Wild-type ERp57 potentiates platelet aggregation and an inactive mutant ERp57 inhibits aggregation, prolongs the tail bleeding time in mice, and inhibits activation of αIIbβ3 and P-selectin expression ex vivo. (A) ERp57 with the 4 active site cysteines mutated to serine (oo-oo) is catalytically inactive. (B) Submaximal aggregation (baseline) was stimulated with collagen or the SFLLRN peptide. The potentiating effect of preincubating the platelets with purified wild-type ERp57 (WT ERp57) in the concentrations indicated is seen. The inhibitory (i) effect of adding the inactive (ii) mutant ERp57 (oo-oo) in the concentrations indicated is also seen. (C) The cumulative data ± SE of at least 3 different experiments, showing potentiation of aggregation by WT ERp57 and inhibition by the mutant ERp57 (oo-oo) relative to the baseline aggregation (i, collagen; ii, SFLLRN). The concentration of WT or mutant ERp57 that provided maximal potentiation or inhibition of responses varied a little between experiments. Fifty to 100nM WT or mutant ERp57 generally gave maximal potentiation and inhibition responses with collagen; 100 to 200nM of WT or mutant ERp57 provided maximal responses with the SFLLRN peptide. In these studies the enzymes were added for 10 minutes before the addition of the agonist. (D) The PBS control, 100 μg of WT ERp57, or 100 μg of the mutant ERp57 (oo-oo) were infused into mice, and the tail bleeding times were recorded up to 30 minutes. Horizontal bars represent mean bleeding times. (E) Results of ex vivo studies of platelets prepared from mice infused with PBS (control) or inactive ERp57 (oo-oo). The platelets were activated with convulxin (500 ng/mL) and activation of (i) αIIbβ3 (measured by binding of the JON/A Ab) and (ii) P-selectin expression were determined (n = 3).