THROMBOSIS RESEARCH Printed in the United
vol. States
7, pp. 401-408, Pergamon Press,
1975 Inc.
STUDIES ON THE CLOTTING MECHANISM OF LIMULUS POLYPHEMUS (") D.Fumarola, N.Pasquetto, P.Telesforo and M.B. Donati Department of Microbiology, University of Bari and Laboratory for Haemostasis and Thrombosis Research, Istituto di Ricerche Farmacologiche 'Mario Negri' Milano, Italy .
(Received
ABSTRACT
17.6.1975; in revised Accepted by Editor H.C.
form 14.7.1975. Godal)
Several substances interacting with the clotting mechanism of mammalians were tested on lysate of Limulus hemolymphe cells. Gelation was observed in the presence of thrombin, reptilase, botropase , thrombin-coagulase and rabbit or human thromboplastin. Acidification experiments suggested that the above mentioned substances are able to activate, like endotoxin, an inactive proenzyme in the lysate to form an enzyme capable to induce the coagulogen gelation . In contrast, only trypsin would be able to transform directly the coagulogen into a gel .
INTRODUCTION The circulating blood cells (amebocytes) of Limulus polvnhemus > an arthropode belonging to subphylum chelicerates, contain material which can be clotted by bacterial endotoxin, whereas the.cell-free 'plasma' of the animal is not clotted (6).
The protein(s) present in the smebocyte lysates
which precipitate(s) during the gelation process has been partially characterised (6,9,10,12). ------------
According to the present concept, endotoxin
(") Presented in part at III Congress of the Italian Society on Haemostasis and Thrombosis, Venezia, September g-10, 1974 .
401
LJM_JLUS POLYPHEWS-COAGULATION
402
Vo1.7,No.3
would activate one or more proenzymes in the amebocyte lysate, the activated enzyme being thereafter responsible for the conversion of the clottable protein (coagulogen) into thepolynerizable form of the molecule (12). The proposed scheme suggests a striking similarity with the last phases of the plasma coagulation mechanism in other animal species, namely the activation by thromboplastin of the inactive precursor, prothrombin, in the active enzyme, thrombin, with subsequent conversion of fibrinogen to fibrin. It seemed therefore interesting to test the reactivity of the Limulus amebocyte lysate to several substances which are known to interact with the components of the clotting mechanism in mammalians.
MATERIALS
Limulus lysate
AND
METHODS
was obtained as lyophylized material from Dr.J. Fine,
City Hospital, Boston, USA, and resuspended freshly before use with pyrogenfree bidistilled water.
All glassware and solutions were sterilized by
autoclaving . Bovine thrombin (Topostasine, Roche, Basel,Switzerland), Reptilase-R Defibrase
and
(Ormonoterapia Richter, Milano, Italy and Pentapharm, Basel,
Switzerland),
Botropase-R
(Ravizza, Muggi6, Italy) , Thrombin-coagulase
(Boehringer Manheim, Menheim, Germany and Biochemia, Milano, Italy) , Staphylocoagulase (Stago, Paris, France) two thromboplastin preparations, one from rabbit brain (Hyland, Costa Mesa, USA) and the other one from human brain (National Thromboplastin Centre, Withington Hospital,Manchester, England), were tested in the lysate system.
Thrombin and thrombin-like
enzymes were used at a concentration which clotted a normal titrated human plasma at 37OC in 18-20
seconds;the thromboplastins and Staphylocoagulase
were used at the concentrations employed for the prothrombin time of human titrated plasma. mine sulphate
Ethanol
at a final concentration of 12% (v/v), 1% prota-
(Organon, Oss, Holland), endotoxin (S. Q-phi lipopolysaccha-
ride W., Difco Labs., Detroit,USA) as a 1 mg/ml solution and trypsin (2.5 mg/ml, Eurobio, Paris, France) were also tested . Protein concentration was measured by the method of Lowry et al. (8). The gelation reaction was monitored in a waterbath at 37'C after addition of 0.1 ml of the test substance to 0.1 ml of Limulus lysate . Results were read after 45 minuted in all the experiments, except
those
in which the gelation time of both acidified and untreated 1YSate was compared.
In the latter experiments the test tubes were CarefullY
503
LIMTJLTJS PoLYPHEMtJS-COAGULATION
Vo1.7,No.3
inclined every 30 seconds. The tensile strenght of the gel was measured by thrombelastography using a Clot-Scanner (Elvi, MilanoJ Italy). To 0.25 ml lysate 0.1 ml thrombin (2 NIH U/ml) was added in the cuvette.
The acidification of
amebocyte
lysate followed by careful neutralization and removal of the precipitated protein, was performed as described by Solum (10) . Inhibition of thrombin-induced gelation was studied with henarin (Liquemin, Roche, Basel, Switzerland, 5 U/ml, f.c.) in the presence of either
saline or a source of heparin cofactor (human citrate normal plasma
previously defibrinated by incubation at56 'C during 3 minutes) . Susceptibility of either coagulogen or formed gel to plasmin was studied by
incubating the lysate during 1 hour at 37OC with plasmin (AB Kabi,
Stockholm, Sweden) at a concentration of 2.5 CTA U/ml,either beforeor
hour
after the addition of thrombin; possible modifications of the clot were monitored visually . The Staphylocoecal Clumping test
was performed as described by Donati
et al. (1) using the kit of Boehringer, .Msnnheim, GmbH, Mannheim,Germany and
Biochemia, Milano, Italy .
RESULTS
The formation of a strong gel was observed upon addition to the lysate of thrombin, reptilase, defibrase, botropase, thrombin-coagulase and both thromboplastin preparations.
No gelation occurred when CaC12 alone was
added to the lysate (Table I) . Substances like ethanol and protamine sulphate,which cause the so-called non enzymatic polymerization of fibrinogen of other species (11) were inactive in the lysate system . The gelation times of the enzymes mentioned above were of the same order as that measured with endotoxin and much slower than that of trypsin, a proteolytic enzyme which causes virtually immediate 'clotting' of the lysate (Table II). The gels formed were all soluble in 5 M urea after 1 hour anddid
not
cause any oscillations in the thromboelastograph . Both the lysates before interaction with the enzymes and the gels subsequently formed were insensitive to plasmin . In contrast to the clottable proteins of most of the animal species tested so far (7), the coagulogen of Limulus was not able to induce
404
LIMULUS
POLYPHEMUS-COAGULATION
Vo1.7,No.3
TABLE I Gelation of amebocyte lysate by different substances
TEST SUBSTANCE 10.0 Thrombin Thrombin-coagulase Reptilase-R Defibrase Botropase-R Thromboplastin (rabbit) Thromboplastin (human ) Staphylocoagulase Trypsin Endotoxin Ethanol Protamine sulphate
LYSATE (m&ml) 5.0 2.0 1.0 3+ 3+ 3+ 3+ 3+ 3+ 3+
3+ 2+ 3+ 3+ 3+ 2+ 3+
3+ 1+ 2+ 2+ 2+ 2+ 3+
3+ 3+
2+ 3+
2+
0.7
0.5
2+
-
1+
-
2+
-
2+
-
Results were expressed conventionally as follows: - : no gelation; 1+ : flocculate; 2+ : loose gel ; 3+ : solid gel . The figures on top of each column represent the concentration of total protein of different dilutions of the original lysate (10 mg/ml) .
TABLE II Effect of the acidification procedure on the gelation time (minutes) of the hemocyte lysate in the presence of several agents
LYSATE
CLOTTING INDUCING AGENT
UNTREATED Endotoxin Try-psin Thrombin Reptilase-R Botropase-R Thrombin-coagulase Rabbit Thromboplastin
staphylococcal clumping.
5 41 5 12 10 10 7
TREATED ,120 41 >120 >120 >120 ,120 >12d
No inhibitor toward the clumping reaction could be
demonstrated in the lysate, since the staphylococcal titre of human plasma was not changed when the latter was diluted using the lysate instead of isotonic saline.
On the other hand lack in the lysate of a cofactor for
clumping was excluded on the basis of the negative results obtained when
LIMULUS
405
POLYPHEMUS-COAGUMTION
human serum was added to the lysate.
Table II shows that,after acidification
and subsequent neutralization , the hemocyte lysate did not gel any more in the presence of endotoxin, of thethrombin-like enzymes and of the thromboplastins.
Only trypsin was able to clot'the treated lysates as rapidly as
the untreated ones .
DISCUSSION
Several studies have indicated that the lysate of amebocytes from Limulus polyphemus
can form a gel in the presence of substances like trypsin or
endotoxin (3,4,6,9,10,12) .
The clottable protein present in the amebocyte
lysate deviates much in its molecular characteristics from those of vertebrate fibrinogens(9, 10) ; very recently, Holme and Solum (5) have also offered electron microscopic evidence that the gel protein of the Limulus is structurally different from mammalian fibrin. of activation of the Limulus
However, an enzymatic mechanism
clotting system has been proposed (12) which is
similar to the last phases of the coagulation mechanism in mammalians. In this study we have shown that thrombin, several thrombin-like enzymes and two thromboplastin preparations, were also able to cause gelation of the amebocyte lysate.
The gels formed were not lysed by plasmin, were soluble
in 5M urea at neutral pH, and did not have any tensile strength as shown by the absence of measurable oscillations in a thromboelastographic record. These results, together with the lack of reactivity of the lysate in the presence of staphylococci , offer further support to'the molecular studies indicating that the coagulogen of Limulus
has many different characteristics
as compared with mammalian fibrinogens . Acidification of the amebocyte lysate followed by neutralization has been described by Solum (10) to result in a precipitation of proteins all of which are of higher molecular weight than that of the coagulogen which is not itself precipitated.
We could confirm that acidification of the lysate and careful
neutralization, followed by removal of the precipitated proteins by centrifugation, prolonged indefinitely the clotting time of the lysate with endotoxin, whereas the clotting time with trypsin was unaltered.
These
findings have been interpreted to mean that the enzyme(s) necessary for clotting of the lysate with endotoxin are inactivated by the acidification procedure, whereas the coagulogen itself is left in solution in a form which can interact directly with trypsin (10) .
The virtually immediate clotting
of both treated and untreated lysate we have observed with trypsin offers
406
L~LUS
Vo1.7,No.3
POLYF'mMUS-COAGULATION
support to the hypothesis of Solum (10) that the polymerization of the converted coagulogen molecule is a very rapid process and that the most timeconsuming part of endotoxin-induced clotting probably is represented by the enzyme activation phase. Moreover, we have observed that also the clotting time of thrombin and of all the other enzymes active on the untreated lysate was not measurable on the supernatant obtained after acidification and neutralization .
The
hypothesis could therefore be put forward that, among the substances tested, thrombin, reptilase, botropase, thrombin-coagulase and the thromboplastins would activate, like endotoxin, the inactive proenzyme to form the enzyme responsible for gelation of the coagulogen; in contrast, only trypsin would have the same activity of the activated enzyme, being able to transform directly the coagulogen into a gel (Table III) .
TABLE III Proposed scheme for the levels of action, in the process of gelation, of the substances tested in this study.
endotoxin or :
thrombin thrombin-coagulase reptilase botropase thromboplastin
I PROENZYME --~-----~--~~-~-------~~-~----~-------ACT~~
ENZYME or trypsin
COAGULOGEN -----4--------> POLYMER
The possibility that the thrombin-like enzymes and the thromboplastins used in this study be contamined with endotoxin has also to be considered; indeed the gelation induced by thrombin was neither inhibited by heparin, as shown in this study, nor by TAME, hirudin or purified antithrombin III (Solum, personal comication)
.
These data would indicate
that thrombin
provokes gelation of the untreated lysate through a mechanism different from its enzymatic activity on fibrinogen.
However, the presence of bacterial
contamination in our system can be reasonably excluded by the negative data obtained in the same experimental conditions with Staphylocoagulase, ethanol,
Vo1.7,No.3
LIxuLUS
POLYPHENUS-COAGULION
protamine sulphate snd CaC12 .
407
Thrombin from four different lots and
thromboplastin from t'hreedifferent kits gave positive gelation tests with the ssme end point in a study reported by Elin and Wollf (2).
Both thrombin
thronboplastin had larger ratios of positivity in the Limulus test as a??_d compared to pirogenicity than endotoxin, thus suggesting that contamination by endotoxin was not the cause of the positive gelation test . ACKNOWLEDGEMENTS The authors wish to thank Dr. N.O.Solum, Oslo, Norway, for helpful criticism and discussions.
Dr. J. Fine, Boston, USA, provided the Limulus
lysate and Dr. L. Poller, Manchester, United Kingdom, provided the human thromboplastin . Thanks are also due to AB Kabi, Stockholm; Pentaphsrm, Basel; Ormonoterepia Richter, Milan0 ; Ravizza, piluggia; Biochemia, Milan0 ; Roche,Basel and Milan0 ; Stago, Paris , who kindly provided the materials used in this study . This work has been supported in part by grant CNR-'73.00400-04of the Italian Research Council .
REFERENCES
1.
Donati M.B., Vermylen J. and Verstraete M. The staphylococcal clumping test for detection of fibrinogen-like material. Stand. J. Haemat. Suppl. 13, 137, 1971 *
2.
Elin R.J. and Wolff S.M. Nonspecificity of limulus amebocyte lysate test : positive reactions with polynucleotides and proteins. J. Infect. Dis. 128 : 349 , 1973 .
3. Fumarola D., Pasquetto N., Telesforo P. and Donati M-B.
11 coagulogeno de1 'Limulus Polyphemus' : un modello di proteina coagulabile di origine cellulare. In : Abstracts of the 3rd. Congress of the Italian Society for Haemostasis
vol. States
7, pp. 401-408, Pergamon Press,
1975 Inc.
STUDIES ON THE CLOTTING MECHANISM OF LIMULUS POLYPHEMUS (") D.Fumarola, N.Pasquetto, P.Telesforo and M.B. Donati Department of Microbiology, University of Bari and Laboratory for Haemostasis and Thrombosis Research, Istituto di Ricerche Farmacologiche 'Mario Negri' Milano, Italy .
(Received
ABSTRACT
17.6.1975; in revised Accepted by Editor H.C.
form 14.7.1975. Godal)
Several substances interacting with the clotting mechanism of mammalians were tested on lysate of Limulus hemolymphe cells. Gelation was observed in the presence of thrombin, reptilase, botropase , thrombin-coagulase and rabbit or human thromboplastin. Acidification experiments suggested that the above mentioned substances are able to activate, like endotoxin, an inactive proenzyme in the lysate to form an enzyme capable to induce the coagulogen gelation . In contrast, only trypsin would be able to transform directly the coagulogen into a gel .
INTRODUCTION The circulating blood cells (amebocytes) of Limulus polvnhemus > an arthropode belonging to subphylum chelicerates, contain material which can be clotted by bacterial endotoxin, whereas the.cell-free 'plasma' of the animal is not clotted (6).
The protein(s) present in the smebocyte lysates
which precipitate(s) during the gelation process has been partially characterised (6,9,10,12). ------------
According to the present concept, endotoxin
(") Presented in part at III Congress of the Italian Society on Haemostasis and Thrombosis, Venezia, September g-10, 1974 .
401
LJM_JLUS POLYPHEWS-COAGULATION
402
Vo1.7,No.3
would activate one or more proenzymes in the amebocyte lysate, the activated enzyme being thereafter responsible for the conversion of the clottable protein (coagulogen) into thepolynerizable form of the molecule (12). The proposed scheme suggests a striking similarity with the last phases of the plasma coagulation mechanism in other animal species, namely the activation by thromboplastin of the inactive precursor, prothrombin, in the active enzyme, thrombin, with subsequent conversion of fibrinogen to fibrin. It seemed therefore interesting to test the reactivity of the Limulus amebocyte lysate to several substances which are known to interact with the components of the clotting mechanism in mammalians.
MATERIALS
Limulus lysate
AND
METHODS
was obtained as lyophylized material from Dr.J. Fine,
City Hospital, Boston, USA, and resuspended freshly before use with pyrogenfree bidistilled water.
All glassware and solutions were sterilized by
autoclaving . Bovine thrombin (Topostasine, Roche, Basel,Switzerland), Reptilase-R Defibrase
and
(Ormonoterapia Richter, Milano, Italy and Pentapharm, Basel,
Switzerland),
Botropase-R
(Ravizza, Muggi6, Italy) , Thrombin-coagulase
(Boehringer Manheim, Menheim, Germany and Biochemia, Milano, Italy) , Staphylocoagulase (Stago, Paris, France) two thromboplastin preparations, one from rabbit brain (Hyland, Costa Mesa, USA) and the other one from human brain (National Thromboplastin Centre, Withington Hospital,Manchester, England), were tested in the lysate system.
Thrombin and thrombin-like
enzymes were used at a concentration which clotted a normal titrated human plasma at 37OC in 18-20
seconds;the thromboplastins and Staphylocoagulase
were used at the concentrations employed for the prothrombin time of human titrated plasma. mine sulphate
Ethanol
at a final concentration of 12% (v/v), 1% prota-
(Organon, Oss, Holland), endotoxin (S. Q-phi lipopolysaccha-
ride W., Difco Labs., Detroit,USA) as a 1 mg/ml solution and trypsin (2.5 mg/ml, Eurobio, Paris, France) were also tested . Protein concentration was measured by the method of Lowry et al. (8). The gelation reaction was monitored in a waterbath at 37'C after addition of 0.1 ml of the test substance to 0.1 ml of Limulus lysate . Results were read after 45 minuted in all the experiments, except
those
in which the gelation time of both acidified and untreated 1YSate was compared.
In the latter experiments the test tubes were CarefullY
503
LIMTJLTJS PoLYPHEMtJS-COAGULATION
Vo1.7,No.3
inclined every 30 seconds. The tensile strenght of the gel was measured by thrombelastography using a Clot-Scanner (Elvi, MilanoJ Italy). To 0.25 ml lysate 0.1 ml thrombin (2 NIH U/ml) was added in the cuvette.
The acidification of
amebocyte
lysate followed by careful neutralization and removal of the precipitated protein, was performed as described by Solum (10) . Inhibition of thrombin-induced gelation was studied with henarin (Liquemin, Roche, Basel, Switzerland, 5 U/ml, f.c.) in the presence of either
saline or a source of heparin cofactor (human citrate normal plasma
previously defibrinated by incubation at56 'C during 3 minutes) . Susceptibility of either coagulogen or formed gel to plasmin was studied by
incubating the lysate during 1 hour at 37OC with plasmin (AB Kabi,
Stockholm, Sweden) at a concentration of 2.5 CTA U/ml,either beforeor
hour
after the addition of thrombin; possible modifications of the clot were monitored visually . The Staphylocoecal Clumping test
was performed as described by Donati
et al. (1) using the kit of Boehringer, .Msnnheim, GmbH, Mannheim,Germany and
Biochemia, Milano, Italy .
RESULTS
The formation of a strong gel was observed upon addition to the lysate of thrombin, reptilase, defibrase, botropase, thrombin-coagulase and both thromboplastin preparations.
No gelation occurred when CaC12 alone was
added to the lysate (Table I) . Substances like ethanol and protamine sulphate,which cause the so-called non enzymatic polymerization of fibrinogen of other species (11) were inactive in the lysate system . The gelation times of the enzymes mentioned above were of the same order as that measured with endotoxin and much slower than that of trypsin, a proteolytic enzyme which causes virtually immediate 'clotting' of the lysate (Table II). The gels formed were all soluble in 5 M urea after 1 hour anddid
not
cause any oscillations in the thromboelastograph . Both the lysates before interaction with the enzymes and the gels subsequently formed were insensitive to plasmin . In contrast to the clottable proteins of most of the animal species tested so far (7), the coagulogen of Limulus was not able to induce
404
LIMULUS
POLYPHEMUS-COAGULATION
Vo1.7,No.3
TABLE I Gelation of amebocyte lysate by different substances
TEST SUBSTANCE 10.0 Thrombin Thrombin-coagulase Reptilase-R Defibrase Botropase-R Thromboplastin (rabbit) Thromboplastin (human ) Staphylocoagulase Trypsin Endotoxin Ethanol Protamine sulphate
LYSATE (m&ml) 5.0 2.0 1.0 3+ 3+ 3+ 3+ 3+ 3+ 3+
3+ 2+ 3+ 3+ 3+ 2+ 3+
3+ 1+ 2+ 2+ 2+ 2+ 3+
3+ 3+
2+ 3+
2+
0.7
0.5
2+
-
1+
-
2+
-
2+
-
Results were expressed conventionally as follows: - : no gelation; 1+ : flocculate; 2+ : loose gel ; 3+ : solid gel . The figures on top of each column represent the concentration of total protein of different dilutions of the original lysate (10 mg/ml) .
TABLE II Effect of the acidification procedure on the gelation time (minutes) of the hemocyte lysate in the presence of several agents
LYSATE
CLOTTING INDUCING AGENT
UNTREATED Endotoxin Try-psin Thrombin Reptilase-R Botropase-R Thrombin-coagulase Rabbit Thromboplastin
staphylococcal clumping.
5 41 5 12 10 10 7
TREATED ,120 41 >120 >120 >120 ,120 >12d
No inhibitor toward the clumping reaction could be
demonstrated in the lysate, since the staphylococcal titre of human plasma was not changed when the latter was diluted using the lysate instead of isotonic saline.
On the other hand lack in the lysate of a cofactor for
clumping was excluded on the basis of the negative results obtained when
LIMULUS
405
POLYPHEMUS-COAGUMTION
human serum was added to the lysate.
Table II shows that,after acidification
and subsequent neutralization , the hemocyte lysate did not gel any more in the presence of endotoxin, of thethrombin-like enzymes and of the thromboplastins.
Only trypsin was able to clot'the treated lysates as rapidly as
the untreated ones .
DISCUSSION
Several studies have indicated that the lysate of amebocytes from Limulus polyphemus
can form a gel in the presence of substances like trypsin or
endotoxin (3,4,6,9,10,12) .
The clottable protein present in the amebocyte
lysate deviates much in its molecular characteristics from those of vertebrate fibrinogens(9, 10) ; very recently, Holme and Solum (5) have also offered electron microscopic evidence that the gel protein of the Limulus is structurally different from mammalian fibrin. of activation of the Limulus
However, an enzymatic mechanism
clotting system has been proposed (12) which is
similar to the last phases of the coagulation mechanism in mammalians. In this study we have shown that thrombin, several thrombin-like enzymes and two thromboplastin preparations, were also able to cause gelation of the amebocyte lysate.
The gels formed were not lysed by plasmin, were soluble
in 5M urea at neutral pH, and did not have any tensile strength as shown by the absence of measurable oscillations in a thromboelastographic record. These results, together with the lack of reactivity of the lysate in the presence of staphylococci , offer further support to'the molecular studies indicating that the coagulogen of Limulus
has many different characteristics
as compared with mammalian fibrinogens . Acidification of the amebocyte lysate followed by neutralization has been described by Solum (10) to result in a precipitation of proteins all of which are of higher molecular weight than that of the coagulogen which is not itself precipitated.
We could confirm that acidification of the lysate and careful
neutralization, followed by removal of the precipitated proteins by centrifugation, prolonged indefinitely the clotting time of the lysate with endotoxin, whereas the clotting time with trypsin was unaltered.
These
findings have been interpreted to mean that the enzyme(s) necessary for clotting of the lysate with endotoxin are inactivated by the acidification procedure, whereas the coagulogen itself is left in solution in a form which can interact directly with trypsin (10) .
The virtually immediate clotting
of both treated and untreated lysate we have observed with trypsin offers
406
L~LUS
Vo1.7,No.3
POLYF'mMUS-COAGULATION
support to the hypothesis of Solum (10) that the polymerization of the converted coagulogen molecule is a very rapid process and that the most timeconsuming part of endotoxin-induced clotting probably is represented by the enzyme activation phase. Moreover, we have observed that also the clotting time of thrombin and of all the other enzymes active on the untreated lysate was not measurable on the supernatant obtained after acidification and neutralization .
The
hypothesis could therefore be put forward that, among the substances tested, thrombin, reptilase, botropase, thrombin-coagulase and the thromboplastins would activate, like endotoxin, the inactive proenzyme to form the enzyme responsible for gelation of the coagulogen; in contrast, only trypsin would have the same activity of the activated enzyme, being able to transform directly the coagulogen into a gel (Table III) .
TABLE III Proposed scheme for the levels of action, in the process of gelation, of the substances tested in this study.
endotoxin or :
thrombin thrombin-coagulase reptilase botropase thromboplastin
I PROENZYME --~-----~--~~-~-------~~-~----~-------ACT~~
ENZYME or trypsin
COAGULOGEN -----4--------> POLYMER
The possibility that the thrombin-like enzymes and the thromboplastins used in this study be contamined with endotoxin has also to be considered; indeed the gelation induced by thrombin was neither inhibited by heparin, as shown in this study, nor by TAME, hirudin or purified antithrombin III (Solum, personal comication)
.
These data would indicate
that thrombin
provokes gelation of the untreated lysate through a mechanism different from its enzymatic activity on fibrinogen.
However, the presence of bacterial
contamination in our system can be reasonably excluded by the negative data obtained in the same experimental conditions with Staphylocoagulase, ethanol,
Vo1.7,No.3
LIxuLUS
POLYPHENUS-COAGULION
protamine sulphate snd CaC12 .
407
Thrombin from four different lots and
thromboplastin from t'hreedifferent kits gave positive gelation tests with the ssme end point in a study reported by Elin and Wollf (2).
Both thrombin
thronboplastin had larger ratios of positivity in the Limulus test as a??_d compared to pirogenicity than endotoxin, thus suggesting that contamination by endotoxin was not the cause of the positive gelation test . ACKNOWLEDGEMENTS The authors wish to thank Dr. N.O.Solum, Oslo, Norway, for helpful criticism and discussions.
Dr. J. Fine, Boston, USA, provided the Limulus
lysate and Dr. L. Poller, Manchester, United Kingdom, provided the human thromboplastin . Thanks are also due to AB Kabi, Stockholm; Pentaphsrm, Basel; Ormonoterepia Richter, Milan0 ; Ravizza, piluggia; Biochemia, Milan0 ; Roche,Basel and Milan0 ; Stago, Paris , who kindly provided the materials used in this study . This work has been supported in part by grant CNR-'73.00400-04of the Italian Research Council .
REFERENCES
1.
Donati M.B., Vermylen J. and Verstraete M. The staphylococcal clumping test for detection of fibrinogen-like material. Stand. J. Haemat. Suppl. 13, 137, 1971 *
2.
Elin R.J. and Wolff S.M. Nonspecificity of limulus amebocyte lysate test : positive reactions with polynucleotides and proteins. J. Infect. Dis. 128 : 349 , 1973 .
3. Fumarola D., Pasquetto N., Telesforo P. and Donati M-B.
11 coagulogeno de1 'Limulus Polyphemus' : un modello di proteina coagulabile di origine cellulare. In : Abstracts of the 3rd. Congress of the Italian Society for Haemostasis
