Amino acid summary of thrombin cleavage sequences.

<p>Amino acids shown in bold and larger font are deviations from the preferred thrombin consensus sequence. The cleavage efficiency compared to the consensus is shown as a percentage.</p

Potential novel thrombin substrates.

<p>Hits holding the consensus P-R-[AGST]-[not DE]-R in an extra-cellular or secreted part are listed alphabetically by name. In the column to the left, <b>A</b> in bold and larger font indicates (presumable) involvement in cell adhesion, <b>B</b> in the nervous system, <b>C</b> in the cardiovascular system, and <b>D</b> in development/differentiation. ED, extra-cellular domain; 7-TM, seven-transmembrane receptor; PBL, peripheral blood leukocytes.</p

Analysis of the cleavage specificity by the use of new types of recombinant protein substrate.

<p>Panel A shows the overall structure of the recombinant protein substrates used for analysis of the efficiency in cleavage by thrombin. In these substrates two thioredoxin molecules are positioned in tandem and the proteins have a His₆-tag positioned in their C termini. The different cleavable sequences are inserted in the linker region between the two thioredoxin molecules by the use of two unique restriction sites, one <i>Bam HI</i> and one <i>SalI</i> site, which are indicated in the bottom of panel A. Panels B to E shows the cleavage of a number of substrates by thrombin, where individual amino acids has been changed from the thrombin consensus sequence. The name and sequence of the different substrates are indicated above the pictures of the gels. The time of cleavage (in minutes) is also indicated above the corresponding lanes of the different gels. The uncleaved substrates have a molecular weight of approximately 25 kDa and the cleaved substrates appear as two closely located bands with a size of 12–13 kDa.</p

Potential novel thrombin substrates.

<p>Hits holding the consensus P-R-[AGST]-[not DE]-R in an extra-cellular or secreted part are listed alphabetically by name. In the column to the left, <b>A</b> in bold and larger font indicates (presumable) involvement in cell adhesion, <b>B</b> in the nervous system, <b>C</b> in the cardiovascular system, and <b>D</b> in development/differentiation. ED, extra-cellular domain; 7-TM, seven-transmembrane receptor; PBL, peripheral blood leukocytes.</p

Amino acid frequency in positions P4 to P4′ of thrombin-susceptible phage sequences.

<p>This analysis is based on the alignments shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031756#pone-0031756-g001" target="_blank">Figure 1A and 1B</a>. For clarity, amino acids are displayed in functional groups, starting to the left with aromatic residues, and ending with acidic residues to the right.</p

Potential novel thrombin substrates.

<p>Hits holding the consensus P-R-[AGST]-[not DE]-R in an extra-cellular or secreted part are listed alphabetically by name. In the column to the left, <b>A</b> in bold and larger font indicates (presumable) involvement in cell adhesion, <b>B</b> in the nervous system, <b>C</b> in the cardiovascular system, and <b>D</b> in development/differentiation. ED, extra-cellular domain; 7-TM, seven-transmembrane receptor; PBL, peripheral blood leukocytes.</p

Comparison of selected studies since 1981 establishing the substrate recognition sequence of thrombin.

<p>Letters in bold indicates investigated positions, residues that were held constant are in parentheses. The preferred amino acids are denoted in the order of preference. Equally favorable residues are indicated by the absence of a slash (/). n.d., not determined; -, not applicable; pNA, para-nitroanilide.</p

Alignment of sequences obtained after five selection rounds with 1 U of thrombin or 0.2 U of thrombin, compared to natural substrates.

<p>Panel A shows the result with 1 U of thrombin, panel B the result with 0.2 U of thrombin and panel C a panel of natural substrates. The P1 residue in natural substrates (after which cleavage occurs) is denoted in parentheses. Substrate sequences refer to <i>Homo sapiens</i> where not indicated otherwise.! marks phage sequences that have LTP<u>R</u>G instead of LTP<u>G</u>G in the N-terminal flank. Residues from the non-randomized phage region are in italics. *, from <i>Rattus norvegicus</i>; IGFBP, insulin-like growth factor-binding protein; PAR, protease-activated receptor. The cleavage site of thrombin in the natural substrates listed in panel C is numbered from the N terminal of the pre-pro protein, from the first methionine. This list of natural substrates is a selection of a few of the most well known substrates of this enzyme. However, the list of potential in vivo substrates is much longer and includes many other proteins such as protein S, TAFI, antithrombin, heparin cofactor II and nexin I.</p

Analysis of the cleavage specificity by the use of new types of recombinant protein substrate.

<p>Panels A to D shows the cleavage of a number of substrates by thrombin, where individual amino acids has been changed from the thrombin consensus sequence. The name and sequence of the different substrates are indicated above the pictures of the gels. The time of cleavage (in minutes) is also indicated above the corresponding lanes of the different gels. Variants 16 is the Protein C cleavage site R211, variant 17 is the Prothrombin site R327, variant 18 is the Prothrombin site R200 and variant 19 is the Fibrinogen A alpha site R35 (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031756#pone-0031756-g001" target="_blank">Figure 1</a>).</p