Polyphosphate binds with high affinity to exosite II of thrombin

Abstract

Background: Polyphosphate (a linear polymer of inorganic phosphate) is secreted from platelet dense granules, and we recently showed that it accelerates factor V activation by thrombin.

Objective: To examine the interaction of polyphosphate with thrombin.

Methods and results: Thrombin, but not prothrombin, altered the electrophoretic migration of polyphosphate in gel mobility assays. Thrombin binding to polyphosphate was influenced by ionic strength, and was evident even in plasma. Two positively charged exosites on thrombin mediate its interactions with other proteins and accessory molecules: exosite I (mainly with thrombin substrates), and exosite II (mainly with certain anionic polymers). Free thrombin, thrombin in complex with hirudin's C-terminal dodecapeptide and gamma-thrombin all bound polyphosphate similarly, excluding exosite I involvement. Mutations within exosite II, but not within exosite I, the Na(+)-binding site or hydrophobic pocket, weakened thrombin binding to polyphosphate as revealed by NaCl dependence. Surface plasmon resonance demonstrated tight interaction of polyphosphate with thrombin (K(d) approximately 5 nm) but reduced interaction with a thrombin exosite II mutant. Certain glycosaminoglycans, including heparin, only partially competed with polyphosphate for binding to thrombin, and polyphosphate did not reduce heparin-catalyzed inactivation of thrombin by antithrombin.

Conclusion: Polyphosphate interacts with thrombin's exosite II at a site that partially overlaps with, but is not identical to, the heparin-binding site. Polyphosphate interactions with thrombin may be physiologically relevant, as the polyphosphate concentrations achievable following platelet activation are far above the approximately 5 nM K(d) for the polyphosphate-thrombin interaction.

Fig. 1

PolyP binds to thrombin but not prothrombin. (A) Gel-shift mobility assay in which 1 µg thrombin (T) or prothrombin (PT) were incubated ± 100 µg polyP₂₅, resolved on native PAGE and stained with toluidine blue to detect polyP. Free polyP₂₅ migrated at the dye front. (B) Thrombin (27 pmol) or prothrombin (143 pmol) were incubated with polyP-zirconia after which fractions were resolved on SDS-PAGE and probed with anti-prothrombin antibody. Lanes: 1, starting material; 2, flow-through; and 3, high salt (1 M NaCl) eluate. (C) Zirconia beads with (+) or without (−) attached polyP₇₅ were incubated with biotin-thrombin in plasma or buffer. Bound thrombin was eluted with high salt buffer, resolved on SDS-PAGE and probed with avidin-peroxidase (representative of four blots).

Fig. 2

PolyP interaction with thrombin is influenced by salt concentration. Thrombin was incubated with polyP-zirconia beads at varying NaCl concentrations, after which the activity of unbound thrombin was quantified in the flow-through (○). Alternatively, thrombin was incubated with polyP-zirconia beads in binding buffer containing 50 mM NaCl, and the activity of bound thrombin was quantified after elution with increasing NaCl concentrations (●). Data represent mean ± S.D. (n = 3).

Fig. 3

Exosite I is not involved in polyP binding to thrombin. (A) α-thrombin (●) or γ-thrombin (▲), both at 27 pmol were incubated with polyP-zirconia beads in binding buffer with varying NaCl concentrations, after which the flow-through was analyzed for thrombin activity. (B) Thrombin (27 pmol) was preincubated for 30 min with vehicle alone (open bars) or with the C-terminal dodecapeptide of hirudin (solid bars; 5 µg/ml), after which the mixtures were incubated with polyP-zirconia beads in binding buffer containing 50 mM or 1 M NaCl. Thrombin activity was quantified in the flow-through. Data represent mean ± S.D. (n = 3).

Fig. 4

Exosite II mutants of thrombin show reduced binding to immobilized polyP. (A) 27 pmol WT (●), H66A (◆), Y71A (◇), E229A (×), or W50A (+) thrombins were incubated with polyP-zirconia beads, and bound enzyme was sequentially eluted with increasing NaCl concentrations (plotted as cumulative thrombin recovery). (B) 27 pmol WT (●), R89A/R93A/E94A (△), R98A (▲), K248A (□), or K252A/D255A/Q256A (■) thrombins were incubated and eluted as in panel A. (C) 27 pmol nM WT, R89A/R93A/E94A, R98A, K248A, or K252A/D255A/Q256A thrombins were incubated with polyP-zirconia beads in binding buffer containing 500 mM NaCl, with unbound thrombin in the flow-through expressed as percent of the starting thrombin concentration. The data shown in (A) and (B) are representative graphs of four separate experiments performed in duplicate and the values shown in (C) represent mean ± S.D. (n = 3). *P< 0.05, **P< 0.005, ***P< 0.0005.

Fig. 5

Some glycosaminoglycans partially compete with polyP for binding to thrombin. (A) 27 pmol thrombin was preincubated with vehicle (V) or glycosaminoglycans (all at 100 µg/ml): DS, dextran sulfate; H, heparin; HS, heparan sulfate; CA, chondroitin sulfate A; CB, chondroitin sulfate B; CC, chondroitin sulfate C; or HA, hyaluronic acid. Mixtures were incubated with polyP-zirconia or zirconia beads only (labeled “no polyP”), after which unbound thrombin in the flow-through was quantified. (B) 27 pmol thrombin was preincubated with buffer alone, or with 55 µg/ml heparin (equivalent to 3 µM) or 130 µM polyP₇₅ (equivalent to 1.8 µM polymer). The mixtures were then incubated with heparin-agarose beads (50 µl) and thrombin activity in the flow-through (open bars) and high salt eluate (closed bars) was quantified.

Fig. 6

PolyP does not interfere with heparin-catalyzed inhibition of thrombin by antithrombin. (A) Residual thrombin activity (A405/min) was measured after incubating 25 nM thrombin with 50 nM antithrombin and 0–1000 U/ml heparin in the absence (○) or presence (●) of 37.5 µM polyP₇₅. The zero point refers to antithrombin inhibition of thrombin without heparin. (B) Residual thrombin activity (A405/min) was measured after incubating 25 nM thrombin with 50 nM antithrombin and 0–375 µM PolyP₇₅ in the absence (○) or presence (●) of 0.01 U/ml heparin. The zero points refer to antithrombin inhibition of thrombin (± heparin) in the absence of polyP₇₅. Data represent mean ± S.D. (n = 3).

Fig. 7

PolyP binds to WT thrombin with high affinity, as measured by surface plasmon resonance. Maximal steady-state binding of polyP₇₅ to biotinylated WT thrombin (●) or K248A mutant thrombin (▲) in 150 mM NaCl was plotted versus polyP₇₅ polymer concentration, to which the single-site ligand binding equation was fitted. Inset shows the same data plotted from 0–50 nM polyP. Data represent mean ± S.D. (n = 3).

Fig. 8

Thrombin-heparin co-crystal structure. Thrombin is rendered as space-filling with Lys and Arg side chains in blue, while heparin is rendered as red wires. Key Lys and Arg residues implicated in heparin and/or polyP binding are indicated by arrows using thrombin numbering, with chymotrypsinogen equivalents in curly braces. Structure is from PDB file 1XMN [31].