Inhibition of ADP-induced platelet shape change and aggregation by o-phthalaldehyde: Evidence for covalent modification of cysteine and lysine residues
| Autor: | Rajinder N. Puri, Robert W. Colman |
|---|---|
| Rok vydání: | 1991 |
| Předmět: |
Blood Platelets
P2Y receptor Adenosine Platelet Aggregation Lysine Biophysics Adenylate kinase Biochemistry Cyclase O-Phthalaldehyde Thrombin medicine Humans Platelet Cysteine Molecular Biology Chemistry Affinity Labels Adenosine Diphosphate Kinetics Spectrometry Fluorescence Ethylmaleimide Platelet Aggregation Inhibitors o-Phthalaldehyde medicine.drug |
| Zdroj: | Archives of Biochemistry and Biophysics. 286:419-427 |
| ISSN: | 0003-9861 |
| DOI: | 10.1016/0003-9861(91)90060-v |
| Popis: | Platelets play a major role in the hemostatic process following vascular injury. Chemical modification of cysteine and/or lysine residues in platelet proteins has been shown to cause loss of platelet aggregation induced by diverse agonists; however, these investigations have not addressed the identity of the specific proteins affected. o -Phthalaldehyde (OPTH) is a unique chemical modification reagent that forms and permits the identification of fluorescent isoindole derivatives with proteins by covalently and simultaneously modifying closely spaced cysteine and lysine residues. We found that OPTH inhibited platelet aggregation induced by ADP, collagen, and U46619 (an analog of prostaglandin H 2 ), but had minimal effect on platelet aggregation induced by thrombin, plasmin, chymotrypsin, A23187 (a calcium ionophore), PMA (phorbol 12-myristate 13-acetate), and PMA + A23187. Since platelet aggregation induced by ADP, collagen, and U46619 has been shown to involve binding of endogenous or exogenous ADP to the platelet receptor, our further studies focused on platelet aggregation induced by ADP. OPTH inhibited ADP-induced shape change and aggregation in a concentration-dependent manner. The second-order rate constant for the inhibition of ADP-induced platelet shape change ( K sc = 1.0 × 10 3 M −1 s −1 ) was lower than that for aggregation ( K agg = 5.4 × 10 3 M −1 s −1 ). Fluorescence excitation and emission spectra of OPTHplatelet adduct exhibited maxima at 346 and 437 nm, respectively, consistent with the formation of an isoindole derivative(s). The nonpenetrating thiol-specific reagent, p -chloromercuribenzenesulfonate (pCMBS) (0.8 m m ), is known to block the inhibition of stimulated adenylate cyclase induced by ADP but not the ADP-induced platelet shape change. The inhibition of ADP-induced platelet shape change ( K sc = 1.5 × 10 3 M −1 s −1 ) by OPTH was not affected by pCMBS. OPTH, at concentrations (15–50 μ m ) that inhibited ADP-induced platelet aggregation and shape change did not raise the intracellular levels of adenosine cyclic 3′,5′-monophosphate (cAMP) in platelets nor did it impair the ability of iloprost (a stable analog of prostaglandin I 2 ) to raise the platelet cAMP level. Thus, OPTH under these conditions did not interact with platelet adenylate cyclase. 5′- p -fluorosulfonylbenzoyladenosine (FSBA) has been previously shown to inhibit ADP-induced platelet shape change and aggregation by covalently modifying aggregin ( M r = 100 kDa), a putative ADP receptor on platelet surface. N -Ethylmaleimide (NEM) also inhibits ADP-induced platelet shape change and aggregation. Preincubation of platelets with OPTH prevented labeling of platelets by [ 3 H]FSBA and [ 3 H]NEM. The results show that OPTH inhibited ADP-induced platelet shape change and aggregation and chemically modifies closely spaced cysteine and lysine residues in aggregin, thus preventing binding of ADP to its receptor. |
| Databáze: | OpenAIRE |
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