Decorsin. A potent glycoprotein IIb-IIIa antagonist and platelet aggregation inhibitor from the leech Macrobdella decora

The discovery, purification, and characterization of decorsin, a protein isolated from the North American leech Macrobdella decora, are described. Decorsin acts as an antagonist of platelet glycoprotein IIb-IIIa (GPIIb-IIIa), and is a potent inhibitor of platelet aggregation. The protein was purified to apparent homogeneity from crude whole leech extracts by treatment with trifluoroacetic acid followed by GPIIb-IIIa affinity chromatography and C18 reverse-phase high performance liquid chromatography. Decorsin was also isolated from a solution of leech ingestate by treatment with trifluoroacetic acid followed by C18 reverse-phase high performance liquid chromatography. The primary sequence of decorsin indicates that the protein is 39 amino acids long and contains 6 cysteine and 6 proline residues, as well as the sequence Arg-Gly-Asp, (RGD), a proposed recognition site of many adhesion proteins. A molecular mass of 4379 was obtained by fast atom bombardment mass spectrometry and is consistent with the mass calculated from the observed sequence. Evidence for an N-3 isoform, lacking the first 3 amino-terminal residues is also presented. Both decorsin and the N-3 isoform inhibit GP IIb-IIIa binding to immobilized fibrinogen with an IC50 of approximately 1.5 nM. Human platelet aggregation induced by ADP is inhibited by decorsin with an IC50 of approximately 500 nM; complete inhibition was observed at less than or equal to 1 microM. Based on overall sequence homology, decorsin does not belong to the family of GPIIb-IIIa protein antagonists that is found in snake venoms (Dennis, M. S., Henzel, W. J., Pitti, R. M., Lipari, M. T., Napier, M. A., Deisher, T. A., Bunting, S., and Lazarus, R. A. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 2471-2475); however the carboxyl-terminal RGD-containing region from residues 27 to 38 of decorsin is approximately 60% homologous with the corresponding region of the snake venom proteins, suggesting that high affinity binding of these proteins to GPIIb-IIIa is defined by this epitope.     

Sequential 1H NMR assignments of kistrin, a potent platelet aggregation inhibitor and glycoprotein IIb-IIIa antagonist.

Sequence-specific nuclear magnetic resonances assignments have been obtained for the protons of kistrin. Kistrin is a small naturally occurring snake venom protein that inhibits platelet aggregation by blocking the interaction of fibrinogen with the membrane-bound glycoprotein IIb-IIIa (GP IIb-IIIa), a receptor from the integrin family. Kistrin has an Arg-Gly-Asp sequence which is believed to form an adhesion recognition sequence that is essential for activity. Therefore, the interaction between kistrin and GP IIb-IIIa may provide important information on the motif used by integrins to recognize their target proteins which bear RGD sequences. Kistrin consists of 68 residues and contains six intramolecular disulfide bonds. Although one-third of the amide protons are protected from exchange with the solvent, there appears to be little or no regular secondary structure. The large number of NOE's between residues separated by two and three positions in the sequence indicates that the protein contains a large number of tightly packed loops. Along with the sequential assignments, this paper also discusses the construction and use of computerized data bases for manipulating NMR results. A strategy for computer-assisted sequential resonance using these data bases is also presented.     

Ornatins: potent glycoprotein IIb-IIIa antagonists and platelet aggregation inhibitors from the leech Placobdella ornata.

The purification and characterization of six isoforms of ornatin, potent glycoprotein IIb-IIIa (GP IIb-IIIa) antagonists and platelet aggregation inhibitors are described. These isoforms were purified from whole leech homogenates of the leech Placobdella ornata, a North American leech commonly known as the turtle leech, by trichloroacetic acid precipitation, Sephadex G-50 size exclusion chromatography, GP IIb-IIIa affinity chromatography, and C18 reverse-phase HPLC. Each of the five completely sequenced isoforms, which range from 41 to 52 residues in length, contains the Arg-Gly-Asp (RGD) sequence, a common recognition sequence in adhesion proteins, as well as 6 cysteine residues; the positions of both of these features are conserved in the primary sequences. The amino acid sequences of ornatin isoforms B, C, D, and E are highly conserved, whereas ornatin A2 and A3 are less similar and lack 9 residues at the N-terminus. The ornatins are approximately 40% identical with decorsin, a GP IIb-IIIa antagonist isolated from the leech Macrobdella decora [Seymour, J. L., Henzel, W. J., Nevins, B., Stults, J. T. & Lazarus, R. A. (1990) J. Biol. Chem. 265, 10143-10147]; furthermore, the RGD sequence and 5 out of 6 cysteine residues are maintained in the same relative positions in both decorsin and ornatin. The ornatin isoforms do not exhibit significant similarity to any members of the snake-venom-derived family of GP IIb-IIIa antagonists [Dennis, M. S., Henzel, W. J., Pitti, R. M., Lipari, M. T., Napier, M. A., Deisher, T. A., Bunting, S. & Lazarus, R. A. (1990) Proc. Natl Acad. Sci. USA 87, 2471-2475] except in the RGD region of these proteins. The ornatin isoforms inhibit the binding of GP IIb-IIIa to immobilized fibrinogen with IC50 values ranging over 2.9-5.3 nM; ornatin isoforms A2, C, and E inhibit ADP-induced human platelet aggregation with IC50 values of about 130, 280, and 440 nM, respectively.     

Triflavin, an antiplatelet Arg-Gly-Asp-containing peptide, is a specific antagonist of platelet membrane glycoprotein IIb-IIIa complex

Triflavin, an antiplatelet peptide containing Arg-Gly-Asp, purified from Trimeresurus flavoviridis venom, inhibits aggregation of human platelets stimulated by a variety of agonists. It blocks aggregation through interference with fibrinogen binding to its specific receptor on the platelet surface membrane in a competitive manner, but it has no apparent effect on intracellular events, such as thromboxane B2 formation, phosphoinositides breakdown and intracellular Ca2+ mobilization of thrombin-activated platelets. In this study, we determined the complete sequence of triflavin, which is composed of a single polypeptide chain of 70 amino acids. Its sequence is rich in cysteine and contains Arg-Gly-Asp at residues 49-51 in the carboxy-terminal domain. Triflavin shows about 68% identity of amino acid sequence with trigramin, which is a specific antagonist of the fibrinogen receptor associated with glycoprotein IIb/IIIa complex. [125I]Triflavin binds to unstimulated and ADP-stimulated platelets in a saturable manner and its Kd values are estimated to be 76 and 74 nM, respectively; the corresponding numbers of binding sites are 31,029 and 34,863 per platelet, respectively. [125I]Triflavin binding is blocked by Gly-Arg-Gly-Asp-Ser in a competitive manner. EDTA, the Arg-Gly-Asp-containing peptides (including naturally occurring polypeptides, trigramin and rhodostomin), and monoclonal antibody, 7E3, raised against GP IIb/IIIa complex, inhibit [125I]triflavin binding to unstimulated and ADP-stimulated human platelets. In conclusion, triflavin specifically binds to fibrinogen receptor associated with GP IIb/IIIa complex and its binding site is located at or near GP IIb/IIIa complex, overlapping with those of 7E3 and another Arg-Gly-Asp-containing polypeptide, rhodostomin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mean platelet volume: Interrelation with platelet aggregation activity and glycoprotein IIb-IIIa and Ib expression levels

Increased mean platelet volume (MPV) is an independent risk factor of thrombotic events in patients with cardiovascular diseases. Interactions of MPV with platelet aggregation activity and contents of glycoprotein (GP) IIb-IIIa (αIIb/β3 integrin, fibrinogen receptor) and GP Ib (von Willebrand factor receptor) have been investigated in this study. The study was performed in a group of healthy volunteers (n = 38) and a group of patients with acute coronary syndrome (ACS, n = 116). Patient’s blood was collected at days 1, 3–5 and 8–12 after ACS development. All patients received acetylsalicylic acid (ASA, inhibitor of thromboxane A2 synthesis) as the antiaggregant therapy and most of them also received clopidogrel (ADP receptor antagonist), except 44 patients who had not taken clopidogrel at day 1 before first blood collection. Aggregation of volunteers’ platelets was stimulated by 1.25, 2.5, 5 and 20 μM ADP, while aggregation of patients’ platelets was stimulated by 5 and 20 μM ADP. GP IIb-IIIa and GP Ib content on the platelet surface was measured using ¹²⁵I-labelled monoclonal antibodies. GP IIb-IIIa and GP Ib genetic polymorphisms were determined in ACS patients. In healthy donors significant correlations between MPV and aggregation levels have been recognized at 1.25 μM and 2.5 μM ADP (correlation coefficient (r) values of 0.396 and 0.373, p < 0.05), while at 5 μM and 20 μM ADP these interactions did not reach the level of statistical significance (r values of 0.279 and 0.205, p > 0.05). Correlations between MPV and aggregation levels were observed at day 1 of ACS in a subgroup of patients receiving ASA but before the beginning of clopidogrel treatment (r values of 0.526, p < 0.001 and 0.368, p < 0.05 for 5 and 20 μM ADP, respectively). Correlations between these parameters were not found during combined treatment of patients with ASA and clopidogrel. Strong direct correlations between MPV and GP IIb-IIIa and GP Ib contents were detected in both healthy donors and ACS patients (at all time points): the r values ranged from 0.439 to 0.647 (p ≤ 0.001 for all correlations). Genetic polymorphisms of GP IIb-IIIa (GP IIIa Leu33Pro) and GP Ib ((?5)T/C (Kozak) and Thr145Met) identified in ACS patients did not affect expression levels of corresponding glycoproteins. The data obtained indicate that increased MPV values correlate with increased platelet aggregation activity and enhanced GP IIb-IIIa and GP Ib expression.     

Inhibition of Fc-receptor dependent platelet aggregation by monoclonal antibodies against the glycoprotein IIb-IIIa complex]

A murine monoclonal antibody (MoAb) VM16a specifically binding to human platelets has been produced. Approximately 56,000 molecules of VM16a bound per platelet at saturation (Kd = 7.9 nM) but no binding to platelets from Glanzmann's thrombasthenia patients was detected. VM16a precipitated two proteins with molecular masses corresponding to those of glycoproteins (GP) IIb and IIIa from solubilized surface-labelled platelets. However, after dissociation of the GPIIb--IIIa complex with EDTA VM16a did not bind to platelets and precipitated nothing from their lysate, thus evidencing that its determinant is complex-dependent. VM16a had no effect on ADP-, thrombin- and ristocetin-induced platelet aggregation but inhibited the aggregation induced by collagen. This inhibitory effect was more pronounced in the presence of plasma. VM16a completely blocked the Fc-receptor-mediated aggregation induced by aggregated human IgG, aggregated murine IgG1 and the previously described MoAb VM58. F(ab')2 fragments of VM16a were also able to inhibit this aggregation by decreasing the rate of aggregation induced by aggregated IgG and by extending the lag phase of VM58-induced aggregation. These results suggest that the platelet Fc-receptor may be topographically associated with the GPIIb-IIIa complex.     

Identification of two structurally and functionally distinct sites on human platelet membrane glycoprotein IIb-IIIa using monoclonal antibodies

Platelet membrane glycoprotein IIb-IIIa exists as a calcium-dependent complex of two large peptides (designated IIb and IIIa) in Triton X-100 solutions, but it remains unknown if these peptides are subunits of one glycoprotein or are actually two individual glycoproteins in the intact platelet membrane. We used crossed immunoelectrophoresis to define the epitopes of two monoclonal antibodies to IIb-IIIa, then used these antibodies to study the structural and functional organization of IIb and IIIa in the platelet membrane. Human platelets solubilized in Triton X-100 were electrophoresed through an intermediate gel containing 125I-monoclonal IgG, then into an upper gel containing rabbit anti-human platelet antibodies. Our previously characterized antibody. Tab, and a new monoclonal antibody, T10, both bound to the immunoprecipitate corresponding to the IIb-IIIa complex. When platelets were electrophoresed after solubilization in 5 mM EDTA, 125I-Tab bound to the dissociated IIb polypeptide, but not to IIIa. In contrast, 125-I-T10 did not react with either IIb or IIIa. Thus, Tab recognizes a determinant on IIb, while T10 recognizes a determinant created only after the association of IIb and IIIa. Gel-filtered platelets from six normal donors bound 50,600 +/- 5,600 125I-T10 molecules/platelet and 47,800 +/- 11,200 125I-Tab molecules/platelet, consistent with IIb-IIIa being a heterodimer. 125I-T10 binding was identical in unactivated platelets and platelets stimulated with 10 microM ADP. However, platelets did not aggregate or bind 125I-fibrinogen until ADP was added. T10, but not Tab or nonimmune mouse antibody, inhibited ADP-induced platelet aggregation and 125I-fibrinogen binding. Our findings suggest that IIb and IIIa exist as subunits of a single membrane glycoprotein in unstimulated platelets. Fibrinogen binding appears to require not only the interaction of IIb and IIIa, but also some additional change occurring after platelet activation.     

Characterization of a potent platelet aggregation inhibitor from Agkistrodon rhodostoma snake venom

By means of CM-Sephadex C-50 column chromatography and gel filtration on Sephadex G-75 and G-50 columns, a potent platelet aggregation inhibitor was purified and characterized. It was a glycoprotein with a molecular weight of 31,000. It was devoid of phospholipase A, ADPase, esterase and fibrino(geno)lytic activities. It inhibited dose-dependently the aggregation of washed platelets induced by collagen, thrombin, sodium arachidonate, platelet activating factor and ionophore A23187 with a similar IC50 (5-10 micrograms/ml). It was also active in platelet-rich plasma, with an IC50 of 10-15 micrograms/ml. The venom inhibitor reduced the elasticity of whole blood clot and inhibited the thrombin-induced clot retraction of platelet-rich plasma. These activities were related to its inhibitory activity on platelet aggregation rather than blood coagulation. The venom inhibitor had various effects on [14C]serotonin release stimulated by aggregation agonists. It had no effect on thromboxane B2 formation of platelets stimulated by sodium arachidonate, collagen and ionophore A23187. The presence of this venom inhibitor prior to the initiation of aggregation was a prerequisite for the maintenance of its maximal activity. It showed a similar inhibitory effect on collagen or thrombin-induced aggregation even when it was added after the platelets had undergone the shape change. High fibrinogen levels partially antagonized its activity. The venom inhibitor completely inhibited the fibrinogen-induced aggregation of alpha-chymotrypsin-treated platelets. It is concluded that this venom inhibitor interferes with the interaction of fibrinogen with fibrinogen receptors, leading to inhibition of aggregation.     

HL 725, an extremely potent inhibitor of platelet phosphodiesterase and induced platelet aggregation in vitro

The new pyrimido-isoquinoline compound HL 725 is an extremely potent inhibitor of the aggregation of human platelets induced in vitro by ADP, collagen, thrombin and epinephrine. The aggregation induced by 0,5 mM arachidonic acid is inhibited about 50% with 50 pM HL 725. Thus the potency of HL 725 is higher than that of prostacyclin, the most active natural inhibitor of aggregation. We hypothesize that HL 725 inhibits the enzymatic degradation of cyclic adenosine 3', 5'-monophosphate (cAMP) in the platelets. In accordance with this proposal is the strong inhibitory action on cAMP phosphodiesterase extracted from human platelets. About 250 pM HL 725 inhibited 50% of the activity of this enzyme at a substrate concentration of 0.5 microM. A marked elevation of cAMP levels in human platelets could be demonstrated after incubation in vitro with 100 nM HL 725.     

Chemical synthesis and structural characterization of the RGD-protein decorsin: a potent inhibitor of platelet aggregation.

Decorsin is a 39-residue RGD-protein crosslinked by three disulfide bridges isolated from the leech Macrobdella decora belonging to the family of GPIIb-IIIa antagonists and acting as a potent inhibitor of platelet aggregation. Here we report the solid-phase synthesis of decorsin using the Fmoc strategy. The crude polypeptide was purified by reverse-phase HPLC in its reduced form and allowed to refold in the presence of glutathione. The homogeneity of the synthetic oxidized decorsin was established by reverse-phase HPLC and capillary zone electrophoresis. The results of amino acid analysis after acid hydrolysis of the synthetic protein, NH2-terminal sequencing and mass determination (4,377 Da) by electrospray mass spectrometry were in full agreement with this theory. The correct pairing of the three disulfide bridges in synthetic decorsin was determined by a combined approach of both peptide mapping using proteolytic enzymes and analysis of the disulfide chirality by CD spectroscopy in the near-UV region. Synthetic decorsin inhibited human platelet aggregation with an IC50 of approximately 0.1 microM, a figure quite similar to that determined utilizing decorsin from natural source. In particular, the synthetic protein was 2,000-fold more potent than a model RGD-peptide (e.g., Arg-Gly-Asp-Ser) in inhibiting platelet aggregation. Thermal denaturation experiments of synthetic decorsin, monitored by CD spectroscopy, revealed its high thermal stability (Tm approximately 74 degrees C). The features of the oxidative refolding process of reduced decorsin, as well as the thermal stability of the oxidized species, were compared with those previously determined for the NH2-terminal core domain fragment 1-41 or 1-43 from hirudin. This fragment shows similarity in size, pairing of the three disulfides and three-dimensional structure with those of decorsin, even if very low sequence similarity. It is suggested that the less efficient oxidative folding and the enhanced thermal stability of decorsin in respect to those of hirudin core domain likely can be ascribed to the presence of the six Pro residues in the decorsin chain, whereas none is present in the hirudin domain. The results of this study indicate that decorsin can be obtained by solid-phase methodology in purity and quantities suitable for structural and functional studies and thus open the way to prepare by chemical methods novel decorsin derivatives containing unusual amino acids or even non-peptidic moieties.     

Anti-idiotypic antibodies against an antibody to the platelet glycoprotein (GP) IIb-IIIa complex mimic GP IIb-IIIa by recognizing fibrinogen.

Binding of the adhesive ligand fibrinogen and the monoclonal antibody PAC1 to platelet glycoprotein (GP) IIb-IIIa is dependent on cell activation and inhibited by Arg-Gly-Asp (RGD)-containing peptides. Previously, we identified a sequence in a hypervariable region of PAC1 (mu-CDR3) that mimics the activity of the antibody. Here we examine whether monoclonal antibodies to this idiotypic determinant in PAC1 can mimic GP IIb-IIIa by binding to fibrinogen. Mice were immunized with a peptide derived from the mu-CDR3 of PAC1. Four antibodies were obtained that recognized fibrinogen as well as a recombinant form of the variable region of PAC1. However, they did not bind to other RGD-containing proteins, including von Willebrand factor, fibronectin, and vitronectin. Several studies suggested that these anti-PAC1 peptide antibodies were specific for GP IIb-IIIa recognition sites in fibrinogen. Three such sites have been proposed: two RGD-containing regions in the A alpha chain, and the COOH terminus of the gamma chain (gamma 400-411). Two of the antibodies inhibited fibrinogen binding to activated platelets, and all four antibodies bound to the fibrinogen A alpha chain on immunoblots. Antibody binding to immobilized fibrinogen was partially inhibited by monoclonal antibodies specific for the two A alpha chain RGD regions. However, the anti-PAC1 peptide antibodies also bound to plasmin-derived fibrinogen fragments X and D100, which contain gamma 400-411 but lack one or both A alpha RGD regions. This binding was inhibited by an antibody specific for gamma 400-411. When fragment D100 was converted to D80, which lacks gamma 400-411, antibody binding was reduced significantly (p less than 0.01). Electron microscopy of fibrinogen-antibody complexes confirmed that each antibody could bind to sites on the A alpha and gamma chains. These studies demonstrate that certain anti-PAC1 peptide antibodies mimic GP IIb-IIIa by binding to platelet recognition sites in fibrinogen. Furthermore, they suggest that the gamma 400-411 region of fibrinogen may exist in a conformation similar to that of an A alpha RGD region of the molecule.     

Kifunensine, a potent inhibitor of the glycoprotein processing mannosidase I

Kifunensine, produced by the actinomycete Kitasatosporia kifunense 9482, is an alkaloid that corresponds to a cyclic oxamide derivative of 1-amino mannojirimycin. This compound was reported to be a weak inhibitor of jack bean alpha-mannosidase (IC50 of 1.2 x 10(-4) M) (Kayakiri, H., Takese, S., Shibata, T., Okamoto, M., Terano, H., Hashimoto, M., Tada, T., and Koda, S. (1989) J. Org. Chem. 54, 4015-4016). We also found that kifunensine was a poor inhibitor of jack bean and mung bean aryl-alpha-mannosidases, but it was a very potent inhibitor of the plant glycoprotein processing enzyme, mannosidase I (IC50 of 2-5 x 10(-8) M), when [3H]mannose-labeled Man9GlcNAc was used as substrate. However, kifunensine was inactive toward the plant mannosidase II. Studies with rat liver microsomes also indicated that kifunensine inhibited the Golgi mannosidase I, but probably does not inhibit the endoplasmic reticulum mannosidase. Kifunensine was tested in cell culture by examining its ability to inhibit processing of the influenza viral glycoproteins in Madin-Darby canine kidney cells. Thus, when kifunensine was placed in the incubation medium at concentrations of 1 microgram/ml or higher, it caused a complete shift in the structure of the N-linked oligosaccharides from complex chains to Man9(GlcNAc)2 structures, in keeping with its inhibition of mannosidase I. On the other hand, even at 50 micrograms/ml, deoxymannojirimyucin did not prevent the formation of all complex chains. Thus kifunensine appears to be one of the most effective glycoprotein processing inhibitors observed thus far, and knowledge of its structure may lead to the development of potent inhibitors for other processing enzymes.     

Isolation and characterization of a chymotryptic fragment of platelet glycoprotein IIb-IIIa retaining Arg-Gly-Asp binding activity.

Platelet membrane glycoprotein (GP)IIb-IIIa exists as a divalent cation-dependent heterodimer which recognizes the Arg-Gly-Asp (RGD) sequence of adhesive proteins. To isolate the RGD binding domain of GPIIb-IIIa we performed proteolysis of GPIIb-IIIa with alpha-chymotrypsin. GPIIb-IIIa was bound to an affinity matrix of GRGDSPK-coupled Sepharose 4B and was then treated with chymotrypsin. After washing the unbound fragments, two discrete polypeptides of 55 and 85 kDa remained bound to the RGD affinity matrix and were specifically eluted by soluble HHLGGAKQAGDV (H12) or by GRGDSP, but not by GRGESP. Immunoblotting with subunit-specific polyclonal antibodies showed that the 55- and 85-kDa fragments were derived from GPIIb and GPIIIa, respectively. Amino-terminal sequencing and immunoblotting using site-specific antibodies indicated that these fragments contained the amino termini of their parent molecules. In the presence of 1 mM Ca2+ and 1 mM Mg2+, these two fragments were maintained as a heterodimer inasmuch as both fragments were immunoprecipitated by the polyclonal anti-GPIIIa antibodies. In contrast, chelating the divalent cations with 5 mM EDTA resulted in the lack of co-immunoprecipitation of the 55-kDa GPIIb fragment. After removal of the H12 peptide, the 55/85-kDa heterodimer bound to immobilized fibrinogen in an enzyme-linked immunosorbent assay by an RGD-dependent mechanism. These findings suggest that the RGD binding domain and structures required for heterodimer maintenance are present within the 55/85-kDa chymotryptic fragment of GPIIb-IIIa.     

Human platelet glycoprotein V: a surface leucine-rich glycoprotein related to adhesion

Human platelet glycoprotein V (Mr 82,000) is a surface glycoprotein and a substrate for thrombin, undergoing proteolytic cleavage by thrombin and releasing a soluble fragment, glycoprotein Vfl (Mr 69,000). It does not appear to be the receptor for thrombin's agonist effect on platelets. A congenital platelet disorder, Bernard-Soulier syndrome, is marked by a deficiency of glycoprotein V and two other surface glycoproteins, Ib-IX. The latter two, Ib-IX, constitute the platelet receptor for von Willebrand factor, mediate arterial platelet adhesion, and contain unique 24-amino acid sequences, termed "leucine-rich glycoprotein" segments. The segments relate to adhesive function and distinguish the leucine-rich glycoprotein family. Surface glycoprotein V is not physically associated with Ib-IX nor does it bind to von Willebrand factor. To date, no common denominator has been found that explains the combined deficiency of glycoproteins V and Ib-IX in Bernard-Soulier syndrome. This study describes the isolation of glycoprotein V/anti-glycoprotein V antibody and the analysis of three glycoprotein V peptides that contain "leucine-rich" sequences. Therefore, glycoprotein V shares the "leucine-rich" structure with platelet glycoproteins Ib-IX and belongs to the family of leucine-rich glycoproteins.     

Studies on the interaction of platelet glycoprotein IIb-IIIa and glycoprotein IV with fibrinogen and thrombospondin: a new immunochemical approach

We have designed a new binding assay based on crossed immunoelectrophoresis that allowed us to test for the relative capacities of platelet membrane glycoprotein IIb-IIIa (GP IIb-IIIa), and glycoprotein IV (GP IV) to bind purified Arg-Gly-Asp (RGD)-containing adhesive proteins. Preformed immune complexes were made by reacting a platelet lysate with murine monoclonal antibodies to GP IV (OKM5 and FA6-152) or to GP IIb-IIIa (AP-2). Upon two-dimensional electrophoretic separation in agarose gels and immunoprecipitation by a polyclonal antibody to mouse IgG, the immobilized complexes containing the desired antigen were further probed with purified 125I-labeled TSP or fibrinogen. Under these conditions, immobilized GP IV was found to specifically bind TSP, whereas it was unreactive with fibrinogen. By contrast, immobilized GP IIb-IIIa demonstrated fibrinogen binding capacity but did not demonstrate any reactivity toward TSP. These observations suggest that the overall structure of the adhesive protein may determine the accessibility of the RGD sequence to its binding site on GP IIb-IIIa.     

Immunochemical characterization of a new platelet specific monoclonal antibody and its use to demonstrate the cytoskeletal association of the platelet glycoprotein IIb-IIIa complex

We describe the production and characterization of a monoclonal antibody specific for platelets. This antibody reacts strongly with human and primate platelets, but does not recognise human monocytes, polymorphonuclear leucocytes, lymphocytes, erythrocytes, leukaemic nor fibroblast cell lines, nor rodent platelets. Immunoprecipitation studies using radiolabelled platelet membrane proteins showed that the monoclonal antibody binds to the platelet membrane glycoprotein IIb-IIIa complex. Affinity chromatography using immobilized monoclonal antibody allows purification of the antigen, but also co-purifies the cytoskeletal proteins actin and myosin.Our results demonstrate immunochemically that although the GP IIb-IIIa complex is an external structure, it is connected through the cell membrane to the microfilament system.     

Reconstitution of the purified platelet fibrinogen receptor. Fibrinogen binding properties of the glycoprotein IIb-IIIa complex

Several lines of evidence indicate that the platelet membrane glycoprotein IIb-IIIa complex (GP IIb-IIIa) is necessary for the expression of platelet fibrinogen receptors. The purpose of the present study was to determine whether purified GP IIb-IIIa retains the properties of the fibrinogen receptor on platelets. Glycoprotein IIb-IIIa was incorporated by detergent dialysis into phospholipid vesicles composed of 30% phosphatidylcholine and 70% phosphatidylserine. 125I-Fibrinogen binding to the GP IIb-IIIa vesicles, as measured by filtration, had many of the characteristics of 125I-fibrinogen binding to whole platelets or isolated platelet plasma membranes: binding was specific, saturable, reversible, time dependent, and Ca2+ dependent. The apparent dissociation constant for 125I-fibrinogen binding to GP IIb-IIIa vesicles was 15 nM, and the maximal binding capacity was 0.1 mol of 125I-fibrinogen/mol of GP IIb-IIIa. 125I-Fibrinogen binding was inhibited by amino sugars, the GP IIb and/or IIIa monoclonal antibody 10E5, and the decapeptide from the carboxyl terminus of the fibrinogen gamma chain. Furthermore, little or no 125I-fibrinogen bound to phospholipid vesicles lacking protein or containing proteins other than GP IIb-IIIa (i.e. bacteriorhodopsin, apolipoprotein A-I, or glycophorin). Also, other 125I-labeled plasma proteins (transferrin, orosomucoid) did not bind to the GP IIb-IIIa vesicles. These results demonstrate that GP IIb-IIIa contains the platelet fibrinogen receptor.     

Batroxostatin, an Arg-Gly-Asp-containing peptide from Bothrops atrox, is a potent inhibitor of platelet aggregation and cell interaction with fibronectin

A potent inhibitor of platelet aggregation and cell adhesion was isolated from the venom of Bothrops atrox. This peptide, referred to as batroxostatin, was composed of 71 amino acids and showed a high degree of homology with other snake venom peptides including trigramin, albolabrin, elegantin and applagin: all 12 cysteines and the RGD sequence (standard one-letter amino acid codes) aligned in the same position. Compared on a molar basis, the anti-platelet aggregation activity of batroxostatin was about 1000-times higher than that of RGDS. In addition, batroxostatin was about 400-times more potent than GRGDS at inhibiting melanoma cell adhesion to fibronectin. Batroxostatin covalently attached to plastic promoted adhesion of melanoma cells. The anti-GP140 antibody, recognizing beta 1 integrins, completely inhibited adhesion of mouse melanoma cells to batroxostatin. This observation, in addition to the inhibitory effect of batroxostatin on the adhesion of chick fibroblasts to fibronectin, suggests that batroxostatin interacts with integrins from both the beta 1 and beta 3 subfamilies.