Immunocytochemical evidence for the translocation of alpha-granule membrane glycoprotein IIb/IIIa (integrin alpha IIb beta 3) of human platelets to the surface membrane during the release reaction.

The localization of glycoprotein (GP) IIb/IIIa (integrin alpha IIb beta 3) in both resting and thrombin-activated platelets was studied immunocytochemically. By the preembedding method where only the GP IIb/IIIa molecules on the surface of platelets were immunostained, the distribution of protein A-colloidal gold label was randomly distributed along the surface membrane of resting platelets at a density of 18.0 +/- 2.7 gold particles/microns of membrane. At 15 s after stimulation by 0.1 U/ml of thrombin in an unstirred platelet suspension, the spheroid-shaped platelets with pseudopodia still had normal numbers of alpha-granules, and the density of gold particles was 19.7 +/- 3.6 particles/microns. At 5 min, the alpha-granules were no longer present because of the release reaction, and the density of gold particles significantly increased (27.0 +/- 3.7 particles/microns; p less than 0.01). In immuno-stained ultra-thin frozen sections, the gold particles were detected not only on the surface membrane, including the open canalicular system (OCS), but also on the alpha-granule membranes of resting platelets. At 30 s after thrombin stimulation the alpha-granules fused with the OCS, resulting in the formation of a swollen OCS, which still had gold particles on its membrane. At 5 min, the gold particles were detected on the membrane of the swollen OCS located near the surface membrane, while very few gold particles were present on the membrane of the OCS in the central part of the platelets. These results demonstrate that alpha-granule membrane GPIIb/IIIa translocates to the surface membrane through the membrane of the OCS.(ABSTRACT TRUNCATED AT 250 WORDS)     

Morphological evidence for the association of plasma membrane glycoprotein IIb/IIIa with the membrane skeleton in human platelets

Summary We examined the association between glycoprotein (GP) IIb/IIIa, a receptor for fibrinogen, and membrane skeletons in both unstimulated and thrombin-activated human platelets. After a treatment with dithiobis succinimidyl propionate (DTSP), a cross-linker, unstimulated and activated platelets were simultaneously extracted and fixed with a fixing solution containing Triton X-100. Also, the localization of GPIIb/IIIa on the plasma membrane was observed by a preembedding staining method of unextracted platelets. In unstimulated platelets, 20–40% of the whole plasma membrane remained in the detergent-extracted samples. Amorphous structures with 10–70 nm in diameters are distributed at 20 to 100-nm intervals on the surface of plasma membrane. Similar structures also were identified in the intact platelets by the immunocytochemical method. By careful inspection, we found that most of the amorphous structures that contained gold particles were connected to the submembrane zone just beneath the plasma membrane. The submembrane zone was identified as the membrane skeleton because actin was detected in the zone. After activation, detergent-insoluble granules were surrounded by dense networks of microfilaments in the central part of platelets. The filaments were identified as actin and became associated with myosin. These results demonstrate that GPIIb/IIIa on the plasma membrane is connected to the membrane skeleton and suggest that, during activation, actin filaments which extend into the cytoplasm from the membrane skeleton increase and form dense networks around Triton-insoluble granules.     

Morphological evidence for the association of plasma membrane glycoprotein IIb/IIIa with the membrane skeleton in human platelets

We examined the association between glycoprotein (GP) IIb/IIIa, a receptor for fibrinogen, and membrane skeletons in both unstimulated and thrombin-activated human platelets. After a treatment with dithiobis succinimidyl propionate (DTSP), a cross-linker, unstimulated and activated platelets were simultaneously extracted and fixed with a fixing solution containing Triton X-100. Also, the localization of GPIIb/IIIa on the plasma membrane was observed by a preembedding staining method of unextracted platelets. In unstimulated platelets, 20-40% of the whole plasma membrane remained in the detergent-extracted samples. Amorphous structures with 10-70 nm in diameters are distributed at 20 to 100-nm intervals on the surface of plasma membrane. Similar structures also were identified in the intact platelets by the immunocytochemical method. By careful inspection, we found that most of the amorphous structures that contained gold particles were connected to the submembrane zone just beneath the plasma membrane. The submembrane zone was identified as the membrane skeleton because actin was detected in the zone. After activation, detergent-insoluble granules were surrounded by dense networks of microfilaments in the central part of platelets. The filaments were identified as actin and became associated with myosin. These results demonstrate that GPIIb/IIIa on the plasma membrane is connected to the membrane skeleton and suggest that, during activation, actin filaments which extend into the cytoplasm from the membrane skeleton increase and form dense networks around Triton-insoluble granules.     

Interaction of fibrinogen with its platelet receptor. Differential effects of alpha and gamma chain fibrinogen peptides on the glycoprotein IIb-IIIa complex

The platelet membrane glycoprotein IIb-IIIa complex (GPIIb-IIIa) recognizes peptides containing the amino acid sequence Arg-Gly-Asp, a sequence present at two locations in the alpha chain of fibrinogen. GPIIb-IIIa also interacts with peptides containing the carboxyl-terminal 10-15 residues of the fibrinogen gamma chain. We found that the alpha chain tetrapeptide, Arg-Gly-Asp-Ser (RGDS), and the gamma chain peptide, Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val (LGGAKQAG-DV), each inhibited fibrinogen binding to ADP-stimulated platelets with Ki values of 15.6 +/- 2.7 and 46.2 +/- 8.2 microM, respectively. Furthermore, the inhibitory effect of the peptides was additive, indicating that they interact with GPIIb-IIIa in a mutually exclusive manner. Mutually exclusive binding suggests that either the alpha and gamma chain peptides bind to identical or overlapping sites on the GPIIb-IIIa complex or that one peptide induces a change in the complex that excludes the other. To differentiate between these possibilities, we compared the ability of RGDS and LGGAKQAGDV to inhibit the binding of fibrinogen and two GPIIb-IIIa complex-specific monoclonal antibodies, A2A9 and PAC-1, to ADP-stimulated platelets. A2A9 and PAC-1 appear to bind to different sites on GPIIb-IIIa because A2A9 binds to both stimulated and unstimulated platelets while PAC-1 only binds to stimulated platelets. RGDS specifically inhibited fibrinogen and PAC-1 binding with nearly identical Ki values of 15.6 +/- 2.7 and 20.2 +/- 3.5 microM, respectively. In contrast, LGGAKQAGDV had a differential effect on fibrinogen and PAC-1 binding, inhibiting PAC-1 binding with a Ki of 116.1 +/- 12.9 microM and fibrinogen binding with a Ki of 46.2 +/- 8.2 microM (p less than 0.005). Furthermore, while RGDS had no effect on the binding of the monoclonal antibody A2A9, LGGAKQAGDV was a partial inhibitor of A2A9 binding to activated platelets. These results suggest that the bindings sites for RGDS and LGGAKQAGDV are spatially distinct. They also suggest that ligand-induced changes in GPIIb-IIIa conformation are likely to be responsible for the mutually exclusive nature of alpha and gamma chain peptide binding.     

Glycoprotein Ib beta is the only phosphorylated major membrane glycoprotein in human platelets.

Platelets were metabolically labelled with 32P and the phosphoproteins examined by two-dimensional non-reduced/reduced gel electrophoresis and isoelectric-focusing/gel electrophoresis. Comparison with similar separations of surface-labelled platelets showed that the only major glycoprotein which is phosphorylated is the beta-subunit of glycoprotein Ib, indicating that this subunit contains a cytoplasmic segment. The identification was confirmed using immunoblotting with an antibody to the beta-subunit. Phosphoserine was the principal phosphorylation site, with some phosphothreonine, but phosphotyrosine was absent. No quantitative or qualitative differences could be detected in the phosphorylation of glycoprotein Ib beta from resting or activated platelets. These results exclude changes in phosphorylation of the major platelet membrane glycoproteins as a method of signal transmission by these receptors.     

Structure of the platelet membrane glycoprotein IIb. Homology to the alpha subunits of the vitronectin and fibronectin membrane receptors

The platelet membrane glycoprotein IIb X IIIa heterodimer complex (GPIIb X IIIa) is the platelet receptor for adhesive proteins, containing binding sites for fibrinogen, von Willebrand factor, and fibronectin on activated platelets. GPIIb X IIIa also appears to be a member of a family of membrane adhesive protein receptors that plays a major role in cell-cell and cell-matrix interactions. GPIb is the larger component of this platelet receptor and is composed of two disulfide-linked subunits. In this report we describe the analysis of cDNA clones for human GPIIb that were isolated from a lambda gt11 expression library prepared using RNA from HEL cells. A total of 3.3 kilobases of cDNA was sequence, revealing a continuous open reading frame encoding both GPIIb subunits. The cDNA encodes 1039 amino acids: 137 constituting the smaller subunit, 871 constituting the larger subunit, and 30 constituting an NH2-terminal signal peptide. No homology was found between the larger and smaller subunits. The smaller subunit contains a 26-residue hydrophobic sequence near its COOH terminus that represents a potential transmembrane domain. Four stretches of 12 amino acids present in the larger subunit are homologous to the calcium binding sites of calmodulin and troponin C. Northern blot analysis using HEL cell RNA indicated that the mature mRNA coding for GPIIb is 4.1 kilobases in size. A comparison of the GPIIb coding region with available cDNA sequences of the alpha-chains of the vitronectin and fibronectin receptors revealed 41% DNA homology and 74% and 63% amino acid homology, respectively. Our data establish the amino acid sequence for the human platelet glycoprotein IIb and provide additional evidence for the existence of a family of cellular adhesion protein receptors.     

Platelet receptor recognition domains on the alpha chain of human fibrinogen: structure-function analysis

We have previously shown that the alpha chain of human fibrinogen interacts directly with ADP-activated human platelets [Hawiger, J., Timmons, S., Kloczewiak, M., Strong, D. D., & Doolittle, R. F. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2068]. Now, we report that platelet receptor recognition domains are localized on two CNBr fragments of the human fibrinogen alpha chain. They encompass residues 92-147 and 518-584, which inhibit 125I-fibrinogen binding to ADP-stimulated platelets. The inhibitory CNBr fragment alpha 92-147 contains the RGD sequence at residues 95-97. Synthetic peptides encompassing this sequence were inhibitory while peptide 99-113 lacking the RGD sequence was inactive. The synthetic peptide RGDF, corresponding to residues alpha 95-98, inhibited the binding of 125I-fibrinogen to ADP-treated platelets (IC50 = 2 microM). However, the peptides containing sequence RGDF, with residues preceding Arg95 or following Phe98, were less inhibitory. It appears that the sequence alpha 95-98 constitutes a platelet receptor recognition domain which is constrained by flanking residues. The second inhibitory CNBr fragment, alpha 518-584, also contains the sequence RGD at positions 572-574. Synthetic peptides overlapping this sequence were inhibitory, while peptides lacking the sequence RGDS were not reactive. Thus, another platelet reactive site on the alpha chain encompasses residues 572-575 containing sequence RGDS. In conclusion, the platelet receptor recognition domains on the human fibrinogen alpha chain in the amino-terminal and in the carboxy-terminal zones contain the ubiquitous cell recognition sequence RGD shared with other known adhesive proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chrysoptin is a potent glycoprotein IIb/IIIa fibrinogen receptor antagonist present in salivary gland extracts of the deerfly

Salivary gland lysates of the deerfly (genus Chrysops) contain chrysoptin, an inhibitor of ADP-induced platelet aggregation, which presumably assists the fly in obtaining a blood meal. Chrysoptin has now been isolated, and its cDNA has been cloned and expressed. Chrysoptin was purified to homogeneity using anion exchange and hydrophobic interaction chromatography and found to be a protein with a molecular mass of 65 kDa as determined by gel electrophoresis. N-terminal amino acid sequencing allowed for the synthesis of degenerate oligonucleotides that led to cloning, from salivary gland specific mRNA, of the cDNA encoding this platelet inhibitor. No RGD sites are present in the predicted sequence. A search of GenBank(TM) did not reveal significant sequence homology between chrysoptin and other proteins. The molecular mass predicted from the cDNA was 59 kDa. Predicted glycosylation and phosphorylation sites may account for this difference in molecular mass, as recombinant chrysoptin expressed in Sf21 cells had a molecular mass of 65 kDa, matching that of the natural protein. Chrysoptin functions by inhibiting the binding of fibrinogen to the fibrinogen/glycoprotein IIb/IIIa receptor on platelets with an IC(50) of 95 pmol. These results reveal that insect salivary glands are a source of fibrinogen receptor antagonists.     

Identification of a nucleotide-binding site on glycoprotein IIb. Relationship to ADP-induced platelet activation

Formalin-fixed platelets have been used to study the binding of adenine nucleotides in order to avoid the complications of nucleotide metabolism and to achieve steady-state binding. Sp-adenosine-5'-(1-thiotriphosphate) (Sp-ATP-alpha-S) binds to platelets at two sites (Kd1 3 nM; 31,000 sites/platelet; Kd2 200 nM; 300,000 sites/platelet) as compared with values for ADP under these conditions (Kd1 30 nM; 25,000 sites/platelet and Kd2 3 microM; 400,000 sites/platelet) (bound/total approximately 0.1). Competition binding experiments showed that both of the ATP-alpha-S sites were accessible to ADP and vice versa. [35S]ATP-alpha-S was photoaffinity cross-linked to unfixed platelets by direct irradiation with ultraviolet light. A single radiolabeled component (120 kDa) was identified and shown to be identical with the alpha subunit of GPIIb based on two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting with anti-GPIIb monoclonal antibodies, by isoelectric focusing (pI 4.5-5.5), by immunoaffinity adsorption using monoclonal anti-GPIIb/IIIa antibodies coupled to Sepharose, and by crossed immunoelectrophoresis. Amino-terminal sequencing of a tryptic fragment labeled with [35S]ATP-alpha-S identified an 18-kDa domain beginning at Tyr-198 in the primary sequence of GPIIb alpha. These studies demonstrate the presence of an adenine nucleotide-binding site on GPIIb alpha.     

Localization of the cross-linking sites of RGD and KQAGDV peptides to the isolated fibrinogen receptor, the human platelet integrin glycoprotein IIb/IIIa. Influence of peptide length.

The non-covalent and Ca(2+)-dependent heterodimer GPIIb/IIIa, formed by platelet glycoproteins IIb (GPIIb) and IIIa (GPIIIa), also known as the integrin alpha IIb beta 3, is the inducible receptor for fibrinogen and other adhesive proteins on the surface of activated platelets. A fraction of the isolated GPIIb/IIIa in solution binds RGD or KQAGDV inhibitory peptides and, upon peptide removal, apparently acquires the capacity to bind fibrinogen ('activated' GPIIb/IIIa) [Du, X., Plow, E. F., Frelinger, A. L., III, O'Toole, T. E., Loftus, J. C. & Ginsberg, M. H. (1991) Cell 65, 409-416]. Photoaffinity labelling was used here to study the ligand binding site(s) of GPIIb/IIIa in solution, for which the peptides CKRKRKRKRRGDV (alpha 1), CGRGDF (alpha 2), CYHHLGGAKQAGDV (gamma 1) and CGAKQAGDV (gamma 2) were synthesized with a photoactivable cross-linker group and a fluorescent reporter group attached to the N-terminal cysteine residue. Contrary to the situation in activated platelets, both GPIIb and GPIIIa were equally labelled by the four peptides and the cross-linking sites were localized by protein chemical analyses of the fluorescently labelled tryptic peptides of both subunits. Thus, the localization of the cross-linking sites in GPIIb varies considerably with the peptide length and is very different from that localization observed in activated platelets: alpha 2 and gamma 2 were found cross-linked to the N-terminal of both the heavy (GPIIbH 42-73) and the light (GPIIbL2 30-75) chains of GPIIb; while the longer peptides alpha 1 and gamma 1 were cross-linked to the C-terminal of GPIIbH within the 696-724 and 752-768 peptide stretches, respectively. On the other hand, the cross-linking sites of the four inhibitory peptides in GPIIIa were found mainly within the proteolysis susceptible region, between the N-terminal (GPIIIa 1-52) and the core (GPIIb 423-622) highly disulphide-bonded domains, observing that the longer the peptide the closer the cross-linking site is to the N-terminal of GPIIIa: alpha 1 at GPIIIa 63-87 and 303-350; gamma 1 at GPIIIa 9-37; alpha 2 at GPIIIa 151-191; and gamma 2 at GPIIIa 303-350. These results led us to the following conclusions. (a) The GPIIIa 100-400 region contributes to the ligand-binding domain in GPIIb/IIIa both in solution and in activated platelets.(ABSTRACT TRUNCATED AT 400 WORDS)     

Autocatalytic cleavage of the EMR2 receptor occurs at a conserved G protein-coupled receptor proteolytic site motif

Post-translational cleavage at the G protein-coupled receptor proteolytic site (GPS) has been demonstrated in many class B2 G protein-coupled receptors as well as other cell surface proteins such as polycystin-1. However, the mechanism of the GPS proteolysis has never been elucidated. Here we have characterized the cleavage of the human EMR2 receptor and identified the molecular mechanism of the proteolytic process at the GPS. Proteolysis at the highly conserved His-Leu downward arrow Ser(518) cleavage site can occur inside the endoplasmic reticulum compartment, resulting in two protein subunits that associate noncovalently as a heterodimer. Site-directed mutagenesis of the P(+1) cleavage site (Ser(518)) shows an absolute requirement of a Ser, Thr, or Cys residue for efficient proteolysis. Substitution of the P(-2) His residue to other amino acids produces slow processing precursor proteins, which spontaneously hydrolyze in a defined cell-free system. Further biochemical characterization indicates that the GPS proteolysis is mediated by an autocatalytic intramolecular reaction similar to that employed by the N-terminal nucleophile hydrolases, which are known to activate themselves by self-catalyzed cis-proteolysis. We propose here that the autoproteolytic cleavage of EMR2 represents a paradigm for the other GPS motif-containing proteins and suggest that these GPS proteins belong to a cell surface receptor subfamily of N-terminal nucleophile hydrolases.     

Localization of a site interacting with human platelet receptor on carboxy-terminal segment of human fibrinogen gamma chain

We report that the 27-residue carboxy-terminal cyanogen bromide fragment of human fibrinogen γ chain inhibits binding of [¹²⁵I]fibrinogen to human platelet receptors and blocks fibrinogen-mediated aggregation of ADP-treated human platelets. The blocking activity of the peptide was preserved after proteolysis of the isolated peptide with staphylococcal protease to generate a mixture of a dodecapeptide and a pentadecapeptide. Trypsin treatment destroyed blocking activity of the isolated peptide. These results indicate that the site responsible for the interaction of human fibrinogen with the platelet receptor resides in the 27-residue carboxy-terminal region of the γ chain.     

Isolation and characterization of a major glycoprotein from milk-fat-globule membrane of human breast milk.

A major periodate--Schiff-positive component from milk-fat-globule membrane of human breast milk has been purified by selectively extracting the membrane glycoproteins, followed by lectin affinity chromatography and gel filtration on Sephadex G-200 in the presence of protein-dissociating agents. The purified glycoprotein, termed epithelial membrane glycoprotein (EMGP-70), has an estimated mol.wt. of 70 000 and yields a single band under reducing conditions on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. The glycoprotein contains 13.5% carbohydrate by weight, with fucose, mannose, galactose, N-acetylglucosamine and sialic acid 17.2, 17.0, 21.1, 7.9 and 36.6% respectively of the carbohydrate moiety. Aspartic and glutamic acid and serine are the major amino acid residues.     

Activation of hepatocyte growth factor by proteolytic conversion of a single chain form to a heterodimer.

Hepatocyte growth factor (HGF) is a heterodimeric protein consisting of a heavy chain and a light chain held by a disulfide bond. These chains are produced by endoproteolytic processing from a single chain precursor. In this study, we examined whether the processing is a prerequisite for the mitogenic activity of HGF on hepatocytes in primary culture. Single chain HGF was proteolytically converted to the heterodimeric form during incubation with hepatocytes and was as mitogenic as the heterodimeric form. When the conversion was inhibited by serine-protease inhibitors, the mitogenic activity of single chain HGF was markedly reduced. Furthermore, a mutant resistant to the proteolytic processing, which was prepared by in vitro mutagenesis, completely lost the mitogenic activity. From these results, we concluded that the single chain form of HGF is endoproteolytically processed by a serine-protease and that this processing is a prerequisite for the mitogenic activity of HGF.     

Mutations within the proteolytic cleavage site of the Rous sarcoma virus glycoprotein define a requirement for dibasic residues for intracellular cleavage.

We investigated the amino acid sequence requirements for intracellular cleavage of the Rous sarcoma virus glycoprotein precursor by introducing mutations into the region encoding the cleavage recognition site (Arg-Arg-Lys-Arg). In addition to mutants G1 (Arg-Arg-Glu-Arg) and Dr1 (deletion of all four codons) that we have reported on previously (L. G. Perez and E. Hunter, J. Virol. 61:1609-1614, 1987), we constructed two additional mutants, AR1 (Arg-Arg-Arg-Arg), in which the highly conserved lysine is replaced by an arginine, and S19 (Ser-Arg-Glu-Arg), in which no dibasic pairs remain. The results of these studies demonstrate that when the cleavage sequence is deleted (Dr1) or modified to contain unpaired basic residues (S19), intracellular cleavage of the glycoprotein precursor is completely blocked. This demonstrates that the cellular endopeptidase responsible for cleavage has a stringent requirement for the presence of a pair of basic residues (Arg-Arg or Lys-Arg). Furthermore, it implies that the cleavage enzyme is not trypsinlike, since it is unable to recognize arginine residues that are sensitive to trypsin action. Substitution of the mutated genes into a replication-competent avian retrovirus genome showed that cleavage of the glycoprotein precursor was not required for incorporation into virions but was necessary for infectivity. Treatment of BH-RCAN-S19-transfected turkey cells with low levels of trypsin resulted in the release of infectious virus, demonstrating that exogenous cleavage could generate a biologically active glycoprotein molecule.     

Isolation and biochemical characterization of the alpha- and beta-subunits of glycoprotein IIb of human platelet plasma membrane.

The alpha- and beta-subunits of glycoprotein IIb (GPIIb) of human platelet plasma membrane were isolated in fully reduced, partially reduced and alkylated, and fully alkylated forms, by size-exclusion chromatography after reduction of pure GPIIb. The sugar moiety of GPIIb alpha accounts for 16.4% of its total weight, whereas that of GPIIb beta accounts for only 10.2%. The molar percentages (per 100 mol of total amino acids) of neuraminic acid and galactose in the alpha-subunit more than double those in the beta-subunit, whereas galactosamine is present only in GPIIb alpha. From the amino acid and sugar compositions the acidic nature of both subunits was confirmed. The Mr values obtained, 114,000 for GPIIb alpha and 22,200 for GPIIb beta, are in very good agreement with those obtained by physical methods. We found by stepwise reduction of pure GPIIb with dithioerythritol that GPIIb alpha and GPIIb beta are joined by a single interchain disulphide bridge, while the remaining half-cystine residues participate in intrachain bonds, six in GPIIb alpha and one in GPIIb beta, the intersubunit disulphide bond being that reduced first. Neither of the two subunits is liberated from isolated plasma membranes when this GPIIb interchain bond is reduced in isolated membranes.     

Complement component C1r mediated cleavage of the heavy chain of the major histocompatibility class I antigens.

Apart from cleaving C1s, we demonstrate for the first time that: 1) at concentrations found in serum, the activated forms of the complement components C1r in addition to C1s can cleave the heavy chain of MHC class I antigens, 2) the cleavage by C1r and C1s is seemingly dependent upon a native configuration of the MHC class I antigen, since heat denaturation of the HLA antigens reduce the cleavage. The proteolytic fragments following C1 cleavage were characterized by precipitation with Con A-Sepharose, anti-MHC class I and anti-beta 2-microglobulin antibodies. The proteolysis of the alpha-chain of MHC class I was shown to take place between the alpha 2- and alpha 3- domains as estimated by the Con A-Sepharose precipitation pattern on SDS-PAGE. The alpha 1/alpha 2 fragment was still shown to interact with beta 2-microglobulin as shown by immunoprecipitation.