Note: Descriptions are shown in the official language in which they were submitted.
S012821J.31
~gent for neutralising the tumour cell-associated
procoaqulant activity
The present invention relates to the use of an
anticoagulant protein from the family of annexines as an
agent for preventing the metastasis of tumours.
The formation of metastases has proved to be a
particular problem in tumour diseases. The mechanism of
this phenomenon, referred to as metastasizing, would
appear to proceed as follows-
tumour cells swamp the circulatory system of the
organism (intravasculation) and are carried in the
bloodstream. These malignant cells interact with the
platelets and the plasma coagulation factors and
activate the haemostatic system. The surface-dependent
procoagulant activity known of tum;our cells results in
the formation of thrombin and finally the fibrin
polymer. Moreover, these same cells also interact with
other host cells. Then the tumour cells surrounded by
clumps of platelets and threads of fibrin become lodged
in capillary vessels. As a result of the formation of
the fibrin web around the surface of the tumour cells
specific tumour antigens are concealed, which would
otherwise cause an immunological reaction resulting in
the destruction of the tumour cells. It is there~ore
precisely this ability of the tumour cell to surround
itself with a fibrin network which is responsible for
the metastatic activity of these cells. Tumour enzymes
then attack the vascular wall proteolytically, enabling
the tumour cells to leave the circulatory system
(extravasculation); secondary tumours are produced.
Clearly, the crucial factor of this mechanism is
the ability of the circulating tumour cells to cause
formation of a fibrin net which serves to lodge the cell
in the capillary vessel and/or escape from the immune
-- 2
defence of the organism. Falanga et al. (Biochemistry
24, 5558-5567 (1985) isolated and characterised a
special cancer procoagulant which was capable of
triggeriny coagulation by activating factor X. Other
cancer procoa~ulants which directly activate factor X
have also been isolated. A summary of the research in
the field of activating the coagulation system by tumour
cells is found in Cancer ~etastasis Reviews 3, 99-116
(1984).
The aim of the present invention was therefore to
provide an active substance directed against the
procoagulant activity of tumour cells.
The formation of fibrin is the last step in a
cascade of enzymatic reactions in which thrombin is
formed, the thrombin finally converting fibrinogen into
~ibrin. Various procoagulant reactions such as, for
example, the activation of prothrombin by factors Xa and
Va are catalysed by phospholipid surfaces to~which~the
clotting factors bind. Not every kind of phospholipid
is capable of stimulating coagulation. The charge on
the phospholipid surface appears to determine the degree
of influence. Negatively charged phospholipids such as
phosphatidylserine have a high procoagulatory effect.
Among the proteins which bind to phospholipids and
interfere with processes dependent on phospholipid
surfaces, there is a family which is Ca2+-dependent in
its bonding to phospholipids.
This family, which is also known as the annexines,
includes in addition to lipocortin I, calpactin I,
protein II, lipocortin III, p67-calelectrin, the
vascular anticoagulant protein (VAC) and IBC, PAP~
PAP I, PP4, endonexine II and lipocortin V. Derivatives
of these proteins also have these properties.
The structural features common to the annexines are
probably the reason for their similar Ca2+ and
phospholipid binding properties. Although this general
quality applies to all annexines, there is a clear
~ ?~
individuality in terms of ~heir affinity ~or Ca2+ and the
various kinds of phospholipids.
The physiological ~UnCtiQnS of the annexines are
concerned with membrane-associated processes. The basic
mechanism of the coagulation-inhibitiny effect of VAC
has been recognised as the inhibition of the catalytic
capacity of phospholipids as a result of the binding of
VAC to their surface, thereby preventiny the formation
of the clot-promoting complex on their surface.
Other annexines are capable of inhibiting blood
clottiny, but VAC appears to be the most effective
inhibitors.
Surprisingly, it has been found that annexines,
especially VAC, inhibit the procoagulant activity of
tumour cells.f
The neutralising effect of annexines on the
procoagulant activity has been demonstrated on various
colon-tumou~ cells. The procoagulant activity of a
certain amount of colon cells was inhi~ited by VAC~ in
dosage-dependent manner (Fig. 2). From the correlation
between the number of cells and the clotting time
(Fig. 1) it has been calculated that 210 nM VAC~
neutralise 99% of the procoagulant activity of 6X106
cells/ml. When colon cells neutralised by VAC~ were
centrifuged in the presence of Ca2+ and then washed with
T~SA, they showed no procoagulant activity in the
coagulation assay. If, however, the liquid contained
EDTA instead of Ca2+, the cells showed normal
procoagulant activity.
These results indicate that VAC~ neutralises the
procoagulant activity associated with tumour cells by
binding, Ca2+-dependently, to surface sites which play a
crucial role in this procoagulant activity. Very
probably, phospholipids also participate in the
procoayulant activity associated with tumour cells
since, up till now, no other surface receptors have been
found for VAC~.
Thus, VAC~ inhibits the procoa~ulant activity of
tumour cells by being bouncl to the sites on the surfaces
of tumour cells which participate in the initiation
and/or propagation of the reactions which lead to
thrombin and finally to the formation of fibrin. Since
not only V~C~ but also the other annexines bind to
phospholipids in the Ca2`~~dependent manner, the results
obtained with VAC~ should also be applicable to these
other annexines.
Owing to the fact that the annexines inhibit the
procoagulant activity inherent in the tumour cells, they
prevent the encapsulation of the tumour cells in a
fibrin net; an absolute necessity for the metastatic
activity of the tumour cells. Because of the
neutralising effect the annexines are therefore capable
of preventing the metastasizing of tumour cells and are
consequently potent active substances with an anti-
metastatic activity. VAC is a~particularly suitable
active substance of this kind.
The objective of the invention is therefore
achieved by the use of an anticoagulant protein selected
from the family of annexines.
The active substances which can be used according
to the invention, particularly V~C, may occur not only
in the free form but also in the form of their salts,
particularly the pharmaceutically acceptable salts.
Since they contain a number of amino acid groups with
free amino groups, the compounds according to the
invention may,~for example, occur in the form of acid
addition salts. The acid addition salts may be, in
particular, physiologic211y acceptable salts with
conventional, therapeutically acceptable acids; examples
of inorganic acids include the hydrohalic acids such as
hydrochloric acid, but also sulphuric and phosphoric or
pyrophosphoric acid; examples of organic acids include,
in particular, sulphonic acids such as benzene- or p-
toluene-sulphonic acid or lower alkanesulphonic acids
such as methanesulphonic, as well as carboxyllc acids
such as acetic, lactic, palmitic and stearic acid,
malic, tartaric, ascorbic and citric acid. Since the
compounds also contain amino acid groups having free
carboxyl yroups, they may also occur as metal salts,
especially alkali metal or alkaline earth metal salts,
e.g. sodium, calcium or magnesium salts, or as ammonium
salts, derived from ammonia or a physiologically
acceptable, organic nitrogenous base~ However, since
they also contain free carboxyl groups and free amino
groups, they may also occur as an internal salt.
The agents accordinq to the invention may be
administered, for example, by parenteral route, e.g.
intravenously, intracutaneously, subcutaneously or
intramuscularly, or topically.
The size of the individual doses and the dosage
plan can best be determined by individual assessment of
the particular case: the methods required to determine
the relevant factors are well known to those skilled in
the art. In normal circumstances, for injection, the
therapeutically effective amount of the drug according
to the invention will be within the range from about
0.005 to about 0.5 mg/kg of body weight. The range from
about 0.01 mg/kg to about 0.1 mg/kg, more especially
from about 0.01 to about 0.05 mg/kg of body weight, is
particularly preferred. The drugs are administered by
intravenous, intramuscular or subcutaneous injection.
Accordingly, pharmaceutical preparations for parenteral
administration in the form of single doses contain about
0.4 to about 7.5 mg of active substance per dose,
depending on the route of administration. The
pharmaceutical compositions according to the invention
may also contain in addition to the active substances a
buffer, e.g. a phosphate buffer, intended to maintain
the pH at between about 3.5 and ~, as well as sodium
chloride, mannitol or sorbitol to achieve an isotonic
solution. They may occur in freeze-dried or dissolved
form, whilst solutions may contain an antibacterially
active preservative, e.g. 0.2 to 0.3% methyl or ethyl 4-
hydroxybenzoate. A preparation for topical use may take
the form of an aqueous solution, lotion or gel, an oily
solution or suspension, or a greasy or, more
particularly, emulsified ointment. A preparation in the
form of an aqueous solution may be obtained, for
example, by dissolving the active substances according
to the invention in an aqueous buffer solution at pH 4
to 6.5 and, if necessary, adding another active
substance, e.g. an antiinflammatory agent, and/or a
polymeric adhesive agent, e.g. polyvinylpyrrolidone,
and/or a preservative. The concentration of active
substance is about 0.1 to about 1.5 mg, preferably 0.25
to 1.0 mg, in 10 ml of a solution or 10 g of a gel.
An oily preparation for topical use is obtained,
for example, by suspending the active substances
according to the invention in an oil, optionally with
the addition of swelling agents such as aluminium
stearate and/or surface-active agents (surfactants), the
HLB (hydrophilic-lipophilic balance) of which is less
than 10, such as fatty acid monoesters of polyhydric
alcohols, e.g. glycerol monostearate, sorbitan
monolaurate, sorbitan monostearate or sorbitan
monooleate. A greasy ointment is obtained, for example,
by suspendiny the active substances according to the
invention in a spreadable grease base, optionally with
the addition of a surfactant having an HLB of less than
10. An emulsified ointment is obtained by triturating
an aqueous solution of the active substances according
to the invention in a soft, spreadable grease base with
the addition of a surfactant having an HLB of less than
10. The topical preparations may also contain
preservatives. The concentration of the active
substance is 0.1 to 1.5 mg, preferably 0.25 to 1.0 mg,
in about 10 g of base.
The invention relates to an agent selected from the
2 ~ d ,~
-- 7
group of annexine~, and the natural.ly occur-~ing
derivatives or analoyues thereof or those prepared
synthetically or by genetlc enyineering, optionally with
excipients and/or carriers and/or stabilisers for
neutralising the tumour cell-associated procoayulant
activity;
agent for neutralising the tumour cell-associated
procoagulant activity, characterised in that the
annexine corresponds to the formula
1 5 10 15
Met Ala Gln Val Leu Arg Gly Thr v21 Thr ASp Phe Pro Gly Plle
3v
Asp Glu Arg Ala Asp Ala XX Thr Leu Arg Lys Ala Met Lys Gly
~ 40 . 45
Leu Gly Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg
Ser Asn Ala Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu
Phe Gly Arg Asp Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly
Lys Phe Glu Lys Leu Ile Val Ala Leu Met Lys Pro Ser Arg Leu
100 105
Tyr Asp Ala Tyr Glu Leu Lys His Ala Leu Lys Gly Ala Gly Thr
110 115 120
Asn Glu Lys Val Leu Thr Glu Ile Ile Ala Ser Arg Thr Pro Glu
~ s~,~ r~
125 130 135
Glu Leu Arg Ala l:le Lys Gln Val Tyr Glu Glu Glu Tyr Gly Ser
140 145 150
Ser Leu Glu Asp Asp Val Val Gly Asp Thr Ser Gly Tyr Tyr Gln
155 160 165
Arg Met Leu Val Val Leu Leu Gln Ala Asn Arg Asp ?ro Asp Ala
170 175 180
Gly Ile Asp Glu Ala Gln Val Glu Gln Asp Ala Gln Ala Leu Phe
185 190 195
Gln Ala Gly Glu Leu Lys Trp Gly Thr~Asp Glu Glu Lys Phe Ile
200 ~ 205 210
Thr Ile Phe Gly Thr Arg Ser Val Ser H~is Leu Arg Lys Val Phe
215 220 225
Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr Ile
230 235 240
Asp Arg Glu Thr Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala Val
245 250 255
Val Lys Ser Ile Arg Ser Ile Pro Al.a Tyr Leu Ala Glu Thr Leu
260 265 270
Tyr Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr Leu Ile
275 280 285
Arg Val Met Val Ser Arg Ser Glu Ile Asp Le~ Phe Asn Ile Arg
290 295 300
Lys Glu Phe Arg Lys Asn Phe Ala Thr Ser Leu Tyr Ser Met Ile
~JL~ et
305 310 315
Lys Gly Asp Thr Ser Gly Asp Tyr L~s Lys Ala Leu Leu Leu Leu
320
Cys Gly Glu Asp Asp *
wherein XX represents Glu or Asp and optionally at
position 1 the methionine is cleaved and the alanine at
position 2 is optionally blocked and/or there may
optionally be aggregations caused by intermolecular
disulphide bridges, for example, between the cysteines
at position 316, or the biologically active variants or
derivatives thereof, optionally with excipients and/or
carriers and/or stabilisers.
The present invention also rel.ates to:
an anticoagulant~;selected from the group of the
annexines and thè naturally occurring derivatives or
analogues thereof or those prepared synthetically or by
genetic engineering, optionally with excipients and/or
carriers and/or stabilisers for pr-eventing the
metastasizing of tumours;
pharmaceutical compositions for preventing the
metastasizing of tumours, characterised in that the
anticoagulant corresponds to the formula
1 5 10 15
Met Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe
Asp Glu Arg Ala Asp Ala XX Thr Leu Arg Lys Ala Met Lys Gl.y
Leu Gly Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg
-- 10 --
S0 55 60
Ser Asn Ala Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu
Phe Gly Arg Asp Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly
~5 go
Lys Phe G].u Lys I,eu Ile Val Ala Leu Met Lys Pro Ser Arg Leu
100 lOS
Tyr Asp Ala Tyr Glu I.eu Lys His Ala Leu Lys Gly Ala Gly Thr
110 llS 120
Asn Glu Lys Val Leu Thr Glu Ile Ile Ala Ser Arg Thr Pro Glu
125 130 135
Glu Leu Arg Ala Ile Lys Gln Val Tyr Glu Glu Glu Tyr Gly Ser
140 145 150
Ser Leu Glu Asp Asp Val Val Gly Asp Thr Ser Gly Tyr Tyr Gln
155 160 165
Arg Met Leu Val Val Leu Leu Gln Ala Asn Arg Asp Pro Asp Ala
170 175 180
Gly Ile Asp Glu Ala Gln Val Glu Gln Asp Ala Gln Ala Leu Phe
185 190 195
Gln Ala Gly Glu I,eu Lys Trp Gly Thr Asp Glu Glu Lys Phe Ile
200 205 210
Thr Ile Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys Val Phe
215 220 225
Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr Ile
3~-?J ~ ~
230 ~35 240
Asp Arg Glu Thr Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala Val
245 250 255
Val Lys Ser Ile Arg Ser Ile Pro Ala Tyr Leu Ala Glu Thr Leu
260 265 270
Tyr Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr I,eu Ile
275 2~0 2~5
Arg Val Met Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg
290 295 300
Lys Glu Phe Arg Lys Asn Phe Ala Thr-Ser Leu Tyr Ser Met Ile
305 310 315
Lys Gly Asp Thr Ser Gly Asp Tyr Lys Lys Ala Leu Leu Leu Leu
320
Cys Gly Glu Asp Asp x
wherein XX represents Glu or Asp and optionally at
position 1 the methionine is cleaved and the alanine at
position ~ is optlonally blocked and/or there may
optionally be aggregations caused by intermolecular
disulphide bridges, for example, between the cysteines
at position 316, or the biologically active variants or
derivatives thereof, optionally with excipients and/or
carriers and/or stabilisers.
I'he present invention also relates to:
the use of an anticoagulant selected from the group of
the annexines and the naturally occurring derivatives or
analogues thereof or those prepared synthetically or by
genetic engineering, optionally with excipients and/or
carriers and/or stabilisers for preventing the
metastasizing of tumours;
Use of an anticoagulant selected from the group of
12 -
annexines and the naturally occurring derivatives or
analogues thereof or tho~e prepared synthetically or by
genetic englneering, optiona]ly with excipients and/or
carriers and/or stabilisers for preventing
metastasizing, wherein the antlcoagulant corresponds to
the formula
1 5 10 ].5
Met Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe
Asp Glu Arg Ala Asp Ala XX Thr Leu Arg Lys Ala l~et Lys Gly
~5
Leu Gly Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg
Ser Asn Ala Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu
Phe Gly Arg Asp Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly
Lys Phe Glu Lys Leu Ile Val Ala Leu Met Lys Pro Ser Arg Leu
-
100 . 105
Tyr Asp Ala Tyr Glu Leu Lys His Ala I.eu Lys Gly Ala Gly Thr
110 115 120
Asn Glu Lys Val Leu Thr Glu Ile Ile Ala Ser Arg Thr Pro Glu
125 130 135
Glu Leu Arg Ala Ile Lys Gln Val Tyr Glu Glu Glu Tyr Gly Ser
140 145 150
Ser Leu Glu Asp Asp Val Val Gly Asp Thr Ser Gly Tyr Tyr Gln
155 160 165
Arg Met Leu Val Val Leu Leu Gln Ala Asn Arg Asp Pro Asp Ala
170 175 1~0
Gly Ile Asp Glu Ala Gln Va]. Glu Gln Asp Ala G].n Ala Leu Phe
185 190 195
Gln Ala Gly Glu Leu Lys Trp Gly Thr Asp Glu Glu Lys Phe Ile
200 205 210
Thr Ile Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys Val Phe
215 220 225
Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr Ile
230 235 240
Asp Arg Glu Thr Ser Gly~Asn Leu Glu Gln Leu Leu Leu Ala Val
245 250 255
Val Lys Ser Ile Arg Ser Ile Pro Ala Tyr Leu Ala Glu Thr Leu
260 265 270
Tyr Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr Leu Ile
275 280 2B5
Arg Val Met Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg
290 2gs 300
Lys Glu Phe Arg Lys Asn Phe Ala Thr Ser Leu Tyr Ser Met I le
305 310 315
Lys Gly Asp Thr Ser Gly Asp Tyr Lys Lys Ala Leu Leu Leu Leu
320
Cys Gly Glu Asp Asp
?
wherein XX represents G]u or Asp and optionally at
position l the methionine is cleaved and the alanine at
position 2 is optionally blocked and/or there may
optional]y ~e aggregations caused by intermolecular
disulphide bridges, for example, between the cysteines
at posi-tion 316, or the biologically active variants or
derivatives thereof;
Use of an anticoagulant selected from the group of
the annexines and the naturally occurring derivatives or
analogues thereof or those prepared by synthetically or
by genetic engineering, for the preparation of a
pharmaceutical composition for preventing the
metastasizing of tumours;
Use of an anticoagulant selected from the group of
the annexines and the naturally occurring derivatives or
analogues thereof or those prepare~ synthetically or by
genetic engineering, for the preparation:of a
pharmaceutical composition for preventing metastasizing,
wherein the anticoagulant corresponds to the formula
l 5 lO 15
Met Ala Gln Val Leu Arg Gly Thr Val Thr Asp Phe Pro Gly Phe
Asp Glu Arg Ala Asp Ala XX Thr Leu Ar~ L~s Ala Met Lys Gly
~5
Leu Gly Thr Asp Glu Glu Ser Ile Leu Thr Leu Leu Thr Ser Arg
Ser Asn Ala Gln Arg Gln Glu Ile Ser Ala Ala Phe Lys Thr Leu
Phe Gly Arg Asp Leu Leu Asp Asp Leu Lys Ser Glu Leu Thr Gly
2~
-
go
Lys Phe Glu Lys Leu Ile Val Ala Leu Met Lys Pro Ser Arg l,eu
100 105
Tyr Asp Ala Tyr Glu Leu Lys His Ala Leu Lys Gly Ala Gly Thr
110 115 120
~Srl ~lU l-ys val Leu lnL ~lu 1l~ lle Ala ~er Ary lnL ~'IO ~lU
125 130 135
Glu Leu Arg Ala Ile Lys Gln Val Tyr Glu Glu Glu Tyr Gly Ser
1~0 145 150
Ser Leu Glu Asp Asp Val Val Gly Asp Thr Ser Gly Tyr Tyr Gln
155 160 1~5
Arg Met Leu Val Val Leu Leu Gln Ala Asn Arg Asp Pro Asp Ala
170 : 175 180
Gly Ile Asp Glu ~la Gln Val Glu Gln Asp Ala Gln Ala Leu Phe
1~5 190 195
Gln Ala Gly Glu Leu Lys Trp Gly Thr Asp G]u Glu Lys Phe Ile
200 205 210
Thr Ile Phe Gly Thr Arg Ser Val Ser His Leu Arg Lys Val Phe
215 220 225
Asp Lys Tyr Met Thr Ile Ser Gly Phe Gln Ile Glu Glu Thr Ile
230 235 240
Asp Arg Glu Thr Ser Gly Asn Leu Glu Gln Leu Leu Leu Ala Val
245 250 255
Val Lys Ser Ile Arg Ser Ile Pro Ala Tyr Leu Ala Glu Thr Leu
- 16 -
260 2~5 270
Tyr Tyr Ala Met Lys Gly Ala Gly Thr Asp Asp His Thr Leu Ile
7.75 280 235
Arg Val Met Val Ser Arg Ser Glu Ile Asp Leu Phe Asn Ile Arg
290 295 300
~yS ~lU i~ne ~rg ~JyS Asn ~ne Ala lhl ~eL LeU lyl .~eL l~le~
305 310 315
Lys Gly Asp Thr Ser Gly Asp Tyr-Lys Lys Ala Leu Leu Leu Leu
320
Cys Gly Glu Asp Asp *
wherein XX represents Glu or Asp and optionally at
position 1 the methionine is cleaved and the alanine at
position 2 is optionally blocked and/or there may
optionally be aggregations caused by intermolecular
disulphide bridges, for example, between the cysteines
at position 316, or the biologically active variants or
derivatives thereof.
The Example which follows is intended to illustrate
the invention without restricting it.
Materials and Methods
VAC~ was prepared analogously to either EPA
0 181 465 or EPA 0 293 567. The following tests were
carried out with VACQ. However, the results should also
apply to the other annexines, especially VAC~.
Colon tumour cells were cultivated in a tissue
flask in RPMI 16~0 supplemented with 10% foetal calves'
serum. The cells were harvested by wiping the bottom of
the flask with a rubber wiper. The collected cells were
washed with a TBSA buffer (50 mM Tris/HCl, 100 mM NaCl,
4.5 mg glucose/ml, 0.5 mg bovine serum albumin/ml, 2 mM
CaCl2, pH 7.9) and finally suspended in this buffer for
the tests. The viability of these cells was tested with
tryptan-blue exclusion. More than 95% of the cells were
viable after this procedure.
Coagulation assay: The procoagulant activity of the
colon cells was measured by adding a certain number of
washed cells to 50 ~1 of citrated platelet-free plasma
(PFP). This mixture was stirred for two minutes at
37'C. Coagulation was initiated by the addition of CaCl2
(the final concentration was 10 mM). The final volume
of the mixture was 500 ~1. Coagulation was observed
turbidimetrica]ly using a "Payton dual wave
aggregometer".
Results
Procoaqulant activitv associated with colon cells:
When washed colon cells were added to citrated PFP
and the plasma was recalcified, a coagulation time was
observed which was dependent on the number of cells
present (Fig. 1). In the absence of the cells the
coagulation time was more than 15 minutes, whereas 8X106
cells/ml reduced this coagulation time to about 2
minutes. This procoagulant activity was caused by the
2 1~ J
- 18 -
intact cells; centrifuging the cells resulted in
coprecipi ation of the procoagulant activity.
Neutrall~ation of the procoaqulant activltY associated
_ith colon cells by VAC~:
The procoagulant activity of a certain number of
colon cells was inhibited by VAC~ in dosage-dependent
manner (Fig. 2). F'rom the correlation between the
number of cells and the coagulation time (Fig. l) it was
calculated that 210 nM of VAC~ neutralised 99% of the
procoagulant activity of 6X106 cells/ml. When colon
cells neutralised by VAC~ were centrifuged in the
presence of Ca2+ and then washed with TBSA, they showed
no procoagulant activity in the coagulation assay. If,
however, the liquid contained EDTA instead of Ca2r, the
cells exhibited normal procoagulant activity.
d.r
-- 19 --
Leqe d accoml~anYinq the dr w~
Fig. 1
Procoagulant activity associated with colon cells.
Citrated platelet-free plasma (PFP) was diluted with
TBSA containing the specified number of colon eells.
After 3 minutes incubation at 37~C, CaCl2 was added
(final concentration 10 mM) and the coagulation time was
recorded.
Fig. 2
Effeet of VAC~ on the proeoagulant activity of eolon
cells. Citrated platelet-free plasma (PFP) was diluted
with TBSA eontaininy the specified amount of VAC~ and a
eertain number of colon eells (up to a final
concentration of 6X106 eells/ml). After 3 minutes
incubation at 37C, eoagulation was started by the
addition of CaCl2 (final eoneentration 10 mM~. The
eoagulation time measured was eompared with Fig. 1 and
extrapolated to the number of procoagulant-aetive eolon
cells.
