AID	Name	Type	Comment
800	Factor XIIa 1536 HTS	screening	Factor XIIa 1536 HTS; hits were confirmed in this assay

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

Target

Factor XII (FXII) is a 80 kDa zymogen found at a concentration of 0.375 uM in plasma, and upon activation by kallikrein at R353, a disulfide-linked two chain molecule called factor XIIa alpha (FXIIa) is generated. FXIIa is also capable of autoactivation by binding to negatively charged surfaces (1). Kallikrein can also cleave other scissile bonds in FXIIa alpha outside of the catalytic domain at R334, R343, and R353, generating FXIIa beta, a 30 kDa enzyme that is no longer able to bind to surfaces, and which activates prekallikrein (PK) to kallikrein, using high molecular weight kininogen (HK) as a cofactor (2, 3). FXIIa is irreversibly inhibited by C-1 inhibitor (C1INH), a 105 kDa plasma SERPIN (4-6).

FXII, PK, HK, C1INH, and factor XI (FXI) have been traditionally placed within the intrinsic pathway of blood coagulation. The intrinsic activation pathway was originally named because its major recognized activation of a coagulation protease leading to thrombin formation, FXIIa activation of FXI to factor XIa (FXIa), occurs within, or intrinsic to the bloodstream. FXI was previously removed from this grouping and the remaining proteins are currently regarded as belonging to the contact activation, or Kallikrein-Kinin pathway, which is defined as proteolytic activation events that occur after collagen exposure from the subendothelium to the circulation (9).

FXIIa cleaves an internal R369-I370 bond in each monomer of FXI, yielding the enzyme FXIa (10). FXIa catalyzes FIX to FIXa activation by cleaving two scissile bonds at R145 and R180 (11). 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 (12). 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 (13).

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 (14, 15). 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.

Deficiencies observed in the contact factor pathway (FXII, HK, or PK) have not shown evidence of abnormal bleeding tendencies, though C1INH deficiency causes hereditary angioedema, a swelling of subcutaneous structures, but not abnormal bleeding. The contact factor pathway, while not recognized to play a direct role in the initiation of the coagulation cascade (although FXIIa activates FVII to FVIIa and FXI to FXIa), is believed to play a more important pathological role in stabilization of the fibrin clot, but which can lead to pathological thrombosis and vascular occlusion. This is consistent with mouse models in which FXII deficient mice showed a severe defect in the formation and stabilization of platelet-rich thrombi, which was corrected by adding human FXIIa (16). Deficiency of FXII has been linked to venous and arterial thrombosis as a risk factor for thrombosis, suggesting a role for FXII as a natural anticoagulant (17).

Contact activation components have also been implicated in modulation of complement proteins, monocyte and neutrophil activities, and thrombin-induced platelet activation. This system has also been shown to have counteradhesive properties against platelet and neutrophil adhesion to endothelial cells, as well as modulating angiogenesis, fibrinolysis, and thrombin formation. Thus, biochemical and molecular binding events mediated by these proteins have important physiological effects especially in disease processes, but their roles are very dependent on the given physiological context and hemostatic state.

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 contact activation proteins as fine modulators of blood coagulation whose inhibition does not cause a bleeding diathesis (18). Thus, drug design against FXIIa using the tools of high throughput chemical inhibitor screening may give rise to novel anticoagulant therapies.

HTS was performed using 217,445 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 with Boc-Gln-Gly-Arg-AMC, as first described by Kawabata et al. (19).


1. R. C. Wiggins, C. C. Cochrane, J Exp Med 150, 1122 (Nov 1, 1979).
2. S. D. Revak, C. G. Cochrane, J Clin Invest 57, 852 (Apr, 1976).
3. S. D. Revak, C. G. Cochrane, B. N. Bouma, J. H. Griffin, J Exp Med 147, 719 (Mar 1, 1978).
4. R. A. Pixley, M. Schapira, R. W. Colman, J Biol Chem 260, 1723 (Feb 10, 1985).
5. M. Silverberg, J. Longo, A. P. Kaplan, J Biol Chem 261, 14965 (Nov 15, 1986).
6. S. Schmidt et al., Mult Scler 10, 243 (Apr, 2004).
7. D. Gailani, G. J. Broze, Jr., Science 253, 909 (Aug 23, 1991).
8. K. Naito, K. Fujikawa, Journal of Biological Chemistry 266, 7353 (1991).
9. D. Gailani, Curr Opin Hematol 1, 347 (Sep, 1994).
10. B. N. Bouma, J. H. Griffin, Journal of Biological Chemistry 252, 6432 (1977).
11. K. Fujikawa, M. E. Legaz, H. Kato, E. W. Davie, Biochemistry 13, 4508 (1974).
12. G. van Dieijen, G. Tans, J. Rosing, H. C. Hemker, J Biol Chem 256, 3433 (Apr 10, 1981).
13. J. P. Miletich, C. M. Jackson, P. W. Majerus, Journal of Biological Chemistry 253, 6908 (1978).
14. K. Bailey, F. R. Bettelheim, L. Lorand, W. R. Middlebrook, Nature 167, 233 (Feb 10, 1951).
15. B. Blomback, M. Blomback, Ann N Y Acad Sci 202, 77 (Dec 8, 1972).
16. T. Renne et al., J Exp Med 202, 271 (Jul 18, 2005).
17. W. M. Halbmayer, C. Mannhalter, C. Feichtinger, K. Rubi, M. Fischer, Wien Med Wochenschr 143, 43 (1993).
18. A. Gruber, S. R. Hanson, Curr Pharm Des 9, 2367 (2003).
19. S. Kawabata et al., European Journal of Biochemistry 172, 17 (Feb 15, 1988).

Materials

Human plasma factor XIIa alpha was purchased from Enzyme Research Laboratories (Cat # HFXIIa 1212a). Substrate Boc-Gln-Gly-Arg-AMC was from Bachem (Cat #I-1595.0050). 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).

Assay

Factor XIIa alpha (3.5 ug/mL) was incubated with Boc-Gln-Gly-Arg-AMC substrate (15 uM) in 10 uL of assay buffer (see above) for 2 hr at room temperature.

IC50 protocol

1.Serial dilute single compounds at 50x concentration in DMSO (16 two-fold dilutions from 2.5 mM to 75 nM)
2.Fill low-volume plate with 4 uL water using Multidrop-micro
3.Add 5 uL assay buffer to columns 1 and 23 using Multidrop-384
4.Add 200 nL of compound (in DMSO from step 1) using 2 transfers of 100 nL with the Evolution 384 pintool (washed with isopropanol after each transfer)
5.Add 1 uL of Boc-Gln-Gly-Arg-AMC substrate (150 uM in 5x assay buffer) using Multidrop-micro
6.Add 5 uL enzyme (7.0 ug/mL in assay buffer) using Multidrop-384
7.Incubate for 2 hr at room temperature
8.Read fluorescence (excitation 355, emission 460) on Envision reader

Data analysis

IC50 plates contained compounds in columns 3-22, controls (enzyme, no compound) in columns 2 and 24, and blanks (no enzyme) in columns 1 and 23. Each column 3-22 contained 16 two-fold dilutions of a single compound, ranging in concentration from 50 uM to 1.5 nM. Percent activity was calculated for each dilution of 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:

% Activity = 100*((signal-blank mean)/(control mean-blank mean))

Dose response curves of percent activity were fit using XLfit equation 205 (four parameter logistic fit with maximum percent activity and minimum percent activity fixed at 100 and 0, respectively).

Activity scoring

The activity score reported here is based on follow-up IC50 testing on compounds that showed >40% inhibition in the primary HTS:

IC50 score = sum (IC50 score #1, IC50 score #2, IC50 score #3).

IC50 scores were calculated as follows:

(1) Score = 5.75 x (pIC50-3), where pIC50 = -log(10) of IC50 in mol/L
(2) For IC50 >50 uM, the score was calculated from percent activity at maximum concentration tested in assay (50 uM):
Score = [5.75 x (0-3)] + [(100-percent activity at max concentration)/1.75]

Activity Outcome

Compounds that gave percent inhibition >40% in the primary HTS were judged to be hits and these compounds were selected for follow-up IC50 testing. IC50 values were determined as described in protocol above. The percent activity at the maximum concentration is reported and can be used to estimate the potency of compounds for which the IC50 values were >50 uM.

Activity outcome is reported as follows:

(1) IC50 <50 uM in all three IC50 determinations = active
(2) IC50 <50 uM in only 1 or 2 out of 3 determinations = inconclusive
(3) IC50 >50 uM, percent inhibition 30-50% at 50 uM = inconclusive
(4) IC50 >50 uM, percent inhibition <30% at 50 uM = inactive

Contributors

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.

Grant Number: X01-MH076406-01