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 mu M calcium ionophore A23187, platelets are stimulated to produce thromboxane B-2 (TxB(2)) by activation of prostaglandin G/H synthase-1 (PGHS-1). TxB(2) concentration, as measured by immunoassay, is maximal at 20-30 min and declines thereafter. Addition of acetysalicylic acid (IC50=2.8 mu M) 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 TxB(2) production. When blood is incubated with 0.01-10 mu g/ml E. coli lipopolysaccharide (LPS), PGHS-2 is induced and TxB(2) levels become detectable at 3 h and continue to increase through 24 h. Using a 5 h incubation with 10 mu g/ml LPS, aspirin (10 mu M added at 0 h), which is rapidly metabolized to salicylic acid, had no effect on 10 mu g/ml LPS-induced TxB(2), but inhibited TxB(2) 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. Diflunisal and nimesulide were >4-fold selective for PGHS-2, and NS-398 was >30-fold selective for PGHS-2.