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

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