PolyP suppresses TP hemolytic activity in serum. (A) P₁ (●) or polyP_>1000 (○) were titrated into the TP assay in the presence of 2% serum and 250 pM C5b,6. The IC₅₀ for polyP is shown by dotted lines and was determined as in Figure 1. Results are representative of more than 5 experiments, each performed in triplicate. (B) 600 µM polyP_>1000 was incubated overnight with 400 U/mL calf intestinal alkaline phosphatase (CIAP) and then added to the TP assay at a final polyP_>1000 concentration of 100 µM. The control represents lysis in the absence of CIAP and polyP, but with an equivalent concentration of CIAP digestion buffer. “CIAP” represents lysis in the presence of CIAP but absence of polyP. The findings indicate that suppression of the TP by polyP requires the integrity of the polymer. Values were normalized to baseline lysis from the control condition. Each column represents quadruplicate data points. (C) 2 mM P₁ and 200 µM polyP_>1000 were added singly or in combination in the TP assay. Values were normalized to baseline lysis from the control condition in which no phosphate was added. Excess monomer could not overcome suppressive properties of polyP. n = 3 independent experiments, each performed in triplicate.