Method for determining the catalytic activity of factor IXa

A method for the determination of factor IXa in a sample solution using a measurable factor IXa substrate and a water-miscible alcohol and measuring the cleavage of the factor IXa substrate as a measure for factor IXa activity is suitable for the direct determination of factor IXa.

The invention concerns a method for determining the catalytic activity of 
factor IXa. The method according to the invention is suitable for finding 
factor IXa inhibitors (screening), for modulating blood coagulation 
(therapeutic application) and for determining factor IX and factor IXa in 
body fluids (diagnostic application). 
Blood plasma proteases play a role in blood coagulation, wound closure by 
fibrin formation as well as in fibrinolysis i.e. clot lysis. After an 
injury the "injury signal" is amplified by the sequential activation 
(specific proteolysis) of inactive proenzymes to form active enzymes which 
initiates blood coagulation and ensures a rapid wound closure. Blood 
coagulation can be initiated by two paths, the intrinsic path in which all 
protein components are present in the blood and the extrinsic path in 
which a membrane protein, the so-called tissue factor plays a critical 
role. 
The molecular mechanism of blood homeostasis (blood coagulation, 
fibrinolysis and the regulation of this equilibrium) and the components 
that are involved in this are comprehensively described in several review 
articles (Furie, B. and Furie, B. C., Cell 53 (1988) 505-518; Davie, E. W. 
et al., Biochem. 30 (1991) 10363-10379; Bergmeyer, H. U. (ed.): Methods of 
Enzymatic Analysis, Vol. V, chapter 3, 3rd ed., Academic Press, New York 
(1983)). 
The factors of the blood coagulation cascade are very complex proteins. As 
a rule they can only be isolated in a complicated manner from the natural 
raw material source, the blood plasma, in a limited amount, with varying 
quality, homogeneity and purity (Van Dam-Mieras, M.C.E. et al., In: 
Bergmeyer, H. U. (ed.), Methods of Enzymatic Analysis, Vol. V, 3rd ed., 
page 365-394, Academic Press, New York (1983)). They play an important 
role in the regulation of blood homeostasis which is the equilibrium 
between blood coagulation, clot formation and dissolution. This 
well-regulated system can become unbalanced by genetic defects such as 
haemophilia A (defective factor VIII) and haemophilia B (defective factor 
IX). Acute disorders can lead to cardiac infarction, embolism and stroke. 
There is therefore a need for substances which can influence the system of 
blood coagulation and fibrinolysis according to the medical requirements. 
For example from blood isolated or recombinantly produced factor VIII or 
factor IX are used to treat haemophilia A and B. tPA (tissue type 
plasminogen activator) and streptokinase (bacterial plasminogen activator) 
are used for example for clot lysis e.g. after cardiac infarction. In 
addition to complex proteins, substances such as hirudin (peptide composed 
of 65 amino acids, thrombin inhibitor), heparin (heteroglycan, cofactor of 
endogenous inhibitors) and vitamin K antagonists (inhibitors of 
.gamma.-carboxylation of Glu residues of the GlA domain) are also used to 
inhibit blood coagulation. However, the available substances are often 
still very expensive (protein factors) and not ideal with regard to their 
medical application (side effects) so that there is a need for medicaments 
which can be used to specifically modulate blood coagulation and clot 
lysis. 
The search for new modulators (activators, inhibitors) of blood 
coagulation, fibrinolysis and homeostasis can for example be carried out 
by screening substance libraries and subsequently improving an identified 
lead structure by drug modelling. For this it is necessary that i) a 
suitable test and ii) the key protein(s) [target(s)] are available in an 
adequate amount and quality for screening and for crystal structure 
investigations (e.g. improvement of the lead structure by the specific 
prediction of changes based on the 3D structure of the protein component 
and lead structure). 
Factor IXa (FIXa) is an interesting target for an inhibitor screening in 
order to find inhibitors to modulate blood coagulation. The known clinical 
picture of haemophilia B (factor IXa defect) warrants the assumption that 
specific factor IXa inhibitors are superior to known thrombin inhibitors 
with regard to the quite considerable pleiotropic side-effects. 
Previously, screening for FIXa inhibitor activity has failed due to the 
availability and extremely low catalytic activity of FIXa. 
The isolation of the inactive serine protease FIX (zymogen) from blood 
plasma and the subsequent activation by proteolysis is difficult, 
time-consuming, expensive and often does not yield the desired amount and 
quality. Thus the plasma concentration of the inactive protease zymogen 
FIX is only 0.5 mg/l (Furie, B. and Furie B. C., Cell 53 (1988) 505-518). 
Moreover the protease preparations isolated from the plasma and activated 
in vitro are often very heterogeneous and unstable. 
The inactive FIX zymogen can for example be activated/converted using 
purified FXIa (Van Dam-Mieras, M.C.E.; Muller, A. D.; van Dieijen, G.; 
Hemker, H. C.: Blood coagulation factors II, V, VII, VIII, IX, X and XI: 
Determination with synthetic substrates. In: Bergmeyer, H. U. (ed.): 
Methods of Enzymatic Analysis, Vol. V, Enzymes 3: Peptidases, Proteinases 
and Their Inhibitors, page 365-394, 3rd ed., Academic Press, New York 
(1983)). 
Blood plasma proteases are complex glycoproteins that belong to the serine 
protease family. They are synthesized in the liver as inactive proenzymes 
(zymogens), secreted into the blood and are activated when required by 
specific proteolysis i.e. by cleavage of one or two peptide bonds. They 
are structurally very similar with regard to the arrangement of their 
protein domains and their composition (Furie, B. and Furie, B. C., Cell 53 
(1988) 505-518). 
According to Furie B. and Furie, B. C. the proteases of the factor IX 
family (factor VII, IX, X and protein C) are composed of 
a propeptide, 
a GLA domain, 
an aromatic amino acid stack domain, 
two EGF domains (EGF1 and EGF2), 
a zymogen activation domain (activation peptide, AP) and 
a catalytic protease domain (CD). 
Furthermore the blood plasma proteases are post-translationally modified 
during secretion: 
11-12 disulfide bridges 
N- and/or O-glycosylation (GLA domain and activation peptide) 
Bharadwaj, D. et al., J. Biol. Chem. 270 (1995) 6537-6542 
Medved, L. V. et al., J. Biol. Chem. 270 (1995) 13652-13659 
cleavage of the propeptide 
.gamma.-carboxylation of Glu residues (GLA domain) 
.beta.-hydroxylation of an Asp residue (EGF domains) 
cleavage of the zymogen region (partially) 
After activation of the zymogens (zymogenic form of the protein) by 
specific cleavage of one or two peptide bonds (cleavage of an activation 
peptide), the enzymatically active proteases are composed of two chains 
which, in accordance with their molecular weight, are referred to as the 
heavy and light chain. In the factor IX protease family the two chains are 
held together by an intermolecular disulfide bridge between the EGF2 
domain and the protease domain. The zymogen-enzyme transformation 
(activation) leads to conformation changes within the protease domain. 
This enables an essential salt bridge necessary for the protease activity 
to form between the N-terminal amino acid of the protease domain and an 
Asp residue within the protease domain. The N-terminal region is very 
critical for this subgroup of serine proteases and should not be modified. 
Only then is it possible for the typical active site of the serine 
proteases to form with the catalytic triad composed of Ser, Asp and His 
[Blow, D. M.: Acc. Chem. Res. 9 (1976) 145-152; Polgar, L.: In: Mechanisms 
of protease action. Boca Raton, Fla., CRC Press, chapter 3 (1989)]. 
Blood plasma proteases can be produced in a classical manner by isolating 
the inactive zymogens from the blood and subsequently activating them or 
they can be produced recombinantly by expressing the corresponding cDNA in 
a suitable mammalian cell line or in yeast. 
The production of coagulation factors by expression/secretion of the 
zymogens or active proteases by means of eukaryotic host/vector systems is 
described for FvII: Hagen, F. S. et al., EP 0 200 421; Pedersen, A. H. et 
al., Biochem. 28 (1989) 9391-9336; FIX: Lin, S.-W. et al., J. Biol. Chem. 
265 (1990) 144-150; FX: Wolf, D. L. et al., J. Biol. Chem. 266 (1991) 
13726-13730, Protein C: Bang, N. U. et al., EP 0 191 606. 
As a rule host cells are used which are able to post-translationally modify 
the coagulation factors like the native enzyme during the secretion 
process. The zymogen-enzyme transformation is then carried out 
subsequently during the downstream processing e.g. by using an activator 
from snake venom in the case of prothrombin or factor X (Sheehan, J. P. et 
al., J. Biol. Chem. 268 (1993) 3639-3645; Fujikawa, K. et al. Biochem. 11 
(1972) 4892-4898). 
For the purpose of zymogen-enzyme activation in vivo (already during 
secretion), the natural zymogen cleavage sites or the entire activation 
peptide were substituted by protease cleavage sites (several adjacent 
basic amino acids) which can be cleaved by specifically cleaving proteases 
that occur naturally in the secretion path of the host cell such as e.g. 
Kex2 (yeast) or E (mammnalian cell lines). (FX: Wolf, D. L. et al., J. 
Biol. Chem. 266 (1991) 13726-13730; Prothrombin: Holly, R. D. and Foster, 
D. C., WO 93/13208). 
The production of variants of coagulation factors (FX: Rezaie, A. R: et 
al., J. Biol. Chem. 268 (1993) 8176-8180); FIX: Zhong, D. G. et al., Proc. 
Natl. Acad. Sci. USA 91 (1994) 3574-3578), mutants (FX: Rezaie, A. R. et 
al., J. Biol. Chem. 269 (1994) 21495-21499; Thrombin: Yee, J. et al., J. 
Biol. Chem. 269 (1994) 17965-17970); FVII: Nicolaisen, E. M. et al., WO 
88/10295) and chimeras e.g. composed as FIX and FX (Lin, S.-W. et al., J. 
Biol. Chem. 265 (1990) 144-150; Hertzberg, M. S. et al., J Biol. Chem. 267 
(1992) 14759-14766) by means of eukaryotic host/vector systems is also 
known. 
However, expression in eukaryotic mammalian cell lines is time-consuming, 
limited with regard to expression output and expensive. In addition 
undesired post-translational modifications can occur. 
The production of blood plasma proteases by expression in prokaryotes and 
subsequent renaturation of the expression product is described by 
Thogersen, H. C. et al. (WO 94/18227). According to this FX variants are 
renatured by means of a cyclic renaturation process in which the inactive 
FX protein is immobilized in a chromatographic column by means of a metal 
chelate complex (poly(His)-affinity handle). A fusion protein is used for 
this composed of a truncated FX variant (EGF1, EGF2 and protease domain), 
an additional FXa protease recognition sequence and an attachment aid at 
the C-terminus of the catalytic domain composed of 6 histidine residues. 
Surprisingly it was found that the catalytic activity of factor IXa with 
respect to substrates can be stimulated by alcohols and hence a very 
sensitive factor IXa test can be constructed in a simple manner. 
Consequently the invention concerns a method for determining factor IXa in 
a sample solution characterized in that a determinable factor IXa 
substrate and an alcohol that is homogeneously miscible with water and 
forms one phase is added to the sample solution and the cleavage of the 
factor IXa substrate is determined as a measure for the factor IXa 
activity. It surprisingly turned out that the catalytic factor IXa 
activity can be increased by more than 20 times by alcohols. As a result 
it is possible to directly determine the factor IXa activity in sample 
solutions. Factor IX can for example be determined in sample solutions, 
preferably body fluids such as plasma after activating factor IX to factor 
IXa by means of Russels viper venom or by the protease isolated from the 
snake venom (RVV-X protease). 
A further subject matter of the invention is the use of the determination 
method according to the invention for factor IXa to screen for substances 
which modulate (inhibit or activate) factor IXa activity. Consequently the 
invention concerns a method for identifying a substance which modulates 
the activity of factor IXa characterized in that 
a) a factor IXa substrate is cleaved by a polypeptide with factor IXa 
activity at a defined concentration in the presence of an alcohol and the 
rate of cleavage of the said substrate is determined as a measure for the 
factor IXa activity, 
b) the said activity is measured in the presence of a test substance, 
c) the activity is compared with and without the test substance and 
d) the difference of the activity is used as a measure for the activity 
modulation by the test substance. 
The inhibition of factor IXa by a test substance is preferably tested. 
Suitable factor IXa substrates are for example factor IXa substrates that 
are described in EP-B 0 034 122. Substrates of the R-Xxx-Gly-Arg-pNA type 
are especially suitable in which Xxx represents a hydrophobic D-amino acid 
and pNA represents a determinable leaving group. R is defined analogously 
to EP-B 0 034 122. Substrates of the general formula I from EP-B 0 034 122 
in which R.sup.3 =R.sup.4 =H are preferred. 
Preferred substrates are tripeptides with a hydrophobic D-amino acid at the 
N-terminus and preferably a cyclohexyl-substituted hydrophobic D-amino 
acid is used. Cyclohexyl-glycine and cyclohexylalanine are particularly 
preferred. 
Further preferred substrates are for example substrates which are also 
cleaved by factor Xa such as e.g. Chromozym X (Boehringer Mannheim GmbH, 
Moc-D-Nle-Gly-Arg-pNA) or Pefachrom tPA (Pentapharm Ltd., Basel, 
MeSO.sub.2 -D-HHT-Gly-Arg-pNA). Commercial (preferably chromogenic or 
fluorogenic) substrates that can be determined optically are preferably 
used. The latter are chromogenic peptides/substrates with a cleavable 
residue that can be easily determined by optical means (the p-nitroaniline 
residue is preferred). The method according to the invention is preferably 
carried out in a buffer solution. All buffers that are effective in a pH 
range of 7-10 (preferably between pH 7.5-9.0) can be used as buffer 
substances. Tris buffer, triethanolamine and Tris-imidazole buffer are 
preferred. 
The determination of FIXa activity and the screening assay are preferably 
carried out at 20-40.degree. C., particularly preferably at room 
temperature and the amount of the cleaved optically determinable group is 
determined photometrically or fluorometrically. The absorbance at 405 nm 
is preferably determined. The enzymatic activity and the kinetic constants 
are determined from the linear initial slope according to the 
Michaelis-Menten equation. 
Factor IX or the ratio of factor IX to factor IXa can be determined 
analogously after activation of factor IX to factor IXa. In order to 
determine factor IX in plasma, factor IX is firstly activated to factor 
IXa by Russels viper venom (preferably 0.2 mg/ml) or RVV-X protease 
(preferably 0.1 mg/ml) in the presence of CaCl.sub.2 (10 mmol/l) at 
20-40.degree. C., preferably at 37.degree. C. After the activation is 
completed (preferably 15 min), ethylene glycol and the cleavable substrate 
are added at concentrations of 20-40% and 0.2-1 mmol/l respectively. 
It has proven to be advantageous for a screening test to add factor IXa at 
a concentration of 0.02-0.5 .mu.mol/l, preferably 0.05 .mu.mol/l and the 
cleavage substrate at a concentration of 0.2-1 mmol/l with a test 
substance concentration in the .mu.mol/l range. Ethylene glycol, ethanol 
or methanol is preferably used as the alcohol. The concentration of the 
alcohol is preferably in the range of 20-40%. Native human factor IXa or 
porcine or bovine factor IXa or recombinantly produced human factor IXa 
can be used as factor IXa. A truncated human recombinant factor IXa that 
is described in EP 96 109 288.9 is particularly preferably used. 
Enzymatically active recombinant factor IXa can be produced by expression 
of a corresponding DNA in prokaryotes, renaturation of the expression 
product and enzymatic cleavage if it is composed of a FIXa serine protease 
domain (catalytic domain) N-terminally linked to an EGF domain (EGF1 
and/or EGF2) and a zymogen activation domain. 
A non-glycosylated, enzymatically active factor IXa composed of the 
following domains is preferably used: 
a) the catalytic FIXa protease domain, N-terminally linked with 
b) a zymogen activation domain, N-terminally linked with 
c) an EGF1 and/or EGF2 domain (preferably EGF2 or EGF1 and EGF2). 
A spacer with up to 50 amino acids is preferably inserted between the 
zymogen activation domain and the EGF domain (or the EGF domains). When 
the zymogenic one chain form according to the invention is cleaved in the 
zymogen activation domain, an active protein is obtained in a two chain 
form. Both chains are linked by an intermolecular disulfide bridge in the 
two chain form. The proteins according to the invention are preferably 
composed of the EGF2 domain, the activation peptide and the catalytic 
domain of factor IX. Such factor IX muteins are described in the European 
Patent Application No. 96 110 959.2. 
Any plasma can be used in the determination method according to the 
invention and citrate plasma is preferred. 
The method can be carried out at neutral or weakly alkaline pH values 
preferably in the pH range between 7.5 and 9.0. Physiologically acceptable 
buffers that are effective in this range can be used as the buffer. In 
addition common stabilizers and preservatives for coagulation tests such 
as bovine serum albumin, merthiolate and such like can also be added. 
In addition the method according to the invention and the reagent according 
to the invention can also contain a surfactant, preferably a non-ionic 
surfactant such as Tween 80.RTM.. In this case the concentration is 
preferably 0.01-1 vol. %. 
A further subject matter of the invention is a reagent for the 
determination of factor IXa which contains an optically measurable 
substrate that can be cleaved by factor IXa, alcohol and buffer in a range 
between pH 7 and 10. 
The following examples, publications, the sequence protocol and the figures 
further elucidate the invention, the protective scope of which results 
from the patent claims. The described methods are to be understood as 
examples which also still describe the subject matter of the invention 
even after modifications.