Factor XIa 1536 HTS
Factor XI (FXI) circulates as a complex with high molecular weight kininogen (HK) in the plasma at a concentration of 5 ug/ml (equivalent to 31.3 nM, dimeric concentration) as a homodimeric glycosylated blood plasma zymogen of approximately 160 kDa, containing monomeric subunits of 80 kDa each (1). Thrombin (2, 3), factor XIa (FXIa) (3), and factor XIIa alpha (FXIIa) (4), all cleave an internal R369-I370 bond in each monomer of FXI, yielding the enzyme FXIa. ..more
BioActive Compounds: 302
Molecular Library Screening Center Network (MLSCN)
Penn Center for Molecular Discovery (PCMD)
Assay Provider: Scott L. Diamond, University of Pennsylvania
MLSCN Grant: X01-MH076406-01
Factor XI (FXI) circulates as a complex with high molecular weight kininogen (HK) in the plasma at a concentration of 5 ug/ml (equivalent to 31.3 nM, dimeric concentration) as a homodimeric glycosylated blood plasma zymogen of approximately 160 kDa, containing monomeric subunits of 80 kDa each (1). Thrombin (2, 3), factor XIa (FXIa) (3), and factor XIIa alpha (FXIIa) (4), all cleave an internal R369-I370 bond in each monomer of FXI, yielding the enzyme FXIa.
After activation from FXI to FXIa, FXIa possesses a heavy chain of 369 residues and a light chain of 238 residues. The heavy chain consists of four apple domains (A1-A4) and the light chain represents a trypsin-like serine protease domain with active site residues at H413, D464, and S557 (1, 5-7). FXIa catalyzes FIX to FIXa activation by cleaving two scissile bonds at R145 and R180 (8). The FIXa generated can catalyze FXa formation on the platelet surface with the active cofactor factor VIIIa (FVIIIa), with FVIIIa increasing the Vmax of FX activation by FIXa by 100,000 fold (9). After activation of sufficient levels of FXa by the consolidation pathway, FXa can go on to form a ternary complex with FVa and prothrombin on the platelet surface, to give sufficient levels of thrombin for activation of fibrinogen to fibrin. Formation of this ternary prothrombinase complex in the presence of phospholipids has been shown to increase the rate of prothrombin to thrombin activation by 300,000-fold more than with FXa and prothrombin alone (10).
Thrombin catalyzes activation of fibrinogen to fibrin, cleaving peptides 14-16 amino acids in length, called fibrinopeptides, from the A alpha and B beta subunits of fibrinogen (11, 12). The fibrin monomers produced by thrombin form a noncovalent meshwork with other fibrin molecules to produce a fibrin clot that stabilizes and retracts the initial platelet plug described above. This stable fibrin clot formation stabilizes the primary platelet plug, and reduces the volume of the plug in order to arrest blood loss.
The role of FXI in thrombosis was investigated using a mouse double knockout of FXI and protein C (PC). First, the single FXI knockout in the mouse did not cause a significant bleeding problem in mice, shown by comparable tail bleeding times between FXI knockout mice and wt mice, as opposed to the FIX knockout mouse which had a tail bleeding time increase of > 5.8-fold over wt mice (13). Additionally, using a FXI deficient mouse model, mice were partially protected from arterial occlusion in a FeCl3 model of thrombosis (13). Thus, by this model, controlling or limiting FXI activity can have a protective effect on hemostasis by preventing pathologic thrombus formation. Further studies of FXI under rapid arterial flow conditions simulating rapid thrombus growth with an anti-FXI antibody that reduced intraluminal thrombus growth did suggest that inhibiting FXI can have a mild, yet controlled antithrombotic effect that may be of significant use clinically (14). A recent report found that increased risk of cerebrovascular events is associated with elevated FXI activity and antigenic levels, implicating FXI as a risk factor in deep venous thrombosis (15). When double knockout mice lacking both FXI and activated protein C (aPC) were examined, the mice possessed greater control over thrombosis than the aPC knockouts, suggesting that FXI deficiency helped these mice to survive past the neonatal period. Thus, the absence of FXI prevented the occurrence of fatal disseminated intravascular coagulation (DIC) in this model (16).
In vivo findings in rodent models of coagulation in addition to clinical data from patients at risk for pathologic cerebrovascular events have given rise to the recognition of FXI as a fine modulator and focal point in blood coagulation whose inhibition does not cause a severe bleeding diathesis, and suggest that drug design using the tools of high throughput chemical inhibitor screening may give rise to novel anticoagulant therapies (17).
HTS was performed using 218,724 compounds of the MLSCN library individually plated into 10ul 1536 compound plates at a concentration of 2.5 mM each, which were diluted 500-fold into 5 ul 1536 well assay plates (final concentration 5 uM each compound). The assay used to test for percent inhibition was a fluorescence assay utilizing hydrolysis of Boc-Glu-Ala-Arg-AMC, as first described by Kawabata et al. (18). Hits with >20% inhibition in both wells of the mixture plates were retested in complete IC50 curves.
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2. D. Gailani, G. J. Broze, Jr., Science 253, 909 (Aug 23, 1991).
3. K. Naito, K. Fujikawa, Journal of Biological Chemistry 266, 7353 (1991).
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7. B. A. McMullen, K. Fujikawa, E. W. Davie, Biochemistry 30, 2056 (Feb 26, 1991).
8. K. Fujikawa, M. E. Legaz, H. Kato, E. W. Davie, Biochemistry 13, 4508 (1974).
9. G. van Dieijen, G. Tans, J. Rosing, H. C. Hemker, J Biol Chem 256, 3433 (Apr 10, 1981).
10. J. P. Miletich, C. M. Jackson, P. W. Majerus, Journal of Biological Chemistry 253, 6908 (1978).
11. K. Bailey, F. R. Bettelheim, L. Lorand, W. R. Middlebrook, Nature 167, 233 (Feb 10, 1951).
12. B. Blomback, M. Blomback, Ann N Y Acad Sci 202, 77 (Dec 8, 1972).
13. X. Wang et al., J Thromb Haemost 3, 695 (Apr, 2005).
14. A. Gruber, S. R. Hanson, Blood 102, 953 (Aug 1, 2003).
15. D. T. Yang, Flanders, M.M., Rodgers, G.M., Blood 106, 2626 (November 16, 2005, 2005).
16. J. C. Chan et al., American Journal of Pathology 158, 469 (Feb, 2001).
17. A. Gruber, S. R. Hanson, Curr Pharm Des 9, 2367 (2003).
18. S. Kawabata et al., European Journal of Biochemistry 172, 17 (Feb 15, 1988).
Human plasma factor XIa was purchased from Enzyme Research Laboratories (Cat # HFXIa 1111a). Substrate Boc-Glu-Ala-Arg-AMC was from Bachem (Cat #I-1575.0050). Assay buffer consisted of 50 mM Tris, pH 7.4, 150 mM sodium chloride, 0.05% Tween 20. 1536-well black plates were from Corning (Item #3728).
Factor XIa (0.27 ug/mL) was incubated with Boc-Glu-Ala-Arg-AMC substrate (15 uM) in 5 uL of assay buffer (see above) for 2 hr at room temperature. HTS was performed using 5 uM compound.
1.Fill 1536 well plate with 4 uL of Boc-Glu-Ala-Arg-AMC substrate (18.75 uM in 1x assay buffer) using Aquamax DW4
2.Add 1 uL assay buffer to columns 1, 2, 45, and 46 using Aquamax DW4
3.Add 10 nL of compound (0.25 mM in DMSO) using 3 transfers of 3.3nl with the Evolution 1536 pintool (washed with water and isopropanol after each transfer)
4.Add 1 uL enzyme (1.35 ug/mL in assay buffer) using Aquamax DW4 to all columns except 1, 2, 45, and 46
5.Incubate for 2 hr at room temperature
6.Read fluorescence (excitation 355, emission 460) on Envision reader
Data were analyzed in IDBS ActivityBase. Each HTS plate a single test compound (5 uM in 0.2% DMSO) in columns 5-44, controls (enzyme, no compound) in columns 3, 4, 47, and 48, and blanks (no enzyme) in columns 1, 2, 45, and 46. HTS percent inhibition was calculated for each compound from the signal in fluorescence units (FU) and the mean of the plate controls and the mean of the plate blanks using the following equation:
% Inhibition = 100*(1-((signal-blank mean)/(control mean-blank mean)))
Activity scores were calculated as follows:
For positive percent inhibition, score = 0.4 x Percent inhibition
For negative percent inhibition, score = 0
Activity outcome is reported as follows:
(1) Percent inhibition >= 40 = active
(2) Percent inhibition < 40 = inactive
Analysis of screening results
HTS plate statistics were as follows:
Number of plates = 183
Median Z-factor = 0.67
Median control percent CV = 8.16
Maximum control percent CV = 17.37
A hit cut-off of 40% inhibition was selected. Based on this cutoff, a hit rate of 0.14% was observed.
This assay was submitted to the PCMD by Scott Diamond, assay development and HTS were conducted by Paul Riley and Sahil Batta, and data were submitted by Paul Riley, Sahil Batta, and Andrew Napper all of the University of Pennsylvania.
** Test Concentration.
Data Table (Concise)