Abstract Number: LPB0101
Meeting: ISTH 2021 Congress
Background: Recent studies have shown that physiological cytosolic ATP concentrations (~1-10 mM) enhance the stability of many intracellular proteins and help keep them soluble. Similar nucleotide concentrations are present in the endoplasmic reticulum (ER) and Golgi. We hypothesize that low mM concentrations of ATP and other nucleotides in the ER and Golgi lumen could stabilize nascent serine proteases (especially, any prematurely activated zymogens) by inhibiting their enzymatic activity prior to secretion.
Aims: Investigate how physiologic concentrations of nucleotides effect serine protease activity.
Methods: We screened 12 serine proteases involved in different cellular processes including blood clotting (tissue factor-factor VIIa, factor Xa, factor XIa, factor XIIa, kallikrein, thrombin), fibrinolysis (plasmin, urokinase, tissue plasminogen activator) and digestion (trypsin, chymotrypsin). Enzymatic activities were measured in the presence of nucleotides (including ADP, ATP, UTP, GTP, CTP). Surface Plasmon Resonance (SPR) with active site-inhibited serine proteases was used to determine KD values for ATP binding.
Results: Low mM concentrations of ATP or ADP strongly inhibited the activity of most serine proteases in our screen. UTP, GTP and CTP showed protease inhibition comparable to ATP. IC50 for inhibition of serine proteases by ATP ranged from 2-11 mM. In-depth kinetic studies for one of the proteases (tissue factor-factor VIIa) revealed that ADP is an uncompetitive inhibitor, with Ki = 0.7-2.5 mM. SPR experiments showed cooperative binding of ATP to serine proteases with KD = 16-23 mM and Hill coefficients = 1.5-1.8.
Conclusions: Our kinetic studies indicate that physiologic concentrations of nucleotides strongly inhibit multiple secreted serine proteases. SPR studies suggest a cooperative binding mode, with more than one ATP binding the protease. Inhibition of serine proteases by nucleotides could serve as a cellular safety mechanism to keep any prematurely activated proteases in the lumen of the ER and Golgi quiescent prior to secretion.
To cite this abstract in AMA style:Birkle F, Wang Y, Collins N, Morrissey JH. Nucleotides Inhibit Serine Proteases Involved in Blood Clotting and Other Cellular Processes [abstract]. Res Pract Thromb Haemost. 2021; 5 (Suppl 2). https://abstracts.isth.org/abstract/nucleotides-inhibit-serine-proteases-involved-in-blood-clotting-and-other-cellular-processes/. Accessed December 3, 2021.
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