study the role of these protein phosphatases in collagen-platelet interaction. The collagen-induced platelet aggregation and the release reaction as measured ATP release were inhibited in a dose-dependent fashion by the addition of okadaic acid, a specific inhibitor of phosphoserine/threonine protein phosphatase 1 and 2A. The inhibition was also observed by the addition of a phosphotyrosine protein phosphatase inhibitor, vanadate. Suboptimal concentrations of both inhibitors together also inhibited collagen-induced platelet aggregation and release reaction in a concentration-dependent fashion. These results suggest that collagen-platelet interaction is modulated by both protein phosphatases.

The effect of platelet surface protein phosphorylation on collagen-platelet interaction.

Phosphorylation of the surface proteins of human platelets by treatment with human plasma kinase and exogenous ATP enhances their aggregation response to collagen. This effect can be reversed by treatment of the phosphorylated platelets with acid phosphatase. Moreover, acid phosphatase treatment of normal human platelets results in a decreased aggregation response to collagen. The increased responsiveness of phosphorylated platelets to collagen is associated with a concomitant increase in Thromboxane A2 formation. These results suggest that the platelet surface protein phosphorylation/dephosphorylation plays an important role in modulating platelet function.

Collagen-platelet interaction: type XI collagen-induced platelet aggregation.

Type XI collagen in its native fibrillar but not in soluble monomeric form mediates human platelet aggregation and release of adenosine triphosphate in a dose-dependent manner. Its action is inhibited by aspirin. Type XI collagen also increased radiolabelled phosphate incorporation into protein bands with molecular weights of 42 KDa and 22 KDa, respectively. In contract, these events were not observed in platelets incubated with type IX collagen. These results suggest that the fibrillar type XI collagen has the same ability as other types of collagen to induce human platelet aggregation.

Collagen-platelet interaction: separate receptor sites for types I and III collagen.

We have isolated a platelet membrane protein of M(r) 47 kDa which is responsible for the interaction of platelets with type III collagen. The 47 kDa protein was purified to apparent homogeneity by type III collagen-Sepharose 2B column chromatography and preparative slab gel electrophoreses. The 47 kDa protein blocked the adhesion of platelets to type III but not to type I collagen. Polyclonal antibodies were obtained from rabbits immunized with the purified 47 kDa protein emulsified in complete Freund's adjuvant. The polyclonal antibodies inhibited the type III collagen but not type I collagen-induced platelet aggregation. The inhibitory effect of the antibodies on type III collagen-induced platelet aggregation was dose-dependent. Cross-inhibition on platelet aggregation studies showed that type I collagen receptor antibodies (M(r) 65 kDa) did not inhibit type III collagen-induced platelet aggregation and type III collagen receptor antibodies did not inhibit type I collagen induced platelet aggregation. These results suggest that type I and type III collagens interact with platelets at separate sites.

Role of nitric oxide synthase in collagen-platelet interaction: involvement of platelet nonintegrin collagen receptor nitrotyrosylation.

Platelets possess the endothelial isoform of nitric oxide synthase (eNOS), which plays an important role in platelet function. Other laboratories, including ours, have reported that nitric oxide (NO) is released upon exposure of platelets to collagen, but the mechanism of the interaction is not yet established. The objective of this study is to examine the possible role of nonintegrin receptor nitrotyrosylation on collagen-induced platelet aggregation. Results of the study show that two platelet proteins with M(r) of 65- and 23-kDa proteins are nitrotyrosylated in a time-dependent manner after the addition of type I collagen. The M(r) 65-kDa protein is identified as the platelet receptor for type I collagen. The recombinant protein of the platelet receptor for type I collagen can also be nitrotyrosylated. The nitrotyrosylated recombinant protein loses its ability to inhibit type I collagen-induced platelet aggregation. In addition, the polyclonal anti-65 kDa immunoprecipitates eNOS suggesting that the platelet nonintegrin receptor for type I collagen is closely linked to the eNOS. These results demonstrate that the inhibitory effect of NO on collagen-induced platelet aggregation may be mediated by the nitrotyrosylation of the 65-kDa receptor.

Role of the recombinant protein of the platelet receptor for type I collagen in the release of nitric oxide during platelet aggregation.

Nitric oxide plays an important role in platelet function and platelets possess the endothelial isoform of nitric oxide synthase. Several reports have indicated that nitric oxide is released upon exposure of platelets to collagen. We have reported that a non-integrin platelet protein of 65 kDa is a receptor for type I collagen. By direct measurement of NO release from washed human platelets suspended in Tyrode buffer with a ISO-NO Mark II, World Precision Instruments, Sarasota, FL, USA, p30 sensor, type I collagen, but not ADP and epinephrine, induces the release of NO in a time-dependent manner. The production of NO is inhibited either by preincubation of type I collagen with the platelet type I collagen receptor recombinant protein or by preincubation of platelets with the antibody to the receptor protein, the anti-65 antibody. However, preincubation of platelets with anti-P-selectin and anti-glycoprotein IIb/IIIa did not affect the release of NO by platelets. These results suggest that the 65 kDa platelet receptor for type I collagen is specifically linked to the generation of NO, and that the 65 kDa platelet receptor for type I collagen plays an important new role in platelet function.

Involvement of phosphoprotein phosphatase 1 in collagen-platelet interaction.

The binding of type I collagen to its receptor initiates platelet aggregation, but the relationship of the receptor to other signal transduction components is not yet established. Correlation of platelet aggregation and anti-type I collagen receptor antibody immunoprecipitation of type I collagen treated [32PO4]-labeled platelets showed that there are two phosphoproteins (M(r) 53 kDa and 21 kDa) that coprecipitated with the 65 kDa platelet type I collagen receptor. In the present investigation, we have identified one of the phosphoproteins. A soluble component the 100,000x g supernatant fraction of 53 kDa protein is recognized by polyclonal anti-PP1 antibody. The activity of the precipitated phosphatase is inhibited by okadaic acid and inhibitor 1, suggesting that it is protein phosphatase 1 (PP 1). Phosphorylation decreases PP 1 activity as was found with [32PO4]-phosphorylase b as the substrate. The immunocoprecipitation of the type-1 collagen receptor and PP 1 inot the result of cross reactivity of the anti-type I collagen receptor antibody with the PP I protein. These results indicate that the platelet type I collagen receptor, PP 1, and unidentified 21 kDa protein are in close association with the platelet type I collagen receptor upon the binding of type I collagen by the receptor.