Regulation of Serine Proteases in Blood Clotting and Beyond

Abstract

Blood clotting is a crucial step in the wound healing process. In a series of proteolytic cleavage reactions, inactive blood clotting factors (zymogens) are transformed into active enzymes. The physiologic activator of blood clotting is the tissue factor – factor VIIa (TF-FVIIa) complex. Upon vascular damage, the integral membrane protein TF is exposed and binds the serine protease FVIIa. TF-FVIIa drives blood clotting through proteolytic cleavage of its two major protein substrates, the zymogens factor IX (FIX) and factor X (FX). The activation of FIX and FX is facilitated by TF residues located adjacent to or within the putative substrate binding site on TF (the TF “exosite”). Previous studies have shown that mutating TF exosite and exosite-adjacent residues leads to a strong decrease in activation of FIX and FX. However, it remains unclear how TF-FVIIa exhibits selectivity between these substrates. We hypothesized that an exosite-adjacent TF serine loop mediates substrate selectivity by the TF-FVIIa complex. Through extensive mutagenesis studies in combination with enzymatic assays, we determined that the length of this TF serine loop affected FIX and FX activation very differently. While FX activation was decreased by up to 200-fold when the serine loop length was changed by just one residue, FIX activation was largely unaffected. The serine loop seems to regulate the TF exosite during FX activation but has no effect on the exosite during FIX activation. The results of this study suggest that the TF-FVIIa complex actively selects between its major protein substrates, which is mediated by a TF serine loop. TF residues are not just involved in TF-FVIIa substrate selectivity but also in substrate recognition. While it is known that mutating TF exosite residues leads to decreased FX activation, it is unclear how the TF exosite interacts with FX. We hypothesized that portions of the FX light chain bind to the TF exosite to facilitate substrate activation. To test this hypothesis, we generated a stable membrane-bound complex comprising TF, FVIIa and a FX mimetic (XK1). XK1 is a hybrid protein that has increased affinity for TF-FVIIa and consists of the FX light chain bound to the Kunitz 1 domain of tissue factor pathway inhibitor. The TF-FVIIa-XK1 complex was generated, validated, and then imaged using negative stain and cryo-electron microscopy (EM). Our preliminary cryo-EM model, the first model of TF-FVIIa bound to a substrate, indicates potential interactions between the TF exosite and FX light chain. These interactions could mediate substrate recognition by the TF-FVIIa complex. Serine proteases are not just important for blood clotting but are also involved in many other cellular processes. Serine proteases are typically synthesized as inactive precursors (zymogens) which remain inactive until they reach their target location. However, in some cases serine proteases can be prematurely activated, leading to severe diseases. We hypothesized that millimolar concentrations of ATP and other nucleotides in the Endoplasmic Reticulum (ER) and Golgi could keep prematurely activated zymogens enzymatically inactive while they transition through these compartments. Our kinetic, binding and structural studies revealed that serine proteases are inhibited at low millimolar concentrations of nucleotides. ADP and ATP act as uncompetitive inhibitors and bind to serine proteases cooperatively. Inhibition of serine protease activity by ATP and other nucleotides may serve as a safety mechanism to prevent cellular damage caused by premature activation of proteases.