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Cardiovascular Research 2000 47(4):819-826; doi:10.1016/S0008-6363(00)00150-4
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

Differential antiplatelet efficacy for various GPIIb/IIIa antagonists

Role of plasma calcium levels

Shaker A. Mousa*, Jeffrey M. Bozarth, Mark S. Forsythe and Andrew Slee

DuPont Pharmaceuticals Co., Wilmington, DE, USA

* Corresponding author. +1-302-695-8418; fax: +1-302-695-7407 shaker.a.mousa{at}dupontpharma.com

Received 15 February 2000; accepted 22 May 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 
Objectives: The present study was undertaken to determine the effects of free ionized calcium influenced by either the anticoagulant used (citrate vs. heparin) or directly varying the calcium levels after treatment of blood with citrate on the antiplatelet efficacy of two classes of GPIIb/IIIa antagonists. Methods: The platelet effects of changes in plasma [Ca++] with the different GPIIb/IIIa antagonists were determined using light transmittance aggregometry, direct binding kinetics, and 125I-fibrinogen binding to activated human platelets. Results: A significantly higher IC50s was shown with heparin (free ionized calcium=1.1 mM) as compared to that with citrate (free ionized calcium=0.12 mM) with class II GPIIb/IIIa antagonists (P<0.01) such as Orbofiban, and Integrilin. In contrast, class I GPIIb/IIIa antagonists such as Roxifiban and Abciximab showed no significant changes in their IC50s in either citrate or heparin. Similar data were shown with other non-calcium chelating anticoagulant such as PPACK as compared to that with heparin. Additionally, similar data were shown with regard to the [Ca++] sensitivity for GPIIb/IIIa antagonists from Class II but not Class I in the changes in IC50 values required for the inhibition of 125I-fibrinogen binding to activated human gel filtered platelets. Additionally, examples from Class I GPIIb/IIIa antagonists such as 3H-active form of Roxifiban showed no significant changes in its platelet binding affinity in response to change in [Ca++]. In contrast, GPIIb/IIIa antagonists from class II such as 3H-active form of Orbofiban demonstrated significant changes (P<0.01) in its platelet binding kinetics and antiplatelet efficacy in response to changes in Ca++ concentrations. Conclusions: These data suggest the impact of the method of blood collection or changes in plasma calcium levels on the antiplatelet efficacy for class II but not class I GPIIb/IIIa antagonists depending on their platelet binding kinetics.

KEYWORDS GPIIb/IIIa, Glycoprotein IIb/IIIa; PRP, platelet-rich plasma; PPP, platelet poor plasma; Ca++, Calcium; ADP, Adenosine diphosphate; TRAP, PRP, Thrombin receptor activating peptide; obtained from citrated blood; cPRP, PRP, obtained from heparinized blood; hPRP; XV459=free acid form of Roxifiban; YZ202=free acid form of Orbofiban


   1 Introduction
Top
 Abstract
 1 Introduction
2 Materials and methods
 3 Results
 4 Discussion
 References
 
Platelet-fibrinogen interaction is a key step in the pathogenesis of coronary artery thrombosis [1–4]. The clinical benefit of aspirin and the more dramatic antithrombotic effect of intravenous antagonists of the platelet surface GPIIb/IIIa receptor underscore the importance of platelet involvement in acute coronary ischemia [5–7]. Platelet GPIIb/IIIa blockade with c7E3 (ReoPro) reduces myocardial infarction and death associated with unstable angina and percutaneous transluminal coronary angioplasty [8,9]. Such therapy, however, may be associated with a risk of hemorrhagic complications. There is considerable person-to-person variability in the number of GPIIb/IIIa receptors and its ligand binding function; in patients with coronary artery disease enhanced platelet GPIIb/IIIa receptor expression may be a marker for increased thrombotic risk. Furthermore, variable inhibition levels of GPIIb/IIIa function may occur after administration of the various GPIIb/IIIa antagonists and more so with the oral form of GPIIb/IIIa antagonists. Light transmittance platelet aggregometry of cPRP obtained from citrated whole blood has been used as a conventional method to measure the degree of ex vivo platelet aggregation inhibition in early clinical studies. Platelet GPIIb/IIIa integrin is known to form a Ca++-dependent functional heterodimer. It was established that both GPIIb and GPIIIa in solution have low-affinity Ca++ binding sites, five in GPIIb and two in GPIIIa [10].

Ex vivo platelet aggregation studies of GPIIb/IIIa antagonists administered either intravenously or orally is intended to reflect potential in vivo effects. Citrated platelet-rich plasma (cPRP) aggregation using light transmittance aggregometry is most commonly used method for assessing pharmacodynamic effects of the various GPIIb/IIIa antagonists in clinical trials. However, the anticoagulant citrate, which chelates calcium, enhanced the potency of certain GPIIb/IIIa antagonists, such as Integrilin (Eptifibatide). This study was undertaken to characterize the antiplatelet (anti-aggregatory) efficacy in citrated, which lower free ionized plasma calcium levels, vs. heparinized blood, which do not affect free ionized plasma calcium concentrations, for various GPIIb/IIIa antagonists having different platelet GPIIb/IIIa binding kinetics. Those GPIIb/IIIa antagonists can be classified based on their platelet binding kinetics into two classes. Class I include GPIIb/IIIa antagonists with relatively high and comparable affinity for both resting and activated platelets and slow platelet dissociation rates. In contrast, class II GPIIb/IIIa antagonists include compounds with relatively lower affinity for resting platelets and fast platelet dissociation rates. Examples of class I include Roxifiban (XV459), DMP 802, XV454 (non-peptide) plus others and class II include Integrilin (cyclic peptide), Orbofiban, Sibrafiban and Tirofiban plus others.

The platelet effects of different GPIIb/IIIa antagonists in response to changes in plasma [Ca++] were determined using light transmittance aggregometry, direct platelet binding kinetics, and 125I-fibrinogen binding to activated human platelets.


   2 Materials and methods
Top
 Abstract
 1 Introduction
 2 Materials and methods
3 Results
 4 Discussion
 References
 
2.1 Reagents
Adenosine 5'-diphosphate (ADP) and other reagents used but not specifically mentioned were obtained from Sigma Chemical Company (St. Louis, MO). Arachidonic acid was purchased from Nu Check prep (Elusian, MN). Thrombin Receptor Agonist Peptide (TRAP) was purchased from Peninsula Laboratories Inc. (Belmont, CA). 125I-Fibrinogen and other radiolabeled GPIIb/IIIa antagonists were obtained from DuPont NEN (Boston, MA). Chimeric 7E3 (c7E3, Abciximab) and 125 I-Abciximab were obtained from Centocor (Malvern, PA). Roxifiban (DMP 754) and its free acid form, XV459 and other GPIIb/IIIa antagonists were synthesized at DuPont Pharmaceuticals Co. (Wilmington, DE). The active free acid form of Roxifiban and Orbofiban were used in all of the in vitro studies described in this text. Radiolabel GPIIb/IIIa antagonists were synthesized at DuPont Pharmaceuticals Co. (Wilmington, DE). A list of 10 GPIIb/IIIa antagonists which are analogs of Roxifiban and DMP802 (class I) and 22 different GPIIb/IIIa antagonists (class II) such as Orbofiban, Sibrafiban, and others were also synthesized at DuPont Pharmaceuticals Co. (Wilmington, DE). See Table 1 for selected examples from class I & class II GPIIb/IIIa antagonists.


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  		Table 1 Platelet dissociation rate for different and selected radiolabeled GPIIb/IIIa antagonists from human plateletsa

 
2.2 Platelet GPIIb/IIIa binding profiles
2.2.1 Platelet binding affinity to activated and resting human platelets
This assay was used to determine a compound's saturable binding to platelets using PRP. Either citrated whole blood (5 ml draw, Vacutainer tubes containing 3.2% sodium citrate) or heparinized whole blood (5 ml draw, Vacutainer tubes containing IU/ml heparin) was collected from healthy, aspirin free, human subjects. Blood was centrifuged for 10 min at 150xg at 22°C using Sorvall RT6000 tabletop Centrifuge (DuPont, Wilmington, DE). PRP was removed, pooled, and platelets were counted using a Coulter T540 Hematology Analyzer. Forty microliters of 3H-active form of Roxifiban (77.5 ci/mMol), 3H-XV454 (23.3 ci/mMol), 3H-DMP802 (25 ci/mMol), 3H-DMP728 (24.3 ci/mMol), 3H-active form of Orbofiban (23.5 ci/mMol) or 125I-Abciximab were added to assay tubes, followed by 50 µl of PRP (1.5x107 platelets). Samples were incubated for 10 min at 22°C. For platelet activation, 100 µl of ADP (10 µM final concentration) was added to all samples followed by incubation for 10 min at 22°C (pH of 7.5). Platelets were harvested through Whatman 934AH GFB filters that had been presoaked (30 min) in 0.2% Polyethylenimine. Filters were washed quickly three times with 5 ml of ice cold saline, removed, and placed into scintillation vials. Six milliliters of DuPont NEN formula 989 per vial was added, vials were allowed to stand for 60 min, shaken, and counted using a liquid scintillation counter. Equilibrium and specific binding affinity for the different radiolabeled GPIIb/IIIa antagonists to activated and resting human platelets was calculated using Scatchard plot. Non-specific binding (about 5–10% of total binding) of radiolabel GPIIb/IIIa antagonists were determined in the presence of 10 mM EDTA.

2.2.2 Dissociation rates
Citrated whole blood (5 ml draw in Vacutainer tubes, 3.2% sodium citrate) was collected from healthy, aspirin-free, human subjects. Blood samples were divided to be used as whole blood or to be centrifuged for 10 min (150xg), the resulting PRP removed, and platelet counts determined to normalize the radiolabeled platelets. Designated individual tubes of whole blood were treated for 60 min, with 0.04 µM of 3H-active form of Roxifiban. Following this 60-min incubation period, the tubes were centrifuged for 10 min (150xg). The resulting 3H-radioligand/PRP was carefully removed and centrifuged an additional 10 min (~250xg). The resulting PPP was removed and the platelet pellet re-suspended (~1.6x108/ml) in fresh PPP. Five-hundred microliter of this suspension was transferred to wells of a 24-well plate (blocked with 5% BSA). To initiate dissociation, 100 µM non-radiolabeled ligand was added to the wells. At designated time points (0, 2, 15, 30, 60, 90, 120 min), the 3H or 125I – bound GPIIb/IIIa antagonists to human resting platelets (PRP) was removed from the wells and centrifuged for 2 min (10,000xg). The resulting platelet pellet was counted using a liquid scintillation counter. CPMs recovered are compared to the Control (t=0) and presented as percent bound per 0.8x108 platelets. Dissociation rate from human resting platelets was carried out for the different intervals for the determination of the t 1/2 (min.) for the dissociation of platelet bound – radiolabeled ligand.

2.3 Antiplatelet efficacy
2.3.1 Platelet aggregation (light transmittance)
Agonist-induced platelet aggregation was measured as change in% light transmission of either citrated (cPRP) or heparinized (hPRP) at platelet count of 2x105 per µl. For studying the effect of GPIIb/IIIa antagonists on platelet aggregation, increasing concentrations were added to PRP for 5 min after which 10 µM TRAP (Peninsula Labs, Belmont, CA) or ADP were added. The aggregation response was measured as the maximum response of the increase in light transmission induced by TRAP or ADP, using PPP to establish 100% light transmission. PRP (200 ul of 2 x108 platelets/ml) were added to each micro test tube, and transmittance was set to 0%. Twenty microliters of the platelet agonist, ADP or TRAP (10 µM final concentration) was added to each tube, and the aggregation profiles were plotted (% transmittance vs. time). Twenty microliters of the active form of Roxifiban or other GPIIb/IIIa antagonists were added at different concentrations for 8 min prior to the addition of ADP or TRAP at 10 µM. Results were expressed as mean IC50± SEM (µM).

2.4 Platelet 125I-fibrinogen binding assay
Human PRP was applied to a sepharose column to prepare gel filtered platelets (GFP) as previously described [11]. Aliquots of GFP (2x108 platelets/ml) along with 1 mM calcium chloride with or without the test agent were added to removable 96-well plates. 125I-fibrinogen (26.5 µCi/mg) was added for 10 min, and the h-GFP was activated by addition of ADP at 10 µM for another 10 min. The 125I-fibrinogen bound to the activated platelet was separated from the free form by centrifugation, and then counted on a gamma counter. Non-specific binding (due to entrapment of 125I-fibrinogen) either in the presence or absence of the inhibitors was shown (in the absence of agonist) to be in the range of 4–6% of total 125I-fibrinogen binding to agonist-activated platelets. Percent inhibition of 125I-fibrinogen binding to activated platelets was calculated by dividing the specific binding in the presence by that of the absence of the inhibitors. For IC50 determination, class I antagonists such as Roxifiban and class II such as Orbofiban or Integrilin were added at various concentrations prior to platelet activation at low calcium level (0.01 mM) and upon the addition of different calcium levels up to normal plasma calcium level (1.0 mM).

2.5 Statistical analysis
The data are expressed as mean±standard error of the mean, unless otherwise specified. Statistical significance of differences between means was determined by single-factor analysis of variance (ANOVA). If means were shown to be significantly different, multiple comparisons by pairs were performed by Tukey test. Probability values<0.05 were selected to indicate statistical significance.


   3 Results
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
4 Discussion
 References
 
3.1 Platelet GPIIb/IIIa binding profiles
3.1.1 Platelet binding affinity to activated vs. resting human platelets
Binding Affinity to Activated vs. Resting Platelets: The active form of Roxifiban binds with high affinity to resting and activated human platelets with a Kd=2.52±0.98, 0.80±0.16 nmol/l, respectively. Both DMP 802 and XV454 demonstrated high affinity to either activated or resting human platelets with a Kd of 0.2–0.50 nmol/l. Similarly, Abciximab demonstrated a comparable affinity for both resting and activated human platelet with a Kd of 9.1±0.5 and 9.5±0.6, respectively along with a slow dissociation rate (t1/2=40 min) (Table 1). In contrast, the active form of Orbofiban binds with relatively lower affinity to activated human platelets (Kd=52 nmol/l) and to resting human platelets (Kd=427 nmol/l).

3.1.2 Platelet dissociation rates
Class II GPIIb/IIIa antagonists such as Integrilin, Tirofiban, Sibrafiban and Orbofiban showed a relatively faster dissociation rates (t1/2 of 5–15 s) from resting human platelets. In contrast, class I GPIIb/IIIa antagonists such as Roxifiban demonstrated a relatively slow dissociation rate (t1/2=8±1 min) from resting human platelets (Table 1).

3.1.3 Effects of calcium levels on the binding kinetics of GPIIb/IIIa antagonists
The binding equilibrium affinity (Kd) of GPIIb/IIIa antagonists from class I such as Roxifiban demonstrated no significant calcium sensitivity in its binding to either resting or activated human platelets (Fig. 1A). In contrast, the platelet binding affinity of antagonists from class II such as Orbofiban demonstrated calcium sensitivity in its binding to either resting or activated human platelets (Fig. 1B).


Figure 1
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  		Fig. 1 Effects of calcium on the binding of 3H-XV459 (Roxifiban) (A) and 3H-YZ202 (Orbofiban) (B) to resting vs. activated human platelets. A lack of effects of calcium on the binding of 3H-Roxifiban to either activated or resting human platelets was demonstrated. In contrast, a two-fold shift in Kd for the binding of 3H-YZ202 was demonstrated when decreasing calcium levels from 1.0 to 0.01 mM. Data represent mean±SD of platelet bound 3H-GPIIb/IIIa antagonists, n=3–4.

 
3.2 Antiplatelet efficacy
3.2.1 Platelet aggregation inhibitory efficacy in citrate vs. heparin
The effects of collecting blood in citrate vs. heparin on the antiplatelet efficacy of 10 different GPIIb/IIIa antagonists (Class I titrators) with high affinity to resting and activated platelets and relatively slow dissociation rates were examined. The same was performed for 22 different GPIIb/IIIa antagonists (Class II non-titrators) with relatively low affinity to resting platelets and faster platelet dissociation rates. Antiplatelet efficacy (IC50) was determined for each compound in citrated platelet rich plasma (cPRP) vs. heparinized PRP (hPRP) obtained from four to five different human subjects using light transmittance aggregometry and ADP or TRAP at 10 uM each. The correlation between the IC50s for the various GPIIb/IIIa antagonists was compared. Data demonstrated a linear correlation between cPRP and hPRP with a slope of 1.1–1.2 (not statistically significantly different) for class I GPIIb/IIIa antagonists (Roxifiban, Abciximab, DMP 802, XV454, and others) see Figs. 2 and 3Go. In contrasts, a slope of 1.9–2.6 in cPRP vs. hPRP (P<0.01) was determined for class II GPIIb/IIIa antagonists (Integrilin, Orbofiban and others) when using ADP or TRAP (Figs. 2 and 3Go).


Figure 2
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  		Fig. 2 Correlation between the platelet aggregation inhibitory efficacy (IC50s) induced by 10 uM ADP for Class I GPIIb/IIIa antagonists’ (A) vs. Class II GPIIb/IIIa antagonists’ (B) in cPRP vs. hPRP. An excellent correlation (r2=0.88–0.87) in the antiplatelet efficacy with a slope of 1.2 reflecting a lack of IC50 differences in cPRP vs. hPRP for Class I GPIIb/IIIa antagonists. In contrast, a significant shift in IC50s (slope=1.9, P<0.01) was demonstrated with Class II GPIIb/IIIa antagonists when switching from citrate to heparin. Class I GPIIb/IIIa antagonists represent compounds having high affinity for activated and resting platelets along with a relatively slow platelet dissociation rates vs. Class II GPIIb/IIIa antagonists which represent compounds with relatively low affinity for resting platelets and faster platelet dissociation rates.

 

Figure 3
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  		Fig. 3 Correlation between the platelet aggregation inhibitory efficacy (IC50s) induced by 10 uM TRAP for Class I (A) vs. Class II GPIIb/IIIa antagonists’ (B) in cPRP vs. hPRP. An excellent correlation (r2=0.99–0.95) in the antiplatelet efficacy with a slope of 1.2 reflecting a lack of IC50 differences in cPRP vs. hPRP for Class I GPIIb/IIIa antagonists. Class I GPIIb/IIIa antagonists (DMP754 analogs) represent compounds having high affinity for activated and resting platelets along with a relatively slow platelet dissociation rates vs. Class II GPIIb/IIIa antagonists which represent compounds with relatively low affinity for resting platelets and faster platelet dissociation rates. In contrast, a significant shift in IC50s (slope=2.6, P<0.01) was demonstrated with Class II GPIIb/IIIa antagonists when switching from citrate to heparin.

 
The effects of citrate vs. heparin on the antiplatelet efficacy of the GPIIb/IIIa antagonists Roxifiban (Class I) vs. Integrilin, Orbofiban or Sibrafiban (Class II) demonstrated a differential sensitivity to changes in free ionized plasma calcium concentrations as a result of the calcium chelation by the anticoagulant citrate (Table 2). Class II but not class I GPIIb/IIIa antagonists demonstrated significant calcium sensitivity in their antiplatelet efficacy with a 1.6–2.9 fold shift.


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  		Table 2 Antiplatelet efficacy of roxifiban (class I) vs. orbofiban, sibrafiban, and integrilin (class II) GPIIb/IIIa antagonists in inhibiting human platelet aggregation in citrate vs. heparina

 
3.2.2 Calcium and efficacy of GPIIb/IIIa antagonists in inhibiting 125I-fibrinogen binding
Citrated blood resulted in the decrease in plasma [Ca++] from 1.12 mM to 0.11 mM. Gel filtered human platelets (GFP) has a residual [Ca++] of 0.01 mM. The effect of adding different [Ca++] on the inhibitory efficacy of Roxifiban, Integrilin and Orbofiban was determined. The effects of different levels of calcium on the antiplatelet efficacy of the GPIIb/IIIa antagonists Roxifiban (Class I) vs. Integrilin or Orbofiban (Class II) in inhibiting 125I-fibrinogen binding to ADP activated human gel purified platelets demonstrated a differential sensitivity to changes in calcium concentrations (Figs. 4 and 5Go). The inhibitory efficacy of Roxifiban was not affected (Fig. 4A). In contrast, the inhibitory efficacy of either Integrilin (Fig. 4B) or Orbofiban (Fig. 4C) was significantly (P<0.01) affected by the change from a lower to normal plasma [Ca++] levels of about 0.1 to 1.0 mM.


Figure 4
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  		Fig. 4 Effects of calcium concentrations on the inhibitory efficacy of XV459 (Roxifiban) (A) vs. Integrilin (B) or YZ202 (Orbofiban) (C) in inhibiting 125 I-fibrinogen binding to ADP activated human gel filtered platelets. A significant shift in the Integrilin (P<0.001) or YZ202 (P<0.01) curves in the presence of 1.0 mM as compared to 0.1 mM calcium was demonstrated. In contrast, a lack of any significant effects of calcium on Roxifiban dose-response was shown. Data represent mean±SD of % inhibition, n=3.

 

Figure 5
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  		Fig. 5 Effects of calcium concentrations on the inhibitory efficacy of Roxifiban vs. Integrilin in the IC50 (uM) for the inhibition of 125 I-fibrinogen binding to ADP (20 uM) activated human gel filtered platelets. A significant (*, P<0.01) shift of about 3 fold in the IC50 for Integrilin in the presence of 1.0 mM as compared to 0.1 mM calcium was demonstrated. In contrast, a lack of any significant effects of calcium (0.01–1.0 mM) on Roxifiban dose-response was shown. Data represent mean±SD of % inhibition, n=3.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
References
 
New targets for antiplatelet therapy have been identified based on a better understanding of the different processes involved in arterial thrombosis. Once endothelial damage occurs, platelet thrombus formation advances in three steps including platelet adhesion, platelet activation, by the various activators from damaged endothelium and from within activated platelets. Various large-scale phase III clinical trials have illustrated the usefulness of intravenous GPIIb/IIIa antagonists in acute myocardial ischemic syndromes [12–14]. The first of such study was the pivotal EPIC trial that enrolled over 2,000 high-risk patients scheduled to undergo coronary intervention. Additionally, other small molecule selective GPIIb/IIIa antagonists, including Integrilin and Tirofiban (AggrastatR) further confirmed the usefulness of GPIIb/IIIa blockade in the treatment and prevention of acute ischemic heart diseases [15,16].

Preclinical and early clinical evaluation of GPIIb/IIIa antagonists relies very much on ex vivo platelet aggregation inhibition assays for dose-findings. For ex vivo platelet aggregation studies, sodium citrate has been the conventional anticoagulant for the collection of whole blood. Unfortunately, the chelation of calcium from PRP by the citrate may result in weakening the GPIIb/IIIa complex, which is a calcium dependent association of the GPIIb and the IIIa subunits. Under such conditions, ex vivo platelet aggregation measurements might result in an artificially higher level of inhibition than what can be achieved in vivo. Intracellular calcium influences the platelet shape change, exposure of GPIIb/IIIa complex, and fibrinogen binding [17]. Additionally, the binding of calcium ions to the four repetitive domains on the GPIIb subunit plays a key role in the stability of the GPIIb/IIIa complex [18]. Earlier clinical trials (IMPACT & IMPACT II) with Integrilin revealed a significant effect of plasma calcium levels on its antiplatelet efficacy [19,20]. The influence of plasma calcium levels as it is affected by the type of anticoagulant used (citrate vs. heparin) on the true in vivo antiplatelet efficacy is a critical factor in determining therapeutic dose regimens with the different GPIIb/IIIa antagonists. Additionally, recent in vivo antithrombotic studies with limited examples of GPIIb/IIIa antagonists revealed a correlation of in vivo antithrombotic efficacy with Formula platelet aggregation inhibition in hPRP vs. cPRP [21,22].

Clinical studies with orally active GPIIb/IIIa antagonists including Xemilofiban, Orbofiban, Sibrafiban, Lotrafiban and LeFradafiban demonstrated oral antiplatelet activity in man upon their administration 2–3 times per day as determined in cPRP using light transmittance aggregometry [23–27]. The active form of Roxifiban have distinct platelet GPIIb/IIIa binding characteristics along with a potent Formula antiplatelet efficacy regardless of the agonist or the anticoagulant used for blood collection (citrate vs. heparin) [28,29].

Class I GPIIb/IIIa antagonists such as Roxifiban equilibrate and titrate to greater extent with human platelets (i.e. its platelet/plasma distribution is ~10/1). Hence its antiplatelet efficacy is not totally dependent on plasma levels. In contrast, class II GPIIb/IIIa antagonists exist in equilibrium between platelet and plasma compartments (i.e. its platelet/plasma ratio of distribution is ~1/1) and its antiplatelet efficacy depend on plasma levels [30].

Recent clinical experiences with oral platelet GPIIb/IIIa antagonists such as Xemilofiban (EXCITE), Orbofiban (OPUS), and Sibrafiban (SYMPHONY) were disappointing [31]. The lack of clinical benefit for the oral agents (Xemilofiban, Orbofiban, and Sibrafiban) as compared to the well documented benefit with the intravenous agents (Abciximab, Integrilin, and Tirofiban) is puzzling. A number of explanations for the discrepancy between the intravenous and oral agents have been suggested [32]. A likely explanation is the lack of a significant and sustained in vivo platelet GPIIb/IIIa blockade due to the overestimation of the antiplatelet efficacy ex vivo as determined in cPRP by about 1.5–3 fold for class II type antagonists. This could be the case especially given the steep dose-response relationship and the low oral bioavilability of these agents.

It appears from this investigation that the dissociation rate (Koff) from human platelets to be the major determining factor affecting the antiplatelet efficacy of a GPIIb/IIIa antagonist in response to changes in plasma [Ca++] levels. Antiplatelet efficacy of GPIIb/IIIa antagonists with a relatively slow platelet dissociation rate (Class I) is not affected by changes in plasma [Ca++] levels. In contrast, antiplatelet efficacy of GPIIb/IIIa antagonists with relatively fast platelet dissociation rate (Class II) is significantly affected by changes in plasma [Ca++] levels.

In conclusion, a comparable inhibitory efficacy for GPIIb/IIIa (class I) antagonists with high affinity for resting platelets and relatively slow dissociation rate (Roxifiban, XV454, and DMP 802, and others) in inhibiting platelet aggregation in either cPRP or hPRP was demonstrated. In contrast, GPIIb/IIIa (class II) antagonists with relatively lower affinity for resting human platelets along with relatively fast platelet dissociation rate such as Integrilin, Orbofiban, Sibrafiban, Tirofiban and others have significantly lower efficacy in inhibiting platelet aggregation in hPRP (non-calcium chelating anticoagulant) as compared to that in cPRP. These data suggest the impact of the method of blood collection on antiplatelet efficacy for some but not all GPIIb/IIIa antagonists depending on their platelet binding kinetics. This might lead to incomplete in vivo antiplatelet efficacy for various GPIIb/IIIa antagonists. Based on this investigation, it is recommended to use non-calcium chelating anticoagulants such as PPACK or hirudin in order to study the true antiplatelet efficacy of GPIIb/IIIa antagonists from class II. Additionally, a whole blood platelet functional assay using the above conditions should allow for a more relevant bedside platelet monitoring.

This data suggest a major differences between different platelet GPIIb-IIIa antagonists which might have major impacts on their clinical efficacy in different thromboembolic disorders.

Time for primary review 28 days.


   References
Top
 Abstract
 1 Introduction
 2 Materials and methods
 3 Results
 4 Discussion
 References
 

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