Evaluation of Platelet Membrane Glycoproteins in Coronary Artery Disease
1999; Lippincott Williams & Wilkins; Volume: 99; Issue: 1 Linguagem: Inglês
10.1161/01.cir.99.1.e1
ISSN1524-4539
AutoresMeinrad Gawaz, Franz‐Josef Neumann, Albert Schömig,
Tópico(s)Coronary Interventions and Diagnostics
ResumoHomeCirculationVol. 99, No. 1Evaluation of Platelet Membrane Glycoproteins in Coronary Artery Disease Free AccessOtherPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessOtherPDF/EPUBEvaluation of Platelet Membrane Glycoproteins in Coronary Artery Disease Consequences for Diagnosis and Therapy Meinrad Gawaz, Franz-Josef Neumann and Albert Schömig Meinrad GawazMeinrad Gawaz From 1 Medizinische Klinik, Klinikum rechts der Isar und Deutsches Herzzentrum, Technische Universität München, München, Germany. , Franz-Josef NeumannFranz-Josef Neumann From 1 Medizinische Klinik, Klinikum rechts der Isar und Deutsches Herzzentrum, Technische Universität München, München, Germany. and Albert SchömigAlbert Schömig From 1 Medizinische Klinik, Klinikum rechts der Isar und Deutsches Herzzentrum, Technische Universität München, München, Germany. Originally published12 Jan 1999https://doi.org/10.1161/01.CIR.99.1.e1Circulation. 1999;99:e1–e11Platelets and Arterial ThrombosisPlatelets play a fundamental role in atherogenesis and development of ischemic complications.123 Under physiological conditions, platelets do not interact with the vessel wall. Injury of vascular intima disrupts the antithrombotic properties of endothelium and exposes the blood to adhesive molecules of the subendothelium. Platelet adhesion to the damaged vessel wall is the first step in hemostasis and thrombosis.4 Platelet adhesion is followed by spreading and activation, resulting in release of granule components and aggregate formation.56 On initial contact, platelet glycoprotein (GP) Ib/V/IX complex binds to von Willebrand factor associated with collagen on the subendothelial surface (Figure 1)45 and thus arrests the platelet on the vessel surface. The collagen receptor α2β1 is an important secondary receptor for platelet adhesion. α2β1-Collagen interaction leads to platelet activation and is critical for the spreading process involving the fibrinogen receptor GP IIb/IIIa to ensure close contact of the spread platelet with the surface.5 Other adhesion receptors, including the fibronectin receptor α5β1 and the laminin receptor α6β1, support and strengthen secondary adhesion (Figure 1). The fibrinogen receptor GP IIb/IIIa is particularly important in platelet-platelet coadhesion, termed aggregation. This requires conformational changes in GP IIb/IIIa that allow binding of soluble fibrinogen to the platelet membrane (Figure 2). Thus, fibrinogen bridging allows formation of platelet aggregates (Figure 2).6789Platelet adhesion and aggregation induce intracellular signaling, which mediates several responses, such as formation and secretion of thromboxane A2 (TXA2), serotonin, and ADP6 (Figure 1). These substances reinforce platelet activation, vasoconstriction, and slowing of blood flow and therefore increase platelet-platelet and platelet–vessel wall interaction.36Platelet Membrane ReceptorsThe most abundant platelet membrane glycoprotein is the β3-integrin GP IIb/IIIa (60 000 to 100 000 per platelet and 1% to 2% of the total platelet protein), the inducible platelet fibrinogen receptor.7 Eighty percent of GP IIb/IIIa is randomly distributed and expressed on the platelet surface in its resting state, and the remaining 20% is located within the surface connecting system (SCS) and in α-granule membranes510 (Figure 3). GP IIb/IIIa stored in this internal pool becomes surface expressed as functional receptor on platelet activation.11 Congenital deficiencies of GP IIb/IIIa in Glanzmann thrombasthenia lead to defective platelet aggregation and enhanced bleeding.5 As is the case with other integrins, GP IIb/IIIa is a heterodimer consisting of an α-subunit (GP IIb) and a β-subunit (GP IIIa)9 (Figure 4). Although the expression of GP IIb/IIIa (αIIbβ3) is limited to megakaryocytes and platelets, the other β3-integrin present on platelets, the vitronectin receptor αvβ3, is more widely distributed and is also found on endothelial and smooth muscle cells.12 αvβ3 shares a common β3-subunit with GP IIb/IIIa but is coupled with a different α-subunit.12 Thus, GP IIb/IIIa antagonists that cross-react with αvβ3 (eg, abciximab) have potential anti-αvβ3 activity that might result in broader pharmacological effects.GP IIb/IIIa on nonstimulated platelets cannot bind soluble, adhesive, RGD-containing glycoproteins such as fibrinogen, von Willebrand factor, vitronectin, or fibronectin (low-affinity state) (Figure 5).9 Stimulation of platelets with agonists7 induces signaling transduction events followed by conformational change within GP IIb/IIIa extracellular domains ("inside-out signaling") that allows binding of fibrinogen (high-affinity state)9 (Figure 5). Distinct amino acid sequences such as RGD or KQAGDV, which are present in the fibrinogen molecule, bind to specific regions of the GP IIb/IIIa complex.913 RGD- and KQAGDV-containing peptides compete with fibrinogen for a common binding site14 (Figure 4). Under certain circumstances, platelets can bind fibrinogen at near-normal affinity but fail to aggregate. These observations suggest a role of "postoccupancy receptor events" in aggregation. Postoccupancy receptor events involve cytoskeletal anchorage and organization of GP IIb/IIIa within the plasma membrane.915Activation-dependent binding of fibrinogen or activation-independent binding of small fibrinogen mimetic peptides to GP IIb/IIIa results in a further conformational change of GP IIb/IIIa (ligand-occupied state). Thus, the RGD sequence in ligands functions both as part of the binding site and as a trigger for secondary conformational changes leading to expression of additional ligand-induced binding sites (LIBS)916 (Figure 5). This leads to receptor clustering and generation of transmembrane cell signaling ("outside-in signaling"), which results in transduction signaling (eg, tyrosine phosphorylation), and irreversible binding of fibrinogen (postoccupancy events) (Figure 5).1216In addition to the β3-integrins αIIbβ3 and αvβ3, 3 β1-integrin receptors have been identified to date on platelets, including a receptor for collagen (α2β1), fibronectin (α5β1), and laminin (α6β1)5 (Table 1). Two additional nonintegrin receptors are also involved in platelet adhesion, the leucine-rich glycoprotein GP Ib/IX/V (receptor for von Willebrand factor) and GP IV (GP IIIb) as a receptor for collagen and thrombospondin (Table 1).5 Congenital deficiencies of GP Ib in Bernard-Soulier syndrome result in a functional defect of platelet adhesion and in an increase in abnormal bleeding.5P-selectin (GMP-140, PADGEM, cluster of determinants [CD]62P) is a membrane glycoprotein located in the α-granules of platelets and in the Weibel-Palade bodies of endothelial cells.5 Platelet activation leads to its fusion with the SCS and expression on the platelet membrane surface.5 P-selectin has been shown to be the major surface receptor for neutrophils and monocytes on thrombin-activated platelets.5 Its expression may facilitate recruitment of leukocytes to sites of thrombosis.4 Lysosomes contain another glycoprotein, GP 53,5 that shares structural similarity to lysosome integral membrane proteins (LIMPs) (Table 1). Moreover, the platelet surface expresses immunoglobulin-type adhesion molecules such as platelet–endothelial cell adhesion molecule (PECAM)-1 and intercellular adhesion molecule (ICAM)-2 (Table 1).5Coronary AtherosclerosisPlatelet α-granules contain a variety of mitogenic growth factors, such as platelet-derived growth factor (PDGF) and transforming growth factor (TGF),6 which might cause migration and proliferation of smooth muscle cells and thus intima proliferation at sites of enhanced platelet activation.1In patients with diabetes, platelet surface expression of P-selectin and microparticle formation were significantly increased.17 The alteration in platelet function in diabetes might result from primary release of larger platelets with enhanced thromboxane formation and increased surface density of platelet membrane glycoproteins.18 Hypercholesterolemia is associated with increased fibrinogen binding to activated platelets19 and an increased degranulation of P-selectin.20 Thus, coronary risk factors might induce early mechanisms of atherogenesis via enhanced systemic activation and degranulation of circulating platelets.Unstable AnginaThe pioneering studies of Willerson and colleagues3 showed that the conversion from stable to unstable angina is associated with platelet aggregation at sites of vascular injury followed by release of mediators that promote vasoconstriction and further platelet aggregation. We found that surface expression of P-selectin and of the activated GP IIb/IIIa complex (LIBS-1) was significantly increased in patients with crescendo unstable angina compared with those with stable angina.21 The alterations in platelet membrane glycoproteins were associated with increased platelet-neutrophil coaggregates and enhanced leukocyte activation.21 Recently, we22 and others23 showed that activated platelets induce the expression and secretion of cytokines in leukocytes. Activated platelets induce the oxidative burst in neutrophils by a fibrinogen-mediated event.24 Furthermore, activated platelets induce surface expression of ICAM-1, interleukin (IL)-6, monocyte chemoattractant protein-1, and IL-8 in cultured endothelial cells via CD40L-mediated mechanisms.252627 Moreover, we found that activated platelets induce activation of the transcription factor nuclear factor-κB (NF-κB) in monocytes22 and in endothelial cells26 through an IL-1–mediated mechanism (Gawaz et al, unpublished data, 1997). NF-κB–regulated gene expression plays a role in systemic inflammatory response.28 Recently, Liuzzo and coworkers showed that patients with unstable angina and elevated systemic C-reactive protein have a poor prognosis,29 whereas aspirin treatment reduces systemic inflammation and improves prognosis.30 Thus, platelets might be a promising pharmacological target for anti-inflammatory treatment in acute coronary syndromes.Acute Myocardial Infarction and ReperfusionReocclusion of the recanalized infarct-related artery after fibrinolysis remains a serious limitation during the acute hospital phase.31 The therapeutic effect of fibrinolysis in acute myocardial infarction (AMI) seems to be limited by increased platelet activation and TXA2 biosynthesis. Thromboxane metabolite excretion is elevated in postinfarction angina, with increased risk for ischemic events.32P-selectin expression is enhanced for days after AMI, which implies enhanced platelet activity.33 Activation of GP IIb/IIIa occurs within 72 hours after fibrinolysis despite treatment with aspirin and heparin.34 This coincides with an increased risk of thrombotic reocclusion of infarct-related vessels.31 We35 showed that fibrinogen receptor activity and P-selectin expression on circulating platelets in AMI decreased early (4 to 8 hours) after direct PTCA, coinciding with decreased peripheral platelet count and increased generation of microparticles. Fibrinogen receptor activity and P-selectin expression increased again 24 to 48 hours after PTCA. Enhanced surface expression of platelet adhesion receptors in the early postinfarction period also seems to influence platelet–endothelial cell adhesion, mediated through vitronectin receptor36 and P-selectin.37 Enhanced platelet–endothelial cell adhesion might contribute to impairment of microcirculation and compromise myocardial salvage during reperfusion.Coronary Angioplasty and StentingIn pioneering studies, Scharf and colleagues38 showed that surface expression of platelet activation markers increases during coronary angioplasty (Table 2). In coronary blood samples obtained at the site of angioplasty, we found enhanced platelet LIBS-1 expression.39 Platelet activation is enhanced for days after coronary interventions.4041 Nonionic as opposed to ionic contrast media causes profound platelet degranulation,42 which indicates that the type of contrast media modulates platelet function. We4344 showed that coronary stenting but not conventional PTCA results in significant platelet activation despite anticoagulation treatment with phenprocoumon and heparin in combination with aspirin. In contrast, platelet fibrinogen receptor activity was significantly reduced in patients treated with combined antiplatelet therapy consisting of ticlopidine and aspirin, which is associated with a reduced incidence of stent thrombosis.44 In addition, we45 found that combined antiplatelet therapy with ticlopidine plus aspirin is superior in inhibiting platelet activation after stenting.Flow Cytometric Functional AssaysFlow cytometry of platelets in whole blood, as first described by Shattil and colleagues,46 enables the evaluation of platelet membrane glycoproteins and is the current method of choice to study functional aspects of platelets.47 Unlike other platelet activation markers (PF4 [platelet factor-4], β-thromboglobulin [βTG], and TXA2), flow cytometry allows the detection of specific activation-dependent modification in the platelet membrane surface (Figure 6).GP IIb/IIIaThe availability of monoclonal antibodies (MAbs) enables us to distinguish between the resting, activated, and ligand-occupied forms of GP IIb/IIIa (Table 2) (Figures 6 and 7). The high-affinity state of activated GP IIb/IIIa can be assessed by PAC-1, which recognizes the ligand binding site in GP IIb/IIIa48 (Figure 7). The ligand-occupied state can be characterized by MAbs specific for LIBS (LIBS epitopes; LIBS-1 or PMI-1) (Figure 7).49 These epitopes are not expressed by resting GP IIb/IIIa or by activated GP IIb/IIIa not bound to ligand (Figure 5). LIBS epitopes decorate both subunits of the receptor, and 1 epitope (PMI-1) has been localized to the carboxy terminus of the heavy chain of GP IIb9 (Figure 4). The advantage of PAC-1 and LIBS antibodies is the detection of activated platelets, stimulated by low doses of agonists such as ADP, when secretion has not occurred.49 In contrast to PAC-1, LIBS-1 binding to GP IIb/IIIa does not interfere with the ligand binding pocket. Another approach to characterize activated platelets is to quantify adhesive proteins bound to activated GP IIb/IIIa5051 by binding of specific MAbs directed against the ligand. A more subtle way to detect ligand binding is to use conformation-dependent antibodies, termed anti-RIBS (receptor-induced binding site), which recognize exclusively receptor-bound fibrinogen that has undergone conformational change49 (Figures 5 and 7). Because GP IIb/IIIa is a promiscuous receptor (Table 1), detecting bound fibrinogen could underestimate the quantity of bound ligand if a significant percentage of receptor is already occupied by other ligands, such as von Willebrand factor. With the use of fluorochrome-conjugated RGD-containing polypeptides such as FITC echistatin, the accessibility of the fibrinogen binding site within the GP IIb/IIIa complex can be evaluated (Figure 7).GP Ib/IX/VIn contrast to activation-dependent MAbs directed to GP IIb/IIIa, binding of GP Ib–specific antibodies to thrombin-activated platelets is decreased owing to internalization of receptor complexes within the SCS (Figure 7).47 Thus, enhanced in vivo activity of thrombin may be monitored by reduction in surface expression of GP Ib.Granula MarkersBinding of anti-thrombospondin and anti–P-selectin antibodies specifically indicates release reaction of α-granules; binding of anti-GP 53 indicates primarily lysosome secretion (Figure 7) (Table 2).5Platelet-Leukocyte AggregatesFlow cytometric evaluation of a single platelet population might underestimate platelet activation in vivo. Analysis of circulating platelet-leukocyte aggregates (Figures 8 and 9) detects activated platelets adhering to circulating leukocytes.225253 Determination of platelet-leukocyte aggregates allows the study of inflammatory aspects of platelet activation. Platelet adhesion to leukocytes triggers activation mechanisms within the leukocytes222324 that result in enhanced expression of adhesion receptors (eg, MAC-1) or shedding of L-selectin on the activated leukocyte surface.21Platelet MicrovesiclesPlatelets have been shown to shed microvesicles during clotting of whole blood and after activation by thrombin, collagen, and the complement protein C5b-9 (Figure 2).5 Microvesicles are particularly rich in anionic phospholipids, GP Ib, GP IIb/IIIa, and receptors for coagulation factors Va, VIII(a), and IXa.554 Thus, microvesicles provide a catalytic surface for the transformation of prothrombin to thrombin. Flow cytometry is a sensitive method to detect platelet microparticles and enables the evaluation of platelet-coagulation interaction.4647Genetic Analysis of Platelet Membrane ReceptorsLittle is known about genetic risk factors of platelet membrane glycoproteins in arterial thrombotic disease. Several platelet membrane glycoproteins are known to be polymorphic, having ≥2 allelic forms present in the human gene pool.55 Restriction fragment length analysis enables the study of polymorphisms of platelet glycoproteins (Figure 10). In addition, with allele-specific antibodies, polymorphism of GP IIb/IIIa can be rapidly determined by flow cytometry (Figure 11).Weiss and colleagues56 demonstrated a high frequency of the PlA2 polymorphism of GP IIIa in patients who became symptomatic before the age of 60. However, the potential link between PlA2 and occurrence of acute coronary syndromes is controversial.57 Recently, Walter and coworkers58 found a strong association between subacute stent occlusion and the PlA2 allele after coronary stenting that was independent of classic risk factors such as reduced left ventricular function or residual dissection.Platelet Analysis and Risk StratificationIncreased platelet reactivity correlates with an increase in mortality and ischemic heart disease. Spontaneous platelet aggregation in vitro predicts coronary events and mortality in survivors of AMI.59 Platelet hyperaggregability in the morning is associated with increased frequency of AMI and sudden cardiac death.60 Mean platelet volume is an independent risk factor for the development of recurrent acute coronary ischemic events in survivors of AMI.61Tschoepe and colleagues41 found that an increased fraction of platelets surface expressing P-selectin or thrombospondin is predictive for occurrence of ischemic events after PTCA. We4362 showed that GP IIb/IIIa levels before stenting were an independent risk factor for stent thrombosis (Figure 12).Monitoring of Antiplatelet TherapyBecause fibrinogen binding to activated platelets is the crucial step in platelet aggregation, several pharmacological agents have been developed to antagonize the binding of fibrinogen to GP IIb/IIIa.1463 Abciximab (c7E3; ReoPro), a GP IIb/IIIa blocker, reduces ischemic events among patients undergoing PTCA.63 Administration of synthetic low-molecular-weight GP IIb/IIIa antagonists such as Integrelin or tirofiban, which might have advantages with respect to rapid reversibility of the antiplatelet effect and reduced immunogenicity,14 was less effective than administration of abciximab in reducing adverse cardiac events after PTCA.63 Thus, although the above-mentioned compounds are all very powerful GP IIb/IIIa blockers, the clinical efficacy might be greatly dependent on their pharmacological characteristics. Because GP IIb/IIIa blockers exhibit an extremely steep dose-response relationship and are characterized by variable individual response, it is necessary to monitor GP IIb/IIIa therapy to establish the optimum degree of platelet inhibition and thereby improve clinical efficacy and reduce the risk of bleeding.In our laboratory, we use flow cytometric techniques and a fluorescein-conjugated disintegrin, echistatin, to evaluate receptor blockade after administration of GP IIb/IIIa antagonists (Figure 7). Both abciximab and Integrelin similarly block echistatin binding to circulating platelets during drug administration (Figures 13 and 14). However, after termination of GP IIb/IIIa antagonist infusion, echistatin binding was rapidly restored within hours in Integrelin-treated patients, whereas normalization of GP IIb/IIIa accessibility in patients receiving abciximab required days (Figures 13 and 14). Thus, evaluation of echistatin binding may be helpful in monitoring long-acting (eg, abciximab) and short-acting (eg, Integrelin) GP IIb/IIIa antagonists. In addition, binding of GP IIb/IIIa inhibitors during or after administration can be easily monitored by a competitive assay with fluorescein-conjugated forms of the inhibitor (Figure 14).In contrast to conventional platelet assays, flow cytometry enables the study of the pharmacodynamic aspects of GP IIb/IIIa antagonists. Binding of small peptide ligands to GP IIb/IIIa produces a conformational change within the receptor molecule resulting in expression of new epitopes, termed LIBS (Figure 5). Du and coworkers64 found that RGD peptides induce proaggregatory conformation of the GP IIb/IIIa complex.Profound thrombocytopenia is one major side effect of GP IIb/IIIa antagonists and occurs independently of the nature of the antagonists (antibody or synthetic).6365 The pathophysiological mechanisms are unclear but might be related to expression of LIBS and thus "intrinsic" GP IIb/IIIa–receptor binding activity ("outside-in signaling").66 One might speculate that a GP IIb/IIIa blocker–induced conformational change of GP IIb/IIIa and the subsequent induction of postoccupancy events might alter the adhesive properties of circulating platelets or that endogenous LIBS-like antibodies bind to the LIBS-positive GP IIb/IIIa ligand complex, thus contributing to immunotype thrombocytopenia. This might particularly be involved during long-term anti-GP IIb/IIIa therapy. As shown in Figure 15, there are significant differences between GP IIb/IIIa antagonists in inducing LIBS expression. Preclinical evaluation of ligand-induced conformation-dependent epitopes (LIBS-1 and PMI-1) might be helpful in disclosing differences in "intrinsic" activities of various GP IIb/IIIa inhibitors and in development and design of new antagonists (Figure 15).Download figureDownload PowerPoint Figure 1. Platelet membrane glycoproteins and primary hemostasis. vWF indicates von Willebrand factor; Fn, fibronectin; Col, collagen; Lam, laminin; and AA, arachidonic acid.Download figureDownload PowerPoint Figure 2. Platelet aggregation. Epi indicates epinephrine; Col, collagen; and Fg, fibrinogen.Download figureDownload PowerPoint Figure 3. Transmission electron microscopy and anti-GP IIb/IIIa immunogold labeling of an unstimulated human platelet. DTS indicates dense tubular system; G, α-granules; and M, mitochondrium. Figure was kindly provided by Prof E. Morgenstern, University of Homburg/Saar, Germany.24Download figureDownload PowerPoint Figure 4. GP IIb/IIIa structure. GP IIb/IIIa is composed of an α-subunit (GP IIb) and a β-subunit (GP IIIa). GP IIb contains 4 putative Ca2+ binding sites. In addition, at least 3 ligand binding regions have been identified within the receptor complex: RGD (amino acid sequence 109 to 171), KGD (211 to 222), and KQAGDV (297 to 314).71316 Conformational change of GP IIb/IIIa receptor complexes leads to exposure of cryptic neoepitopes. LIBS epitopes (•). C indicates carboxy terminus; N, amino terminus.Download figureDownload PowerPoint Figure 5. Functional states of GP IIb/IIIa.Download figureDownload PowerPoint Figure 6. Whole-blood flow cytometry of platelet membrane glycoproteins. Platelets are identified in whole blood by size and platelet-specific CD42b antigen in the forward scatter versus CD42 immunofluorescence (phycoerythrin fluorescence) plot (top left). Other constitutively expressed membrane antigens (CD41, top right) or activation-dependent antigens (CD62P and LIBS-1; bottom panels) can be evaluated simultaneously by use of a second specific MAb conjugated to fluorescein. Representative immunofluorescence histograms are depicted (light lines indicate nonstimulated and bold lines indicate ADP [20 μmol/L]-activated platelets).Download figureDownload PowerPoint Figure 7. Immunological detection of platelet membrane glycoproteins.Download figureDownload PowerPoint Figure 8. Platelet-leukocyte aggregates. In the confocal mode (top), adhering platelets to leukocytes are seen that correspond to CD41-positive areas in the fluorescent mode (bottom).Download figureDownload PowerPoint Figure 9. Flow cytometric analysis of platelet-leukocyte aggregates. Leukocytes are identified in whole blood by size and leukocyte-specific CD14 antigen in the forward scatter versus CD14 immunofluorescence (phycoerythrin fluorescence) plot (left). Platelet-specific CD42 immunofluorescence present on CD14-positive leukocytes is used as an index of platelet-leukocyte aggregates (right). Simultaneously, activation-dependent leukocyte-specific markers can be evaluated by use of a third fluorescence (middle). Representative immunofluorescence histograms are depicted (light lines indicate nonstimulated and bold lines indicate ADP [20 μmol/L]-activated platelets).Download figureDownload PowerPoint Figure 10. Restriction fragment length analysis of platelet GP IIIa PlA1/2 diallele. MWM indicates molecular weight marker.Download figureDownload PowerPoint Figure 11. Flow cytometric characterization of the diallelic PlA1/PlA2 polymorphism. Depicted are representative histograms of MAb binding SZ21 to intact platelets of a homozygous PlA1/PlA1 and PlA2/PlA2 and a heterozygous PlA1/PlA2 individual. MAb SZ21 binds primarily to PlA1 phenotype of GP IIIa.Download figureDownload PowerPoint Figure 12. Surface expression of platelet membrane glycoproteins and risk of subacute stent thrombosis. For further description, see Reference 43.Download figureDownload PowerPoint Figure 13. Effect of administration of GP IIb/IIIa antagonists on ligand accessibility to GP IIb/IIIa. Binding of FITC-conjugated echistatin to platelets before, during, and after administration of the GP IIb/IIIa antagonists abciximab and Integrelin is shown. Patients with unstable angina were treated with an abciximab (12-hour) or Integrelin (72-hour) infusion, according to published protocols.63Download figureDownload PowerPoint Figure 14. Binding of FITC echistatin and FITC c7E3 to platelets collected from patients before and 8 to 12, 24, and 48 hours after starting administration of abciximab. Depicted are representative histograms of binding of FITC-conjugated c7E3 and echistatin to circulating platelets before, during, and after administration of abciximab in a patient with unstable angina.Download figureDownload PowerPoint Figure 15. Induction of LIBS on platelets treated with GP IIb/IIIa antagonists. Gel-filtrated platelets were incubated with the GP IIb/IIIa antagonists GRGDSP, γ-chain (KQAGDV) peptide H12, abciximab, Integrelin, tirofiban, or lamifiban for 30 minutes in the presence of MAb FITC-LIBS-1 or FITC-PMI-1, respectively. Binding of LIBS-1 (top) and PMI-1 (bottom) was quantified by platelet flow cytometry.66 Table 1. Platelet Membrane GlycoproteinClassificationElectrophoretic ClassificationCluster of DeterminantsNumber of Receptor CopiesLigand/CounterreceptorIntegrinsα2β1GP Ia/IIaCD49b1000Collagenα5β1GP Ic/IIaCD49c1000Fibronectinα6β1GP Ic′/IIaCD49f1000LamininαIIbβ3GP IIb/IIIaCD41-CD6160 000–100 000FibrinogenFibronectinvon Willebrand factorVitronectinαvβ3GP αv/IIIaCD51-CD61100VitronectinFibrinogenFibronectinLeucin-rich glycoproteinsGP Ib/V/IXCD42a-b-c25 000von Willebrand factorGP IV (GP IIIb)CD3615 000–25 000CollagenThrombospondinSelectinsP-selectinCD62P12 000PSGL-1Immunoglobulin-like adhesion receptorsICAM-2CD1025000LFA-1PECAM-1CD313000?Lysosome-integral membrane proteinsGP53CD633000? Table 2. Platelet-Specific MAbsPrototypic AntibodyAntibody SpecificityPlatelet Membrane Glycoprotein4F10 PAC-1Complex-specific fibrinogen-binding site of the activated GP IIb/IIIa receptor complexGP IIb/IIIa GP IIIaLIBS-1, LIBS-2, LIBS-6Ligand-induced binding siteGP IIIaPMI-1Ligand-induced binding siteGP IIbSZ21Binds primarily to P1A1 allele of GP IIIaGP IIIaRIBS-1Receptor-induced binding siteFibrinogenTSP-1ThrombospondinSZ12P-selectinThis study was supported by grants from the Deutsche Forschungsgemeinschaft (Ga 381/1-1 and 1-2; Ga 381/2-1; and Ne 540/1-2). We thank Drs Andreas Ruf, Karlheinz Peter, Melchior Seyfarth, and Silja Rüdiger for critical reading of the manuscript. The authors are grateful to Dr Mark Ginsberg for helpful discussions and for generously supporting us with material.FootnotesCorrespondence to Meinrad Gawaz, MD, 1 Medizinische Klinik, Klinikum rechts der Isar und Deutsches Herzzentrum, Technische Universität München, Lazarettstraße 36, 80636 München, Germany. E-mail [email protected] References 1 Ross R. The pathogenesis of atherosclerosis: a perspective for the 1990's. Nature.1993; 362:801–809.CrossrefMedlineGoogle Scholar2 Fuster V, Badimon L, Badimon J, Chesebro JH. The pathogenesis of coronary artery disease and the acute coronary syndromes. N Engl J Med.1992; 326:242–250.CrossrefMedlineGoogle Scholar3 Willerson JT, Golino P, Eidt J, Campbell WB, Buja M. Specific platelet mediators and unstable coronary artery lesions: experimental evidence and potential clinical implications. Circulation.1989; 80:198–205.CrossrefMedlineGoogle Scholar
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