The role of branching glycan of human alpha1-acid glycoprotein in enantioselective binding to basic drugs as studied by capillary electrophoresis

The role of the branching glycan structure of human alpha1-acid glycoprotein (AGP) in the interaction with basic drugs was investigated in terms of enantioselectivity in binding ability. AGP was separated by concanavalin A lectin affinity chromatography into two subfractions, the unretained AGP (UR-AGP) which has no biantennary glycan chain and the retained AGP (R-AGP) which possesses biantennary oligosaccharide chain(s). The unbound concentrations of propranolol (PRO) enantiomers and verapamil (VER) enantiomers in UR-AGP solution and R-AGP solution were determined by high-performance frontal analysis combined with capillary electrophoresis. It was found that (S)-PRO is bound to UR-AGP and R-AGP more strongly than (R)-PRO, whereas the reverse applies to VER enantiomers, and that such enantioselectivity is common to these proteins. This suggests that the branching type of glycan chains of AGP does not play significant role in the chiral recognition in binding these basic drugs.     

普萘洛尔对映异构体诱导HUVEC细胞的蛋白质表达谱差异

手性药物只能通过严格的手性识别才能选择性地与特定生物大分子相互作用,在药动学、药效学等方面上表现出手性特征.以非选择性β肾上腺素能受体阻滞剂普萘洛尔（PRO）的对映异构体R(+)/S(-)-PRO为模型药物,分别作用于人脐静脉内皮细胞（HUVEC）,提取全细胞蛋白质,经双向电泳、MALDI-TOF-MS、SWISSPROT数据库分析鉴定差异表达蛋白质;共筛选出22个差异表达蛋白质点,鉴定了HSP86、HSP84、GRP75、KLC18、KBTB2、TGM2、GBLP、GCNT2、RAB36、KLH34等10种蛋白质.研究表明,PRO对映异构体可引起广泛的基因表达改变,涉及信号分子、代谢酶、骨架蛋白、伴侣蛋白等,且具有显著的手性特征,这可能与PRO显著的手性生物学特征有紧密联系,但仍需开展进一步深入研究,以明确产生PRO手性生物学特征的多种途径和机制.蛋白质组学技术为深入了解药物的手性生物学特征及其作用机制提供了新的思路和策略,对手性药物开发和临床合理用药有着重要的意义.     

Strategy Approach for Direct Enantioseparation of Hyoscyamine Sulfate and Zopiclone on a Chiral αl‐Acid Glycoprotein Column and Determination of Their Eutomers: Thermodynamic Study of Complexation

Rapid and simple isocratic high‐performance liquid chromatographic methods with UV detection were developed and validated for the direct resolution of racemic mixtures of hyoscyamine sulfate and zopiclone. The method involved the use of α_l‐acid glycoprotein (AGP) as chiral stationary phase. The stereochemical separation factor (?) and the stereochemical resolution factor (R_s) obtained were 1.29 and 1.60 for hyoscyamine sulfate and 1.47 and 2.45 for zopiclone, respectively. The method was used for determination of chiral switching (eutomer) isomers: S‐hyoscyamine sulfate and eszopiclone. Several mobile phase parameters were investigated for controlling enantioselective retention and resolution on the chiral AGP column. The influence of mobile phase, concentration and type of uncharged organic modifier, ionic strength, and column temperature on enantioselectivity were studied. Calibration curves were linear in the ranges of 1–10 µg mL^‐1 and 0.5–5 µg mL^‐1 for S‐hyoscyamine sulfate and eszopiclone, respectively. The method is specific and sensitive, with lower limits of detection and quantifications of 0.156, 0.515 and 0.106, 0.349 for S‐hyoscyamine sulfate and eszopiclone, respectively. The method was used to identify quantitatively the enantiomers profile of the racemic mixtures of the studied drugs in their pharmaceutical preparations. Thermodynamic studies were performed to calculate the enthalpic ΔH and entropic ΔS terms. The results showed that enantiomer separation of the studied drugs were an enthalpic process. Chirality 28:49–57, 2016. © 2015 Wiley Periodicals, Inc.     

Mechanisms of retention of pyrrolidinyl norephedrine on immobilized alpha(1)-acid glycoprotein

The HPLC separation of the R,S and S,R enantiomers of pyrrolidinyl norephedrine on immobilized alpha-1 glycoprotein (AGP) was investigated. Conditions for the separation were varied using a premixed mobile phase containing an ammonium phosphate buffer and an organic modifier. The influence of mobile phase pH, ionic strength, organic modifier composition, modifier type, and temperature on the chiral selectivity and retention were investigated. The presented data demonstrate that independent phenomena govern the enantioselectivity and retention. Retention is a function of both ion exchange equilibria and hydrophobic adsorption. Thermodynamic data derived from van't Hoff plots illustrates that while enantioselectivity is also enthalpically driven, the magnitude of the enthalpy term is governed by pH. Enantioselectivity has little dependence on ionic strength. Hydrophobic interactions appear to foster hydrogen bonding interactions; the two appear to be mutually responsible for chiral selectivity. The chiral selectivity decreases as the pH is decreased and increases with mobile phase buffer strength.     

Improved chromatographic performance of a modified human albumin based stationary phase

Derivatization of the free cys₃₄ in human serum albumin (HSA) anchored to a silica matrix has been performed by in situ reaction with ethacrynic acid. This modification, which is reported to occur under physiological conditions, gives rise in practice to a new column with different binding properties with respect to the column based on the native protein. Significant differences were observed in the binding of drugs known to bind to site I, (R)-(S)-warfarin and phenylbutazone, and to site II, 1,4-benzodiazepin-2-ones and nonsteroidal anti-inflammatory agents. In particular, the chromatographic retentions markedly decreased for most of the drugs, and, in the case of chiral compounds, significant differences were often observed in the behavior of the two enantiomers, with higher values of enantioselectivity obtained for some of the examined compounds. Furthermore, the noncovalent binding of ethacrynic acid to the protein modifies the binding properties of the albumin. Chirality 9:335–340, 1997. © 1997 Wiley-Liss, Inc.     

Plasma protein binding study of oxybutynin by high-performance frontal analysis

Plasma protein binding of oxybutynin (OXY) was investigated quantitatively and enantioselectively using high-performance frontal analysis (HPFA). An on-line HPLC system which consists of HPFA column, extraction column and analytical column was developed to determine the unbound concentrations of OXY enantiomers in human plasma, in human serum albumin (HSA) solutions, and in human alpha1-acid glycoprotein (AGP) solutions. OXY is bound in human plasma strongly and enantioselectively. The bound drug fraction in human plasma containing 2-10 microM (R)- or (S)-OXY was higher than 99%, and the unbound fraction of (R)-OXY was 1.56 times higher than that of (S)-isomer. AGP plays the dominant role in this strong and enantioselective plasma protein binding. The total binding affinities (nK) of (R)- and (S)-OXY to AGP were 6.86 x 10(6) and 1.53 x 10(7) M(-1), respectively, while the nK values of (R)- and (S)-OXY to HSA were 2.64 x 10(4) and 2.19 x 10(-4) M(-1), respectively. The binding affinity of OXY to AGP is much higher than that to HSA, and shows high enantioselectivity (SIR ratio of nK values is 2.2). It was found that both enantiomers are bound competitively at the same binding site on an AGP molecule. The binding property between OXY and low density lipoprotein (LDL) was investigated by using the frontal analysis method incorporated in high-performance capillary electrophoresis (HPCE/FA). It was found the binding is non-saturable and non-enantioselective.     

Selective binding of coumarin enantiomers to human alpha1-acid glycoprotein genetic variants

Coumarin-type anticoagulants, warfarin, phenprocoumon and acenocoumarol, were tested for their stereoselective binding to the human orosomucoid (ORM; AGP) genetic variants ORM 1 and ORM 2. Direct binding studies with racemic ligands were carried out by the ultrafiltration method; the concentrations of free enantiomers were determined by capillary electrophoresis. The binding of pure enantiomers was investigated with quinaldine red fluorescence displacement measurements. Our results demonstrated that all investigated compounds bind stronger to ORM 1 variant than to ORM 2. ORM 1 and human native AGP preferred the binding of (S)-enantiomers of warfarin and acenocoumarol, while no enantioselectivity was observed in phenprocoumon binding. Acenocoumarol possessed the highest enantioselectivity in AGP binding due to the weak binding of its (R)-enantiomer. Furthermore, a new homology model of AGP was built and the models of ORM 1 and ORM 2 suggested that difference in binding to AGP genetic variants is caused by steric factors.     

Measurement of the (R)- and (S)-isomers of warfarin in patients undergoing anticoagulant therapy.

An achiral/chiral high-performance liquid chromatographic system for the analysis of total warfarin together with the (R)- and (S)-enantiomers in clinical samples has been developed. The achiral analysis is achieved using a C8 column, which is coupled to a chiral stationary phase, alpha 1-acid glycoprotein (AGP), thereby allowing for analysis of warfarin isomers without interfering serum peaks. A 0.015 M phosphate buffer mobile phase with 15% v/v propan-2-ol (pH 7.0) was used on the C8/AGP system. UV analysis at 308 nm was used for quantitation of total warfarin on the C8 column and fluorescence (excitation 300 nm, emission 390 nm) detection was employed for isomer quantitation on the AGP. Retention time of total warfarin on the C8 column was 5.95 min, while that of the (S)- and (R)-warfarin on the AGP column was 10.38 and 12.69 min, respectively. Peak resolution of the warfarin isomers was 1.64. All serum samples were subjected to solid-phase extraction. Data from two patients in a single dose study indicate that a two-compartmental model could represent the warfarin concentration-time data with enterohepatic circulation. In some patients studied during steady state therapy, concentrations of (S)-warfarin were greater than (R)-warfarin indicating that the clearance of the former is slower in these patients.     

Bi-directional chiral inversion of ketoprofen in CD-1 mice

The R enantiomers of some of the 2-arylpropionic acid non-steroidal antiinflammatory drugs (NSAIDs) are known to undergo metabolic chiral inversion to their more pharmacologically active antipodes. This process is drug and species dependent and usually unidirectional. The S to R chiral inversion, on the other hand, is rare and has been observed, in substantial extents, only for ibuprofen in guinea pigs and 2-phenylpropionic acid in dogs. After i.p. administration of single doses of racemic ketoprofen or its optically pure enantiomers to male CD-1 mice and subsequent study of the concentration time-course of the enantiomers, we noticed substantial chiral inversion in both directions. Following racemic doses, no stereoselectivity in the plasma-concentration time courses was observed. After dosing with optically pure enantiomer, the concentration of the administered enantiomer predominated during the absorption phase. During the terminal elimination phase, however, the enantiomers had the same concentrations. Our observation is suggestive of a rapid and reversible chiral inversion for ketoprofen enantiomers in mice. Chirality 9:29–31, 1997. © 1997 Wiley-Liss, Inc.     

Enhanced expression of α1-acid glycoprotein and fucosylation in hepatitis B patients provides an insight into pathogenesis

Altered glycosylation and concentration of α1-acid glycoprotein has been known to be related to the pathogenesis of the hepatic diseases. The present study investigated enhanced fucosylation of AGP in the sera of chronic hepatitis B (HBV-CH) and hepatitis B cirrhosis (HBV-LC) patients by high performance anion exchange chromatography and by ELISA using fucose binding Aleuria aurantia lectin. The concentration of AGP determined by ELISA using monoclonal anti-human AGP (mAb-AGP) showed high level of AGP in HBV-CH and HBV-LC patients. This was further judged by association constant (K _A) measured by surface plasmon resonance analysis. There was no apparent linkage variation of sialic acid among different patient groups when tested with two sialic acid binding lectins viz., Maackia amurensis agglutinin (MAA, NeuAc α2-3-) and Sambucus nigra agglutinin (SNA, NeuAc α2-6-) respectively. There was no change of oligosaccharide branching in HBV-CH in comparison to controls whereas a slight change was observed in HBV-LC using ConA. The above results suggest that the changes in concentration of AGP and fucosylation have a prognostic value of hepatitis diseases and it could be possible to use AGP as diagnostic marker besides clinical examination and routine laboratory investigation.     

Stereoselective protein binding of alprenolol in the renal diseased state

The investigation was undertaken to study the stereoselective protein binding of alprenolol in renal disease patient sera, compared to that in the sera of healthy volunteers. The in vitro stereoselective protein binding of beta-blockers was determined in undiluted serum and in isolated alpha(1)-acid glycoprotein (AGP) solutions by ultrafiltration. The stereoselctive serum protein binding of alprenolol, a beta-adrenergic blocking agent, in healthy volunteers was significantly altered in renal disease patients. We investigated the effects of AGP concentration and endogenous substances, including uremic toxins, on the stereoselective protein binding of alprenolol in renal disease patients. A good correlation between the unbound (R)/(S) ratio (F(R)/F(S) ratio), an apparent index of stereoselectivity in alprenolol serum binding and AGP concentration in serum, was found. However, stereoselective protein binding was not influenced by endogenous substances. This result can be explained by the difference in binding affinities of (R) and (S)-isomers of alprenolol to AGP. We conclude that the stereoselective protein binding of alprenolol in healthy volunteers and renal disease patients varies as a result of changes in AGP concentration. Accordingly, these findings might be useful in alprenolol therapy in renal disease patients.     

Synthesis and absolute configuration of the stereoisomers of [2-(1-diethylaminopropyl)] 1-hydroxy-1,1′-bicyclohexyl-2-carboxylate,a muscarinic antagonist

The compound [2-(1-diethylaminopropyl)] 1-hydroxy-1,1′-bicyclohexyl-2-carboxylate 1 is a muscarinic antagonist characterized by the presence of three chiral atoms and eight possible stereoisomers. The binding affinities to the five cloned m₁–m₅ muscarinic receptors of the stereoisomers of 1 were previously investigated and proved to be related to the chirality of the molecules. The eight isomers are prepared through the synthesis of their racemates followed by chemical resolution as (+) and (−) tartrate or (+) and (−) dibenzoyltartrate salts. The isomers with cis-configuration of OH and COOH substituents of the cyclohexane are also obtained by coupling optically active (1S, 2S) or (1R,2R)-1-hydroxy-[1,1′-bicyclohexyl]-2-carboxylic acid with (S)- or (R)-1-diethylamino-2-propanol. Chiral GC and HPLC methods are used to determine their optical purity. The absolute configurations of the four cis- and four trans-isomers are established by stereospecific synthesis and X-ray crystallographic data. Chirality 9:713–721, 1997. © 1997 Wiley-Liss, Inc.     

Detailed structural features of glycan chains derived from alpha1-acid glycoproteins of several different animals: the presence of hypersialylated, O-acetylated sialic acids but not disialyl residues

We analyzed carbohydrate chains of human, bovine, sheep, and rat alpha1-acid glycoprotein (AGP) and found that carbohydrate chains of AGP of different animals showed quite distinct variations. Human AGP is a highly negatively charged acidic glycoprotein (pKa = 2.6; isoelectic point = 2.7) with a molecular weight of approximately 37,000 when examined by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and contains di-, tri-, and tetraantennary carbohydrate chains. Some of the tri- and tetraantennary carbohydrate chains are substituted with a fucose residue (sialyl Lewis x type structure). In sheep AGP, mono- and disialo-diantennary carbohydrate chains were abundant. Tri- and tetrasialo-triantennary carbohydrate chains were also present as minor oligosaccharides, and some of the sialic acid residues were substituted with N-glycolylneuraminic acid. In rat AGP, very complex mixtures of disialo-carbohydrate chains were observed. Complexity of the disialo-oligosaccharides was due to the presence of N, O-acetylneuraminic acids. Triantennary carbohydrate chains carrying N,O-acetylneuraminic acid were also observed as minor component oligosaccharides. We found some novel carbohydrate chains containing both N-acetylneuraminic acid and N-glycolylneuraminic acid in bovine AGP. Interestingly, triantennary carbohydrate chains were hardly detected in bovine AGP, but diantennary carbohydrate chains with tri- or tetrasialyl residues were abundant. Furthermore the major sialic acid in these carbohydrate chains was N-glycolylneuraminic acid. It should be noted that these sialic acids are attached to multiple sites of the core oligosaccharide and are not present as disialyl groups.     

Conformation selectivity in the binding of diazepam and analogues to alpha1-acid glycoprotein

Diazepam, a 1,4-benzodiazepine lacking chiral centre, exists in an equimolar mixture of two chiral conformers. Induced circular dichroism spectra for the binding of diazepam and its 3,3-dimethyl substituted analogues to alpha1-acid glycoprotein (AGP) revealed that opposite to human serum albumin, AGP preferably binds the P-conformers. Accordingly, slightly favoured binding of (R)-enantiomers of 3-alkyl derivatives having P-conformation was found. In case of 3-acyloxy derivatives, however, AGP preferably binds the (S)-enantiomers. Studies with the separated genetic variants of AGP proved similar binding affinities, but markedly different conformation selectivities. For diazepam bound by the F1-S variant, a P/M selectivity of about 2 could be estimated.     

Mechanism of asymmetric hydrogenation by rhodium complexes with unsymmetrical P-chirogenic bisphosphine ligands

Using a series of the rhodium complexes with (1S,2S)-1-(R(1))methylphosphino-2-(R(2))(R(3))phosphinoethane (R(1), R(2) and R(3) = 1-adamantyl, t-butyl, cyclohexyl, cyclopentyl, methyl; abbreviated as unsymmetrical BisP*), very high enantioselectivities were observed when the di- or tri- substituted and tetra-substituted dehydro-alpha-amino acid derivatives were used as the substrates. The main factor to give high enantioselectivity is the repulsive interaction between the functional groups of the substrate and the bulky substituents of the unsymmetrical BisP*. Since the unsymmetrical BisP* has two independent chiral phosphorous atoms in the vicinity of the active site, the higher enantioselectivity than those by the C2 symmetric BisP* complexes can be obtained. Moreover, the fine-tuning to obtain extremely high enantioselectivity may be possible by changing the combination of the substituents on the two phosphorous atoms of the unsymmetrical BisP*.     

Enantioselective plasma protein binding of propafenone: mechanism, drug interaction, and species difference

The interaction of propafenone (PPF) enantiomers with human plasma, human serum albumin (HSA), alpha(1)-acid glycoprotein (AGP), as well as with plasma from rat, rabbit, and cow was investigated using indirect chiral high performance liquid chromatography (HPLC) and ultrafiltration techniques. The stronger binding of the S-PPF found in human plasma was due to AGP. Two classes of binding sites in AGP were identified: one with high-affinity and small binding capacity (K(1(S)) = 7.65 x 10(6) M(-1), n(1(S)) = 0.50; K(1(R)) = 2.81 x 10(6) M(-1), n(1(R)) = 0.46), which revealed stereoselectivity; the other with low-affinity and high-binding capacity (n(2(S)) K(2(S)) = 9.95 x 10(3) M(-1); n(2(R)) K(2(R)) = 9.74 x 10(3) M(-1)). The binding to HSA was found to be weak and not enantioselective (nK(S) = 2.08 x 10(3) M(-1), nK(R) = 2.05 x 10(3) M(-1)). The interaction between enantiomers observed in human plasma was confirmed as a competitive type interacting at the high-affinity site in AGP. The binding mode of both enantiomers with AGP was mainly hydrophobic bond. PPF enantiomers had higher-binding affinity for the F-S variant of human AGP. Drug-drug binding interaction studies showed that verapamil, diazepam, nifedipine, furosemide, nitrendipine, and nimodipine did not affect the binding of PPF enantiomers except quinidine and aprindine at the therapeutic concentration. Comparative studies indicated considerable species-dependent binding stereoselectivity between plasma of the four species investigated.     

Marked enantioselective protein binding in humans of ketorolac in vitro: Elucidation of enantiomer unbound fractions following facile synthesis and direct chiral hplc resolution of tritium-labelled ketorolac

The protein binding of the enantiomers of the nonopiate analgesic, ketorolac, was investigated in vitro using human plasma and solutions of human serum albumin (HSA) at physiological pH and temperature. In order to detect the very low levels of unbound enantiomers in protein solutions, tritium-labelled rac-ketorolac was synthesised by regiospecific isotopic exchange of the parent drug with tritiated water as the isotope donor. Radio-chemical purification of this compound by reversed-phase HPLC followed by direct resolution using a chiral α₁-acid glycoprotein (Chiral-AGP) HPLC column afforded labelled enantiomers of high specific activity. The in vitro use of (R)- and (S)-[³H₄]ketorolac enabled reproducible radiometric detection of enantiomers in protein solution ultrafiltrate. The unbound fractions of (R)- and (S)-ketorolac [fu(R) and fu(S), respectively] were determined when drug was added to various plasma or albumin solutions as either the separate enantiomers or as the racemate. Over an enantiomeric plasma concentration range of 2.0—15.0 μg/ml, fu(S) (mean range: 1.572—1.795%) was more than 2-fold greater (P < 0.001) than fu(R) (mean range: 0.565—0.674%). Both fu(R) and fu(S) were constant over this concentration range, and each was unaffected by the presence of the corresponding antipode (P > 0.05). At a concentration of 2.0 μg/ml in 40.0 g/liter fatty acid-free HSA, fu(R) and fu(S) were approximately 0.5 and 1.1%, respectively, and both values declined with increasing concentrations of the long chain fatty acid, oleic acid. We have previously shown that the pharmacokinetics of ketorolac in humans are markedly enantioselective and suggest in this report that these differences are largely the result of substantial differences in the protein binding of ketorolac enantiomers. These findings stress the importance of monitoring the unbound concentrations of the enantiomers of chiral drugs if correct interpretations are to be made of enantioselective pharmacokinetic data. © 1994 Wiley-Liss, Inc.     

Study on the sorption properties of alpha1-acid glycoprotein (AGP)-based stationary phase modified by organic solvents

An alpha(1)-acid glycoprotein, immobilized on silica (Chiral-AGP) is one of the most widely used chiral stationary phases for the enantiomeric separation of a wide variety of chiral drugs with several applications in the biological and clinical field. The aim of this work was to study the sorption properties of the AGP-based stationary phase, which may have crucial importance for enantioselectivity. New binding data to the mechanism of the chromatographic separation are presented. The sorption of both organic solvents, i.e., acetonitrile and dioxane, shows remarkable pH dependency. A fluorescence quenching study was carried out to elucidate structural changes of AGP in the presence of acetonitrile using 2,2,2-trichloroethanol as fluorescence quencher.     

Reversed-phase LC resolutions of chiral antiarrhythmic agents via derivatization with homochiral isothiocyanates

The search for new antiarrhythmic agents has been intense, because the established drugs for the treatment of cardiac arrhythmias are neither uniformly effective nor well-tolerated. Among the recently introduced new antiarrhythmic agents are tocainide (TOC), mexiletine (MEX), flecainide (FLE), and propafenone (PRO). Each of these drugs is a chiral amine used clinically as the racemic mixture. We have examined the high-performance liquid chromatographic chiral resolution of the above four drugs via derivatization with homochiral derivatizing agents (HDAs). The amino functionality of the drugs was reacted with four homochiral isothiocyanates, 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (TAGIT), (R)-alpha-methylbenzyl isothiocyanate (RAMBI), (S)-1-(1-naphthyl)ethyl isothiocyanate (SNEIT), and (R)-1-(2-naphthyl)ethyl isothiocyanate (RBEIT). Complete separation of the two peaks (resolution factor R = 1.5) was achieved with all four HDAs for TOC, with TAGIT, RBEIT, and RAMBI for MEX, with TAGIT and SNEIT for PRO, and only with TAGIT for FLE. SNEIT was used to develop analytical procedures for the determination of the enantiomeric composition of TOC in human urine and blood serum. The four HDAs offer several advantages over many other HDAs and should be useful in studies of enantioselective drug action and disposition.     

Resolution and adrenergic activities of the optical isomers of 4-[1-(1-naphthyl)ethyl]-1H-imidazole.

Recently we synthesized a naphthalene analog of medetomidine, 4-[1-(1-naphthyl)ethyl]-1H-imidazole hydrochloride (1), and found it to be highly potent in adrenergic systems. The separation of optical isomers of this naphthalene analog was achieved by using the isomers of tartaric acid. The optical purities of the isomers were determined by HPLC using a chiral column. Using X-ray analysis the (+)-isomer was determined to have the S absolute configuration. It has been reported that the (+)-isomer of medetomidine (2) is the most potent enantiomer on alpha 2-adrenergic receptors. There were both qualitative and quantitative differences in biological activities of the optical isomers of 1 in alpha 1- and alpha 2-adrenergic receptor systems of guinea pig ileum and human platelets. (+)-(S)-1, but not (-)-(R)-1 was a selective agonist of alpha 2-mediated responses in ileum whereas (-)-(R)-1 was more potent than (+)-(S)-1 as an inhibitor of alpha 2-mediated platelet aggregation.