Note: Descriptions are shown in the official language in which they were submitted.
213
- 1 -
The invention relates to a procedure for the photo-
metric determination of the activated partial thromboplastin
time tAPTT) and to a reagent sui.able~for this purpose.
Determination of the act;vated partial thrombo-
plastin ti~e (APTT) is, in add;tion to the prothroMb;n
time (thromboplastin t;me, Quick value), the test of coagu-
lation which is carried out most -Frequently. The A?TT per~
mits conclusions to be drawn about the behav;or of the en-
dogenous pathway of coagulation. Another important use of
this test is for monitoring heparin therapyn The APTT is
sens;tive to lligh molecular ~eight kininogen, prekalli-
krein and factors XIIr X~ IXr VIII, V and I~ as well as to
the ;nhibitors of coagulation, especially antithrombin III
under heparin therapy and fibrinogen cleavage products
(antithrombin VI), all of which prolong the hPTT.
Reasents for the determination of the APTT essen-
tially contain phospholip;ds and a suitable activator of
the "contact phase". By contact activation, factor XII is
activatedr and this then activates factor XI and prekalli-
krein. Due to the lipids and calcium ions contained in the
reagent~ activation of the entire endogenous pathl~ay then
occurs, and this terminates in formation of a fibrin clot~
The time equired for this to occur is the variable mea
sured.
Inorganic materials are employed as activators of
the contact phase, preferably celite or kaolin. Ellag1c
- . $
-- 2
acid is aiso used (U.S. Patent 3,486~981 and German Offen-
legungsschrf;t 2,9l5,310). As an optically transparent
reagent, ellagic acid has advantages for use in optical
measur;ng procedures for detect;ng the formation of the
clot. ~owever, according to Bock et al., Biochemistry 20,
7258-7266 (1932), the active species is a complex of me~al
ions and ellagic acid, which is insoluble in water. Another
activator is dextran sulfate.
i;owever, these activators of the contact phase are
non-physiological and are also diff;cult to standardize.
For example, the coagulation time oF a kaolin~activated
APTT depends to a quite considerab;e extent on the particle
size of the act;va~or. Moreover, some coagulation factors
are so strongly adsorbed on surface~active materials of
this type, that the latter are used as adsorbents for the
prepara~ion of the contact factors. However, in addition~
the colnpos-,tion of the phospholip;d contained in a reagen~
of this type is essential for the result~ Furthermore, the
lensth of the preincubation time, after which the actual
reaction leading to the forma-ion of the clot is started by
"recalcification", plays an inportant part, since activated
coagulation factors are inhib;ted by inhibitors in the
plasma, especially antithrombin II~.
SuLfatidcs are regarded as being physiological
activators of coagulation (Fujikawa e~ al., Biochem. 19
(1980), 1~22-133~, and rans and Griffin~ Blood, 59 (19S2),
69-75). Sulfatides belong to the class of glycosphingo-
lipids and they conta;n a suLfate group on the
galactose ring~ Various species belong to this group of
~L~;5~ 13
_ 3
compounds, and ~hey differ in the nature of the fatty acid
chain. They can be detected in all tissues, in the mem-
branes, and in espec;ally large amounts in the bra;n, from
which they can be obta;ned in a very pure form.
Sulfatides are more effective than kaolin for the contact
activation of plasma.
The end point of conventional determinations of
APTT is the measurement of a fibrin clot~ Following the
-ForMation of a clot of this type is possible only with
technical difficulty. Quite a number of devices have been
developed~ and these make use of a variety of mechanical,
electrical or optical procedures.
Since the introduction of chromogenic substrates
for coaguLation factors, attempts have also been made
to emp~oy them for the determinat;on of the coagulation
enzymes. The advantage of chromogenic substrates is the
poss;b;lity of straightforward standardisation of the low
molecular weight substrates~ in contrast to the complexO
h;gh molecular weight natural substrates. The problem of
measuring the -format;on of a clot no longer ar;sesO
Moreover, chromogenic substrates have already been
employed for carrying out "global tests". Thus, Yamada
and Meguro, Thrombos. Res. 15 (1979), 351-358, describe a
method for measur;ng the APTT. Th;s is based on activa-
tion of the endogenous pathway us;ng ellagic ac;d in the
presence of phospholipid~ calcium and the chromogen;c
thrombin substrate H-D-Phe-P;p-Arg-pNA (p 2238). The
d;sad~arltages of this procedure are the use of a non-
physiological activatc,r, the lack of spec;fic;ty of the
3L~ 3
-- 4 --
chromogenic substrate~ and the extremely long measurement
ti~es (normal figure about 7~4 minutes)~ 9nly incomple~e
data has been published on ~he sensitivity to the indivi~
dual cl~tt ing f ~ctors .
Another method is described by P. Aiyappa, Annu
r~ew York ~cad. Sci. (1~1), pages ~12-~21. In this tech-
n;que of measurement~ plasma is activated by ellagic acid
in the presence of phospholipid and calcium ions fcr 5
minutes~ and the thrombin formed in this time is measured
using a chromo~enic substrate~ This method can lead to the
'ormation of ,ibrin, and active thrombin undergoes ;nclusion
;n th;s. Moreover, thromb;n can be further deactivated
by inhibition. On the other hand, it is possible that~
within the selected ;ncubation time, the activation of patho
logical plasmas takes place e;ther not at all or only ;ncom-
pletely, although they are actually capable of this~ even
though slowlyO The procedure described by Aiyappa cannot
be used to measure coagulation in deficient plasmas~
It has now been ~ound~ surprisingly~ that it is
poss;ble to elim;nate the essential disadvantages of the
described APTT methods with chromogenic substrates by
using a sulfatide as the physiological activator in com-
bination with a highly specific thromb;n substrate~
Thus the invention relates to a procedure for the
determination of the activated partial thromboplastin time
using an activator, a coagulation-active phospholip;d or
mixture of phospholipids~ calcium ions and a chromogenic
substrate ,or thrombin, ~Jh,ch comprises the activator
being a sulfa~ide or a mix~ure of sulfatides.
~Z~ 3
The invention also relates to a reagent for the
determination of activated partial thromboplastin time~
comprising a sulfatide, a phospl1olip;d~ a soluble calcium
salt and a chromogen;~c substrate in the freeze-dried form.
U~ilisable sulfatides are commercially available,
for example from Serva, Sigma or Supelco~ It is advan-
tageous tc use sulfatides which have~ on thin~layer chroma-
tography on silica gel~ an RF of 0.2-003~ preferably 0.25,
with a mixture of chloroform/methanol/wa~.er 65:25:4 as the
mobile phase and an RF of 0.25~0.35~ preferably 0.31~
using chloroform/methanol 40:15. However, i~ is also pos-
s;ble to obtain sulfatides of this type by processes which
have been described, for example by Hara and Radin, Anal.
e;ochem. 100, 36~-370 (197~), or from dried acetone extract
of brain~ t~lorthwhile test concentrations are betweerl 0~1 and
5Q ~g/ml. The sulfatide can initially be suspended at a
concentration of 0.01 g/l in 50 mmol/l of ~EPES buffer, pH
7.6, and this suspension used for prepar;ng the reagent.
The phospholipids which can be used are the animal
and veget-7ble lipids employed in conventional reagents~ It
is particularly advantageous to use lipids from human plate-
lets or an extract from human placenta. It is ;mportant
that these lipids have no thromboplastic activity, so that
the exogenous path~lay is not also activated.
The chromogenic substrate for thrombin must be speci~i.c
for this enzyme to be possible tG employ it in this test,
sînce. of course, the intention is to measure thrornbin
selective'y in the presence of the other coagulation
factors which are likew;se activated~ Chromo~yM tr~
~'~5~ 3
TH (Tos-Gly-Pro-Arg-p~A) and S 2160 (Bz-Phe-Val-Arg-pNA~
are relatively non-specific and also indicate other coagu-
lation factors~ such as kallikrein or factors Xa and XIIa.
S 2238 (HD-Phe-Pip-Arg-pNA) is rr,ore suitable, althou~h its
spec;ficity for the contact factors is again not very high.
Apart from para-nitroanilides, peptides having other chromo-
phores are also suitable~ for example, coumarin derivatives~
where tne hydrolysis is followed by measur;ng the fluores-
cence.
The most suitable chrornogenic pep~ides for a global
test, such as APTT, are thrombin substrates sucl7 as are des~
cribed in German Patent Application 3,244,~030.8. The
chromophoric derivative they contain is 5~amino~2~nitro--
henzoic ac;d.
A cornpound of the general formula I
X-P~o -Arg-Ni~-~f-~o2 ( 1 )
C0-NH R
~here R is C1_5-alkyl or -CH~CH(CH3~2~COOC~l3, and
X is H-D-Phe-, Boc- Gly- or Tosyl-Gly-, is preferably ~sed.
The substrate is preferably employed at very low
concentration. The best results are obtained with
50 ~umol/l when H-D-Phe-Pro-Ar~ANB~~isopropylamide is
used. The calcium concentration should be between 1 and
10 mmol/l preferably 5 mmol/l.
The sulfatide, phospholipid, chromogenic substrate
and the calciurn salt, preferably ~a'c1um chloride~ are d;s-
solved or suspended at pH 7.2-~.5 in a bu-ffer~ such as
HEPES or Tris. IrQidazole, glycyl~lycine or triethat1olalnine
buffers can also be used~
The procedure according to the invent;on ;s advan-- -
tas~ously carried out at 25 37C b~ m;x;ng a plasma sample
w;th the reagent at this temperature in a thermostated cell.
The reaction is followed at 405~410 nm in a photometer.
~ 1ith a typical normal plasma the extinction remains
constant for some time and then increases greatly ~lithin a
few seconds. The variable measured is the time necessary
to reach a spec;f;ed d;fference in ext;nct;on, -ror example
0.1. In principle~ k;netic evaluation of the exponentiai
curve is a!so poss;ble~ The reaction of a specified amount
of substrate ;s analogous to that in a conventional APTT~
where the activated thrombin reacts with a specified amount
of fibrinogen and thus induces the clot. Example 1 shows
that the new procedure in the preFerred 'orrn ind;cates
cnanges in the activity of each individual factor ;n tne
endogenous pathway.
In contrast to the convention APTT~ in ~hich~ a,.er a
certain fixed activation time, the actual reaction leading
to the clot is started by add;ng CaCl2, it ;s possible for
CaCl2 to be added From the st3rt -in the new procedure;
and this leads to there being one pipetting step less
(monoreagent). However, in principle~ a test system analo-
gous to the procedure used hitherto is also possible with
the procedure accordirlg to the invention~ With this ob-
jective, the chrornogenic peptide is added~ tc~etller with
CaCl2, after a speci~;ed preincubation time. In this
case~ it has proved to be favourable to add a little CaCl2
(about 1 l~mol/l~-to the sulfatide reagent everl duril~g
æ~3
the pre;ncubat;on time, and to start the actual reaction by
addition of more CaCl2 solution~ The optimal final con
centration in this case is again 5 mmol/l of CaCl2.
The use o-f sulfatides for the contact activation of
plasma, combined with chromogenic substrates, has not hitherto
been disclosedO Manuf2cturers of reagents recommend that
the sulfatides be stored below 0C. rreeze drying
sulfatides together with the other necessary constituents
o-f the reagent does not lead to a stable product. The APTT
is always drastically increased.
Surprisingly~ a stable and effective reagent is
obtained by addition of serurn albumin, ge1atin or degraded arld che~i-
cally crosslinked collage~. A co~c~ntration of only about 0.1-1% ~llows
a lyophilisate of high activity to be produced,
As with conventional reagents~ it is possible, in
combination with deFic;ent plasma to determir)e the indivi~
dual factors of the endogenous pathway. For this purpose,
ttle plasma sample w;ll be d;lu.ed ;n order o eliminate the
effect of the other coagulation factors, or it is ernployed
;n an excess of the deficient plasma used.
Addition of aminoac;ds, especially glu~amine~
asparagine or glutamic acid, increases the sensitivity of
the reagent to heparin.
Abbreviations:
ANBA 5-amino-2-nitrobenzoic acid
Arg Arsinine
Gly Glycine
l-IEPES N-2-hydroxyethylpiperazinyl-N'-2-ethanesulfonic
acid
.,
_ 9 ~
Abrre~iations continued:
Phe Phenylalanine
Pip Pipecolic acid
Pro Proline
Tos Toluenesulfonyl
Tris Tr;s(hydro~ymethyl)aminonlethane
The invention is illustrated by the Example ~hich
follows.
5~2-~
' ~ 10 -
Example :
Factor sensit;vity of the new reagent
Reagent~ 0.1 ml of 3mmol/l H-D-Phe-Pro Arg-ANB~-isopropyl-
~amide chromogen;c substrate (S 82 107)
0.5 ml of Fibraccel R 1:1000 tcoagulation-
active homologous phosphol;p;d com-
plex, Behr;ngwerke AG)
0.5 ~l of sulfatide solution (supplied by
Supelco; RF 0025 with CHCl3/MeOH
H20 6S:25:~ on silica gel) 0~01 g/l
5.0 ml of buffer (HEPES, NaCl, Ca2~: 25, 50,
S mmol/l; pH 7~6~ 0~25% HaemaccelR
tGerman patent 1~11S~7~2 and 1,153,134)
ixture: 100 ~l of plasma or deficient plasma ~Behr;ngwerke),
congen;tal F VII-def;c;ent plasma
1 ml of reagen~
37C, measurement o-F the t;me unt;l E~05 nm = 0~1.
Def;cient plasma Seconds
Factor II 1,200
Factor V 1,200
Factor VIII 378
Factor IX 498
Factor X ~48
Fac'.or XI 396
Factor XII 336
HMWK (high molecular ~eight kin1nogen) 396
Kailikrein 720
Pooled plasma 170
