Thrombin 1536 HTS
Prothrombin, a 72 kDa blood zymogen (plasma concentration = 2 uM, (1)) is converted to thrombin by factor Xa (FXa) in the prothrombinase complex on platelets by cleavage of R271 and R320. Thrombin then further processes itself by cleavage at R155 and R284 in order to remove prothrombin fragment 1 and fragment 2, which contain the kringle 1 and 2 domains, leaving fully active alpha-thrombin (2). more ..
BioActive Compounds: 557
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
Prothrombin, a 72 kDa blood zymogen (plasma concentration = 2 uM, (1)) is converted to thrombin by factor Xa (FXa) in the prothrombinase complex on platelets by cleavage of R271 and R320. Thrombin then further processes itself by cleavage at R155 and R284 in order to remove prothrombin fragment 1 and fragment 2, which contain the kringle 1 and 2 domains, leaving fully active alpha-thrombin (2). alpha-thrombin goes to activate other coagulation factors that are involved in its own production, including the cofactors factor V and factor VIII, as well as activating platelets through the protease activated receptor-1 (PAR-1) (3).
With respect to formation of the secondary blood clot, alpha-thrombin catalyzes activation of fibrinogen to fibrin, cleaving peptides 14-16 amino acids in length, called fibrinopeptides, from the Aalpha and Bbeta subunits of fibrinogen (4). Fibrinogen (plasma concentration = 10.6 uM (5)) is a dimeric protein consisting of three pairs of disulfide-bonded polypeptide chains, referred to as Aalpha, Bbeta, and gamma, which form a complex of a total MW of approximately 340 kDa (6).
Specifically, the sites uncovered by the release of the fibrinopeptides, which are located in central regions of their respective chains, allow for noncovalent multimer formation of fibrin polymers by interactions with complementary central and outer regions of other fibrin monomers, forming an extensive fibrin meshwork. In this way alpha-thrombin produces a fibrin clot that is stabilized by the crosslinking activity of FXIIIa, which is also activated by alpha-thrombin from its inactive zymogen, factor XIII (7). The fibrin monomers produced by alpha-thrombin form a noncovalent meshwork with other fibrin molecules to produce a fibrin clot that stabilizes the primary platelet plug already in place at the site of injury, and reduces the volume of the plug in order to arrest blood loss (8).
HTS was performed using 217,350 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). This assay was performed as a counterscreen to compare with a separate HTS campaign to isolate inhibitors of the strong protein-protein interactions among fibrin monomers responsible for fibrin gel formation. Since any inhibitors of the proteolytic activity of alpha-thrombin would inhibit an assay to detect fibrin formation, this counterscreen will identify direct alpha-thrombin inhibitors. The assay used to test for percent inhibition was a fluorescence assay utilizing hydrolysis of Boc-Val-Pro-Arg-AMC, as first described by Morita, et al. (9).
1. F. C. McDuffie et al., Thromb Res 16, 759 (1979).
2. T. Morita, C. M. Jackson, J Biol Chem 261, 4015 (Mar 25, 1986).
3. E. Di Cera, Q. D. Dang, Y. M. Ayala, Cell Mol Life Sci 53, 701 (Sep, 1997).
4. K. Bailey, F. R. Bettelheim, L. Lorand, W. R. Middlebrook, Nature 167, 233 (Feb 10, 1951).
5. Y. Takeda, J Clin Invest 45, 103 (Jan, 1966).
6. H. A. Scheraga, Biophys Chem 112, 117 (Dec 20, 2004).
7. J. E. Folk, S. I. Chung, Adv Enzymol Relat Areas Mol Biol 38, 109 (1973).
8. L. L. Shen, J. Hermans, J. McDonagh, R. P. McDonagh, M. Carr, Thromb Res 6, 255 (Mar, 1975).
9. T. Morita, H. Kato, S. Iwanaga, K. Takada, T. Kimura, J Biochem 82, 1495 (Nov, 1977).
Human alpha-thrombin was purchased from Haematologic Technologies Inc. (Cat #HCT-0020). Substrate Boc-Val-Pro-Arg-AMC was from Bachem (Cat #I-1120.0050). Assay buffer consisted of 50 mM Tris, pH 7.5, 150mM NaCl, 0.05% Tween 20. 1536-well black plates were from Corning (Item #3728).
alpha-thrombin (5.5 ng/mL) was incubated with Boc-Val-Pro-Arg-AMC substrate (15 uM) in 5 uL of assay buffer (see above) for 30 min 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 = 182
Median Z-factor = 0.80
Median control percent CV = 5.89
Maximum control percent CV = 17.60
A hit cut-off of 40% inhibition was selected. Based on this cutoff, a hit rate of 0.26% was observed.
This assay was submitted to the PCMD by Scott Diamond, assay development and HTS were conducted by Paul Riley and Abhishek Phatarphekar, and data were submitted by Paul Riley, Abhishek Phatarphekar, and Andrew Napper all of the University of Pennsylvania.
** Test Concentration.
Data Table (Concise)