Ability of Different Glycoprotein IIb-IIIa Ligands to Support Platelet Aggregation Induced by Activating Antibody CRC54

The ability of different ligands of glycoprotein (GP) IIb-IIIa (alphaIIb/beta3-integrin) to support platelet aggregation stimulated by activating anti-GP IIb-IIIa monoclonal antibody (monoAB) CRC54 has been investigated. Antibody CRC54 stimulated aggregation of washed platelets not only in the presence of fibrinogen, the main GP IIb-IIIa ligand, but also in the presence of von Willebrand factor (vWF). Unlike these ligands, fibronectin failed to support CRC54-induced aggregation. Fibrinogen and vWF dependent platelet aggregation was completely suppressed by GP IIb-IIIa antagonists--preparations Monafram (F(ab')2 fragments of monoAB that blocked GP IIb-IIIa receptor activity) and aggrastat (RGD-like peptidomimetic). However, aggregation stimulated in the presence of vWF was also completely inhibited by monoAB AK2 directed against GP Ib and capable of blocking its binding with vWF. CRC54-induced aggregation of platelets from patient with GP Ib deficiency in the presence of vWF was significantly lower than aggregation of platelets from normal donors and was not inhibited by anti-GP Ib antibody but still blocked by GP IIb-IIIa antagonist Monafram. Monafram also suppressed CRC54-stimulated platelet adhesion to plastic-adsorbed fibrinogen, vWF, and fibronectin. Unlike CRC54-induced platelet aggregation supported by fluid phase vWF, CRC54-induced adhesion to adsorbed vWF was not affected by anti-GP Ib antibody. Aggregation induced by CRC54 in the presence of fibrinogen and vWF was only partially suppressed by prostaglandin E1, an inhibitor of platelet activation, and was associated with serotonin release from platelet granules only when Ca2+ concentration was decreased from 1 mM (physiological level) to 0.1 mM. The data indicate that vWF supports CRC54-induced platelet aggregation via interaction with two receptors--GP IIb-IIIa and GP Ib. Aggregation induced by CRC54 in the presence of vWF or fibrinogen is only partially dependent on platelet activation and is accompanied with granule secretion only at low Ca2+ concentrations.     

Selective recognition of adhesive sites in surface-bound fibrinogen by glycoprotein IIb-IIIa on nonactivated platelets

We demonstrate that unstimulated platelets attach to immobilized fibrinogen in a selective process mediated by the membrane glycoprotein (GP) complex IIb-IIIa (alpha IIb beta 3). The initial attachment, independent of platelet activation, is followed by spreading and irreversible adhesion even in the presence of activation inhibitors. Using fibrinogen fragments derived from plasmin digestion, we found that unstimulated platelets do not attach to immobilized fragment E, which contains an Arg-Gly-Asp sequence at A alpha 95-97, and adhere to fragments X and D, both containing the gamma 400-411 dodecapeptide adhesion sequence, less efficiently than to intact fibrinogen. Thus, the carboxyl terminus of the A alpha chain, missing in the "early" fragment X used in these studies, appears to be involved in the interaction of fibrinogen with unstimulated platelets. In contrast, activated platelets adhere to immobilized fibrinogen and fragments X, D, and E in a time-dependent and equivalent manner. Although activated platelets adhere to immobilized vitronectin, fibronectin, and von Willebrand factor through GP IIb-IIIa, unstimulated platelets fail to adhere to vitronectin and have only a limited capacity to adhere to fibronectin and von Willebrand factor. These results demonstrate that GP IIb-IIIa on unstimulated platelets displays a recognition specificity for attachment to immobilized adhesive proteins that is distinct from that seen following platelet activation. Thus, unstimulated platelets selectively interact with fibrinogen, and the initial attachment is followed by spreading and irreversible adhesion in the absence of exogenous agonists. This process may be regulated by plasmin cleavage of the fibrinogen A alpha chain and may play an important role during normal hemostasis and during the pathological development of thrombotic vascular occlusions.     

A conformation-dependent epitope of human platelet glycoprotein IIIa.

This study explores conformational states of human platelet glycoprotein IIIa (GP IIIa) and possible mechanisms of fibrinogen receptor exposure. D3GP3 is an IgG1, kappa monoclonal antibody generated against purified GP IIIa and found to be specific for GP IIIa by immunoprecipitation and Western blot analysis. The binding of D3GP3 to resting platelets caused fibrinogen binding (approximately 5,000 molecules/platelet) and platelet aggregation but not secretion. Platelets express 40,000-50,000 GP IIb-IIIa molecules in their surface membranes. However, resting platelets only bound approximately 5,000 D3GP3 molecules/platelet. D3GP3 binding to platelets could be increased 2-3-fold by dissociation of the GP IIb-IIIa complex with 5 mM EDTA or by occupying the fibrinogen receptor with either RGDS peptides or fibrinogen. Platelet stimulation with ADP in the absence of fibrinogen did not cause increased D3GP3 binding above control levels. These data suggest that 1) GP IIb-IIIa can exist in multiple conformations in the platelet membrane, 2) D3GP3 binding to GP IIIa can expose the fibrinogen receptor, 3) the binding of either RGDS peptides or fibrinogen causes exposure of the D3GP3 epitope, and 4) platelet activation in the absence of ligand does not induce the same conformational changes in GP IIb-IIIa as does receptor occupancy by RGDS peptides or fibrinogen.     

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.     

The occupancy of glycoprotein IIb-IIIa complex modulates thrombin activation of human platelets

Platelet membrane glycoprotein (GP IIb-IIIa), besides its activity as adhesive protein receptor, displays a number of properties supporting its involvement in the mechanisms of transduction of the activation signal. Recently we have observed that GP IIb-IIIa ligands, mostly fibrinogen, inhibit Ca2+ movement and cytoskeleton reorganization caused by mild platelet activation. These findings led us to investigate the effect of GP IIb-IIIa ligands on agonist-induced platelet responses, with particular attention to the two major messenger generating systems, involving the activation of phospholipase C and the inhibition of cAMP production. In this paper we demonstrate that the occupancy of the major adhesive protein receptor on the platelet surface modulates the phosphatidylinositol cycle decreasing the amount of IP3, IP2 and IP produced after mild platelet activation as well as the pattern of protein phosphorylation. The platelet cAMP content of activated platelets was also affected and kept higher when evaluated under the same experimental conditions. Our data provide evidence for a role of fibrinogen binding in regulating the degree of activation of circulating platelets.     

Function of glycoprotein Ib alpha in platelet activation induced by alpha-thrombin.

We have obtained evidence that selective inhibition of high affinity thrombin-binding sites located in the amino-terminal domain of the membrane glycoprotein (GP) Ib alpha results in impaired platelet activation, as shown by abrogation or reduction of the following responses induced in normal platelets by exposure to less than 1 nM alpha-thrombin: (i) increase in intracellular ionized calcium concentration ([Ca2+]i), (ii) release of dense granule content, (iii) binding of fibrinogen, (iv) aggregation. An anti-GP Ib monoclonal antibody, LJ-Ib 10, which does not inhibit von Willebrand factor binding to platelets, obliterated the high affinity alpha-thrombin-binding sites on normal platelets. Isotherms of alpha-thrombin binding to normal platelets treated with saturating amounts of the antibody were virtually identical to those obtained with platelets from a patient with classical Bernard-Soulier syndrome. In parallel with decreased binding of the agonist, this antibody caused 50% inhibition of the maximal extent of platelet aggregation and 90% inhibition of ATP release induced by 0.3 nM alpha-thrombin. By inhibiting alpha-thrombin binding to GP Ib, the antibody prevented the activation of platelets exposed to low concentrations of the agonist, as demonstrated by abrogation of the increase in intraplatelet ionized calcium concentration induced in control platelets by 0.18 nM alpha-thrombin; under these conditions, fibrinogen binding was inhibited by 84%. Therefore, there is a correlation between occupancy of the high affinity sites for alpha-thrombin on GP Ib alpha and platelet activation, secretion, and aggregation, suggesting that GP Ib alpha is part of an alpha-thrombin receptor relevant for platelet function.     

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.     

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.     

Platelet adhesion receptors and (patho)physiological thrombus formation

In thrombus formation associated with hemostasis or thrombotic disease, blood platelets first undergo a rapid transition from a circulating state to an adherent state, followed by activation and aggregation. Under flow conditions in the bloodstream, this process potentially involves platelet-platelet, platelet-endothelium, platelet-subendothelial matrix, and platelet-leukocyte interactions. Specific adhesion receptors on platelets mediate these interactions, by engaging counter-receptors on other cells, or noncellular ligands in the plasma or matrix. The glycoprotein (GP) Ib-IX-V complex on platelets initiates adhesion at high shear stress by binding the adhesive ligand, von Willebrand Factor (vWF). GP Ib-IX-V may also mediate platelet-endothelium or platelet-leukocyte adhesion, by recognition of P-selectin or Mac-1, respectively. Other membrane glycoproteins, such as the collagen receptor GP VI, may trigger platelet activation at low shear rates. Engagement of GP Ib-IX-V or GP VI leads ultimately to platelet aggregation mediated by the integrin, alphaIIbbeta3 (GP IIb-IIIa). This review will focus on recent advances in understanding structure-activity relationships of GP Ib-IX-V, its role in initiating thrombus formation, and its emerging relationships with other vascular cell adhesion receptors.     

Regulation of glycoprotein IIb-IIIa receptor function studied with platelets permeabilized by the pore-forming complement proteins C5b-9.

Recent evidence suggests that the cytoplasmic domains of platelet glycoprotein (GP) IIb-IIIa are involved in the agonist-initiated transformation of this integrin into a receptor for fibrinogen. To identify intracellular reactions that regulate the receptor function of GP IIb-IIIa, membrane-impermeable agonists and antagonists were introduced into the platelet by permeabilizing the plasma membrane with the pore-forming complement proteins C5b-9. Platelet responses were then analyzed by flow cytometry. Non-lytic concentrations of C5b-9 caused permeabilization of the platelet plasma membrane, as determined by uptake of a water-soluble fluorescent tracer dye. The complement pores were large enough to permit the entry of fluorescein isothiocyanate (FITC)-labeled oligopeptides in a size-dependent manner. Under conditions of low external Ca2+, C5b-9 treatment per se did not activate GP IIb-IIIa, as measured by binding of the activation-dependent antibody FITC-PAC1. However, FITC-PAC1 binding to C5b-9-permeabilized platelets was stimulated by a thrombin receptor agonist acting at the cell surface and by guanosine 5'-O-(thiotriphosphate), a membrane-impermeable activator of G proteins. Permeabilization also permitted the entry of cyclic AMP and the peptide, RFARKGALRQKNV, a pseudo-substrate inhibitor of protein kinase C. Each of these inhibited agonist-induced FITC-PAC1 binding to permeabilized platelets but not to intact platelets. Agonist-induced GP IIb-IIIa activation in permeabilized platelets was also inhibited by tyrphostin-23, a protein tyrosine kinase inhibitor. Thus, C5b-9 can be used to permeabilize the plasma membrane to permit the selective entry of small peptides and other bioactive compounds into permeabilized platelets. Results obtained with these platelets indicate that GP IIb-IIIa receptor function is regulated by a network of signaling reactions involving G proteins, serine/threonine kinases, and tyrosine kinases.     

The IIb-IIIa glycoprotein complex that mediates platelet aggregation is directly implicated in leukocyte adhesion

Evidence is presented that the IIb-IIIa glycoprotein complex, which functions as the receptor for fibrinogen on platelets and is central to platelet aggregation, is expressed on the surface of leukocytes where it may function as a receptor for fibronectin. F(ab')2 fragments of a monoclonal antibody, 25E11, raised against activated large granular lymphocytes, inhibited killing by natural killer cells, blocked the binding of fibronectin-coated particles by monocytes, and stimulated neutrophils to exhibit increased antibody-dependent killing. Immunoprecipitation studies of leukocytes and platelets, and the ability of 25E11 to inhibit platelet aggregation, identified the antigen as an epitope on the IIb-IIIa complex. This glycoprotein thus constitutes the first example of a receptor mediating both platelet aggregation and leukocyte adhesion.     

Unique pathway of thrombin-induced platelet aggregation mediated by glycoprotein Ib

Thrombin plays a central role in normal and abnormal hemostatic processes. It is assumed that alpha-thrombin activates platelets by hydrolyzing the protease-activated receptor (PAR)-1, thereby exposing a new N-terminal sequence, a tethered ligand, which initiates a cascade of molecular reactions leading to thrombus formation. This process involves cross-linking of adjacent platelets mediated by the interaction of activated glycoprotein (GP) IIb/IIIa with distinct amino acid sequences, LGGAKQAGDV and/or RGD, at each end of dimeric fibrinogen molecules. We demonstrate here the existence of a second alpha-thrombin-induced platelet-activating pathway, dependent on GP Ib, which does not require hydrolysis of a substrate receptor, utilizes polymerizing fibrin instead of fibrinogen, and can be inhibited by the Fab fragment of the monoclonal antibody LJIb-10 bound to the GP Ib thrombin-binding site or by the cobra venom metalloproteinase, mocarhagin, that hydrolyzes the extracellular portion of GP Ib. This alternative alpha-thrombin pathway is observed when PAR-1 or GP IIb/IIIa is inhibited. The recognition sites involved in the cross-linking of polymerizing fibrin and surface integrins via the GP Ib pathway are different from those associated with fibrinogen. This pathway is insensitive to RGDS and anti-GP IIb/IIIa antibodies but reactive with a mutant fibrinogen, gamma407, with a deletion of the gamma-chain sequence, AGDV. The reaction is not due to simple trapping of platelets by the fibrin clot, since ligand binding, signal transduction, and second messenger formation are required. The GP Ib pathway is accompanied by mobilization of internal calcium and the platelet release reaction. This latter aspect is not observed with ristocetin-induced GP Ib-von Willebrand factor agglutination nor with GP Ib-von Willebrand factor-polymerizing fibrin trapping of platelets. Human platelets also respond to gamma-thrombin, an autoproteolytic product of alpha-thrombin, through PAR-4. Co-activation of the GP Ib, PAR-1, and PAR-4 pathways elicit synergistic responses. The presence of the GP Ib pathway may explain why anti-alpha-thrombin/anti-platelet regimens fail to completely abrogate thrombosis/restenosis in the cardiac patient.     

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.     

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.     

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.     

Binding interactions of kistrin with platelet glycoprotein IIb-IIIa: Analysis by site-directed mutagenesis

The binding interactions between platelet fibrinogen receptor, glycoprotein (GP) IIb-IIIa, and kistrin, a snake venom disintegrin protein that contains the adhesion site recognition sequence Arg-Gly-Asp (RGD) and potently inhibits platelet aggregation, have been investigated by site-directed mutagenesis of a synthetic kistrin gene. Kistrin was expressed as a fusion protein in Escherichia coli under control of the alkaline phosphatase promoter. This construction included the stII signal sequence to direct secretion to the periplasmic space and one synthetic (Z) domain of Staphylococcal protein A to allow affinity purification using IgG Sepharose. Kistrin was cleaved from the Z-domain by site-specific proteolysis using a mutant subtilisin BPN' and purified by reverse-phase HPLC. This approach facilitated the rapid purification of a set of 43 alanine replacement mutants whose relative affinity for GP IIb-IIIa was measured by competition with immobilized kistrin and by inhibition of platelet aggregation in human platelet-rich plasma. Alanine replacements at R49, G50, and D51 led to weaker inhibitors of platelet aggregation by 90-fold, 2-fold, and >200-fold, respectively. The conservative D51E mutant was still >100-fold less potent whereas R49K had a minor effect (1.8-fold), implying the critical nature of the aspartate for high affinity binding. However, mutations outside of the RGD region led to proteins indistinguishable from kistrin, suggesting no substantial secondary binding interactions. Furthermore, reduced kistrin is not active. We therefore propose that a favorable conformation of the RGD region alone is responsible for the high affinity binding of kistrin to GP IIb-IIIa. © 1993 Wiley-Liss, Inc.     

Identification of aspartic acid 514 through glutamic acid 542 as a glycoprotein Ib-IX complex receptor recognition sequence in von Willebrand factor. Mechanism of modulation of von Willebrand factor by ristocetin and botrocetin.

As the first step in hemostasis, the binding of von Willebrand factor (vWF) to the platelet membrane glycoprotein (GP) Ib-IX complex is essential for platelet adhesion at high-shear blood flow. This interaction in vivo requires the prior binding of vWF to the subendothelial matrix, a process which exposes a normally cryptic binding site on vWF for the GP Ib-IX complex. This process can be mimicked in vitro by modulators such as ristocetin or the snake venom protein botrocetin or by desialation of vWF. We have previously localized the GP Ib binding site on vWF to a monomeric dispase fragment which extends from Leu-480/Val-481 to Gly-718 in the primary sequence of mature vWF [Andrews, R. K., Gorman, J. J., Booth, W. J., Corino, G. L., Castaldi, P. A., & Berndt, M. C. (1989) Biochemistry 28, 8326-8336]. This fragment also contains a distinct binding site for botrocetin. Analysis of synthetic peptides corresponding to hydrophilic stretches of sequence within this fragment indicated that the sequence Asp-514-Glu-542 represents a major adhesive sequence involved in receptor recognition. This peptide inhibited both the ristocetin- and botrocetin-mediated binding of vWF to either platelets or purified GP Ib-IX complex (IC50 approximately 50-200 microM) as well as the asialo-vWF- and bovine vWF-dependent agglutination of platelets. Both the N- and C-terminal halves of the peptide were inhibitory but less so than the intact peptide. This peptide also inhibited botrocetin binding to vWF, suggesting that botrocetin modulates vWF-GP Ib interaction by binding in close proximity to the vWF adhesion sequence.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mambin, a potent glycoprotein IIb-IIIa antagonist and platelet aggregation inhibitor structurally related to the short neurotoxins.

The purification, complete amino acid sequence, functional activity, and structural modeling are described for mambin, a platelet glycoprotein GP IIb-IIIa antagonist and potent inhibitor of platelet aggregation from the venom of the Elapidae snake Dendroaspis jamesonii (Jameson's mamba). Mambin is 59 residues in length and contains four disulfide linkages and an RGD amino acid sequence found in protein ligands that bind to GP IIb-IIIa. Mambin inhibits ADP-induced platelet aggregation (IC50 = 172 +/- 22 nM) and inhibits the binding of purified platelet fibrinogen receptor GP IIb-IIIa to immobilized fibrinogen (IC50 = 3.1 +/- 0.8 nM). Mambin has very little sequence similarity to the Viperidae family of platelet aggregation inhibitors, except for the RGD-containing region in the protein. However, mambin does have ca. 47% similarity to the short-chain postsynaptic neurotoxins found in other Elapidae venoms, which do not contain the RGD sequence and do not act as GP IIb-IIIa antagonists. On the basis of its circular dichroism spectrum, mambin has a beta-sheet structure characteristic of the neurotoxins. Molecular modeling of the mambin sequence onto the erabutoxin b structure predicts a very similar structure within the entire protein except for the loop containing the RGD sequence. Mambin may therefore represent a genetic hybrid of neurotoxic and hemotoxic proteins found in snake venoms.     

Functional modulation of the isolated glycoprotein Ib binding domain of von Willebrand factor expressed in Escherichia coli

We have expressed in Escherichia coli the domain of von Willebrand factor (vWF) containing the binding site for platelet glycoprotein (GP) Ib and used it to study the regulation of vWF-platelet interaction. The recombinant fragment, comprising residues 445-733 of the mature vWF subunit and designated rvWF445-733, did not have the native conformation of the corresponding domain in the intact molecule because, in order to prevent formation of random aggregates, the seven cysteine residues in the sequence were reduced and alkylated. Unlike native vWF, rvWF445-733 bound to GP Ib in the absence of any modulator, suggesting that the lack of disulfide bonds and/or carbohydrate side chains within this domain may expose platelet interaction sites. In the presence of two modulators, the glycopeptide ristocetin and the snake protein botrocetin, rvWF445-733 inhibited native vWF binding to GP Ib as well as platelet aggregation mediated by vWF, suggesting that both the fragment and the native molecule interact with the same site on platelets. This conclusion was also supported by the observation that the recombinant fragment competed with the binding to platelets of an anti-GP Ib monoclonal antibody known to inhibit vWF binding. Botrocetin formed a complex with rvWF445-733, but the affinity of this interaction was approximately 25-fold lower than with native vWF. However, the complexes of botrocetin with either rvWF445-733 or multimeric native vWF bound to GP Ib with similar dissociation constant. Therefore, conformational attributes of vWF regulate its affinity for botrocetin, but once the complex is formed, interaction with GP Ib is independent of native vWF conformation. These findings provide insights into the regulation of vWF-platelet interaction.