Factor XIIa Mixture HTS
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 more ..
BioActive Compounds: 94
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 62,107 compounds of the MLSCN library combined as mixtures of 10 orthogonally pooled compounds totaling 2.5 mM, which were diluted 50-fold into 10 ul 384 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).
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200 plates containing 64,000 compounds from the MLSCN library were arranged in two 10 x 10 grids of 100 plates each. Sets of ten plates were pooled as follows to give 20 mixture plates per 100 single compound plates:
Plate 1_2_3_4_5_6_7_8_9_10 pooled to mixture plate 1-10
Plate 11_12_13_14_15_16_17_18_19_20 pooled to mixture plate 11-20
Plate 21_22_23_24_25_26_27_28_29_30 pooled to mixture plate 21-30
Plate 31_32_33_34_35_36_37_38_39_40 pooled to mixture plate 31-40
Plate 41_42_43_44_45_46_47_48_49_50 pooled to mixture plate 41-50
Plate 51_52_53_54_55_56_57_58_59_60 pooled to mixture plate 51-60
Plate 61_62_63_64_65_66_67_68_69_70 pooled to mixture plate 61-70
Plate 71_72_73_74_75_76_77_78_79_80 pooled to mixture plate 71-80
Plate 81_82_83_84_85_86_87_88_89_90 pooled to mixture plate 81-90
Plate 91_92_93_94_95_96_97_98_99_100 pooled to mixture plate 91-100
Plates were also pooled 'vertically':
Plates 1, 11, 21, 31, 41, 51, 61, 71, 81, 91 pooled to mixture plate 1-91
Plates 2, 12, 22, 32, 42, 52, 62, 72, 82, 92 pooled to mixture plate 2-92
And so on until mixture plate 10-100.
For example, the single compound in well A3 in Plate 1 is mixed with 9 compounds in well A3 in mixture plate 1-10, and also with 9 different compounds in well A3 in mixture plate 1-91.
The concentration of each mixture was 2.5 mM in DMSO (250 uM per compound). Pintool transfer into the HTS assay gave a final mixture concentration of 50 uM in 2% DMSO (5 uM per compound).
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).
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. HTS was performed using 50 uM compound mixture (5 uM each of 10 compounds).
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 mixture (2.5 mM in DMSO) using Evolution pintool
4.Add 1 uL of with Boc-Gln-Gly-Arg-AMC substrate (150 uM in 5x assay buffer) using Multidrop-micro
5.Add 5 uL enzyme (7.0 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 contained compound mixtures (50 uM in 2% DMSO) in columns 3-22, controls (enzyme, no compound mixture) in columns 2 and 24, and blanks (no enzyme) in columns 1 and 23. HTS percent inhibition was calculated for each compound mixture 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)))
Assignment of percent inhibition to individual compounds---Mixture percent inhibition data were retrieved in IDBS SARgen, together with the 10 compounds contained in each mixture. The data were rearranged in Excel using VLookup functions such that each row contained a single compound and the two percent inhibition values corresponding to the location of the compound in the mixture plates.
Activity scoring is complicated by the fact that the two percent inhibition values associated with each compound in fact represent two different mixtures of 10 compounds. Thus simple averaging of the two values is not meaningful. Instead each compound was assigned a percent inhibition score based on the lower of the two percent inhibition values. This system appropriately scores hits showing activity in both mixtures, but avoids assignment of an erroneously high score to inactives that shared one location with an active compound.
The activity score reported here is based on percent inhibition observed in the primary HTS (see above):
Percent inhibition scores were calculated from the lower of the two percent inhibition values 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)