Paradoxical inhibition of ﬁbrinogen binding and potentiation of a -granule release by speciﬁc types of inhibitors of glycoprotein IIb–IIIa

Objective: To determine whether glycoprotein (GP) IIb–IIIa inhibitors can paradoxically augment activation of platelets, activation of GP IIb–IIIa, a -granule degranulation, and lysosome release were induced after exposure of platelets to GP IIb–IIIa inhibitors. Methods: ADP-induced platelet activation was assessed after exposure of platelets to Abciximab, or to a non-peptide ligand, the free acid of Orboﬁban (Orboﬁban ). Activation of GP IIb–IIIa was detected based on binding of ﬂuorochrome labeled ﬁbrinogen or a labeled a monoclonal antibody, PAC-1. a -Granule degranulation was detected based on surface expression of P-selectin and lysosome release was detected based on surface expression of CD63. Results: Despite signiﬁcant inter-individual variability in inhibition of ﬁbrinogen binding in response to each of the GP IIb–IIIa inhibitors used, a concentration dependent decrease in ﬁbrinogen binding was seen with each agent in samples from each subject. Binding of PAC-1 was inhibited in a parallel manner. Abciximab increased ADP-induced P-selectin expression. Orboﬁban did not alter ADP-induced P-selectin expression. Neither agent altered ADP-induced CD63 expression. When a platelets were exposed to Abciximab and Orboﬁban , both Abciximab and Orboﬁban were found in the a -granules (by confocal a a microscopy), consistent with potentiation of agonist-induced release of a -granular products associated with uptake of proteins. Conclusions: Speciﬁc types of GP IIb–IIIa inhibitors can paradoxically augment agonist-induced release of a -granules despite inhibiting agonist-induced ﬁbrinogen binding. (cid:211)


Introduction
products by adherent platelets in vivo may exacerbate thrombosis by supplying coagulation factors V/ Va and Glycoprotein (GP) IIb-IIIa inhibitors inhibit aggrega-fibrinogen [5]. Furthermore, the release of growth factors tion of platelets by inhibiting binding of fibrinogen to the (such as platelet derived growth factor and transforming activated conformer of GP IIb-IIIa [1][2][3]. We have growth factor b) present in a-granules may exacerbate observed a phenomenon in which potentiation of agonist-atherogenesis [5]. induced a-granule release occurs in platelets that take up Three types of GP IIb-IIIa inhibitors have been deproteins such as fibrinogen [4]. Accordingly, this study veloped for intravenous use; (1) a chimeric Fab fraction of was designed to determine whether the same thing occurs an antibody (Abciximab [ReoPro®]), (2) peptide ligands with inhibitors of GP IIb-IIIa and hence whether such (e.g. eptifibatide [Integrilin®]), and (3) non-peptide ligagents can paradoxically augment ADP-induced a-granule ands (e.g. tirofiban [Aggrastat®]). The administration of degranulation reflected by the surface expression of P-GP IIb-IIIa inhibitors in conjunction with coronary anselectin. Determination of the effects of antiplatelet agents gioplasty is effective in reducing the incidence of cardiac on multiple components of activation is necessary because events (myocardial infarction, death, and need for urgent despite inhibition of aggregation the release of a-granular revascularization) [6][7][8][9]. Treatment of patients with unstable angina or non-Q-wave myocardial infarction with GP IIb-IIIa inhibitors reduces the incidence of subsequent cardiac events [10][11][12]. Adjunctive therapy with GP IIb-of Factor XIIa without effect on other coagulation factors IIIa inhibitors in patients being treated with thrombolytic [16], was used as the anticoagulant because we have agents has increased early coronary patency [13,14]. The shown that the activation of platelets is altered by convendevelopment of orally active preparations (such as Or-tional anticoagulants such as citrate [15]. Platelets were bofiban) should facilitate longer term treatment and may exposed to GP IIb-IIIa inhibitors for 5-10 min before reduce further the incidence of cardiac events.
initiation of assays. To prevent clotting of blood during We have developed an assay in which the activation of relatively prolonged exposure of the samples to Abcixplatelets is induced in minimally altered whole blood by imab, blood was anticoagulated with 0.4 nM recombinant agonists such as adenosine diphosphate (ADP) and de-tick anticoagulant peptide (rTAP, kindly provided by Dr. tected with the use of flow cytometry [4,15]. It entails the George P. Vlasuk, Corvas International), 3 anti-IIa U / ml of use of whole blood; provides sensitive and specific de-recombinant hirudin (Sigma), or 1 anti-Xa U / ml of tection of selected components of platelet activation; enoxaparin (Rhone-Poulenc Rorer) in addition to CTI. utilizes physiologic concentrations of agonists; and permits analysis of the fixed platelets with flow cytometry long 2.3. Analysis of platelets by flow cytometry after sample acquisition. The assay permits assessment of diverse components of platelet activation: (1) Activation of Activation of platelets in response to adenosine diphos-GP IIb-IIIa can be identified by the binding of fibrinogen phate (ADP) was determined with respect to surface or an activation dependent antibody such as PAC-1; (2) expression of P-selectin (a-granule degranulation), surface a-granule degranulation can be identified by the surface expression of CD63 (lysosome release), and with respect expression of P-selectin; and (3) lysosome release can be to binding of fibrinogen or PAC-1 (activation of surface GP identified by the surface expression of CD63. IIb-IIIa). Surface expression of P-selectin was delineated In the present study, platelets were exposed to Abcix-with a phycoerythrin (PE) conjugated monoclonal antibody imab, a chimeric Fab fragment, or to the free acid of (anti-CD62, Becton Dickinson). Surface expression of Orbofiban (Orbofiban ), a non-peptide inhibitor, in vitro.
CD63 was determined with a PE conjugated monoclonal a Platelet function in response to exposure to each agent was antibody (anti-CD63, Immunotech, Marseille, France). characterized by flow cytometry.
Activation of GP IIb-IIIa was determined with fluorescein isothiocyanate (FITC) labeled PAC-1 (Becton Dickinson) or FITC-fibrinogen. Fibrinogen was labeled with celite 2. Methods FITC (Calbiochem, La Jolla, CA) as previously described [17]. Labeling of fibrinogen with celite FITC preserves the 2.1. Patient population functional activity of fibrinogen and does not alter binding of fibrinogen to activated platelets [17]. Platelets were In protocols approved by the University of Vermont identified on the basis of size and the binding of a PerCP Institutional Review Board, samples were obtained from conjugated IgG directed against glycoprotein IIIa (antihealthy subjects who had not ingested aspirin or other CD61, Becton Dickinson). Anti-CD61 binds to glycopronon-steroidal anti-inflammatory agents for at least 10 days.
tein IIIa regardless of the activation state and does not After informed consent had been obtained, blood was interfere with binding of fibrinogen. obtained by peripheral venipuncture from all subjects.
Assays were initiated by addition of 5 ml of whole blood Three groups of subjects were recruited; (1) ten subjects to polypropylene tubes containing HEPES-Tyrode's buffer whose blood was used for determination of the effect of (5 mM HEPES, 137 mM NaCl, 2.7 mM NaHCO , 0.36 3 Abciximab on fibrinogen binding and CD62 expression, mM NaH PO , 2 mM CaCl , 4 mM MgCl , and 5 mM (2) ten subjects whose blood was used for determination of dextrose, pH 7.4) and PerCP conjugated anti-CD61 (0.46 the effect of Orbofiban on fibrinogen binding and CD62 mg / ml) in addition to selected concentrations of ADP (0, a expression, and (3) five subjects whose blood was used for 0.2, 1, 2 mM). Activation of platelets was determined with determination of the effect of Abciximab and Orbofiban FITC conjugated fibrinogen (0.1 mg / ml) and PE conjua on PAC-1 binding and CD63 expression. gated anti-CD62 (1.15 mg / ml) or with FITC conjugated PAC-1 (2 mg / ml) and PE conjugated anti-CD63 (0.6 2.2. Collection of blood samples mg / ml). After 15 min platelets were fixed and red blood cells were lysed with Optilyse C (Immunotech). The Phlebotomy was performed with a two syringe tech-association of antibodies with platelets was detected with nique in which the first 3 ml of blood were discarded. the use of a fluorescence-activated cell sorter (Becton Blood to be analyzed with flow cytometry was drawn into Dickinson). To quantify non-specific binding, control syringes containing corn trypsin inhibitor (CTI, 32 mg / ml) samples containing non-fractionated mouse IgG conjugated alone and in combination with selected concentrations of with PE or FITC and FITC conjugated albumin (for Abciximab (0.01, 0.1, 1, 2, 4 and 10 mg / ml) or Orbofiban fibrinogen binding) were assayed for each subject. To a (1, 10, 50, 100, and 250 ng / ml). CTI, a specific inhibitor confirm that an increase in the intensity of each activation Fig. 1. ADP-induced P-selectin expression (left) and fibrinogen binding (right) in blood obtained from ten healthy subjects. Blood was anticoagulated with corn trypsin inhibitor (CTI, a specific inhibitor of factor XIIa) and exposed to 0, 1, 2, or 4 mg / ml of Abciximab (ReoPro) for 5-10 min before platelet assays were initiated. Surface expression of P-selectin (reflecting a-granule degranulation) and fibrinogen binding were determined with the use of flow cytometry. Values are means6SEM. Fibrinogen binding in response to 0.2 and 1 mM ADP was inhibited (P,0.001) by each concentration of ReoPro. Fibrinogen binding in response to 2 mM ADP was inhibited (P,0.001) by 2 and 4 mg / ml of ReoPro. Increased P-selectin expression in response to 1 and 2 mM ADP was seen (P,0.01) after exposure of blood to 2 and 4 mg / ml of ReoPro. dependent fluorochrome was caused by increased associa-min. The slide was washed a second time, and then a tion of the specific fluorochrome with platelets, two FITC-conjugated anti-CD62 was applied for 30 min. The additional controls were performed. In the first, blood was Abxicimab treated platelets were subsequently exposed to exposed to anti-CD61 PerCP to mark platelets, albumin-a mouse monoclonal anti-CD62 antibody for 15 min and FITC, and anti-CD62 PE in addition to 2 mM ADP. In the then applied to a glass microscopic slide. The slide was second, blood was exposed to anti-CD61 PerCP to mark washed, and an Alexa 568 conjugated goat anti-mouse IgG platelets, fibrinogen-FITC, and IgG PE in addition to 2 (Molecular Probes, Eugene, OR) was applied for 30 min. mM ADP. With respect to CD63, values reported are the After additional washes had been performed on both sets, percentage of platelets expressing CD63 in excess of samples were air-dried, and a cover-slip was applied with binding seen in the absence of agonist.
1% n-propyl gallate (Sigma) in 50% glycerol:50% PBS. The slides were evaluated with the use of a BioRad 2.4. Confocal laser scanning microscopy (Hercules CA) MRC 1000 confocal scanning laser system equipped with a krypton / argon laser mounted on an The association with and localization in platelets of Olympus BX50 microscope. Abciximab and Orbofiban were determined as previously Platelets were imaged with the use of a 1003 phase a described [4]. Abciximab was labeled with celite FITC, contrast oil immersion lens (N.A.51.3) and an electronic and blood anti-coagulated with CTI was exposed to 10 zoom factor of 2.4. Phase contrast (non-confocal) images mg / ml of FITC-Abciximab or 250 ng / ml of Orbofiban were acquired with the use of a transmitted light detector a for 15 min. Platelets were fixed, and red blood cells were attachment. For platelets that were exposed to Abciximab, lysed with Optilyse C. The plasma membranes of the FITC-Abciximab was visualized with the use of 488 nm platelets were rendered permeable by addition of 0.1% laser excitation, and P-selectin was visualized with 568 nm Triton X-100 (Sigma, St. Louis). The Orbofiban treated laser excitation. For Orbofiban -treated platelets, the Alexa a a platelets were subsequently exposed to a mouse mono-568 conjugated secondary antibody localizing Orbofiban a clonal anti-Orbofiban antibody (provided by Searle). The was visualized with the use of 568 nm laser excitation, and platelet suspension was incubated for 15 min and then FITC-anti-CD62 was visualized with 488 nm laser excitaapplied to a glass microscopic slide for 15 min. The slide tion. was washed, and an Alexa 568 conjugated goat anti-mouse Co-localization of Abciximab and P-selectin was de-IgG (Molecular Probes, Eugene, OR) was applied for 30 lineated with the use of the multiply function in Macro Fig. 2. P-selectin expression and fibrinogen binding induced by 1 mM ADP in blood exposed to 10 mg / ml of Abciximab for up to 4 h. Blood was anticoagulated with CTI and 0.4 nM recombinant tick anticoagulant peptide. Increased ADP-induced P-selectin expression that was apparent from 10 min to 2 h after exposure to Abciximab was not seen after 4 h of exposure. Fibrinogen binding was inhibited throughout the entire interval. Results presented are from a representative experiment with blood from one healthy subject. Similar results were seen with blood from each of three subjects and when blood was anticoagulated with 3 anti-IIa U / ml of hirudin and 1 anti-Xa U / ml of enoxaparin.
Programming Language Software (BioRad). This function tion, the resulting image displays color only in areas where multiplies each pixel in the active display box by the fluorescence was present in both images permitting cocorresponding pixel of a second image. After multiplica-localization of two fluorochromes. Fig. 3. ADP-induced CD63 expression (left) and PAC-1 binding (right) in blood obtained from five healthy subjects. Blood was anticoagulated with corn trypsin inhibitor (CTI, a specific inhibitor of factor XIIa) and exposed to 0, 0.01, 0.1, 1, or 4 mg / ml of Abciximab (ReoPro) for 5-10 min before platelet assays were initiated. Surface expression of CD63 (reflecting lysosome release) and PAC-1 binding were determined with the use of flow cytometry. Values are means6SEM. PAC-1 binding in response to all concentrations of ADP was inhibited (P,0.001) by 1 and 4 mg / ml of ReoPro. CD63 expression was not altered by exposure of platelets to ReoPro.

Analysis of data
Values are means6SEM. Significance of differences was determined by analysis of variance. Differences between treatments were determined with the use of Student-Newman-Keuls tests. Significance was defined as P,0.05.

Effects of Abciximab on platelet activation
Exposure of blood from ten subjects to 1, 2, and 4 mg / ml of Abciximab inhibited ADP-induced fibrinogen binding in a concentration dependent manner (Fig. 1). Each concentration of Abciximab inhibited (P,0.001) fibrinogen binding induced by 0.2, 1, and 2 mM ADP. By contrast, the ADP-induced P-selectin expression paradoxically increased after exposure of blood to Abciximab in a concentration dependent manner (Fig. 1). For example, P-selectin expression was increased (P,0.01) by exposure of blood to 2 and 4 mg / ml of Abciximab in combination with 1 and 2 mM ADP.
To determine whether exposure to Abciximab for up to 4 h resulted in a similar increase in ADP-induced Pselectin expression, blood was anticoagulated with rTAP in addition to CTI to prevent clotting of blood through 4 h. Increased P-selectin expression was seen from 10 min through 2 h after exposure to 10 mg / ml of Abciximab. However, it did not persist with 4 h of exposure (Fig. 2). Thus, the paradoxically induced degranulation appears to have a prompt onset and relatively prompt offset. Similar results were seen with blood that had been anticoagulated with recombinant hirudin and enoxaparin (data not shown). To determine whether exposure to Abciximab potenbinding was assayed with the use of flow cytometry in whole blood anticoagulated with CTI. Blood was exposed to Abciximab (ReoPro -0, tiated the activation of GP IIb-IIIa, Abciximab (0.01, 0.1, 1, 2, or 4 mg / ml) for 5-10 min before addition of agonists. Values reflect 1, and 4 mg / ml) was added to blood from five subjects and the ratio of results with exposure of platelets to the inhibitor (1, 2, and 4 activation of GP IIb-IIIa was detected with PAC-1. In mg / ml of ReoPro) with respect to those under control conditions (no addition, lysosome release was detected based on surface ReoPro) expressed as percentages. Results for each individual healthy expression of CD63. Abciximab did not activate per se nor subject are identified with the same symbol. Concentration dependent decreases in fibrinogen binding were apparent in each subject. did it potentiate the activation of GP IIb-IIIa (Fig. 3). PAC-1 binding in response to 0.2 mM ADP decreased with each concentration of Abciximab (P,0.001). Abciximab did not alter ADP-induced release of lysosomes (Fig. 3).
of blood to Orbofiban did not alter the threshold for a Inter-individual variability of inhibition of fibrinogen ADP-induced a-granule degranulation (surface expression binding was apparent after exposure of blood to Abcix-of P-selectin). Parallel results were seen with the free acid imab (Fig. 4). Nevertheless, the inhibition was concen-of Xemilofiban, another non-peptide ligand of the activated tration dependent in each individual subject.
conformer of GP IIb-IIIa (data not shown).
To determine whether exposure to Orbofiban potena 3.2. Effects of Orbofiban on platelet activation tiated the activation of GP IIb-IIIa, Orbofiban (1, 10, and a 100 ng / ml) was added to blood from five subjects. The exposure of blood from ten healthy subjects to the Activation of GP IIb-IIIa was detected with the use of active metabolite of Orbofiban inhibited (P,0.001) ADP-PAC-1 and lysosome release was detected based on surface a induced binding of fibrinogen in a concentration dependent expression of CD63. Orbofiban did not activate per se nor a fashion (Fig. 5). Unlike results with Abciximab, exposure did it potentiate the activation of GP IIb-IIIa (Fig. 6). A non-significant trend toward increased release of lyso-of P-selectin (a-granule degranulation) induced by ADP or somes was seen with 100 ng / ml of Orbofiban (Fig. 6).
by the thrombin receptor agonist peptide (TRAP) [4]. a Similar to the case with Abciximab, substantial inter-Because one mechanism potentially responsible is an individual variation was seen in ADP-induced fibrinogen increased total mass of protein in a-granules, platelets binding in response to a given concentration of Orbofiban .
were exposed to FITC-Abciximab and Orbofiban before a a Nevertheless, a concentration dependent decrease in fibrin-fixation and visualized with confocal laser scanning microogen binding was seen in each individual subject (Fig. 7).
scopy. Both Orbofiban and Abciximab co-localized with a P-selectin in a-granules of quiescent platelets (Fig. 8).

Uptake of Abciximab and of Orbofiban by platelets
Thus, if protein and peptide uptake stimulates a-granule release as implicated by results in previous studies [4,18], We have previously shown that exposure of platelets to the exposure of platelets to a protein or a peptide inhibitor fibrinogen leads to its uptake into a-granules and an of GP IIb-IIIa may be a sufficient condition to induce the associated decrease in the threshold for surface expression phenomenon.

Discussion
The development of GP IIb-IIIa inhibitors has improved the care of patients with acute coronary syndromes [6][7][8][9][10][11][12][13][14]. Characterization of the effects of these agents on selected components of platelet activation is likely to facilitate optimal implementation of therapy [19,20]. We used flow cytometry to determine whether exposure of platelets from healthy subjects to GP IIb-IIIa inhibitors altered a-granule degranulation and lysosome release in addition to blocking the binding of fibrinogen to the activated conformer of GP IIb-IIIa. As would be expected based on their mechanism of action, GP IIb-IIIa inhibitors did not inhibit activation of platelets but rather blocked binding of fibrinogen to previously activated conformers of GP IIb-IIIa.
We found that ADP-induced P-selectin expression was increased after exposure of blood to Abciximab but not to Orbofiban . P-selectin expression under basal conditions a (no ADP) was low (,2% of platelets) and was not affected by exposure to Abciximab or Orbofiban . Further, we a found that exposure of platelets to Abciximab and to Orbofiban did not increase binding of the activation a dependent antibody, PAC-1, to GP IIb-IIIa. Accordingly, neither Abciximab nor Orbofiban activated platelets per a se. By contrast, exposure of platelets to Abciximab augmented ADP-induced P-selectin expression. These results are consistent with those observed after exposure of platelets to fibrinogen [4]. We found that the exposure of platelets to increased concentrations of fibrinogen potentiated agonist-induced P-selectin expression associated with the uptake of fibrinogen into a-granules. Further, we have found that the exposure of platelets to a peptide inhibitor of GP IIb-IIIa, Eptifibatide, potentiates also agonist-induced P-selectin expression [18]. a-granules of quiescent platelets after exposure of platelets Fig. 8. The association of FITC-Abciximab (ReoPro) and Orbofiban with quiescent platelets in blood anticoagulated with CTI (32 mg / ml). Platelets were a exposed to FITC-ReoPro (10 mg / ml) for 15 min (micrographs on left). The platelets were fixed in Optilyse C and exposed subsequently to 0.1% Triton X-100 and anti-P-selectin IgG. After washing, platelets were exposed to a secondary Alexa-conjugated anti-mouse IgG. The micrograph on the top (A) shows a representative platelet imaged with fluorescence (excitation 488 nm) demonstrating FITC-ReoPro (green signal). The micrograph in the center (C) depicts the same platelet visualized with fluorescence (excitation 568 nm) demonstrating P-selectin expression and localizing a-granules (red signal). The micrograph on the bottom (E) shows the same platelet viewed by multiplying fluorescent images (excitation 488 and 568 nm) and accordingly, the extent of co-localization of ReoPro-FITC and P-selectin (yellow color). The yellow color shows localization of ReoPro in a-granules. The micrographs on the right depict the association of Orbofiban with a quiescent platelet. Platelets were exposed to 250 ng / ml of Orbofiban for 15 min. Subsequently the a a platelets were fixed with Optilyse C and exposed to 0.1% Triton X-100 and anti-Orbofiban IgG. After washing, platelets were exposed to a secondary Alexa-conjugated anti-mouse IgG. P-selectin and thus a-granules were identified with FITC-anti-CD62. The micrograph on the top (B) shows a representative platelet imaged with fluorescence (excitation 568 nm) demonstrating Orbofiban (red signal). The micrograph in the center (D) depicts the a same platelet visualized with fluorescence (excitation 488 nm) localizing a-granules (green signal). The micrograph on the bottom (F) shows the same platelet viewed by multiplying fluorescent images (excitation 488 and 568 nm) and accordingly, the extent of co-localization of Orbofiban and P-selectin a (yellow color). Bar510 mm, all images were obtained at the same magnification.
to the agents in whole blood, Orbofiban is a not a protein other non-peptide ligands for GP IIb-IIIa, Xemilofiban a and does not contribute to the overall mass of protein in and Tirofiban, do not potentiate ADP-induced P-selectin a-granules. Accordingly, the lack of an effect of expression [18]. By contrast, Abciximab, an Fab fragment Orbofiban on ADP-induced P-selectin expression, despite of IgG, and Eptifibatide, a peptide inhibitor of GP IIb-IIIa, a its uptake into a-granules is consistent with the mechanism potentiated agonist-induced a-granule degranulation [18]. we have proposed. In addition, we have found that two These observations are consistent with the mechanism proposed because the uptake of both Abciximab and they suggest that increased uptake of proteins into a-Eptifibatide contributes to the overall mass of protein in granules potentiates agonist-induced degranulation. a-granules.
Despite the efficacy of inhibition of fibrinogen binding The increased ADP-induced P-selectin expression seen demonstrated when data from each group of subjects were after exposure of platelets to Abciximab was maximal analyzed in aggregate, marked inter-individual variability during the first 2 h and was not apparent after 4 h of was observed. However, a concentration dependent deincubation of platelets in whole blood in vitro. This crease in fibrinogen binding was observed in samples from suggests that the potentiation of ADP-induced P-selectin each individual subject. Thus, our results suggest that after expression may have a relatively rapid offset, consistent identification of a target level of inhibition of fibrinogen with recent observations in patients undergoing coronary binding, titration of dose should be readily accomplished angioplasty [21]. In addition, our results were obtained with the use of conventional pharmacokinetic principles. with blood from healthy subjects not taking aspirin or Potent anti-platelet agents are potentially pivotal in other medications. The studies reported here were designed improving the treatment of patients with coronary artery to determine whether differential effects of selected types disease. Their availability has highlighted the need for of GP IIb-IIIa inhibitors were present. Subsequent studies improved assessment of diverse components of platelet in blood from patients with coronary disease and in function. The development of readily available, accurate, subjects ingesting other medications, particularly anti-and targeted assays of platelet function should facilitate platelet agents such as aspirin are required. In addition, in optimal titration of individual pharmacologic agents. In vivo studies with more prolonged exposure to selected addition, such assays should permit enhanced characterizaagents should elucidate potential clinical implications.
tion of diverse and potentially paradoxical effects of a A second possible explanation for the increased ADP-given agent on platelet function that can facilitate its induced P-selectin expression seen with Abciximab is a optimal clinical utilization. partial agonist effect of Abcixmab. Peter and colleagues found that Abciximab, particularly at low concentrations, can potentiate the activation of GP IIb-IIIa and hence the