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Effect of calcium phosphate on thrombin and on its sensitivity to heparin and antithrombin-III
THROMBOSIS RESEARCH Printed
in
the
United States
vol. 9, pp. 123-131, 1976 Pergamon Press, Inc.
EFFECT OF CALCIUM YHOSYHATE ON THROMBIN AND ON ITS SENSITIVITY TO HEPARIN AifD ANTITHROMBIN-III Raymund Machovich Medical School, First Department Of Pledicine, 1389 Budapest, Hungary
Postgraduate
(Received
20.4.1976; in revised Accepted
by Editor
M.
form
2.6.1976.
Kopec)
ABSTRACTThrombin was inactivated by antithrombin-III and the rate of inactivation was accelerated by heparin either in 0.04 M sodium phosphate buffer or in 0.04 M Tris-HCl buffer, at pH 7.4. Calcium chloride, at a final concentration of 1 mM, influenced slightly thrombin inactivation by both antithrombin and antithrombin plus heparin in Tris-HCl buffer, whereas in phosphate buffer the sensitivity of enzyme to antithrombin and heparin decreased. Thrombin showed also increased stBbility against the inactivating effect of heat at 54 C in sodium phosphate buffer containing calcium chloride. These findings suggest that thrombin bound to calcium phosphate is extremely stable against inactivation by antithrombin and heparin, while its clotting activity does not change. This nature of enzyme may play a role in predisposition for thrombosis during arteriosclerosis. INTRODUCTION Thrombin plays a key role in thrombosis and hemostasis. Its main function is to catalyse the conversion of fibrinogen to fibrin (1). Under physiological conditions, thrombin formed in blood circulation can be inactivated by antithrombinIII (Z-5). It is also known that heparin accelerates the complex formation between enzyme and its inhibitor (5-6,19). It has been proposed that "heparin acts to accelerate inhibitor function by binding to antithrombin and inducing an allosteric modification in it" (6). Other data suggest that heparin 123
124
may affect
CALCIUM
enzyme
PHOSPHATE
inactivation
AND THROMBIN
by antithrombin
Vo1.9,No.Z
through
a
thrombin and heparin interaction as well, since hepsrin binds tightly to thrombin but not at the active site (8-10). Namely, the binding of heparin to thrombin may induce a conformational change in the enzyme, facilitating the complex formation of thrombin with antithrombin (7).
It has also been documen-
ted that many factors influence thrombin-heparin interaction and enzyme inactivation by antithrombin (ll-12,20). Among the possible factors which may interfere with thrombin, antithrombin and heparin, we examined the effect of calcium chloride in Iris-HCl buffer and in sodium phosphate buffer on activity and stability of thrombin, as well as on its sensitivity to heparin and antithrombin-III. MATERIALS AND METHODS Thrombin was prepared from commercial bovine thrombin (Topostasin, La Roche) by chromatography on Sulfoethyl-Sephadex C-50 column according to the method of Lundblad (14) and gel-filtered on Sephadex G-25 equilibrated with 0.05 M TrisHCl buffer or 0.05 M sodium phosphate buffer, at pH 7.4. The specific activity of purified thrombin was about 1500-2000 NIH units per mg protein before gel-filtration. Thrombin and antithrombin activity were assayed as published earlier (7). Thrombin activity was calculated and expressed in NIH units as detailed elsewhere (7). Antithrombin-III was partially purified from human plasma as published earlier (12) and dialyzed against 0.1 M sodium chloride. Protein was determined according to the method of Iowry et al (15). Sulfoethyl-Sephadex C-50 and Sephadex G-25 were purchased from Pharmacia. Heparin was obtained from G. Richter Ltd. and dissolved in 0.1 M sodium chloride. Fibrinogen was the product of KABI (human, Grade L.). Other chemicals were purchased from Reanal Fine Chemicals, Budapest. RESULTS Thrombin was not inactivated by incubation at 37'C for 5 min in 0.04 M Tris-HCl buffer at pH 7.4 either in the presence or absence of calcium chloride (Fig. 1). Antithrombin
CALCIUM
Vol.9,No.2
PHOSPHATE
AND THROMBIN
125
inactivated about 50 percent of enzyme in a 2 min incubation. If calcium chloride was also added, at a final concentration of 1 mM, the rate of thrombin inactivation did not change. Heparin, at a C.01 unit per ml final concentration, increased the rate of enzyme inactivation by antithrombin, i.e. about 80 percent of thrombin activity was already lost in a 1 min incubation. The results with heparin were not altered in the presence of 1 mN calcium chloride. Calcium chloride increased
-
1
0
0
”
A
-
I
1
1
I
min
I
1
5
FIG. 1 Thrombin inactivation by antithrombin-III and heparin in Tris-HCl buffer. 0.5 fig thrombin was incubated either alone or with 50 fig antithrombin protein or with antithrombin plus 0.0025 units heparin, in 0.2 ml reaction mixture containing 8 moles TrisHCl buffer pH 7.4 and 10 ,umoles sodium chloride either in the presence or the absence of 0.25 ,ugoles calcium chloride, respectively. After incubation at 37 C for varying periods, 500 ,ug fibrinogen in 0.05 ml volume was added and clotting time measured with the Hyland Clotek System. plus calcium chloride, 0-o thrombin alone, e-0 plus calcium chlob--------d plus antithrombin, _ ride and antithrombin, cF------_-0 plus antithrombin and heparin, _ plus calcium chloride, heparin ard. antithrombhn
126
CALCIUM PHOSPHATE AND THROMBIN
Vol.g,No.2
slightly thrombin activity, however it did not change the rate of enzyme inactivation either by antithrombin or by antithrombin plus heparin. Thrombin behaved differently in 0.04 M sodium phosphate buffer at pH 7.4 (Fig. 2). Although, the enzyme showed some inactivation in the presence of 1 mM calcium chloride, it was not altered by a further 5 min incubation. Without calcium chloride, antithrombin or antithrombin plus heparin affected thrombin activity in a similar way as in Tris-HCl buffer, i.e. about 50 percent of enzyme activity was inhibited by antithrombin in a 2 min incubation, whereas in the presence of heparin (at the same concentration as in Tris-HCl buffer) about 90 percent of enzyme activity was also lost in a 1 min incubation. If 1 mM calcium chloride was also present, thrombin inactivation by both antithrombin and antithrombin plus heparin changed. Namely, even after 8 min incubation, only 30 percent of thrombin was inactivated by antithrombin. Although heparin resulted in moderate inactivation, the effect was expressed only at a four-times higher heparin level. Accordingly, thrombin was slightly inactivated by antithrombin in the presence of 1 mM calcium chloride and 0.04 M sodium phosphate buffer at pH 7.4. These results suggested that calcium phosphate formed in the solution containing 1 mM calcium chloride and 0.04 M sodium phosphate at pH 7.4, bound thrombin thereby protecting it against antithrombin inactivation. To check this possibility, thrombin was mixed with the system mentioned, centrifuged at 30.000 x g for 30 min at 20°C and its activity determined in both the supernatant and the precipitate. As it can be seen from Table 1, after centrifugation, the supernatant of phosphate buffer containing calcium chloride too, showed no enzyme activity, whereas thrombin activity slightly.changed in the precipitate resuspended, as well as in the supernatant of the reaction mixture containing no calcium chloride. Namely, almost 100 percent of enzyme was bound to calcium phosphate. That is, a thrombin and calcium phosphate interaction can be suggested which does not-essentially influence the proteolytic activity of the enzyme (the conversion of fibrinogen).
CALCIUM
Vol.g,No.2
PHOSPHATE
127
AND THROMBIN
Similar experiments with antithrombin-III revealed that the interaction of calcium phosphate and antithrombin-III is out of question. As to the other nature of thrombin bound to calcium phosphate, it is clear from the results presented in Fig. 3 that the stability of the enzyme against the inactivating effect of heat also changed. Namely, thrombin was easily denatured at 54'C either in 0.05 M Tris-HCl buffer or in 0.05 M sodium phosphate buffer at pH 7.4. In phosphate buffer, about 80 percent of enzyme activity was lost in a 3 min incubation.
1.0
0.5 v) .Z C 3
I f
O!
4
min
8
FIG. 2 Tbrombin inactivation by antitbrombin-III and heparin in sodium phosphate buffer. Experiments were carried out as detailed in Fig. 1, except the Tris-HCl buffer, i.e. 8 moles sodium phosphate buffer, pH 7.4 was used for enzyme assay. plus calcium chloride, O_ thrombin alone, 0-e plus antithrombin, b------_-i plus calcium chloplus antithrombin and heride and antithrombin, cF--------_cI plus calcium chloride, antiparin (0.0025 units), _ plus calcium thrombin and heparin (0.0025 units), +-a chloride, antithrombin and heparin (0.01 unit).
128
CALCIUM
PHOSPHATE
AND
THROMBIN
Vol.g,No.2
If the reaction mixture contained calcium chloride too, at 1 mM final concentration, the rate of thrombin inactivation decreased, i.e. even after a 7 min incubation, approximately 50 percent of enzyme remained. In Tris-HCl buffer, calcium chloride acted in another way. It did not result in thrombin protection against heat inactivation. More than 50 percent of thrombin (either alone or with calcium chloride) was inactivated in about 1 min. Accordingly, thrombin bound to calcium phosphate is extremely stable against heat denaturation as well. TAElX 1 Binding of thrombin to calcium Fhosphate 18 NIH units thrombin was dissolved in 2 ml final volume containing 0.05 li sodium phosphate buffer, pi17.4 either in the presence or absence of 1 mM calcium chloride. 0.05 ml aliquots of reaction mixture were immediately assayed for clotting time. Thereafter the plastic tubes were centrifuged at 30.000 x g for 30 min at 20°C and the enzyme activity determined again from the supernatant as well as from the resuspended precipitate. The results are presented as NIH units per ml.
1 mM CaC12 absence
presence
Before centrifugation
8.4
7.0
After centrifugation supernatant
7.8
(1
precipitate
6.8
It is also interesting to note that a special behaviour of phosphate ions can be detected, i.e. 'thrombin seems to be more stable in phosphate buffer than in Tris-HCl buffer at pH 7.4. Similar results were also published by others for different proteins (16-17). However these observations need additional experimentation, since the clotting time of thrombin as well as the inactivation of thrombin by antithrombin-III and heparin depend on the ionic strength too (12,20).
Vol.g,No.2
CALCIUM PHOSPHATE AND THROMDIN
FIG. 3 Heat inactivation of thrombin in sodium phosphate buffer and in Tris-HC1 buffer at pH 7.4. 7 A.I~@; thrombin was incubated at 54'C either alone or with 0.5 voles calcium chloride in 0.55 ml final volume containing 25 jumoles sodium phosphate buffer or 25 moles Tris-HCl buffer, respectively. At varying periods between 0 and 10 min 0.05 ml aliquots were taken and determined for thrombin activity as detailed previously (10). Thrombin was gel-filtered on Sephadex G-25 equilibrated with 0.05 M sodium phosphate buffer, pH 7.4 or with 0.05 M Tris-HCl buffer, pH 7.4, respectively, immediately before heat inactivation experiments. _ thrombin alone in phosphate buffer, A-A thrombin and calcium chloride in phosphate buffer, 0-a thrombin alone in Tris-HCl buffer, ?? -m thrombin and calcium chloride in Tris-HCl buffer DISCUSSION The findings that thrombin bound to calcium phosphate can be hardly inactivated by its physiological inhibitor (antithrombin-III) may have importance under some pathological conditions. Since calcium phosphate can form in the wall of blood vessels during arteriosclerosis (13,18), it may inter-
129
130
CALCIUM PHOSPHATE AND THROMBIN
Vol.g,No.2
fere with thrombin when it forms in blood circulation. Such a bound form of enzyme cannot be inactivated by antithrombin, while it may catalyse the conversion of fibrinogen to fibrin, thereby possibly leading to predisposition for thrombosis. Whether the role of heparin in this reaction is the releasing of enzyme molecules from the bound form or it is another mechanism, needs further experimentation.
I am grateful to Mrs Therese Fazekas for the excellent technical assistance. RZFERERCES 1.
MAGNUSSON, S. Thrombin and prothrombin. In: Enz.ymes 3rd
Ed. 3, 278, 1971. 2. SEEGERS, W. H., WARNER, E. D., BRIMKHOUS, K. M. and %ITH, H. P. The use of purified thrombin as an hemostatic agent. Science 89, 86, 1939. 3. ABILDGAARD, U. Binding of thrombin to antithrombin-III. Stand. J. Clin. Lab. Invest. 24, 23, 1969. 4. ROSENBERG, R. D. and DAMUS, P. S. The purification and mechanism of action of human antithrombin-heparin cofactor. J. Biol. Chem. 248, 6490, 1973. 5. YIN, E. T., WESSLER, S. and STOLL, I?. J. Identity of plasma-activated factor X inhibitor with antithrombin and heparin cofactor. J. Biol. Chem. 246, 3712, 1971. 6. DAMUS, I?. S., HICKS, M. and ROSENBERG, R. D. Anticoagulant action of heparin. Nature 246, 355, 1973. R. Mechanism of action of heparin through 7. IUr.ACHOVICH, thrombin on blood coagulation. Biochim. Biophss. Acta 412, 13, 1975. 8. LI, E. H. H., ORTON, CH. and FEIRMAN, R. D. The interaction of thrombin and heparin. Proflavine dye binding studies. Biochemistry 13, 5012, 1974. 9. MACHOVICH, R., BLASKd, GY. and FALOS, A. L. Action of heparin on thrombin-antithrombin reaction. Biochim. Bionhys. &&
379, 193, 1975.
Vol.9,No.2
CALCIUM PHOSPHATE AND THROMBIN
131
10. MACHOVICH, R., BLASKC, GY. and ARANYI, P. The interaction of thrombin and heparin. Heat inactivation kinetics. Thrombos. Res. 7, 253, 1975.
11. MACHOVICH, R. Heparin sensitive and nonsensitive forms of thrombin. Biochim. Biophys. Acta 400, 62, 1975. 12. MACHOVICH, R., BLASK6, GY., HIMER AGBJES and SZIKLA, K. Effect of sodium and potassium ions on the human antithrombin-heparin cofactor. Thrombos. Res. 7, 305, 1975.
13. SCHETTiZR, F. G. and BOYD, G. S. (ED.) Atherosclerosis Elsevier Publishing Company, p. 13, 1969. 14. ILJXDBLAD, R. L. A rapid method for the purification of bovine thrombin. Biochemistry 10, 2501, 1971. 15. LOWRY, 0. II., ROSEBROUGH, N. J., FARR, A. L. and RANDALL, R. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265, 1951. 16. WICKETT, R., LI, H. J. and ISENBERG, J. Salt effects of histone IV conformation. Biochemistry 11, 2952, 1972. 17. FURBERG, S. and SOLBAKK, J. On the stereochemistry of the interaction between nucleic acids and basic protein side chains. J. Acta Chem. Stand. B. 28, 481, 1974. 18. FLEISCH, H. and NEUMAN, W. F. Quantitative aspects of nucleation in calcium phosphate precipitation. Amer. Chem. Sot. 82, 996, 1960. 19. GERENDAS, M., PALOS, A. L. and CSEFKO, J. Heparin effect and thrombin inactivation. Ante eadern Arch. Biol. Hung. 19, 191, 1949. 20. GODAL, H. C. The assay of heparin in thrombin Stand. Clin. Lab. Invest. 13, 153, 1961.
SyStem.
in
the
United States
vol. 9, pp. 123-131, 1976 Pergamon Press, Inc.
EFFECT OF CALCIUM YHOSYHATE ON THROMBIN AND ON ITS SENSITIVITY TO HEPARIN AifD ANTITHROMBIN-III Raymund Machovich Medical School, First Department Of Pledicine, 1389 Budapest, Hungary
Postgraduate
(Received
20.4.1976; in revised Accepted
by Editor
M.
form
2.6.1976.
Kopec)
ABSTRACTThrombin was inactivated by antithrombin-III and the rate of inactivation was accelerated by heparin either in 0.04 M sodium phosphate buffer or in 0.04 M Tris-HCl buffer, at pH 7.4. Calcium chloride, at a final concentration of 1 mM, influenced slightly thrombin inactivation by both antithrombin and antithrombin plus heparin in Tris-HCl buffer, whereas in phosphate buffer the sensitivity of enzyme to antithrombin and heparin decreased. Thrombin showed also increased stBbility against the inactivating effect of heat at 54 C in sodium phosphate buffer containing calcium chloride. These findings suggest that thrombin bound to calcium phosphate is extremely stable against inactivation by antithrombin and heparin, while its clotting activity does not change. This nature of enzyme may play a role in predisposition for thrombosis during arteriosclerosis. INTRODUCTION Thrombin plays a key role in thrombosis and hemostasis. Its main function is to catalyse the conversion of fibrinogen to fibrin (1). Under physiological conditions, thrombin formed in blood circulation can be inactivated by antithrombinIII (Z-5). It is also known that heparin accelerates the complex formation between enzyme and its inhibitor (5-6,19). It has been proposed that "heparin acts to accelerate inhibitor function by binding to antithrombin and inducing an allosteric modification in it" (6). Other data suggest that heparin 123
124
may affect
CALCIUM
enzyme
PHOSPHATE
inactivation
AND THROMBIN
by antithrombin
Vo1.9,No.Z
through
a
thrombin and heparin interaction as well, since hepsrin binds tightly to thrombin but not at the active site (8-10). Namely, the binding of heparin to thrombin may induce a conformational change in the enzyme, facilitating the complex formation of thrombin with antithrombin (7).
It has also been documen-
ted that many factors influence thrombin-heparin interaction and enzyme inactivation by antithrombin (ll-12,20). Among the possible factors which may interfere with thrombin, antithrombin and heparin, we examined the effect of calcium chloride in Iris-HCl buffer and in sodium phosphate buffer on activity and stability of thrombin, as well as on its sensitivity to heparin and antithrombin-III. MATERIALS AND METHODS Thrombin was prepared from commercial bovine thrombin (Topostasin, La Roche) by chromatography on Sulfoethyl-Sephadex C-50 column according to the method of Lundblad (14) and gel-filtered on Sephadex G-25 equilibrated with 0.05 M TrisHCl buffer or 0.05 M sodium phosphate buffer, at pH 7.4. The specific activity of purified thrombin was about 1500-2000 NIH units per mg protein before gel-filtration. Thrombin and antithrombin activity were assayed as published earlier (7). Thrombin activity was calculated and expressed in NIH units as detailed elsewhere (7). Antithrombin-III was partially purified from human plasma as published earlier (12) and dialyzed against 0.1 M sodium chloride. Protein was determined according to the method of Iowry et al (15). Sulfoethyl-Sephadex C-50 and Sephadex G-25 were purchased from Pharmacia. Heparin was obtained from G. Richter Ltd. and dissolved in 0.1 M sodium chloride. Fibrinogen was the product of KABI (human, Grade L.). Other chemicals were purchased from Reanal Fine Chemicals, Budapest. RESULTS Thrombin was not inactivated by incubation at 37'C for 5 min in 0.04 M Tris-HCl buffer at pH 7.4 either in the presence or absence of calcium chloride (Fig. 1). Antithrombin
CALCIUM
Vol.9,No.2
PHOSPHATE
AND THROMBIN
125
inactivated about 50 percent of enzyme in a 2 min incubation. If calcium chloride was also added, at a final concentration of 1 mM, the rate of thrombin inactivation did not change. Heparin, at a C.01 unit per ml final concentration, increased the rate of enzyme inactivation by antithrombin, i.e. about 80 percent of thrombin activity was already lost in a 1 min incubation. The results with heparin were not altered in the presence of 1 mN calcium chloride. Calcium chloride increased
-
1
0
0
”
A
-
I
1
1
I
min
I
1
5
FIG. 1 Thrombin inactivation by antithrombin-III and heparin in Tris-HCl buffer. 0.5 fig thrombin was incubated either alone or with 50 fig antithrombin protein or with antithrombin plus 0.0025 units heparin, in 0.2 ml reaction mixture containing 8 moles TrisHCl buffer pH 7.4 and 10 ,umoles sodium chloride either in the presence or the absence of 0.25 ,ugoles calcium chloride, respectively. After incubation at 37 C for varying periods, 500 ,ug fibrinogen in 0.05 ml volume was added and clotting time measured with the Hyland Clotek System. plus calcium chloride, 0-o thrombin alone, e-0 plus calcium chlob--------d plus antithrombin, _ ride and antithrombin, cF------_-0 plus antithrombin and heparin, _ plus calcium chloride, heparin ard. antithrombhn
126
CALCIUM PHOSPHATE AND THROMBIN
Vol.g,No.2
slightly thrombin activity, however it did not change the rate of enzyme inactivation either by antithrombin or by antithrombin plus heparin. Thrombin behaved differently in 0.04 M sodium phosphate buffer at pH 7.4 (Fig. 2). Although, the enzyme showed some inactivation in the presence of 1 mM calcium chloride, it was not altered by a further 5 min incubation. Without calcium chloride, antithrombin or antithrombin plus heparin affected thrombin activity in a similar way as in Tris-HCl buffer, i.e. about 50 percent of enzyme activity was inhibited by antithrombin in a 2 min incubation, whereas in the presence of heparin (at the same concentration as in Tris-HCl buffer) about 90 percent of enzyme activity was also lost in a 1 min incubation. If 1 mM calcium chloride was also present, thrombin inactivation by both antithrombin and antithrombin plus heparin changed. Namely, even after 8 min incubation, only 30 percent of thrombin was inactivated by antithrombin. Although heparin resulted in moderate inactivation, the effect was expressed only at a four-times higher heparin level. Accordingly, thrombin was slightly inactivated by antithrombin in the presence of 1 mM calcium chloride and 0.04 M sodium phosphate buffer at pH 7.4. These results suggested that calcium phosphate formed in the solution containing 1 mM calcium chloride and 0.04 M sodium phosphate at pH 7.4, bound thrombin thereby protecting it against antithrombin inactivation. To check this possibility, thrombin was mixed with the system mentioned, centrifuged at 30.000 x g for 30 min at 20°C and its activity determined in both the supernatant and the precipitate. As it can be seen from Table 1, after centrifugation, the supernatant of phosphate buffer containing calcium chloride too, showed no enzyme activity, whereas thrombin activity slightly.changed in the precipitate resuspended, as well as in the supernatant of the reaction mixture containing no calcium chloride. Namely, almost 100 percent of enzyme was bound to calcium phosphate. That is, a thrombin and calcium phosphate interaction can be suggested which does not-essentially influence the proteolytic activity of the enzyme (the conversion of fibrinogen).
CALCIUM
Vol.g,No.2
PHOSPHATE
127
AND THROMBIN
Similar experiments with antithrombin-III revealed that the interaction of calcium phosphate and antithrombin-III is out of question. As to the other nature of thrombin bound to calcium phosphate, it is clear from the results presented in Fig. 3 that the stability of the enzyme against the inactivating effect of heat also changed. Namely, thrombin was easily denatured at 54'C either in 0.05 M Tris-HCl buffer or in 0.05 M sodium phosphate buffer at pH 7.4. In phosphate buffer, about 80 percent of enzyme activity was lost in a 3 min incubation.
1.0
0.5 v) .Z C 3
I f
O!
4
min
8
FIG. 2 Tbrombin inactivation by antitbrombin-III and heparin in sodium phosphate buffer. Experiments were carried out as detailed in Fig. 1, except the Tris-HCl buffer, i.e. 8 moles sodium phosphate buffer, pH 7.4 was used for enzyme assay. plus calcium chloride, O_ thrombin alone, 0-e plus antithrombin, b------_-i plus calcium chloplus antithrombin and heride and antithrombin, cF--------_cI plus calcium chloride, antiparin (0.0025 units), _ plus calcium thrombin and heparin (0.0025 units), +-a chloride, antithrombin and heparin (0.01 unit).
128
CALCIUM
PHOSPHATE
AND
THROMBIN
Vol.g,No.2
If the reaction mixture contained calcium chloride too, at 1 mM final concentration, the rate of thrombin inactivation decreased, i.e. even after a 7 min incubation, approximately 50 percent of enzyme remained. In Tris-HCl buffer, calcium chloride acted in another way. It did not result in thrombin protection against heat inactivation. More than 50 percent of thrombin (either alone or with calcium chloride) was inactivated in about 1 min. Accordingly, thrombin bound to calcium phosphate is extremely stable against heat denaturation as well. TAElX 1 Binding of thrombin to calcium Fhosphate 18 NIH units thrombin was dissolved in 2 ml final volume containing 0.05 li sodium phosphate buffer, pi17.4 either in the presence or absence of 1 mM calcium chloride. 0.05 ml aliquots of reaction mixture were immediately assayed for clotting time. Thereafter the plastic tubes were centrifuged at 30.000 x g for 30 min at 20°C and the enzyme activity determined again from the supernatant as well as from the resuspended precipitate. The results are presented as NIH units per ml.
1 mM CaC12 absence
presence
Before centrifugation
8.4
7.0
After centrifugation supernatant
7.8
(1
precipitate
6.8
It is also interesting to note that a special behaviour of phosphate ions can be detected, i.e. 'thrombin seems to be more stable in phosphate buffer than in Tris-HCl buffer at pH 7.4. Similar results were also published by others for different proteins (16-17). However these observations need additional experimentation, since the clotting time of thrombin as well as the inactivation of thrombin by antithrombin-III and heparin depend on the ionic strength too (12,20).
Vol.g,No.2
CALCIUM PHOSPHATE AND THROMDIN
FIG. 3 Heat inactivation of thrombin in sodium phosphate buffer and in Tris-HC1 buffer at pH 7.4. 7 A.I~@; thrombin was incubated at 54'C either alone or with 0.5 voles calcium chloride in 0.55 ml final volume containing 25 jumoles sodium phosphate buffer or 25 moles Tris-HCl buffer, respectively. At varying periods between 0 and 10 min 0.05 ml aliquots were taken and determined for thrombin activity as detailed previously (10). Thrombin was gel-filtered on Sephadex G-25 equilibrated with 0.05 M sodium phosphate buffer, pH 7.4 or with 0.05 M Tris-HCl buffer, pH 7.4, respectively, immediately before heat inactivation experiments. _ thrombin alone in phosphate buffer, A-A thrombin and calcium chloride in phosphate buffer, 0-a thrombin alone in Tris-HCl buffer, ?? -m thrombin and calcium chloride in Tris-HCl buffer DISCUSSION The findings that thrombin bound to calcium phosphate can be hardly inactivated by its physiological inhibitor (antithrombin-III) may have importance under some pathological conditions. Since calcium phosphate can form in the wall of blood vessels during arteriosclerosis (13,18), it may inter-
129
130
CALCIUM PHOSPHATE AND THROMBIN
Vol.g,No.2
fere with thrombin when it forms in blood circulation. Such a bound form of enzyme cannot be inactivated by antithrombin, while it may catalyse the conversion of fibrinogen to fibrin, thereby possibly leading to predisposition for thrombosis. Whether the role of heparin in this reaction is the releasing of enzyme molecules from the bound form or it is another mechanism, needs further experimentation.
I am grateful to Mrs Therese Fazekas for the excellent technical assistance. RZFERERCES 1.
MAGNUSSON, S. Thrombin and prothrombin. In: Enz.ymes 3rd
Ed. 3, 278, 1971. 2. SEEGERS, W. H., WARNER, E. D., BRIMKHOUS, K. M. and %ITH, H. P. The use of purified thrombin as an hemostatic agent. Science 89, 86, 1939. 3. ABILDGAARD, U. Binding of thrombin to antithrombin-III. Stand. J. Clin. Lab. Invest. 24, 23, 1969. 4. ROSENBERG, R. D. and DAMUS, P. S. The purification and mechanism of action of human antithrombin-heparin cofactor. J. Biol. Chem. 248, 6490, 1973. 5. YIN, E. T., WESSLER, S. and STOLL, I?. J. Identity of plasma-activated factor X inhibitor with antithrombin and heparin cofactor. J. Biol. Chem. 246, 3712, 1971. 6. DAMUS, I?. S., HICKS, M. and ROSENBERG, R. D. Anticoagulant action of heparin. Nature 246, 355, 1973. R. Mechanism of action of heparin through 7. IUr.ACHOVICH, thrombin on blood coagulation. Biochim. Biophss. Acta 412, 13, 1975. 8. LI, E. H. H., ORTON, CH. and FEIRMAN, R. D. The interaction of thrombin and heparin. Proflavine dye binding studies. Biochemistry 13, 5012, 1974. 9. MACHOVICH, R., BLASKd, GY. and FALOS, A. L. Action of heparin on thrombin-antithrombin reaction. Biochim. Bionhys. &&
379, 193, 1975.
Vol.9,No.2
CALCIUM PHOSPHATE AND THROMBIN
131
10. MACHOVICH, R., BLASKC, GY. and ARANYI, P. The interaction of thrombin and heparin. Heat inactivation kinetics. Thrombos. Res. 7, 253, 1975.
11. MACHOVICH, R. Heparin sensitive and nonsensitive forms of thrombin. Biochim. Biophys. Acta 400, 62, 1975. 12. MACHOVICH, R., BLASK6, GY., HIMER AGBJES and SZIKLA, K. Effect of sodium and potassium ions on the human antithrombin-heparin cofactor. Thrombos. Res. 7, 305, 1975.
13. SCHETTiZR, F. G. and BOYD, G. S. (ED.) Atherosclerosis Elsevier Publishing Company, p. 13, 1969. 14. ILJXDBLAD, R. L. A rapid method for the purification of bovine thrombin. Biochemistry 10, 2501, 1971. 15. LOWRY, 0. II., ROSEBROUGH, N. J., FARR, A. L. and RANDALL, R. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265, 1951. 16. WICKETT, R., LI, H. J. and ISENBERG, J. Salt effects of histone IV conformation. Biochemistry 11, 2952, 1972. 17. FURBERG, S. and SOLBAKK, J. On the stereochemistry of the interaction between nucleic acids and basic protein side chains. J. Acta Chem. Stand. B. 28, 481, 1974. 18. FLEISCH, H. and NEUMAN, W. F. Quantitative aspects of nucleation in calcium phosphate precipitation. Amer. Chem. Sot. 82, 996, 1960. 19. GERENDAS, M., PALOS, A. L. and CSEFKO, J. Heparin effect and thrombin inactivation. Ante eadern Arch. Biol. Hung. 19, 191, 1949. 20. GODAL, H. C. The assay of heparin in thrombin Stand. Clin. Lab. Invest. 13, 153, 1961.
SyStem.