McMaster 1

University/ Department of Pathology and Molecular 2

Medicine/ Canadian Blood Services

Library screening and combinatorial approaches identify an alpha-1

proteinase inhibitor variant with enhanced specificity for factor XIa
M. HAMADA1, V. BHAKTA2, A. NOUANESENGSY1, J. LAPIERRE1, D. PERRUZZA1, and W. SHEFFIELD1,2

INTRODUCTION
• Α1-Proteinase Inhibitor (α1-PI, α1-Antitrypsin): Most abundant
member of the serpin proteinase inhibitor superfamily in human
plasma, inhibits neutrophil elastase, other proteinases
• α1-PI M358R (at P1 of Reactive Centre Loop, RCL, “Pittsburgh”)
converted α 1-PI to inhibit thrombin, other coagulation proteinases
(e.g. Factor XIa, FXIa) [1] but not a good antithrombotic agent [2]
• Serpin RCLs (P13 – P3’) contribute to inhibitor specificity
• FXIa: Emerging target for counter-thrombosis; ↓ FXI protects from
thromboembolic disease without a strong bleeding diathesis
AIMS
A B C
Figure 3. Serpin-proteinase complex formation.
ABOVE: Coomassie Blue-stained gels of reactions of 1.0 µM α1-PI variants (identified
above the lanes) reacted with 0.1 µM proteinase (FXIa or FIIa [thrombin] for 5 min at
37°C. (A) Reaction of single motif variants and α1-PI M358R with FXIa. (B) Reaction of
single motif variants with FIIa (thrombin). (C) Reaction of motif-combining variants
Construct C and Construct D with FXIa or FIIa (identified above the lanes). NOTE:
Constructs A and B demonstrated more substrate behavior than C or D and were not
further characterized. M, molecular weight standards (in kDa): 200; 150; 120; 100; 85;
70; 60; 50 (greater intensity); 40; 30; 25; and 20.

• To biopan phage display or bacterial libraries hypervariable at α1-

PI RCL positions P13-P8, P7-P3, and P2-P3’ with FXIa
• To express and characterize most abundant variants from 3
sectors Figure 4. Rate constants for
• To combine 1,2, or 3 selected sequence motifs, and determine inhibition of other proteinases.
the most specific FXIa inhibitor of these candidates LEFT: Mean K2 values (n=6 ± SD) are
shown, for Construct C (grey) or α1-PI
M358R (black) for the proteinases
METHODS identified on the x axis.

• Phage display libraries constructed using T7Select system, fuses

α1-PI M358R to phage T7 coat protein 10B [3]
• Biopanned using FXIa, biotinylated anti-FXI IgG, streptavidin-
coated magnetic beads
• 5 rounds of biopanning → (deep) sequence to ID RCL codons
• Transferred candidates to glutathione sulfotransferase (GST)
bacterial expression system, cleaved GST-API fusions on
Table 1. Kinetic characterization of single
glutathione-agarose column to elute soluble, His-tagged α1-PI motif and combination Constructs C and D. Figure 5. Structural
variants, polished on nickel-chelate agarose or DEAE-sepharose ABOVE: k2 values for the α1-PI variants identified under
consequences of
• Kinetic characterization of inhibition of chromogenic substrate “Protein” versus FXIa or thrombin, the selectivity (k2 for
FXIa/k2 for thrombin, and the SI are shown. Data values
Construct C
amidolysis, determined second order rate constant (k2) for FXIa, are the mean of 4 to 9 determinations ± SD. Construct substitutions. The crystal
thrombin (FIIa), Activated Protein C (APC), Kallikrein (Kal), factor C is bolded because it has the highest selectivity forstructure of α1-PI M358R (PDB
1OO8; α1-PI Pittsburgh in the
FXIa over thrombin of the tested proteins. For Construct
Xa (FXa) and factor XIIa (FXIIa) (pseudo-first order conditions) native conformation) was
D no inhibition of thrombin was detected at a molar
and stoichiometry of inhibition (SI) for FXIa ratio. of 100:1 (α1-PI: proteinase). manipulated in Pymol to focus
on the RCL P5 – P5’, with amino
CONCLUSIONS acid residues identified below
the image. M358R, red, other
RESULTS 1. Phage display and biopanning with FXIa
residues grey- highlighted in
upper panel; lower panel,
Figure 1. Schematic diagram of identified α1-PI M358R variants with increased variant residues in Construct C
approach specificity for FXIa without negative selection. unshaded, unaltered residues
brown. Lower panel made by
RIGHT: “Kettle ball” model of α1-PI 2. Combining sequence motifs from different
introducing Construct C
shows RCL protruding from body of sectors of the RCL yielded further cooperative substitutions into the RCL in
protein (red). Motifs tested by phage increases in selectivity. Pymol (Schrödinger, Inc.), with
display are teal (P13-P8, EAAGAM,
3. Glu (E) substitutions at P3 and P3’ likely drive overlap of RCL sidechain
P12 fixed as A) or yellow (P7-P3); P2- electron densities energy-
P3’ (P2-P3’, PRSIP, grey, P2-P1’ fixed the increased specificity of Construct C.
minimized. Altered residues on
as PRS) was screened using microtiter 4. Next steps: Test Construct C in animal models. ribbon diagram of loop, purple,
plate assays of bacterial lysates. conserved residues, green.
Constructs A – D (below “kettle ball”)
combine motifs as shown. REFERENCES
[1]. Scott CF et al. Alpha-1-antitrypsin-Pittsburgh. A potent inhibitor of human plasma factor XIa,
kallikrein, and factor XIIf. J Clin Invest. 1986;77(2):631-4.
[2]. Harper JL et al. Recombinant antitrypsin Pittsburgh undergoes proteolytic cleavage during E. coli
sepsis and fails to prevent the associated coagulopathy in a primate model. Thromb
Haemost.1998;80(5):816-21.
[3]. de Souza LR et al. Serpin Phage Display: The Use of a T7 System to Probe Reactive Center
Figure 2. Motif sequences Loop Libraries with Different Serine Proteinases. Methods Mol Biol. 2018;1826:41-64.

LEFT: The most abundant and active inhibitory sequences

from the three sectoral screens are shown, aligned to P13-P3’ ACKNOWLEDGEMENTS
of α1-PI M358R, as well as Constructs C and D. Bolded Grant-In-Aid G-19-0026318 from the Heart and Stroke Foundation of Canada to WS
residues differ from α1-PI M358R.
Programmatic funding: Canadian Blood Services, Health Canada (federal government of Canada).

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