Roles of Mac-1 and glycoprotein IIb/IIIa integrins in leukocyte-platelet aggregate formation: stabilization by Mac-1 and inhibition by GpIIb/IIIa blockers.

Circulating platelet-leukocyte hetero-aggregates play an important role in acute cardiovascular events and hypersensitivity reactions. The association involves the receptor families of selectins and integrin. The objective of this study was to investigate the role of CD11b/CD18 integrin (Mac-1) in hetero-aggregate formation and search for a counter-receptor on platelets ready to interact with Mac-1. As a model of leukocytes, Mac-1 presenting Chinese hamster ovary (CHO) cells were used to evaluate the role of Mac-1 in hetero-aggregate formation. The amount of CHO cell-bound active and inactive platelets was measured by flow cytometry, while the counter-receptors on platelets were identified via using blocking antibodies. We observed significant platelet adhesion on Mac-1-bearing cells when platelet-rich plasma or activated platelets were present. Inactive platelets did not adhere to Mac-1-bearing cells. Addition of fibrinogen, a ligand of Mac-1 significantly increased platelet binding. CD40L was demonstrated to act similarly on Mac-1. Inhibition of platelet GpIIb/IIIa completely abolished CHO cell-platelet aggregation. In our study, we have shown for the first time that Mac-1 mediates the formation of hetero-aggregates without selectin tethering when Mac-1 ligands such as fibrinogen or CD40L are present and blockers of platelet GpIIb/IIIa are able to diminish this interaction.

Circulating annexin V positive microparticles in patients after successful cardiopulmonary resuscitation.

INTRODUCTION: Ischemia/reperfusion after cardiopulmonary resuscitation (CPR) induces systemic inflammatory response and activation of endothelium and coagulation, resulting in a post-cardiac arrest syndrome. We analysed circulating (annexin V+) microparticles and their conjugates in resuscitated patients. METHODS: 36 patients after successful resuscitation, 20 control patients with stable cardiac disease and 15 healthy subjects were included prospectively. Two blood samples were drawn, one immediately and one 24 hours after return of spontaneous circulation (ROSC) to detect (annexin V+) monocyte-derived microparticles (MMPs) or procoagulant (annexin V+) platelet-derived microparticles (PMPs) and conjugates of endothelial-derived (annexin V+) microparticles (EMPs) with monocytes (EMP-MC) or platelets (EMP-PC). Measurements were performed by flow cytometric analysis. Additionally, the effect of isolated microparticles on cultured endothelial cells was assessed by ELISA. RESULTS: MMPs were significantly elevated immediately after ROSC compared to the cardiological control group (control; p < 0.01) and healthy subjects (healthy; p < 0.05) and persisted to be elevated in the following 24 hours after CPR (p < 0.05 vs. control and healthy, respectively). Procoagulant PMPs increased within the first 24 hours after ROSC (p < 0.01 vs. control and p < 0.005 vs. healthy). Conjugates of EMP with monocytes and platelets were both significantly elevated immediately after CPR (EMP-MC: p < 0.05 vs. control and p < 0.05 vs. healthy; EMP-PC: p < 0.05 vs. control and p < 0.05 vs. healthy), while only EMP-MC showed persisting high levels within 24 hours after CPR (p < 0.05 vs. control and p < 0.01 vs. healthy). MMP levels of ≥ 1.0/µL 24 hours after CPR predicted adverse outcome at 20 days (p < 0.05). Furthermore, isolated microparticles circulating in CPR patients early after ROSC led to enhanced endothelial apoptosis ex vivo compared to those of the healthy controls (p < 0.005). CONCLUSIONS: Resuscitated patients show substantially increased levels of different (annexin V+) microparticles and their conjugates immediately and 24 hours after cardiopulmonary resuscitation, suggesting an early onset of inflammation, an ongoing endothelial activation and a procoagulatory state. Additionally, microparticles of CPR patients may contribute to endothelial apoptosis. These results point to an involvement of microparticles in the development of the post-cardiac arrest syndrome.

Severe endothelial injury and subsequent repair in patients after successful cardiopulmonary resuscitation.

INTRODUCTION: Ischemia and reperfusion after cardiopulmonary resuscitation (CPR) induce endothelial activation and systemic inflammatory response, resulting in post-resuscitation disease. In this study we analyzed direct markers of endothelial injury, circulating endothelial cells (CECs) and endothelial microparticles (EMPs), and endothelial progenitor cells (EPCs) as a marker of endothelial repair in patients after CPR. METHODS: First we investigated endothelial injury in 40 patients after CPR, 30 controls with stable coronary artery disease (CAD), and 9 healthy subjects, who were included to measure CECs and EMPs. In a subsequent study, endothelial repair was assessed by EPC measurement in 15 CPR, 9 CAD, and 5 healthy subjects. Blood samples were drawn immediately and 24 hours after ROSC and analyzed by flow cytometry. For all statistical analyses P < 0.05 was considered significant. RESULTS: There was a massive rise in CEC count in resuscitated patients compared to CAD (4,494.1 +/- 1,246 versus 312.7 +/- 41 cells/mL; P < 0.001) and healthy patients (47.5 +/- 3.7 cells/mL; P < 0.0005). Patients after prolonged CPR (>or=30 min) showed elevated CECs compared to those resuscitated for <30 min (6,216.6 +/- 2,057 versus 2,340.9 +/- 703.5 cells/mL; P = 0.13/ns). There was a significant positive correlation of CEC count with duration of CPR (R2= 0.84; P < 0.01). EMPs were higher immediately after CPR compared to controls (31.2 +/- 5.8 versus 19.7 +/- 2.4 events/microL; P = 0.12 (CAD); versus 15.0 +/- 5.2 events/microL; P = 0.07 (healthy)) but did not reach significance until 24 hours after CPR (69.1 +/- 12.4 versus 22.0 +/- 3.0 events/microL; P < 0.005 (CAD); versus 15.4 +/- 4.4 events/microL; P < 0.001 (healthy)). EPCs were significantly elevated in patients on the second day after CPR compared to CAD (1.16 +/- 0.41 versus 0.02 +/- 0.01% of lymphocytes; P < 0.005) and healthy (0.04 +/- 0.01; P < 0.005). CONCLUSIONS: In the present study we provide evidence for a severe endothelial damage after successful CPR. Our results point to an ongoing process of endothelial injury, paralleled by a subsequent endothelial regeneration 24 hours after resuscitation.

Conformation-specific blockade of the integrin GPIIb/IIIa: a novel antiplatelet strategy that selectively targets activated platelets.

Platelet activation causes conformational changes of integrin GPIIb/IIIa (alpha(IIb)beta3), resulting in the exposure of its ligand-binding pocket. This provides the unique possibility to design agents that specifically block activated platelets only. We used phage display of single-chain antibody (scFv) libraries in combination with several rounds of depletion/selection to obtain human scFvs that bind specifically to the activated conformation of GPIIb/IIIa. Functional evaluation of these scFv clones revealed that fibrinogen binding to human platelets and platelet aggregation can be effectively inhibited by activation-specific scFvs. In contrast to clinically used GPIIb/IIIa blockers, which are all conformation unspecific, activation-specific GPIIb/IIIa blockers do not induce conformational changes in GPIIb/IIIa or outside-in signaling, as evaluated by ligand-induced binding-site (LIBS) exposure in flow cytometry or P-selectin expression in immunofluorescence microscopy, respectively. In contrast to the conformation-unspecific blocker abciximab, activation-specific scFvs permit cell adhesion and spreading on immobilized fibrinogen, which is mediated by nonactivated GPIIb/IIIa. Mutagenesis studies and computer modeling indicate that exclusive binding of activation-specific scFv is mediated by RXD motifs in the heavy-chain complementary-determining region (CDR) 3 of the antibodies, which in comparison with other antibodies forms an exceptionally extended loop. In vivo experiments in a ferric-chloride thrombosis model of the mouse carotid artery demonstrate similar antithrombotic potency of activation-specific scFv, when compared with the conformation-unspecific blockers tirofiban and eptifibatide. However, in contrast to tirofiban and eptifibatide, bleeding times are not prolonged with the activation-specific scFvs, suggesting lower bleeding risks. In conclusion, activation-specific GPIIb/IIIa blockade via human single-chain antibodies represents a promising novel strategy for antiplatelet therapy.

A mechanistic model for paradoxical platelet activation by ligand-mimetic alphaIIb beta3 (GPIIb/IIIa) antagonists.

OBJECTIVE: Integrins are attractive therapeutic targets. Inhibition of integrin alphaIIb beta3 effectively blocks platelet aggregation. However, limitations with intravenous alphaIIb beta3 antagonists and failure of oral alphaIIb beta3 antagonists prompted doubts on the current concept of ligand-mimetic integrin blockade. METHODS AND RESULTS: Evaluating P-selectin expression on platelets by flow cytometry, we report a mechanism of paradoxical platelet activation by ligand-mimetic alphaIIb beta3 antagonists and define three requirements: (1) Induction of ligand-bound conformation of alphaIIb beta3, (2) receptor clustering, (3) prestimulation of platelets. Conformational change is inducible by clinically used ligand-mimetic alphaIIb beta3 antagonists, RGD-peptides, and anti-LIBS antibodies. In a mechanistic experimental model, clustering is achieved by crosslinking integrins via antibodies, and preactivation is induced by low-dose ADP. Finally, we demonstrate that platelet adhesion on collagen represents an in vivo correlate of platelet prestimulation and receptor clustering, in which the presence of ligand-mimetic alphaIIb beta3 antagonists results in platelet activation as detected by P-selectin, CD63, and CD40L expression as well as by measuring Ca2+-signaling. Blockade of the ADP receptor P2Y12 by AR-C69931MX and clopidogrel inhibits alphaIIb beta3 antagonist-induced platelet activation. CONCLUSION: These findings can explain limitations of ligand-mimetic anti-alphaIIb beta3 therapy. They describe potential benefits of concomitant ADP receptor blockade and support a shift in drug development from ligand-mimetic toward allosteric or activation-specific integrin antagonists.

Targeting ligand-induced binding sites on GPIIb/IIIa via single-chain antibody allows effective anticoagulation without bleeding time prolongation.

OBJECTIVE: Therapeutic anticoagulation is widely used, but limitations in efficacy and bleeding complications cause an ongoing search for new agents. However, with new agents developed it seems to be an inherent problem that increased efficiency is accompanied by an increase in bleeding complications. We investigate whether targeting of anticoagulants to activated platelets provides a means to overcome this association of potency and bleeding. METHODS AND RESULTS: Ligand-induced binding sites (LIBS) on fibrinogen/fibrin-binding GPIIb/IIIa represent an abundant clot-specific target. We cloned an anti-LIBS single-chain antibody (scFv(anti-LIBS)) and genetically fused it with a potent, direct factor Xa (fXa) inhibitor, tick anticoagulant peptide (TAP). Specific antibody binding of fusion molecule scFv(anti-LIBS)-TAP was proven in flow cytometry; anti-fXa activity was demonstrated in chromogenic assays. In vivo anticoagulative efficiency was determined by Doppler-flow in a ferric chloride-induced carotid artery thrombosis model in mice. ScFv(anti-LIBS)-TAP prolonged occlusion time comparable to enoxaparine, recombinant TAP, and nontargeted mutant-scFv-TAP. ScFv(anti-LIBS)-TAP revealed antithrombotic effects at low doses at which the nontargeted mutant-scFv-TAP failed. In contrast to the other anticoagulants tested, bleeding times were not prolonged by scFv(anti-LIBS)-TAP. CONCLUSIONS: The novel clot-targeting approach of anticoagulants via single-chain antibody directed against a LIBS-epitope on GPIIb/IIIa promises effective anticoagulation with reduced bleeding risk.

Single-chain antibodies for the conformation-specific blockade of activated platelet integrin alphaIIbbeta3 designed by subtractive selection from naive human phage libraries.

Binding of fibrinogen to platelet integrin alphaIIbbeta3 mediates platelet aggregation, and thus inhibition of alphaIIbbeta3 represents a powerful therapeutic strategy in cardiovascular medicine. However, the currently used inhibitors of alphaIIbbeta3 demonstrate several adverse effects like thrombocytopenia and bleeding, which are associated with their property to bind to non-activated alphaIIbbeta3. To circumvent these problems, we designed blocking single-chain antibody-fragments (scFv) that bind to alphaIIbbeta3 exclusively in its activated conformation. Two naive phage libraries were created: a natural phage library, based on human lymphocyte cDNA, and a synthetic library, with randomized VHCDR3. We performed serial rounds of subtractive panning with depletion on non-activated and selection on activated alphaIIbbeta3, which were provided on resting and ADP-stimulated platelets and CHO cells, expressing wild-type or mutated and thereby activated alphaIIbbeta3. In contrast to isolated, immobilized targets, as generally used for phage display, this unique cell-based approach for panning allowed the preservation of functional integrin conformation. Thereby, we obtained several scFv-clones that demonstrated exclusive binding to activated platelets and complete inhibition of fibrinogen binding and platelet aggregation. Interestingly, all activation-specific clones contained an RXD pattern in the HCDR3. Binding studies on transiently expressed point mutants and mouse-human domain-switch mutants of alphaIIbbeta3 indicate a binding site similar to fibrinogen. In conclusion, we generated human activation-specific scFvs against alphaIIbbeta3, which bind selectively to activated alphaIIbbeta3 and thereby potently inhibit fibrinogen binding to alphaIIbbeta3 and platelet aggregation.

Fibrin-targeted direct factor Xa inhibition: construction and characterization of a recombinant factor Xa inhibitor composed of an anti-fibrin single-chain antibody and tick anticoagulant peptide.

We investigated whether the direct fXa inhibitor tick anticoagulant peptide (TAP) can be N-terminally coupled to a clot-targeting, single-chain antibody specific for fibrin (scFv(59D8)). Due to its unique position at the convergence point of the intrinsic and extrinsic pathways early in the coagulation cascade, factor Xa (fXa) represents an attractive therapeutic target. In contrast to indirect inhibitors, direct fXa inhibitors effectively inhibit clot-bound and prothrombinase-associated fXa. Targeting of direct fXa inhibitors to clots promises to enhance local anticoagulative potency and to reduce systemic anticoagulation which potentially results in less bleeding complications.TAP is a highly potent fXa inhibitor. Since its N-terminus is essential for anti-fXa activity, it was a challenging question, whether TAP will be active as a N-terminally coupled fusion molecule. Two step affinity chromatography with Ni(2+) and beta(15-22)-peptide of human fibrin results in a pure 36 kDa protein, which was tested for its targeting function and anti-fXa activity. The recombinant fusion did not destroy the function of the fusion partners. Antibody binding function was on a par with the parent molecule. TAP activity was partially reduced, arguing that a free N-terminus is not required for anti-fXa activity, but is important for maximal potency. In human whole blood clots, scFv(59D8)-TAP revealed anticoagulative properties at concentrations (200 to 500 nM) where non-targeted TAP did not reveal anticoagulative activity at all. In summary, scFv(59D8)-TAP constitutes a promising new anticoagulant with fibrin-targeted factor Xa inhibition. The production in E. coli and the established purification methods are a solid basis for a modern, large scale production at low cost and reproducible activity.

The GP IIb/IIIa inhibitor abciximab (c7E3) inhibits the binding of various ligands to the leukocyte integrin Mac-1 (CD11b/CD18, alphaMbeta2).

Cross-reactivity with integrins other than glycoprotein IIb/IIIa (GP IIb/IIIa) is discussed as a potential reason for the overall clinical benefits of the GP IIb/IIIa-blocking antibody-fragment abciximab. We evaluated whether abciximab binds to the leukocyte integrin Mac-1, whether it inhibits binding of the distinct ligands and thereby may modulate inflammation, cell proliferation and coagulation. Binding of fluorescence-labelled abciximab to phorbolmyristate acetate-stimulated monocytes and to a monocytic cell line (THP-1) could be detected in flow cytometry. The binding of fibrinogen, the inactivated complement factor 3b (iC3b), and the coagulation factor X to Mac-1 could be inhibited by abciximab (10 microg/ml) in vitro. As a functional consequence, the conversion of factor X to factor Xa mediated by Mac-1, as detected by the chromogenic substrate SZ-2222, was impaired by abciximab. Adhesion of THP-1 cells to immobilized intercellular adhesion molecule 1 (ICAM-1) and to fibrinogen was reduced significantly by abciximab. Fibrinogen-mediated cell aggregation was also impaired. In conclusion, we describe binding of abciximab to Mac-1 on stimulated monocytes. Thereby, abciximab inhibits binding of the ligands fibrinogen, ICAM-1, iC3b and factor X. Furthermore, we demonstrated that Mac-1-dependent conversion from factor X to factor Xa is impaired by abciximab, arguing for the direct modulation of the coagulation cascade by abciximab. Overall, the inhibition of Mac-1 could provide additional clinical benefits of abciximab beyond the well-described blockade of GP IIb/IIIa.

Mac-1 directly binds to the endothelial protein C-receptor: a link between the protein C anticoagulant pathway and inflammation?

OBJECTIVE: The endothelial protein C-receptor (EPCR) is an endothelial transmembrane protein that binds protein C and activated protein C (APC) with equal affinity, thereby facilitating APC formation. APC has anticoagulant, antiapoptotic and antiinflammatory properties. Soluble EPCR, released by the endothelium, may bind activated neutrophils, thereby modulating cell adhesion. EPCR is therefore considered as a possible link between the anticoagulant properties of protein C and the inflammatory response of neutrophils. In the present study, we aimed to provide proof of concept for a direct binding of EPCR to the ß2-integrin Mac-1 on monocytic cells under static and physiological flow conditions. MEASUREMENTS AND MAIN RESULTS: Under static conditions, human monocytes bind soluble EPCR in a concentration dependent manner, as demonstrated by flow cytometry. Binding can be inhibited by specific antibodies (anti-EPCR and anti-Mac-1). Specific binding was confirmed by a static adhesion assay, where a transfected Mac-1 expressing CHO cell line (Mac-1+ cells) bound significantly more recombinant EPCR compared to Mac-1+ cells blocked by anti-Mac-1-antibody and native CHO cells. Under physiological flow conditions, monocyte binding to the endothelium could be significantly blocked by both, anti-EPCR and anti-Mac-1 antibodies in a dynamic adhesion assay at physiological flow conditions. Pre-treatment of endothelial cells with APC (drotrecogin alfa) diminished monocyte adhesion significantly in a comparable extent to anti-EPCR. CONCLUSIONS: In the present study, we demonstrate a direct binding of Mac-1 on monocytes to the endothelial protein C receptor under static and flow conditions. This binding suggests a link between the protein C anticoagulant pathway and inflammation at the endothelium side, such as in acute vascular inflammation or septicaemia.

Functionalized magnetic resonance contrast agent selectively binds to glycoprotein IIb/IIIa on activated human platelets under flow conditions and is detectable at clinically relevant field strengths.

Recent progress in molecular magnetic resonance imaging (MRI) provides the opportunity to image cells and cellular receptors using microparticles of iron oxide (MPIOs). However, imaging targets on vessel walls remains challenging owing to the quantity of contrast agents delivered to areas of interest under shear stress conditions. We evaluated ex vivo binding characteristics of a functional MRI contrast agent to ligand-induced binding sites (LIBSs) on activated glycoprotein IIb/IIIa receptors of human platelets, which were lining rupture-prone atherosclerotic plaques and could therefore facilitate detection of platelet-mediated pathology in atherothrombotic disease. MPIOs were conjugated to anti-LIBS single-chain antibodies (LIBS-MPIO) or control antibodies (control MPIO). Ex vivo binding to human platelet-rich clots in a dose-dependent manner was confirmed on a 3 T clinical MRI scanner and by histology (p < .05 for LIBS-MPIO vs control MPIO). By using a flow chamber setup, significant binding of LIBS-MPIO to a platelet matrix was observed under venous and arterial flow conditions, but not for control MPIO (p < .001). A newly generated MRI contrast agent detects activated human platelets at clinically relevant magnetic field strengths and binds to platelets under venous and arterial flow conditions, conveying high payloads of contrast to specific molecular targets. This may provide the opportunity to identify vulnerable, rupture-prone atherosclerotic plaques via noninvasive MRI.

Single-chain antibodies as new antithrombotic drugs.

Antibodies are the most rapidly growing class of human therapeutics and the second largest class of drugs after vaccines. At present, several antibodies are approved for therapeutic use in diverse clinical settings, including oncology, chronic inflammatory diseases, transplantation, infectious diseases, and cardiovascular medicine. These approved antibody therapeutics include unmodified immunoglobulin G molecules, radioimmunoconjugates, antibody-drug conjugates, and fragment antigen-binding molecules. At least 150 additional antibodies are in clinical development. A major strength of therapeutic antibodies is their established properties as a drug class with high success rates from clinical trials to regulatory approvals. Much of the experience gained from the generation and optimization of one antibody is applicable to other antibodies. Antibody fragments are a subclass with growing clinical importance. This review focuses on single-chain antibodies as the smallest possible format for recombinant antibodies, and their use as antithrombotic drugs. We describe different antibody formats, the current applications of antibody fragments, and their generation by cloning from hybridoma cell lines as well as their selection from antibody libraries. We review the use of antibody fragments for thrombus targeting using fibrin and platelet-specific single-chain antibodies in combination with anticoagulants and thrombolytic agents as antithrombotic drugs.

Subtractive single-chain antibody (scFv) phage-display: tailoring phage-display for high specificity against function-specific conformations of cell membrane molecules.

Phage-display of antibody libraries is a powerful tool to select antibodies for specific epitopes. We describe a strategy for selecting highly specific scFv-clones that discriminate between various conformational states of cell surface receptors. This approach adapts the M13 pIII phage-display technology toward a cell suspension-based strategy, which allows panning against complex, multimeric, fully functional cell membrane epitopes without alteration of structure due to purification or immobilization. As the functional properties are preserved, phage can be specifically depleted or selected for neo-epitopes exposed after physiological alterations of the targeted molecules. This subtractive strategy allows highly specific selection for single-chain antibodies directed against functionally regulated epitopes on the cell surface molecules that can be tailored for diagnostic and therapeutic applications. Using this protocol, activation-specific single-chain antibodies can be obtained within 4-6 weeks.

Generation of activation-specific human anti-alphaMbeta2 single-chain antibodies as potential diagnostic tools and therapeutic agents.

The leukocyte integrin Mac-1 (alpha(M)beta(2)) plays a pivotal role in inflammation and host defense. Upon leukocyte activation, Mac-1 undergoes a conformational change exposing interaction sites for multiple ligands. We aimed to generate single-chain antibodies (scFv's) directed against activation-specific Mac-1 ligand-binding sites. Using human scFv phage libraries, we developed subtractive strategies with depletion of phages binding to nonactivated Mac-1 and selection of phages binding to activated Mac-1, using monocytes as well as CHO cells transfected with native or mutated, activated Mac-1. Three scFv clones demonstrated exclusive binding to activated Mac-1. Mac-1 binding of the ligands fibrinogen, heparin, and ICAM-1, but not C3bi, was inhibited. Using alanine substitutions, the paratope was identified within the heavy chain HCDR3s of the scFv's. The epitope was localized to Lys(245)-Arg(261) of the alpha(M) I-domain. In a pilot study with septicemic patients, we provide initial support for the use of these scFv's as markers of monocyte activation and as potential diagnostic tools. Potential therapeutic use was tested in adhesion assays under static and flow conditions demonstrating the selective blockade of activated monocytes only. Furthermore, scFv HCDR3-derived peptides retain selectivity for the activated integrin, providing a unique template for the potential development of inhibitors that are specific for the activated Mac-1.

Reversibility versus persistence of GPIIb/IIIa blocker-induced conformational change of GPIIb/IIIa (alphaIIbbeta3, CD41/CD61).

Clinically used GPIIb/IIIa blockers are ligand mimetics, and thereby their binding can induce conformational changes of the platelet integrin GPIIb/IIIa. Since the reversibility of these conformational changes may be an important determinant of potential adverse effects of GPIIb/IIIa blockers, we produced a new monoclonal antibody (anti-LIBS-mAb), and by using its binding properties, we investigated the conformational changes of GPIIb/IIIa during the binding and especially the dissociation of GPIIb/IIIa blockers. Production of monoclonal antibody (mAb) clones was performed using purified GPIIb/IIIa in a high affinity conformation and using activated platelets. Clone anti-LIBS-145-mAb was chosen, since it allowed the sensitive probing of eptifibatide-induced conformational changes of GPIIb/IIIa. On resting and activated platelets and on GPIIb/IIIa-expressing Chinese hamster ovary cells, anti-LIBS-145-mAb binding returned to background binding after dissociation of eptifibatide, indicating a complete reversibility of the eptifibatide-induced conformational change. Furthermore, with the mixing of eptifibatide-preincubated and nonincubated cells, a fast reversibility could be demonstrated. However, when fibrinogen was present in a physiological concentration, the GPIIb/IIIa blocker-induced conformation was partially retained after the dissociation of eptifibatide and to the same extent binding of fibrinogen and the activation-specific mAb Pac-1 was induced. In conclusion, a fast reversibility of the conformational change of GPIIb/IIIa after dissociation of GPIIb/IIIa blockers could be demonstrated as an intrinsic property of the GPIIb/IIIa receptor. This mechanism prevents general platelet aggregation after dissociation of ligand mimetic GPIIb/IIIa blockers. Nevertheless, in the presence of fibrinogen this reversibility is not complete, which may explain some of the side effects of GPIIb/IIIa blockers, especially those of the oral GPIIb/IIIa blockers.

Distinct roles of ligand affinity and cytoskeletal anchorage in alphaIIbbeta3 (GP IIb/IIIa)-mediated cell aggregation and adhesion.

Platelet integrin alphaIIbbeta3 (GP IIb/IIla) is functionally modulated by changes in ligand affinity or in cytoskeletal anchorage. CHO cells transfected with wild-type/mutated alphaIIbbeta3 allow the dissection of the relative contributions of the two regulatory mechanisms in alphaIIbbeta3-mediated adhesion and aggregation. Mutations included a truncation of the cytoplasmic domain of the beta-subunit, resulting in a loss of cytoskeletal anchorage of alphaIIbbeta3, and a VGFFK-deletion of the alpha-subunit, resulting in a permanent high affinity state. alphaIIbbeta3-mediated cell aggregation is dependent on the high affinity state but only partially on the cytoskeletal anchorage of alphaIIbbeta3. In contrast, alphaIIbbeta3-mediated cell adhesion is dependent on the cytoskeletal anchorage but only partially on the high affinity state of alphaIIbbeta3. Thus, the functional evaluation of mutated alphaIIbbeta3 implies a differential role of affinity state and cytoskeletal anchorage for alphaIIbbeta3-mediated cell adhesion and aggregation.