Ca2+-dependent binding of a synthetic Arg-Gly-Asp (RGD) peptide to a single site on the purified platelet glycoprotein IIb-IIIa complex

The platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa) is a member of the integrin receptor family that recognizes adhesive proteins containing the Arg-Gly-Asp (RGD) sequence. In the present study the binding characteristics of the synthetic hexapeptide Tyr-Asn-Arg-Gly-Asp-Ser (YNRGDS, a sequence present in the fibrinogen alpha-chain at position 570-575) to purified GP IIb-IIIa were determined by equilibrium dialysis. The binding of 125I-YNRGDS to GP IIb-IIIa was specific, saturable, and reversible. The apparent dissociation constant was 1.0 +/- 0.2 microM, and the maximal binding capacity was 0.92 +/- 0.02 mol of 125I-YNRGDS/mol of GP IIb-IIIa, indicating that GP IIb-IIIa contains a single binding site for RGD peptides. The binding of 125I-YNRGDS to purified GP IIb-IIIa showed many of the characteristics of fibrinogen binding to activated platelets: the binding was inhibited by fibrinogen, by the monoclonal antibody A2A9, and by the dodecapeptide from the C terminus of the fibrinogen gamma-chain. In addition, the binding of 125I-YNRGDS to GP IIb-IIIa was divalent cation-dependent. Our data suggest that two divalent cation binding sites must be occupied for YNRGDS to bind: one site is specific for calcium and is saturated at 1 microM free Ca2+, whereas the other site is less specific and reaches saturation at millimolar concentrations of either Ca2+ or Mg2+. The results of the present study support the hypothesis that the RGD domains within the adhesive proteins are responsible for their binding to GP IIb-IIIa.     

Modulation of an RGDS binding site on the platelet membrane glycoprotein IIb-IIIa complex

Fibronectin, von Willebrand factor, and fibrinogen each bind to the glycoprotein IIb-IIIa complex on activated platelets via an arg-gly-asp-ser (RGDS) sequence present within the adhesive proteins. Both the IIb and IIIa polypeptides of the IIb-IIIa complex on thrombin activated platelets are specifically and extensively labeled by a radiolabeled, photoactivatable arylazide derivative of the RGDS sequence when the labeling is performed in the presence of concentrations of Ca++ or Mg++ approaching 0.5 mM. In contrast, labeling of unactivated platelets, ADP activated platelets, or thrombin activated platelets in the presence of low concentrations of divalent cations resulted in restriction of labeling to the IIb polypeptide of the complex.     

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.     

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.     

Dynamics of platelet glycoprotein IIb-IIIa receptor expression and fibrinogen binding. II. Quantal activation parallels platelet capture in stir-associated microaggregation.

There is broad agreement that platelet aggregation is generally dependent on fibrinogen (Fg) binding to the glycoprotein (GP) IIb-IIIa receptor expressed on the activated platelet surface. We therefore compared rates and extents of aggregation and of fibrinogen receptor expression and specific Fg binding to activated platelets, as a function of ADP concentration. Human citrated platelet-rich plasma (diluted 10-fold) was stirred with adenosine diphosphate (ADP) for 10 s or 2 min to measure rates and extent of aggregation, respectively, determined from the decrease in the total number of particles. The number of fibrinogen receptors and bound Fg were measured from mean fluorescence values obtained with FITC-labeled IgM monoclonal antibody PAC1 and the IgG monoclonal antibody, 9F9, respectively, using flow cytometry as presented in part I (Frojmovic et al., 1994). Because flow cytometric and aggregation measurements were routinely determined at room temperature and 37 degrees C, respectively, we also compared and found temperature-independent initial rates of aggregation. The fraction of platelets with fluorescence values above one critical threshold value, corresponding to maximally "activated" platelets (P*), increased with increasing activator concentration and correlated linearly with the fraction of platelets recruited into aggregates for ADP (r > 0.9). Aggregation was not rate-limited by fibrinogen receptor expression or by Fg binding. It appears that each platelet expresses its maximal Fg receptors at a critical ADP concentration, i.e., occupancy of ADP receptors. This, in turn, leads to rapid Fg occupancy and capture of such "quantally activated" platelets into aggregates.     

Large-scale purification of active platelet integrin glycoprotein IIb-IIIa

Glycoprotein IIb-IIIa is an abundant platelet receptor of the integrin family that plays a primary role in platelet aggregation. It exists on the platelet surface predominantly in a resting or inactive conformation that is converted to an active binding competent conformation upon platelet activation. There is much interest in studying the difference between active and inactive GP IIb-IIIa, developing therapeutic agents targeted towards GP IIb-IIIa and developing diagnostic assays for antibodies that recognize epitopes on GP IIb-IIIa. We present here the development of a large-scale process for purifying active GP IIb-IIIa from human platelets. The procedure results in 25mg batch sizes of high purity and activity. Additionally, the effects of detergent concentration and impurities such as IgG on ELISA assays are examined.     

A receptor-induced binding site in fibrinogen elicited by its interaction with platelet membrane glycoprotein IIb-IIIa.

The interaction of fibrinogen with membrane glycoprotein GPIIb-IIIa regulates platelet aggregation. This ligand:integrin receptor interaction elicits conformational changes in GPIIb-IIIa as evidenced by the induction of ligand-induced binding sites which are recognized by antibodies that react selectively with the occupied receptor. The dynamic nature of these conformational changes is now demonstrated by the identification and characterization of a receptor-induced binding site (RIBS) elicited in fibrinogen bound to GPIIb-IIIa. A monoclonal antibody to fibrinogen, anti-Fg-RIBS-I, failed to bind to nonstimulated platelets in the presence or absence of fibrinogen. However, when platelets were stimulated with an agonist, the antibody reacted with platelet-bound fibrinogen even in the presence of a marked excess of unbound fibrinogen. A key element of the RIBS epitope has been precisely localized to residues 373-385 of the gamma chain of fibrinogen. Conformational elements also are important in defining the epitope. Fab fragments of the antibody inhibited platelet aggregation. As these fragments also inhibited fibrin polymerization, a commonality between these two diverse functions of fibrinogen in thrombus formation is indicated. In general, antibodies to RIBS and ligand-induced binding site provide unique probes for characterizing ligand:receptor interactions.     

Isolation and characterization of staphylocoagulase chymotryptic fragment. Localization of the procoagulant- and prothrombin-binding domain of this protein

Several strains of Staphylococcus aureus secrete a protein, staphylocoagulase, that binds stoichiometrically to human prothrombin, resulting in a coagulant complex designated staphylothrombin. In the present study, staphylocoagulase was digested with alpha-chymotrypsin and the resulting fragments were isolated by gel filtration. One fragment (Mr 43,000) exhibited a high affinity for human prothrombin (Kd = 1.7 X 10(-9) M), which is comparable to the affinity observed using intact staphylocoagulase (Kd = 4.6 X 10(-10) M). A complex of the Mr 43,000 fragment and prothrombin possessed both clotting and amidase activity essentially identical to that observed in a complex of intact staphylocoagulase and prothrombin. A second fragment (Mr 30,000) exhibited weaker affinity for prothrombin (Kd = 1.2 X 10(-7) M). While clotting activity was not observed with a complex of this fragment and prothrombin, it nonetheless possessed a weak amidase activity. A third fragment (Mr 20,000) was found to bind to prothrombin, but the resultant complex did not exhibit clotting or amidase activity. Amino-terminal sequence analyses of these staphylocoagulase fragments revealed that the Mr 43,000 fragment constitutes the amino-terminal portion of staphylocoagulase and also contains the Mr 30,000 and 20,000 fragments. Moreover, the amino-terminal sequence of the Mr 20,000 fragment was identical to that observed for the Mr 30,000 fragment. From these results, we conclude that the functional region of staphylocoagulase for binding and activation of human prothrombin is localized in the amino-terminal region of the intact bacterial protein.     

Dynamics of platelet glycoprotein IIb-IIIa receptor expression and fibrinogen binding. I. Quantal activation of platelet subpopulations varies with adenosine diphosphate concentration.

We have previously reported that maximal platelet activation with adenosine diphosphate (100 microM ADP) causes rapid expression of all GPIIb-IIIa receptors for fibrinogen (FgR) (< 1-3 s), measured with FITC-labeled PAC1 by flow cytometry. We have extended these studies to examine the effects of ADP concentration on the graded expression and Fg occupancy of GPIIb-IIIa receptors. Human citrated platelet-rich plasma, diluted 10-fold with Walsh-albumin-Mg+2 (2 mM), was treated with ADP (0.1-100 microM). The rates of GPIIb-IIIa receptor expression or Fg binding were measured in unstirred samples by flow cytometry, using FITC-labeled monoclonal antibodies (mAb) PAC1 and 9F9, respectively, from on-rates, using increasing times between mAb and ADP additions. Fibrinogen receptors were all expressed rapidly at low (1 microM) or high (100 microM) ADP (few seconds), whereas Fg occupancy was 50% of maximal by about 2 min. The maximal extent of GPIIb-IIIa receptor expression and Fg occupancy was determined from maximal binding (Flmax) at 30 min incubation with PAC1 or 9F9. On-rates and maximal extents of binding for either PAC1 or 9F9 probes showed identical [ADP]-response profiles ("KD" approximately 1.4 +/- 0.1 microM). However, Flmax studies showed bimodal histograms consisting of "resting" (Po) and maximally "activated" (P*) platelets for both PAC1 and 9F9 binding, with the fraction of "activated" platelets increasing with ADP concentration. The data best fit a model where platelet subpopulations are "quantally" transformed from Po to P*, expressing all GPIIb-IIIa receptors, rapidly filled by Fg, but "triggered" at critical ADP concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fibronectin-binding properties of the purified platelet glycoprotein IIb-IIIa complex

Fibronectin binds to specific receptors on the surface of washed, thrombin-activated platelets. Evidence suggests that these receptors are closely associated with the platelet glycoprotein IIb-IIIa complex (GP IIb-IIIa). To determine whether GP IIb-IIIa itself can form a platelet receptor for fibronectin, we used a filtration assay to examine the interaction of purified fibronectin with purified GP IIb-IIIa incorporated into phospholipid vesicles. 125I-Fibronectin binding to the phospholipid vesicles required the presence of incorporated GP IIb-IIIa and was specific, time-dependent, reversible, saturable, and divalent cation-dependent (Mg2+ greater than Ca2+). The dissociation constant for 125I-fibronectin binding to the GP IIb-IIIa-containing vesicles in the presence of 2 mM MgCl2 was 87 nM. Proteins or peptides that inhibit 125I-fibronectin binding to whole platelets also inhibited 125I-fibronectin binding to the GP IIb-IIIa vesicles. Thus, specific 125I-fibronectin binding was inhibited by excess unlabeled fibrinogen or fibronectin, the anti-GP IIb-IIIa monoclonal antibody 10E5, the decapeptide from the carboxyl terminus of the fibrinogen gamma-chain, and the tetrapeptide Arg-Gly-Asp-Ser from the cell-binding domain of fibronectin. In contrast to results obtained using whole platelets, unlabeled fibronectin inhibited 125I-fibronectin binding to the GP IIb-IIIa vesicles. These results show that 125I-fibronectin binds directly to purified GP IIb-IIIa with most of the previously reported properties of 125I-fibronectin binding to washed, thrombin-stimulated platelets. Thus, GP IIb-IIIa has the potential to function as a platelet receptor for fibronectin as well as for fibrinogen.     

Determination of the putative binding site for fibronectin on platelet glycoprotein IIb-IIIa complex through a hydropathic complementarity approach

We have applied the principle of complementary hydropathy to the prediction of the binding site for fibronectin (FN) and for the alpha-chain of fibrinogen in the platelet receptor complex glycoprotein (GP) IIb-IIIa. Since both ligands bind to it through their respective RGDS (Arg-Gly-Asp-Ser) domains and since both have been cloned, we were able to deduce the amino acid sequence of the binding site from the nucleotide sequence coding for RGDS in both proteins. The deduced peptides were very similar. Antibodies raised against a synthetic peptide WTVPTA (Trp-Thr-Val-Pro-Thr-Ala) deduced from the cloned rat FN RGDS domain block ADP-mediated platelet aggregation; this block can be overcome by additional fibrinogen. In Western blots of whole cell platelet extracts run under reducing conditions, this antibody binds to a 108-kDa band. It also binds to affinity-purified GP IIIa. Furthermore, it reacts strongly with GP IIIa immunoprecipitated by a commercially available anti-GP IIb-IIIa monoclonal antibody. Binding of affinity-purified GP IIb-IIIa complex to fibronectin is inhibited by the 110-kDa FN fragment. Similar inhibitions can be effected by WTVPTA (Trp-Thr-Val-Pro-Thr-Ala) and GAVSTA (Gly-Ala-Val-Ser-Thr-Ala) predicted from the rat and human fibronectin nucleotide sequences, respectively. GAGSTA (Gly-Ala-Gly-Ser-Thr-Ala) and GARSTA (Gly-Ala-Arg-Ser-Thr-Ala) related to the human peptide but with discrepant hydropathies are noninhibitory.     

Role of platelet membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins

Treatment of platelets with thrombin was shown previously to induce rapid changes in tyrosine phosphorylation of several platelet proteins. In this report, we demonstrate that a variety of agonists which induce platelet aggregation also stimulate tyrosine phosphorylation of three proteins with apparent molecular masses of 84, 95, and 97 kD. Since platelet aggregation requires the agonist-induced activation of an integrin receptor (GP IIb-IIIa) as well as the binding of fibrinogen to this receptor, we examined the relationship between tyrosine phosphorylation and the function of GP IIb-IIIa. When platelets were examined under conditions that either precluded the activation of GP IIb-IIIa (prior disruption of the complex by EGTA at 37 degrees C) or the binding of fibrinogen (addition of RGDS or an inhibitory mAb), tyrosine phosphorylation of the 84-, 95-, and 97-kD proteins was not observed. However, although both GP IIb-IIIa activation and fibrinogen binding were necessary for tyrosine phosphorylation, they were not sufficient since phosphorylation was observed only under conditions in which the activated platelets were stirred and allowed to aggregate. In contrast, tyrosine phosphorylation was not dependent on another major platelet response, dense granule secretion. Furthermore, granule secretion did not require tyrosine phosphorylation of this set of proteins. These experiments demonstrate that agonist-induced tyrosine phosphorylation is linked to the process of GP IIb-IIIa-mediated platelet aggregation. Thus, tyrosine phosphorylation may be required for events associated with platelet aggregation or for events that follow aggregation.     

A spontaneous mutation of integrin alpha IIb beta 3 (platelet glycoprotein IIb-IIIa) helps define a ligand binding site.

This work characterizes a mutant integrin alpha IIb beta 3 (glycoprotein (GP) IIb-IIIa) from a thrombasthenic patient, ET, whose platelets fail to aggregate in response to stimuli. The nature of defect was defined by the reduced ability of synthetic peptide ligands, corresponding to the carboxyl terminus of the fibrinogen gamma chain (gamma 402-411) and Arg-Gly-Asp (RGD), to increase the binding of the occupancy-dependent anti-LIBS1 antibody to mutant alpha IIb beta 3 and the reduced binding of mutant alpha IIb beta 3 to an immobilized RGD peptide. In addition, ET's platelets failed to bind the ligand-mimetic monoclonal anti-alpha IIb beta 3, PAC1. DNA sequence analysis of amplified ET genomic DNA revealed a single G----A base change which encoded substitution of R214 by Q in mature beta 3. Introduction of this point mutation into recombinant wild type alpha IIb beta 3 expressed in Chinese hamster ovary cells reproduced the ET platelet alpha IIb beta 3 deficits in binding of fibrinogen, mAb PAC1, and synthetic peptide ligands. Furthermore, substitution of R214 by Q in the synthetic peptide containing the sequence of beta 3(211-222) resulted in decreased ability of this peptide to block fibrinogen binding to purified alpha IIb beta 3. These findings suggest that substitution of beta 3 R214 by Q is responsible for the functional defect in alpha IIb beta 3 and that R214 is proximal to or part of a ligand binding domain in alpha IIb beta 3.     

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.     

Isolation and characterization of platelet glycoprotein IV (CD36)

Platelet glycoprotein IV (GPIV, Mr 88,000), which is immunologically related to the leukocyte differentiation antigen CD36, has been isolated from both intact and trypsinized platelet membranes by a series of steps involving (i) phase partitioning in Triton X-114, (ii) ion exchange chromatography on DEAE-cellulose, (iii) lectin affinity chromatography on wheat germ agglutinin-Sepharose, and (iv) size exclusion chromatography on Ultrogel AcA-44. The homogenous product contained 26% carbohydrate (sialic acid, Gal, Man, GalNAc, GlcNAc), of which approximately two-thirds were in alkali-labile O-glycosidic linkages. A rabbit polyclonal antibody raised against purified GPIV gave a single band on immunoblot and on immunoprecipitation from solubilized, 3H-labeled platelet membranes indicating its monospecificity. The antibody gave a strongly positive reaction with platelets on flow cytofluorimetry further confirming the surface location of GPIV. Immunoblotting and flow cytometry also identified GPIV-like molecules on the surface of U937, HEL, and C32 cells but not on HT1080 fibroblasts. Amino acid analysis showed values comparable with those deduced from the cloning data for GPIb, GPIIb, and GPIIIa. Automated Edman degradation allowed the identification of the sequence of the first 36 residues of the NH2-terminal domain. G-X-D-R-N-X-G-L-I-A-G-A-V-I-G-A-V- L-A-V-F-G-G-I-L-M-P-V-G-D-L-P-X-Q-K-F. There are no identifiable homologies between the NH2-terminal domain and other known protein sequences. Following a hydrophilic hexapeptide, there is a hydrophobic sequence of 23 amino acids (underlined) that is of the size and composition expected for a transmembrane domain. Since the NH2-terminal domain lacks tyrosine, but GPIV may be readily iodinated in intact platelets, this suggests that GPIV may have a configuration expressing other extramembranous domains.     

Fibrinogen binding to purified platelet glycoprotein IIb-IIIa (integrin alpha IIb beta 3) is modulated by lipids.

Soluble fibrinogen binding to the glycoprotein IIb-IIIa complex (integrin alpha IIb beta 3) requires platelet activation. The intracellular mediator(s) that convert glycoprotein IIb-IIIa into an active fibrinogen receptor have not been identified. Because the lipid composition of the platelet plasma membrane undergoes changes during activation, we investigated the effects of lipids on the fibrinogen binding properties of purified glycoprotein IIb-IIIa. Anion exchange chromatography of lipids extracted from platelets exposed to thrombin or other platelet agonists resolved an activity that increased fibrinogen binding to glycoprotein IIb-IIIa. A monoester phosphate was important for activity, and phosphatidic acid coeluted with the peak of activity. Purified phosphatidic acid dose-dependently promoted a specific interaction between glycoprotein IIb-IIIa and fibrinogen which possessed many but not all of the properties of fibrinogen binding to activated platelets. Phosphatidic acid appeared to increase the proportion of fibrinogen binding-competent glycoprotein IIb-IIIa complexes without altering their affinity for fibrinogen. The effects of phosphatidic acid were a result of specific structural properties of the lipid and were not mimicked by other phospholipids. Lysophosphatidic acid, however, was a potent inducer of fibrinogen binding to glycoprotein IIb-IIIa. These results demonstrate that specific lipids can affect fibrinogen binding to purified glycoprotein IIb-IIIa and suggest that the lipid environment has the potential to influence fibrinogen binding to its receptor.     

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.     

Isolation and characterization of a 33,000-dalton fragment of complement Factor B with catalytic and C3b binding activity

Factor B is the zymogen of the catalytic site bearing subunit Bb of the C3/C5 convertase of the alternative pathway of complement. In this study, the location of the C3b binding site and the catalytic site within the Bb subunit were investigated. When human Factor B was treated with porcine elastase, fragments with respective molecular weights of 36,000, 35,000, 33,000, 31,000, and 25,000 were generated. Binding studies showed that only the 33,000-dalton fragment was capable of binding to C3b. The 33,000-dalton fragment was purified using fast protein liquid chromatography and found to be part of the Bb fragment upon testing with monoclonal antibody 15-6-19-1. Amino-terminal amino acid sequence analysis of the 33,000-dalton fragment placed it in the C-terminal half of Bb. The fragment expressed esterolytic activity as evidenced by cleavage of the synthetic substrate N alpha-acetyl-glycyl-L-lysine methyl ester and restored alternative pathway activity in Factor B-depleted serum. Its hemolytic activity was approximately 60-fold lower than that of Factor B. Comparative binding studies in the presence of metal ions using zymosan-C3b showed that the 33,000-dalton fragment bound to C3b with higher affinity than Factor B. Addition of the fragment to human serum inhibited alternative pathway activation by rabbit erythrocytes due to its high affinity for C3b and its low hemolytic activity compared to Factor B. These results show that the C-terminal 33,000-dalton portion of Bb contains not only the enzymatic site of Bb but also a C3b binding site which confers hemolytic activity upon the fragment. The observation that the fragment inhibited alternative pathway activation suggests that a synthetic peptide may be constructed that exhibits negative regulator activity in the alternative pathway.     

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.     

Monoclonal antibody AG-1 initiates platelet activation by a pathway dependent on glycoprotein IIb-IIIa and extracellular calcium.

The biochemical responses of intact human platelets to the monoclonal antibody (mAb) AG-1 were investigated. AG-1 is a murine IgG mAb that recognizes a series of platelet membrane glycoproteins (Gp) from M(r) 21,000 to 29,000, one of which is the M(r) 24,000 (p24) receptor for anti-CD9 mAbs. AG-1 causes platelet aggregation and secretion. Platelets binding AG-1 demonstrate a dose- and time-dependent breakdown of phosphatidylinositol 4,5-bisphosphate (PIP2), production of diacylglycerol, and generation of phosphatidic acid (PA). These events are associated with the activation of protein kinase C (PKC), an increase in intracellular calcium, and fibrinogen binding. Platelet PA generation and PKC activation in response to AG-1 are inhibited by mAbs to platelet GpIIb-IIIa or by extracellular EGTA, but not by a mAb to platelet GpIb or by inhibiting platelet Na+/H+ exchange with 5-(N-ethyl-N-isopropyl)amiloride. Platelet cytoplasmic free calcium ([Ca2+]i) is elevated in response to AG-1, and this elevation is inhibited by mAbs to GpIIb-IIIa, an RGDS peptide or by chelating extracellular calcium. These results suggest that AG-1 binding to a unique platelet-surface glycoprotein initiates platelet responses through the activation of PIP2-specific phospholipase C, and that this occurs through a signal pathway that is dependent on GpIIb-IIIa and extracellular calcium.