The Extended Cleavage Specificity of Human Thrombin

Abstract

Thrombin is one of the most extensively studied of all proteases. Its central role in the coagulation cascade as well as several other areas has been thoroughly documented. Despite this, its consensus cleavage site has never been determined in detail. Here we have determined its extended substrate recognition profile using phage-display technology. The consensus recognition sequence was identified as, P2-Pro, P1-Arg, P1′-Ser/Ala/Gly/Thr, P2′-not acidic and P3′-Arg. Our analysis also identifies an important role for a P3′-arginine in thrombin substrates lacking a P2-proline. In order to study kinetics of this cooperative or additive effect we developed a system for insertion of various pre-selected cleavable sequences in a linker region between two thioredoxin molecules. Using this system we show that mutations of P2-Pro and P3′-Arg lead to an approximate 20-fold and 14-fold reduction, respectively in the rate of cleavage. Mutating both Pro and Arg results in a drop in cleavage of 200–400 times, which highlights the importance of these two positions for maximal substrate cleavage. Interestingly, no natural substrates display the obtained consensus sequence but represent sequences that show only 1–30% of the optimal cleavage rate for thrombin. This clearly indicates that maximal cleavage, excluding the help of exosite interactions, is not always desired, which may instead cause problems with dysregulated coagulation. It is likely exosite cooperativity has a central role in determining the specificity and rate of cleavage of many of these in vivo substrates. Major effects on cleavage efficiency were also observed for residues as far away as 4 amino acids from the cleavage site. Insertion of an aspartic acid in position P4 resulted in a drop in cleavage by a factor of almost 20 times.

Massively parallel enzyme kinetics reveals the substrate recognition landscape of the metalloprotease ADAMTS13

“…Such methods include engineering deletion mutants (3), use of competitive ligands (4,5), and site-directed mutagenesis (6,7). In contrast to these techniques, substrate phage display is a highthroughput, unbiased approach to studying protease substrate specificity (8)(9)(10). In this method, a library consisting of 10 6 -10 9 independent phage clones, each expressing a unique potential substrate on its surface, is panned for multiple rounds with a protease, and the cleaved or uncleaved phages after each reaction are removed and amplified for subsequent rounds of selection.…”

Ratiometric Activatable Cell‐Penetrating Peptides Provide Rapid In Vivo Readout of Thrombin Activation

“…Angewandte Communications 326 www.angewandte.org uncleaved probe, in which FRET was intact (Figure 2, blue), from cleaved probe, in which FRET was disrupted (Figure 2, red). Images confirmed that RACPP PPRSFL (10) and RACPP DPRSFL (5) are cleaved by thrombin in a time-dependent manner and that RACPP PPRSFL (10) is selective for thrombin. An MMP cleavable RACPP PLGC(Me)AG (20) was also shown, as a control that was not cleaved by any of the procoagulation enzymes.…”

“…Mice were fed a 0.5 % cholesterol diet (Harlan Laboratories) for 3-6 months. [30] Intraoperative imaging of atherosclerotic plaques was performed 2.5 h after intravenous injection of 10 nmol of RACPP DPRSFL (5). Prior to imaging, animals were anesthetized with ketamine/xylazine (100 mg kg À1 , 10 mgkg À1 ) and the carotid arteries were exposed.…”

“…Further optimization should also be possible, because several recent reports describe potential new thrombin-selective substrates that could be incorporated into RACPPs to attempt to increase both sensitivity and specificity. [5,25] Thrombin activation is dynamically regulated in clotting blood and continues to be active even when bound to fibrin after the clot has formed. Previous work with a near-infrared fluorescent (NIRF) dequenching probe demonstrated diffuse and rapid thrombin activation within 12 minutes of tail clipping, although thrombin specificity was not tested with pharmacological inhibitors.…”

“…It is responsible for the proteolytic cleavage and activation of multiple coagulation factors including Factor V, VIII, XI, as well as fibrinogen and protein C. [1][2] Thrombin also cleaves and activates protease-activated receptors (PARs) which are highly expressed on platelets, endothelial cells, myocytes, and neurons. [3][4][5] Thrombin is a major therapeutic target for thrombosis and stroke intervention/prevention through indirect inhibitors such as heparin or warfarin, and direct inhibitors hirudin (divalent), and argatroban (monovalent). [6,7] In addition to its role in thrombosis and stroke, [8][9][10][11] thrombin is reported as a relevant player in cardiovascular disease, [12,13] renal injury, [14] and cancer.…”