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(Circulation. 1997;96:1488-1494.)
© 1997 American Heart Association, Inc.

Articles

Effect of Ca²⁺ on GP IIb-IIIa Interactions With Integrilin

Enhanced GP IIb-IIIa Binding and Inhibition of Platelet Aggregation by Reductions in the Concentration of Ionized Calcium in Plasma Anticoagulated With Citrate

David R. Phillips, PhD; Willie Teng, BSc; Ann Arfsten, MSc; Lisa Nannizzi-Alaimo, BSc; Melanie M. White, BSc; Celia Longhurst, PhD; Sanford J. Shattil, MD; Anne Randolph, PhD; Joseph A. Jakubowski, PhD; Lisa K. Jennings, PhD; ; Robert M. Scarborough, PhD

From COR Therapeutics, South San Francisco, Calif (D.R.P., W.T., A.A., L.N.-A., A.R., R.M.S.); University of Tennessee, Memphis (M.M.W., C.L., L.K.J.); Lilly Research Laboratories, Indianapolis, Ind (J.A.J.); and Scripps Research Institute (S.J.S.).

	Abstract

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Background Integrilin (eptifibatide), a potent inhibitor of the fibrinogen binding function of GP IIb-IIIa, has been shown to reduce the thrombotic complications of angioplasty and of acute coronary syndromes. The present study was designed to determine whether the reduced Ca²⁺ concentrations in plasma anticoagulated with citrate affect Integrilin binding to GP IIb-IIIa and the ex vivo pharmacodynamic measurements for this drug.

Methods and Results Lower concentrations of Integrilin were found to inhibit platelet aggregation in plasma anticoagulated with citrate (for ADP, mean±SD IC₅₀=140±40 nmol/L, n=6; Ca²⁺=40 to 50 µmol/L) than with PPACK (IC₅₀=570±70 nmol/L, P<.0001, n=6; Ca²⁺ 1 mmol/L). Chelation of Ca²⁺ with EDTA or citrate caused a similar degree of enhancement in the inhibitory activity of Integrilin. Measurements of D3 LIBS epitope expression showed that the enhanced inhibitory activity was caused by enhanced GP IIb-IIIa occupancy by Integrilin. Citrate anticoagulation decreased the amounts of Integrilin required to inhibit the binding of PAC1, a monoclonal antibody that mimics the GP IIb-IIIa binding activity of fibrinogen. Reduced Ca²⁺ also increased Integrilin inhibition of the binding of biotinylated fibrinogen to purified, immobilized GP IIb-IIIa.

Conclusions These data suggest that citrate anticoagulation removes Ca²⁺ from GP IIb-IIIa and enhances the apparent inhibitory activity of Integrilin. This finding indicates that the inhibitory activity of Integrilin is overestimated in blood samples collected with citrate, suggesting that it may be possible to achieve greater antithrombotic efficacy beyond that observed in clinical trials to date with Integrilin.

Key Words: calcium • drugs • fibrinogen • glycoproteins • platelets

	Introduction

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Recent clinical trials have established that antagonists of fibrinogen binding to GP IIb-IIIa are effective in reducing thrombotic complications induced by coronary angioplasty.^{1 2 3 4 5} The most widely studied GP IIb-IIIa antagonists are parenteral agents that have been administered by infusion before coronary intervention and continuing for up to 36 hours after the procedure. Although all clinical trials show that GP IIb-IIIa inhibition reduces acute thrombotic complications, some variability in other end points has been observed. Thus, it is reasonable to question whether all trials have been performed with the same level and duration of GP IIb-IIIa antagonism and inhibition of platelet aggregation.

Integrilin, a cyclic heptapeptide, is a GP IIb-IIIa antagonist that blocks platelet aggregation and has been found to reduce the incidence of ischemic clinical events in acute coronary indications.^{3 6 7} Integrilin, like other antagonists of GP IIb-IIIa, functions by blocking the binding of the adhesive proteins fibrinogen and von Willebrand factor to GP IIb-IIIa on the surface of activated platelets.^{1 7 8} GP IIb-IIIa antagonists are effective antithrombotics because the binding of adhesive proteins to GP IIb-IIIa is required for platelet aggregation and arterial thrombus formation.^{9 10} Pharmacodynamic measurements of the potential in vivo activity of Integrilin and other parenteral inhibitors have been based primarily on their effects on ex vivo platelet aggregation, although receptor occupancy has been determined in patients receiving the monoclonal antibody abciximab.¹¹ Ex vivo platelet aggregation analyses are routinely performed in blood anticoagulated with citrate,^{3 5 6} an agent that prevents in vitro clotting by reducing the ionized calcium concentration from the 1 mmol/L level found in circulating blood to 40 to 50 µmol/L.¹² Thus, the Ca²⁺ concentrations are markedly reduced in most routine pharmacodynamic measurements of platelet aggregation inhibitors.

GP IIb-IIIa reversibly binds Ca²⁺ and other divalent cations at its five divalent cation binding sites; the affinities of these sites predict that all are occupied at physiological levels of Ca²⁺.^{13 14} Reductions in the amount of divalent cations bound to GP IIb-IIIa can dramatically affect its structure and function. For example, suspension of platelets in solutions containing ionized calcium concentrations of 40 to 50 µmol/L as achieved in citrate-anticoagulated blood can cause the removal of Ca²⁺ from GP IIb-IIIa^{15 16} and induce a loss of fibrinogen-binding activity.¹⁷ At <1 µmol/L Ca²⁺ at 37°C, GP IIb dissociates from GP IIIa within the plane of the plasma membrane,¹⁸ and both subunits lose their structure.¹⁹ Although it has been well established that the binding of adhesive proteins and peptide ligands to GP IIb-IIIa does not occur at ionized calcium concentrations <1 µmol/L, recent studies have established that peptide ligands harboring the Arg-Gly-Asp (RGD) sequence, which are also antagonists of GP IIb-IIIa, displace Mn²⁺ from GP IIb-IIIa when Mn²⁺ is used to occupy the divalent cation binding sites²⁰ and that Ca²⁺ increases the rate of dissociation of RGD ligands from GP IIb-IIIa.²¹ From this finding, D'Souza and coworkers²⁰ have suggested that divalent cations and competitive antagonists may compete for binding to GP IIb-IIIa. An extension of these observations is that partial reductions in the amount of divalent cations bound to GP IIb-IIIa may actually enhance the binding of some of the GP IIb-IIIa antagonists.²² The present study examines the effect of Ca²⁺ on the inhibitory activity of Integrilin and demonstrates that the ionized calcium concentrations achieved in blood anticoagulated with citrate markedly enhance the inhibitory activity of Integrilin. This finding indicates that pharmacodynamic measurements of the platelet aggregation inhibitory activity of Integrilin is enhanced by collection of blood samples with citrate as the anticoagulant.

	Methods

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Platelet Aggregation
PRP was prepared from blood drawn from healthy human volunteers either into sodium citrate solution (51 mL of blood into 9 mL of 3.8% sodium citrate) to make citrate-anticoagulated blood or into PPACK (60 mL blood into 126 µL of 25 mg/mL PPACK, Calbiochem-Novabiochem) for PPACK-anticoagulated blood. Platelet aggregation in PRP samples was determined with a platelet aggregometer (Chrono-log Corp) as described,²³ initiating aggregation either by the addition of ADP (20 µmol/L final concentration, Sigma Chemical Co) or TRAP (SFLLRN-NH2, 5 µmol/L final concentration, Peninsula Laboratories) to 0.50 mL of stirred PRP samples (1000 to 1200 rpm) at 37°C. Integrilin (solution for intravenous administration) was added 2 minutes before agonist. The Integrilin platelet aggregation IC₅₀ was determined from full dose-response curves. Each value is reported as the average±SD from values determined on the platelet preparations from different donors.

Purified GP IIb-IIIa–Fibrinogen Binding
The binding of purified fibrinogen to purified GP IIb-IIIa, immobilized on microtiter plates, was performed as previously described.²⁴ The indicated concentrations of Integrilin were included in each incubation. The Integrilin ligand binding IC₅₀ was calculated as the average from replicate dose-response curves.

Ca²⁺ Determinations
The ionized calcium concentration in PRP samples was determined with an ion-selective electrode (Orion 93-20) coupled to a single-junction reference electrode (Orion 90-01).¹² Standard curves were generated with the Orion calcium activity standard solution that were linear on a logarithmic scale from 10 mmol/L down to 80 µmol/L and curvilinear below this value. All readings were made after a 2-minute incubation that was required to achieve stability.

D3 Binding
Binding of the D3 antibody after Integrilin induction was determined by a modification of a published procedure.²⁵ Integrilin was added to 50-µL aliquots of PRP (platelet count previously adjusted to 250 000/µL with autologous platelet-poor plasma) and incubated for 2 minutes, followed by a 1-hour incubation with PE-conjugated D3. Binding of PE-D3 was analyzed with a Becton-Dickinson FACSCaliber. Baseline D3 binding was measured in the absence of added Integrilin; nonspecific IgG binding was determined by use of PE–mouse IgG. The Quantum Series (Flow Cytometry Standards Corp) was used to quantify fluorescence intensity. The Quick Cal (Flow Cytometry Standards Corp) program was used to standardize the linear regressions for the construction of calibration plots. From a full dose-response curve, the PE-D3 maximum mean fluorescence intensity value was determined, converted to the corresponding number of MESF, and designated 100% occupancy. For each dose of Integrilin added, the mean fluorescence intensity value was also converted to MESF units and the percent of maximum calculated and reported as percent occupancy.

PAC1 Binding
PAC1 antibody was labeled with FITC. PAC1 binding was performed in PRP samples treated with 20 µmol/L ADP.²⁶

	Results

Top Abstract Introduction Methods Results Discussion References

 
Aggregation
Fig 1 illustrates the platelet aggregation inhibitory activity of 500 nmol/L Integrilin in PRP treated with 20 µmol/L ADP from blood anticoagulated with citrate or PPACK. Although this concentration of drug resulted in complete inhibition of ADP-induced platelet aggregation in the presence of citrate, only 15% to 30% inhibition was observed in the PPACK-anticoagulated blood. To quantify the effect of anticoagulant on the inhibitory activity of Integrilin, the IC₅₀ for Integrilin inhibition of platelet aggregation was determined for each of the anticoagulants (Table). The concentration required to inhibit ADP-induced aggregation by 50% was more than 4-fold higher for plasma anticoagulated with PPACK (mean±SD, IC₅₀=570±70 nmol/L; n=6) than for plasma anticoagulated with citrate (IC₅₀=140±40 nmol/L, P<.0001; n=6). The IC₅₀ for heparin-anticoagulated PRP was similar to that of PPACK-anticoagulated PRP (data not shown). It has been observed that higher concentrations of GP IIb-IIIa antagonists are required to inhibit platelet aggregation induced by thrombin, a more potent platelet agonist.^{22 27 28} To determine whether the anticoagulants also impact the inhibitory activity of Integrilin on aggregation induced by activation of the thrombin receptor, we measured the ability of Integrilin to inhibit aggregation induced by TRAP, a synthetic peptide (SFLLRN-NH₂) that activates thrombin receptors to induce platelet aggregation in PRP without clotting fibrinogen.²⁹ Although ADP-induced aggregation required a 4-fold- higher Integrilin concentration to inhibit aggregation by 50% in PRP anticoagulated with PPACK compared with citrate (P<.0001), TRAP-induced aggregation required a 7.5-fold-higher Integrilin concentration in PPACK-anticoagulated PRP (P<.0001). TRAP-induced aggregation also required a higher Integrilin concentration for inhibition than did ADP-induced aggregation (1.4-fold for citrate, P=.095; 2.7-fold for PPACK, P=.0002).

View larger version (17K): [in this window] [in a new window]   Figure 1. Effect of Integrilin on ADP-induced platelet aggregation measured in PRP samples from blood anticoagulated with citrate or with PPACK. Aggregation was induced by addition of 20 µmol/L ADP (final concentration), as indicated by dotted line, to PRP anticoagulated with citrate or PPACK and treated with 500 nmol/L Integrilin. Control used PPACK-anticoagulated PRP in absence of added Integrilin: a similar control tracing was obtained with PPACK-anticoagulated blood (data not shown).

View this table: [in this window] [in a new window]   Table 1. Effect of Citrate and PPACK Anticoagulation on Integrilin Pharmacodynamic Measurements

Receptor Occupancy
To determine the effect of anticoagulant on Integrilin binding to GP IIb-IIIa, Integrilin induction of the D3 epitope on the GP IIb-IIIa of platelets in plasma was measured. Previous studies have established that the binding of fibrinogen, RGD-containing peptides, or Integrilin to GP IIb-IIIa induces the expression of an LIBS neoepitope on GP IIb-IIIa that can be detected by the D3 antibody.^{25 30} As shown in the Table, Integrilin induced the expression of D3 binding in PRP anticoagulated by PPACK with an IC₅₀=420±37 nmol/L (n=3) and in PRP anticoagulated by citrate with an EC₅₀=105±5 nmol/L (P=.0001, n=3). Because these data reflected the amount of Integrilin bound to GP IIb-IIIa on unstimulated platelets, these observations indicate that Integrilin has greater binding to GP IIb-IIIa in blood anticoagulated with citrate than with PPACK.

PAC1 Binding
The monoclonal antibody PAC1 has binding properties that mimic the binding of fibrinogen to GP IIb-IIIa in that (1) both require platelet activation before binding can be observed, (2) both have similar divalent cation requirements for binding, and (3) the binding of both is inhibited by similar antagonists.²⁶ The effect of anticoagulant on Integrilin inhibition of PAC1 binding was measured in an effort to determine whether enhanced Integrilin binding in citrate resulted in enhanced inhibition of ligand binding to ADP-activated platelets. The Table shows that a higher concentration of Integrilin was required to inhibit PAC1 binding to platelets activated with ADP in PRP anticoagulated with PPACK (EC₅₀=143±87 nmol/L; n=3) than with citrate (EC₅₀=31.7±9.0 nmol/L, P=.092; n=3), demonstrating that the enhanced Integrilin inhibition of aggregation in citrate is due to enhanced inhibition of ligand binding to the activated platelets.

Calcium Concentrations
Although blood contains 2.2 mmol/L total calcium, the ionized calcium concentration is approximately one half this amount, or 1.1 to 1.2 mmol/L. Although PPACK or heparin anticoagulation maintains this concentration of Ca²⁺, the Ca²⁺ concentration in citrate-anticoagulated PRP is reduced to 40 to 50 µmol/L.¹² To determine whether the difference in the inhibitory activity of Integrilin in citrate versus non–Ca²⁺-chelating anticoagulants was due to the difference in Ca²⁺ concentration or to a specific effect of the anticoagulant, the concentration of ionized calcium in PRP anticoagulated with heparin was titrated with either citrate or EDTA. Fig 2 shows that chelation of Ca²⁺ with either agent caused a similar degree of enhancement of Integrilin inhibition of ADP to induced platelet aggregation at all Ca²⁺ concentrations (EC₅₀=60 µmol/L). Because two different Ca²⁺ chelators showed a similar enhancement of the inhibitory activity of Integrilin, whereas the inhibitory activity was similar in PRP anticoagulated by two independent antithrombin strategies (eg, heparin and PPACK), these data suggest that citrate enhances the apparent inhibitory activity of Integrilin by reducing the concentration of ionized calcium and not because of a nonchelating effect of citrate.

View larger version (17K): [in this window] [in a new window]   Figure 2. Effect of calcium ion concentration on Integrilin inhibition of ADP-induced platelet aggregation. PRP samples from blood collected in heparin were incubated with various amounts of citrate or EDTA to achieve indicated concentrations of ionized calcium. IC50 for Integrilin inhibition was determined by adding various concentrations of Integrilin before induction of platelet aggregation with 20 µmol/L ADP to allow calculation of IC50 at each concentration of ionized calcium (data are representative of three trials).

Purified Protein Binding
We next determined whether the Ca²⁺ concentrations achieved in PPACK- and citrate-anticoagulated PRP affected the ability of Integrilin to inhibit direct fibrinogen–GP IIb-IIIa interactions. This was determined by measuring Integrilin inhibition of the binding of biotinylated fibrinogen to purified GP IIb-IIIa with a solid-phase binding assay that measures the binding of fibrinogen to GP IIb-IIIa immobilized on microtiter wells.²⁴ Previous studies have established that this assay, which uses purified proteins, mimics the binding of soluble fibrinogen to GP IIb-IIIa on the surface of activated platelets and correspondingly reflects the binding of inhibitors to the activated form of GP IIb-IIIa.^{7 24} As shown in Fig 3, Integrilin inhibits the binding of fibrinogen to GP IIb-IIIa in 1 mmol/L Ca²⁺ with an IC₅₀ of 63±34 nmol/L (n=6). When this same measurement is conducted in 50 µmol/L Ca²⁺, Integrilin becomes a more potent inhibitor with an IC₅₀ of 8.7±1.2 nmol/L (P=.003, n=6). Thus, reduction of the ionized calcium concentration enhances Integrilin inhibition of fibrinogen binding to GP IIb-IIIa.

View larger version (20K): [in this window] [in a new window]   Figure 3. Effect of Ca2+ concentration on Integrilin inhibition of fibrinogen binding to GP IIb-IIIa by a solid-phase microtiter assay. Biotinylated fibrinogen dissolved in buffers containing either 50 µmol/L or 1 mmol/L CaCl2 was incubated in microtiter wells coated with purified GP IIb-IIIa in the presence of the indicated concentration of Integrilin. Average values for bound fibrinogen (n=6) were detected with antibiotin antibody conjugated to alkaline phosphatase.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The present study demonstrates that a decrease in the ionized calcium from the 1 mmol/L concentration normally found in blood to <100 µmol/L, as is found in citrate-anticoagulated blood, enhances the GP IIb-IIIa binding of Integrilin and increases its apparent platelet aggregation inhibitory activity. Citrate is perhaps the most widely used anticoagulant for analysis of the pharmacodynamics of platelet aggregation inhibitors; the ionized calcium concentrations achieved with this anticoagulant (40 to 50 µmol/L) are too low to support the catalytic activities of coagulation factors yet are sufficient to support platelet aggregation. The present data indicate, however, that use of citrate-anticoagulated blood grossly overestimates the platelet aggregation inhibitory activity of Integrilin.

Citrate Effect
The present study was designed to determine the mechanism by which citrate enhances the platelet inhibitory activity of Integrilin. The IC₅₀ of 570 nmol/L for inhibition of ADP-induced platelet aggregation in PPACK-anticoagulated PRP was assumed to be indicative of the activity of Integrilin in PRP at normal plasma concentrations of Ca²⁺, because similar IC₅₀ values were observed for ADP-induced platelet aggregation in PRP anticoagulated with heparin or with hirudin (data not shown). Because chelation of divalent cations with either citrate or EDTA caused a similar enhancement of the inhibitory activity of Integrilin, it would appear that the enhanced activity in citrate was due to chelation of divalent cations and not to a direct activity of citrate on platelets or on fibrinogen. In support of this conclusion, Integrilin also showed enhanced inhibition of the binding of fibrinogen to purified GP IIb-IIIa at 50 µmol/L Ca²⁺ compared with that observed at 1 mmol/L Ca²⁺, a binding reaction observed with purified proteins in the absence of added chelator. We have also observed that Integrilin has enhanced inhibition of ADP-induced aggregation of washed platelets, with added fibrinogen suspended in the absence of added Ca²⁺ compared with the same platelet preparation suspended in the presence of 1 mmol/L Ca²⁺ (data not shown). When PAC1 binding to activated platelets was measured, enhanced Integrilin inhibition in PRP anticoagulated with citrate compared with PPACK was observed. The relevance of this observation is that PAC1 binds to GP IIb-IIIa in a manner similar to the binding of soluble fibrinogen in that both require prior activation of platelets, have a similar requirement on divalent cations, and have reduced binding in the presence of GP IIb-IIIa antagonists.^{26 31} Finally, the presence of citrate was also found to lower the EC₅₀ for Integrilin-induced expression of the D3 LIBS epitope compared with that observed with PPACK-anticoagulated PRP. For certain ligands, expression of the D3 epitope is a function of GP IIb-IIIa receptor occupancy.³² Taken together, these data establish that the effect of the reduced concentration of divalent cations caused by citrate chelation is to increase the binding of Integrilin to both unactivated and activated forms of GP IIb-IIIa and thus enhance the apparent platelet inhibitory activity of Integrilin.

Ca²⁺–GP IIb-IIIa
Five divalent cation binding sites have been observed on GP IIb-IIIa,^{13 14} and all of these are occupied when this integrin is exposed to buffers containing 1 mmol/L Ca²⁺. Four divalent cation binding sites have been attributed to sequences encoding the classic calcium-binding domains called the EF hands on GP IIb^{33 34} ; the remaining site is believed to be at the MIDAS domain on GP IIIa.^{35 36 37} Although the precise affinities of each of the sites for divalent cations have not been determined, it is known that suspension of platelets in media containing 20 to 100 µmol/L Ca²⁺ removes Ca²⁺ from GP IIb-IIIa on the platelet surface.^{15 16 38} Previous studies have established that divalent cations bound to GP IIb-IIIa are required for GP IIb-IIIa to bind fibrinogen, enabling this bivalent adhesive protein to mediate platelet aggregation.³⁹ Furthermore, a variety of experimental approaches, including cross-linking of peptide ligands to GP IIb-IIIa, inhibition of GP IIb-IIIa binding activity by peptides, fibrinogen-binding activities of GP IIb-IIIa peptides, and blocking activities of GP IIb-IIIa monoclonal antibodies, have established that the ligand binding sites on GP IIb-IIIa are in close proximity to the divalent cation binding sites on this protein.^{20 39 40} Therefore, it is somewhat paradoxical that less than saturating Ca²⁺ concentrations would actually enhance the binding and inhibitory activity of Integrilin. Insight into this issue may be found in the observations of D'Souza et al,²⁰ who showed that peptide ligands (Arg-Gly-Asp–containing peptides were used in their study) actually displaced Ca²⁺ from GP IIb-IIIa upon binding, leading these authors to postulate a "displacement hypothesis," which predicts that selected ligands and divalent cations may actually compete for overlapping sites on GP IIb-IIIa. In this capacity, Ca²⁺ increases the rate of ligand dissociation from GP IIb-IIIa.²¹ The importance of this model to the present study is that it predicts that partial removal of divalent cations from GP IIb-IIIa may enhance ligand binding.

An extension of these observations is that partial reductions in the amount of divalent cations may enhance the binding of other ligands to GP IIb-IIIa. Previous studies by Collen et al²² showed that the cyclic nonapeptide TP9201, which contains an RGD sequence, has >10-fold enhanced inhibition of either ADP- or collagen-induced aggregation in human PRP anticoagulated with citrate versus with heparin. On the basis of the observations reported here, it is possible to speculate that the enhanced activity of TP9201 may also be due to enhanced binding in the low-ionized divalent-cation environment afforded by citrate anticoagulation. In another example, Hu et al⁴¹ showed that Ca²⁺ will displace vitronectin from Vß3, an integrin that has a ß-subunit identical to that of GP IIb-IIIa and binds vitronectin at its RGD sequence. It remains to be determined whether other integrin inhibitors based on the RGD binding motif also have enhanced binding after calcium chelation.

Platelets are known to be activated by multiple agonists, including ADP, thrombin, collagen, and high shear, all of which may be involved in vivo in inducing platelet aggregation and thrombus formation. The present study shows that platelet aggregation induced by activation of the thrombin receptor required greater concentrations of Integrilin to inhibit aggregation than did aggregation induced by ADP. This was expected, because other GP IIb-IIIa antagonists have shown similar properties when platelets are activated by more potent agonists.^{22 27 28} The GP IIb-IIIa within platelet -granules is known to be surface expressed, partially with bound fibrinogen, after platelet stimulation by thrombin but not with ADP,^{42 43} and it is likely that this is the cause for increased requirements on Integrilin to effectively inhibit aggregation. Because TRAP-induced platelet aggregation showed a similar citrate enhancement on the requirements for Integrilin inhibition, we conclude that Ca²⁺ affects aggregation by the two agonists similarly.

Mechanism
The above considerations provide a mechanism for the ability of citrate and other calcium chelators (eg, EDTA) to enhance the inhibitory activity of Integrilin in blocking platelet aggregation in PRP. Citrate chelation reduces the Ca²⁺ concentration of PRP to 40 to 50 µmol/L, partially removing Ca²⁺ from the divalent cation binding sites on GP IIb-IIIa.^{16 38} Although the reduced Ca²⁺ lowers the affinity of fibrinogen for GP IIb-IIIa on the surface of activated platelets,¹⁷ sufficient binding persists to allow for platelet aggregation. Reduced Ca²⁺ simultaneously increases the binding of Integrilin, possibly because Integrilin and Ca²⁺ occupy overlapping sites on GP IIb-IIIa. The increased binding of Integrilin and the decreased binding of fibrinogen together serve to increase the inhibitory activity of Integrilin, both in blocking the binding of purified fibrinogen to purified GP IIb-IIIa in buffers containing 50 µmol/L Ca²⁺ compared with buffers containing 1 mmol/L Ca²⁺ and in blocking fibrinogen binding to platelets and platelet aggregation in PRP anticoagulated with citrate compared with PPACK.

Clinical Implications
Inherent in the analysis of the clinical activities of GP IIb-IIIa antagonists is the development of ex vivo analytical procedures to accurately reflect pharmacodynamics of this class of compounds. Such information can be used to optimize antagonist concentrations, determine differences between classes of antagonists, optimize the duration of exposure in the various clinical settings, and determine the impact of thrombosis on the clinical disease. The recently completed IMPACT II trial showed that Integrilin infusions during angioplasty caused a 40% reduction in acute abrupt closure and reduced the clinical end points of the trial at 30 days by 18%.⁴⁴ The dosing regimens of Integrilin used in IMPACT II included a bolus of 135 µg/kg followed by infusions of 0.5 or 0.75 µg · kg^-1 · min^-1 in the two treatment arms. These infusions yielded steady-state plasma concentrations of 350 to 500 nmol/L, which are expected to inhibit ADP-induced platelet aggregation by 70% to 80% in PRP samples anticoagulated in citrate: greater inhibition of aggregation was achieved immediately after the bolus (the present study and References 3 and 6). In view of the data presented here, it is expected that at normal plasma concentrations of Ca²⁺, the steady-state plasma levels of Integrilin achieved in IMPACT II would inhibit aggregation by <30% and would approach only 50% receptor occupancy. It thus appears likely that collection of blood in citrate as the anticoagulant increases the amount of Integrilin bound to GP IIb-IIIa as measured by D3 binding and enhances the apparent inhibitory activity of Integrilin over that observed when plasma levels of Ca²⁺ are maintained. Safety considerations played a major role in determining the dosing regimens in early clinical trials with Integrilin and other GP IIb-IIIa inhibitors.^{3 6} The doses studied in IMPACT II, a study with more than 4000 patients, have been shown to be quite safe. The further insights into the pharmacodynamic measurements of Integrilin provided by the present data indicate that higher Integrilin doses in patients may impart an increased therapeutic benefit. It is currently unknown whether the observed effects of citrate on the apparent potency of Integrilin will be observed with other GP IIb-IIIa antagonists. The efficacy seen in IMPACT II suggests that even at <80% receptor occupancy, antithrombotic activity can be expected in this class of compounds.

	Selected Abbreviations and Acronyms

 

GP = glycoprotein LIBS = ligand-induced binding site MESF = molecules of equivalent soluble fluorochrome PE = phycoerythrin PPACK = Phe-Pro-Arg chloromethyl ketone PRP = platelet-rich plasma TRAP = thrombin receptor agonist peptide

	Acknowledgments

 
We gratefully acknowledge Ginny Wyss and Barbara Utterbach for initiating these studies.

	Footnotes

 
Reprint requests to David R. Phillips, PhD, Principal Research Scientist, COR Therapeutics, 256 E Grand Ave, South San Francisco, CA 94080.

Received November 26, 1996; revision received March 24, 1997; accepted March 26, 1997.

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