Factor XIa Single Well 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: 94
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 on a total of 33,068 compounds of the MLSCN library, 23,017 of which were not in the mixture HTS plates used previously. These compounds were plated as single components of 384 well plates at 0.5 mM stock concentration, and were diluted 50-fold into 10 ul 384 well assay plates (final concentration 10 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).
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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, 0.02% Tween 20. Low-volume 384-well black plates were from Corning (Item #3676).
Factor XIa (0.23 ug/mL) was incubated with Boc-Glu-Ala-Arg-AMC substrate (15 uM) in 10 uL of assay buffer (see above) for 2 hr at room temperature. HTS was performed using 10 uM compound.
1.Fill low-volume plate with 4 uL water using Multidrop-micro
2.Add 5 uL assay buffer to columns 1 and 23 using Multidrop-384
3.Add 200 nL of compound (0.5 mM in DMSO) using Evolution pintool
4.Add 1 uL of Boc-Glu-Ala-Arg-AMC substrate (150 uM in 5x assay buffer) using Multidrop-micro
5.Add 5 uL enzyme (0.46 ug/mL in assay buffer) using Multidrop-384
6.Incubate for 2 hr at room temperature
7.Read fluorescence (excitation 355, emission 460) on Envision reader
Data were analyzed in IDBS ActivityBase. Each HTS plate a single test compound (10 uM in 2% DMSO) in columns 3-22, controls (enzyme, no compound) in columns 2 and 24, and blanks (no enzyme) in columns 1 and 23. 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)))
The activity score reported here is based on percent inhibition observed in the primary HTS (see above). Compounds were selected for confirmation by dose response testing with IC50 determination if the percent inhibition score was greater than 40.
Percent inhibition scores were calculated from the percent inhibition value associated with each compound as follows:
(1) For percent inhibition between 0 and 100, score = percent inhibition
(2) For negative percent inhibition, score = 0
This assay was submitted to the PCMD by Scott Diamond, assay development and HTS were conducted by Paul Riley, and data were submitted by Andrew Napper and Paul Riley, all of the University of Pennsylvania.
** Test Concentration.
Data Table (Concise)