Fig 1.
Regions surrounding cleavage sites for thrombin in a panel of important target molecules.
Panel A shows the regions flanking the activating sites in natural substrates of thrombin. The amino acid sequences flanking both N-terminally and C-terminally of the cleavage sites for thrombin in FV, FVIII, PAR1,3 and 4, α & β fibrinogen and Protein C are depicted. Sequences are shown in a one-letter code and cleavage site numbering is based on the mature protein without signal peptide. The negatively charged amino acids are marked in red and thrombin cleavage sites are shown by arrows. Panels B-E shows schematic figures of FVIII, FV, fibrinogen and protein C showing the cleavage sites for thrombin (depicted by scissors with numbered residue).
Fig 2.
Schematic 3-D models of human thrombin.
Panel A shows a schematic 3D structure of human thrombin with the charge density illustrated with blue for basic or positively charged regions and red for negatively charged or acidic regions. The color intensity reflects the charge intensity. Panel B shows a schematic 3D structure of human thrombin where some of the positively charged amino acid positions studied by site directed mutagenesis in exosites I and II are marked in blue. The active site serine is shown in green and the histidine and aspartic acids are hidden under an extending loop and therefore not visible in this angle of the molecule. Two extended surface loops, the 60 loop and the γ-loop are shown in yellow and orange, respectively. Thrombin structure from PDB, code 1DM4 run using UCSF Chimera v1.8 and annotated in Adobe Illustrator CS5.
Fig 3.
Analyses of the minimal sites for three thrombin cleavage sites in FVIII by the use of recombinant protein substrates.
Panel A shows the overall structure of the recombinant protein substrates used for analysis. In these substrates, two thioredoxin (trx) 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 trx molecules with the use of two unique restriction sites, one BamHI and one SalI site, which are indicated in the bottom of panel A. Panels B shows a schematic representation of a cleavage reaction. 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. Panel C shows a comparative analysis of the cleavage efficiency of the thrombin consensus sequence with the P4-P4’ sequences from three cleavage sites in FVIII. 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 their corresponding lanes. In panel D we show an SDS-PAGE density summary of cleaved substrates. All protein gels were analyzed using Image Quant TL 1D gel density software (v8.1) from GE Life Science (Piscataway, NJ USA) or the UN-SCAN-IT Gel Analysis Software from Silk Scientific Inc. (Orem, Utah USA). Individual bands from the full-length constructs (top bands) were analyzed with manual lane editing and minimum profile. Bands were detected automatically and gating adjusted to compare cleavage over the time course. Figures show percentage cleavage of the original construct (time point 0 minutes). Standard deviation of the time points are shown (mean +- standard deviation). Statistical analyses were performed using the Mann-Whitney test with two-tailed P value.
Fig 4.
The importance of exosite interactions for the cleavage efficiency of cleavage sites in FVIII.
The name and sequence of the substrates are indicated above the gel pictures. The time of cleavage (in minutes) is also indicated above their corresponding lanes on the gel. Panels A-C shows the results for the individual cleavage sites in FVIII, R372, R740 and R1689, respectively. Panels D, E and F shows the results from a scanning of the individual gels with corresponding percentages for a more easy evaluation of the result.
Fig 5.
The importance of exosite interactions for the cleavage efficiency of cleavage sites in FV.
The name and sequence of the substrates are indicated above the gel pictures. The time of cleavage (in minutes) is also indicated above their corresponding lanes on the gel. Panels A shows the results from the analysis of the minimal sites for FV, R709, R1018 and R 1545. Panels B, D and F shows the results from a scanning of the individual gels with corresponding percentages for a more easy evaluation of the result. Standard deviation of the time points are shown (mean +- standard deviation). Statistical analyses were performed using the Mann-Whitney test with two-tailed P value. **p value = 0.0079, *p value = 0.0119, ns, not significant.
Fig 6.
The importance of exosite interactions for the cleavage efficiency of cleavage sites in fibrinogen α chain (panel A), β chain (panel C) and protein C (panel E).
The name and sequence of the substrates are indicated above the gel pictures. The time of cleavage (in minutes) is also indicated above their corresponding lanes on the gel. Panels B, D and F shows the results from a scanning of the individual gels with corresponding percentages for a more easy evaluation of the result.
Fig 7.
Schematic 3-D models of human thrombin showing the position of the N-terminal region of fibrinogen α chain.
Panel A shows a space-filling model with the alpha chain peptide in purple. Panel B shows the interaction between thrombin (ribbon structure in beige) and the N-terminal region of fibrinogen α chain (ball and stick structure in purple) in detail. The same orientation as panel A is shown with the catalytic residues His57, Asp102 and Ser195 together with the S1 pocket residues Asp 189, Gly216 and 226 in green. Thrombin structure from PDB, code 1DM4 run using UCSF Chimera v1.8 and annotated in Adobe Illustrator CS5.
Fig 8.
Analyses of the importance of Phe8, Gly12 and three negatively charged residues in the N-terminal region of fibrinogen α chain for the cleavage by thrombin.
The name and sequence of the substrates are indicated above the gel pictures. The time of cleavage (in minutes) is also indicated above their corresponding lanes on the gel. The mutations are marked in green in the sequences above each gel. Panels B and D shows the results from a scanning of the individual gels with corresponding percentages for a more easy evaluation of the result.