Human whole blood assays for inhibition of prostaglandin G/H synthases-1 and-2 using A23187 and lipopolysaccharide stimulation of thromboxane B2 production

Abstract

When freshly drawn, heparinized human whole blood is incubated with 50μM calcium ionophore A23187, platelets are stimulated to produce thromboxane B₂ (TxB₂) by activation of prostaglandin G/H synthase-1 (PGHS-1). TxB₂ concentration, as measured by immunoassay, is maximal at 20–30 min and declines thereafter. Addition of acetylsalicylic acid (IC₅₀=2.8μM) or other nonsteroidal antiinflammatory drugs (NSAIDs) 15 min or 4.5 h prior to 30 min stimulation with ionophore results in concentration dependent inhibition of TxB₂ production. When blood is incubated with 0.01–10 μg/ml E. coli lipopolysaccharide (LPS), PGHS-2 is induced and TxB₂ levels become detectable at 3h and continue to increase through 24 h. Using a 5 h incubation with 10 μg/ml LPS, aspirin (10 μM added at 0 h), which is rapidly metabolized to salicylic acid, had no effect on 10 μg/ml LPS-induced TxB₂, but inhibited TxB₂ production by ionophore A23187 added at 4.5 h, through acetylation of preexisting PGHS-1. In a 5 h assay, NSAIDs added at 0 h were compared for inhibition of TxB₂ production stimulated by addition of ionophore A23187 at 4.5 h (PGHS-1), or by addition of LPS at 0 h (PGHS-2). Most NSAIDs were more potent against PGHS-1 than PGHS-2. Diclofenac, naproxen and flufenamic acid were equipotent or slightly selective for PGHS-2. Diffunisal and nimesulide were >4-fold selective for PGHS-2, and NS-398 was >30-fold selective for PGHS-2.