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Calcium-dependent binding of α2-plasmin inhibitor to fibrin
IXROMBOSIS Pergamon
RESEARCH 16; 279-282 Printed Press Ltd.1979.
BRIEF
in Great
COMMUNI
Brixain
CATI
(3 S
CALCIUM-DEPENDENT BINDING OF a,-PLASMIN INHIBITOR TO FIBRIN Yoichi Sakata, Keiko Tateno, Taro Tamaki and Nobuo Aoki Institute of Hematology and Department of Medicine, Jichi Medical School, Tochigi-Ken 329-04 Japan
(Received
6.6.1979.
Accepted
by
Editor
S.
Iwanaga)
INTRODUCTION A proteinase inhibitor termed as az-proteinase inhibitor or az-plasmin inhibitor (cQPI) (1, 2) is very unique among various inhibitors in plasma in that it inhibits fibrinolysis more efficiently than expected from its inhibitory activity on plasmin (3, 4). The efficient inhibition of fibrinolysis by a2PI was attributed to the inhibition of binding of plasminogen to fibrin by aeP1 (3) in addition to the rapid inactivation of plasmin formed during plasminogen activation (1). We herein present data to suggest an additional mechanism of efficient inhibition of fibrinolysis by a,PI, that is the binding of a,PI to fibrin when fibrin is formed. The binding was dependent on calcium ions and seemed to be covalent.
MATERIALS AND METHODS Plasma and serum Blood freshly drawn from antecubital veins was divided. One portion was immediately mixed with the powder of disodium ethylenediaminetetraacetate dihydrate (EDTA) (Wako Pure Chemical Ind., Osaka) in a ratio of 1.86 mg EDTA to 1 ml blood and was centrifuged at 2,000 g for 20 min to prepare plasma. Another portion was allowed to stand in a glass test tube for 2 hours at room temperature, and serum was separated from the clot by centrifugation. Serum was also obtained by clotting 1 ml of EDTA-plasma with 50 ~1 of thrombin (20 u/ml) or thrombin-calcium mixture (thrombin 20 u/ml, CaC12 0.5 M).
Key Words:
a,-plasmin inhibitor, fibrinolysis, factor XII I, cross#link, Binding of a2-plasmin inhibitor 279
Fibrinogen A plasminogen- free fibrinogen solution was prepared by removir;g 2la;minogen from human Cohn's fraction I (Green Cross Corp., Osaka) with lysineSepharose (5). Fibrinogen was dissolved in barbital buffered saline (0.005 M Barbital-acetic acid-O.14 M NaCl, pH 7.4) and the concentration was adjusted ;;r;,;i; (w/v) clottable protein. Purified thrombin was prepared from bovine thrombin preparation (Parke & Davis Comp., Detroit) according to the method of Lundblad (6), and was stored as a 200 u/ml solution in 50 "s glycerol at -20°C. Inhibitor proteins cr2PIwas purified by the method previously described (1). The concentration of a2PI was determined by single radial immunodiffusion using monospecific antiserum to a2PI and a purified a,PI preparation with a known concentration as a standard (7). Radioiodination of azP1 Purified azP1 was radioiodinated by the method of Thorell and Johansson (8) using lactoperoxidase (Calbiochem., LaJolla, Cal.) and Na lz51 (17 Ci/mg) (New England Nuclear, Boston, Mass.). The labeled a2PI preparation had the radioactivity of 1.1 x 10' cpm/ug. Specific activity of a2PI before and after radioiodination were 1136 u/A2a0 and 1050 u/AZBO, respectively. The labeled azP1 was mixed with fivefold volumes of nonlabeled azP1 to obtain 1.5 mg/ml a,PI solution before the study. Quantitation of the binding of a2PI to fibrin 130 ~1 of fibrinogen (1.5 %) were mixed with 40 ul of radiolabeled CQPI (1.5 mg/ml) and 800 ~1 of buffered saline containing various concentrations of calcium chloride in a counting vial for radioactivity. Subsequently, 20 ~1 of thrombin (ZOO u/ml) was added, and the mixture was incubated at 25°C for 1 hour. After counting radioactivity, the mixture was centrifuged at 2000 g for 20 min. The supernatant was removed, and the clot was transfered onto a millipore filter (Filter type G.S., pore size 0.22 vm, Millipore Corp., Bedford, Mass.) placed on a suction funnel. The clot was drained Off on the filter by a sucking force, and was further washed with buffered saline totaling to 80 ml. Afterwards, the clot was counted for radioactivity. Radioactivity was counted by Auto Well Gamma System, Aloka JDC-752 (Aloka co., Tokyo). The amount of a2PI bound to fibrin was calculated from the radioactivity remaining in the washed clot and expressed as a percentage of the original total radioactivity in the clotting mixture. The studies were carried in triplicate. The original radioactivity in the clotting mixture was 80817 2 4747 cpm.
RESULTS Difference a,PI concentration in plasma was all the time higher than that in serum of the same individual. When 11 normal individuals were examined, the mean concentrations of a,PI in plasma and in serum were 6.9 r 0.6 mg/lOO ml and 5.3 + 0.7 mg/lOO ml, respectively. The difference was 1.6 + 0.4 mg/lOO ml or 23.6 r 6 % of the concentration in plasma. azP1 concentration in serum prepared by clotting plasma with thrombin and calcium ions was compared with that in serum prepared by clotting plasma with thrombin only. When 9 pairs of the serum samples were examined, the mean concentrations in the former and the latter sera were 4.2 it0.6 mg/lOO ml and 6.3 + 0.6 mg/lOO ml, respectively. The difference was 2.1 i 0.6 mg/lOO ml or 32.9 + 7.8 % o f the concentration in serum prepared in the absence of
Vo1.16,N0.1/2
BINDI?X
OF d2PI
TO
FIBRIX
281
calcium ions. Thus, sera prepared in the presence of calcium ions had on average 33 I lower concentrations of azPI than sera prepared in the absence of calcium ions. Binding of a2PI to fibrin in the presence of calcium In the absence of calcium ions only 1.6 2 0.17 X of aTPI in the clotting mixture remained bound to fibrin after washing with buffered saline. However, the binding was increased when calcium ion was present in the clotting mixture. In the presence of 5 mM calcium chloride 16 2 0.6 % of azP1 in the clotting mixture were bound to fibrin when it clotted. The increase of calcium concentration up to 50 mM did not change appreciably the ratio between fibrin-bound a2PI and free azPI. The bound_azPI could not be eluted from the fibrin with 6aminohexanoic acid (20 mM), tranexamic acid (5 mM), EDTA (20 mM) or 2M urea.
DISCUSSION When a2PI concentration in serum prepared from spontaneously clotted blood was compared with that in plasma, serum was found to contain less amount of azP1 than plasma. When sera were prepared by clotting plasma with thrombin in the presence or the absence of calcium ions, azPI concentration in serum formed in the presence of calcium ions was less than that in serum prepared in the absence of calcium ions. These findings suggest that azP1 is specifically adsorbed to fibrin when blood is clotted in the presence of calcium ions. This was further supported by the finding that radiolabeled azP1 was bound to fibrin when fibrin was formed by thrombin and calcium ions. Without calcium ions binding of a2PI to fibrin was only 1.6 X on average, whereas the binding was 16 Z when calcium ions were present. This value was less than the difference of azP1 concentrations between plasma and serum (24 %) or between serum prepared by thrombin-calcium and serum prepared by thrombin only (33 %). This may imply that another factor is necessary for the binding in addition to calcium ions and the factor is present in a larger amount in plasma than in fibrinogen preparation (Cohn's fraction I). The bound a2PI could not be eluted from fibrin even with a high concentration of urea, suggesting that the binding is covalent. Furthermore, the binding seems to be mediated by thrombin and calcium ions. These findings suggest that the factor responsible for the binding may be blood coagulation factor XIII. Factor XIII activated by thrombin and calcium ions (9) might be catalizing the crosslinking of a2PI to fibrin. This interesting subject is now under investigation in our laboratory. Whatever mechanism may be, the binding of apPI to fibrin would localize and concentrate azP1 strategically on fibrin molecules so that fibrin would be protected efficiently from plasmin-catalized fibrinolysis
ACKJK'WLEDGEMENTS This work was supported in part by Scientific Research Grants from the Ministry of Education of the Government of Japan and by Yamanouchi Foundation for Research on Metabolic Disorders.
282
REFERENCES MOROI, M. and AOKI, Ii. Isolation and characterization of az-plasmin inhibitor from human plasma. A novel proteinase inhibitor which inhibits activator-induced clot lysis. J. Biol. Chem. 251:5956-5965, 1976.
2)
AOKI, N. Natural inhibitors of fibrinolysis. 21:267-286, 1979.
Prog. Cardiovasc. Dis.
3)
AOKI, N., MOROI, M. and TACHIYA, K. Effects of a*-plasmin inhibitor on fibrin clot lysis. Its comparison with az-macroglobulin. Thrombos. Haemostas. 39:22-31, 1978.
4)
AOKI, N., SAITO, H., KAMIYA, T., KOIE, K., SAKATA, Y. and KOHAKURA, M. Congenital deficiency of a*-plasmin inhibitor associated with severe hemorrhagic tendency. J. Clin. Invest. 63:877-884, 1979.
5)
MATSUDA, M., IWANAGA, S. and NAKAMURA, S. A simple, large scale method for preparation of plasminogen-free fibrinogen. Thromb. Res. 1:619-630, 1972.
6)
LLJNDBLAD,R. L. A rapid method for the purification of bovine thrombin and the inhibition of the purified enzyme with phenylmethylsulfonyl fluoride. Biochemistry 10:2501-2506, 1971.
7)
AOKI, N. and YAMANAKA, T. The crz-plasmin inhibitor levels in liver diseases. Clin. Chim. Acta 84:99-105, 1978.
8)
THORELL, 3. 1. and JOHANSSON, B. G. Enzymatic iodination of polypeptides with "'1 to high specific activity. Biochim. Biophys. Acta 251:363-369, 1971.
9)
LORAND, L. The fibrin-stabilizing factor system of blood plasma. Ann. N.Y. Acad. Sci. 202:6-30, 1972.
RESEARCH 16; 279-282 Printed Press Ltd.1979.
BRIEF
in Great
COMMUNI
Brixain
CATI
(3 S
CALCIUM-DEPENDENT BINDING OF a,-PLASMIN INHIBITOR TO FIBRIN Yoichi Sakata, Keiko Tateno, Taro Tamaki and Nobuo Aoki Institute of Hematology and Department of Medicine, Jichi Medical School, Tochigi-Ken 329-04 Japan
(Received
6.6.1979.
Accepted
by
Editor
S.
Iwanaga)
INTRODUCTION A proteinase inhibitor termed as az-proteinase inhibitor or az-plasmin inhibitor (cQPI) (1, 2) is very unique among various inhibitors in plasma in that it inhibits fibrinolysis more efficiently than expected from its inhibitory activity on plasmin (3, 4). The efficient inhibition of fibrinolysis by a2PI was attributed to the inhibition of binding of plasminogen to fibrin by aeP1 (3) in addition to the rapid inactivation of plasmin formed during plasminogen activation (1). We herein present data to suggest an additional mechanism of efficient inhibition of fibrinolysis by a,PI, that is the binding of a,PI to fibrin when fibrin is formed. The binding was dependent on calcium ions and seemed to be covalent.
MATERIALS AND METHODS Plasma and serum Blood freshly drawn from antecubital veins was divided. One portion was immediately mixed with the powder of disodium ethylenediaminetetraacetate dihydrate (EDTA) (Wako Pure Chemical Ind., Osaka) in a ratio of 1.86 mg EDTA to 1 ml blood and was centrifuged at 2,000 g for 20 min to prepare plasma. Another portion was allowed to stand in a glass test tube for 2 hours at room temperature, and serum was separated from the clot by centrifugation. Serum was also obtained by clotting 1 ml of EDTA-plasma with 50 ~1 of thrombin (20 u/ml) or thrombin-calcium mixture (thrombin 20 u/ml, CaC12 0.5 M).
Key Words:
a,-plasmin inhibitor, fibrinolysis, factor XII I, cross#link, Binding of a2-plasmin inhibitor 279
Fibrinogen A plasminogen- free fibrinogen solution was prepared by removir;g 2la;minogen from human Cohn's fraction I (Green Cross Corp., Osaka) with lysineSepharose (5). Fibrinogen was dissolved in barbital buffered saline (0.005 M Barbital-acetic acid-O.14 M NaCl, pH 7.4) and the concentration was adjusted ;;r;,;i; (w/v) clottable protein. Purified thrombin was prepared from bovine thrombin preparation (Parke & Davis Comp., Detroit) according to the method of Lundblad (6), and was stored as a 200 u/ml solution in 50 "s glycerol at -20°C. Inhibitor proteins cr2PIwas purified by the method previously described (1). The concentration of a2PI was determined by single radial immunodiffusion using monospecific antiserum to a2PI and a purified a,PI preparation with a known concentration as a standard (7). Radioiodination of azP1 Purified azP1 was radioiodinated by the method of Thorell and Johansson (8) using lactoperoxidase (Calbiochem., LaJolla, Cal.) and Na lz51 (17 Ci/mg) (New England Nuclear, Boston, Mass.). The labeled a2PI preparation had the radioactivity of 1.1 x 10' cpm/ug. Specific activity of a2PI before and after radioiodination were 1136 u/A2a0 and 1050 u/AZBO, respectively. The labeled azP1 was mixed with fivefold volumes of nonlabeled azP1 to obtain 1.5 mg/ml a,PI solution before the study. Quantitation of the binding of a2PI to fibrin 130 ~1 of fibrinogen (1.5 %) were mixed with 40 ul of radiolabeled CQPI (1.5 mg/ml) and 800 ~1 of buffered saline containing various concentrations of calcium chloride in a counting vial for radioactivity. Subsequently, 20 ~1 of thrombin (ZOO u/ml) was added, and the mixture was incubated at 25°C for 1 hour. After counting radioactivity, the mixture was centrifuged at 2000 g for 20 min. The supernatant was removed, and the clot was transfered onto a millipore filter (Filter type G.S., pore size 0.22 vm, Millipore Corp., Bedford, Mass.) placed on a suction funnel. The clot was drained Off on the filter by a sucking force, and was further washed with buffered saline totaling to 80 ml. Afterwards, the clot was counted for radioactivity. Radioactivity was counted by Auto Well Gamma System, Aloka JDC-752 (Aloka co., Tokyo). The amount of a2PI bound to fibrin was calculated from the radioactivity remaining in the washed clot and expressed as a percentage of the original total radioactivity in the clotting mixture. The studies were carried in triplicate. The original radioactivity in the clotting mixture was 80817 2 4747 cpm.
RESULTS Difference a,PI concentration in plasma was all the time higher than that in serum of the same individual. When 11 normal individuals were examined, the mean concentrations of a,PI in plasma and in serum were 6.9 r 0.6 mg/lOO ml and 5.3 + 0.7 mg/lOO ml, respectively. The difference was 1.6 + 0.4 mg/lOO ml or 23.6 r 6 % of the concentration in plasma. azP1 concentration in serum prepared by clotting plasma with thrombin and calcium ions was compared with that in serum prepared by clotting plasma with thrombin only. When 9 pairs of the serum samples were examined, the mean concentrations in the former and the latter sera were 4.2 it0.6 mg/lOO ml and 6.3 + 0.6 mg/lOO ml, respectively. The difference was 2.1 i 0.6 mg/lOO ml or 32.9 + 7.8 % o f the concentration in serum prepared in the absence of
Vo1.16,N0.1/2
BINDI?X
OF d2PI
TO
FIBRIX
281
calcium ions. Thus, sera prepared in the presence of calcium ions had on average 33 I lower concentrations of azPI than sera prepared in the absence of calcium ions. Binding of a2PI to fibrin in the presence of calcium In the absence of calcium ions only 1.6 2 0.17 X of aTPI in the clotting mixture remained bound to fibrin after washing with buffered saline. However, the binding was increased when calcium ion was present in the clotting mixture. In the presence of 5 mM calcium chloride 16 2 0.6 % of azP1 in the clotting mixture were bound to fibrin when it clotted. The increase of calcium concentration up to 50 mM did not change appreciably the ratio between fibrin-bound a2PI and free azPI. The bound_azPI could not be eluted from the fibrin with 6aminohexanoic acid (20 mM), tranexamic acid (5 mM), EDTA (20 mM) or 2M urea.
DISCUSSION When a2PI concentration in serum prepared from spontaneously clotted blood was compared with that in plasma, serum was found to contain less amount of azP1 than plasma. When sera were prepared by clotting plasma with thrombin in the presence or the absence of calcium ions, azPI concentration in serum formed in the presence of calcium ions was less than that in serum prepared in the absence of calcium ions. These findings suggest that azP1 is specifically adsorbed to fibrin when blood is clotted in the presence of calcium ions. This was further supported by the finding that radiolabeled azP1 was bound to fibrin when fibrin was formed by thrombin and calcium ions. Without calcium ions binding of a2PI to fibrin was only 1.6 X on average, whereas the binding was 16 Z when calcium ions were present. This value was less than the difference of azP1 concentrations between plasma and serum (24 %) or between serum prepared by thrombin-calcium and serum prepared by thrombin only (33 %). This may imply that another factor is necessary for the binding in addition to calcium ions and the factor is present in a larger amount in plasma than in fibrinogen preparation (Cohn's fraction I). The bound a2PI could not be eluted from fibrin even with a high concentration of urea, suggesting that the binding is covalent. Furthermore, the binding seems to be mediated by thrombin and calcium ions. These findings suggest that the factor responsible for the binding may be blood coagulation factor XIII. Factor XIII activated by thrombin and calcium ions (9) might be catalizing the crosslinking of a2PI to fibrin. This interesting subject is now under investigation in our laboratory. Whatever mechanism may be, the binding of apPI to fibrin would localize and concentrate azP1 strategically on fibrin molecules so that fibrin would be protected efficiently from plasmin-catalized fibrinolysis
ACKJK'WLEDGEMENTS This work was supported in part by Scientific Research Grants from the Ministry of Education of the Government of Japan and by Yamanouchi Foundation for Research on Metabolic Disorders.
282
REFERENCES MOROI, M. and AOKI, Ii. Isolation and characterization of az-plasmin inhibitor from human plasma. A novel proteinase inhibitor which inhibits activator-induced clot lysis. J. Biol. Chem. 251:5956-5965, 1976.
2)
AOKI, N. Natural inhibitors of fibrinolysis. 21:267-286, 1979.
Prog. Cardiovasc. Dis.
3)
AOKI, N., MOROI, M. and TACHIYA, K. Effects of a*-plasmin inhibitor on fibrin clot lysis. Its comparison with az-macroglobulin. Thrombos. Haemostas. 39:22-31, 1978.
4)
AOKI, N., SAITO, H., KAMIYA, T., KOIE, K., SAKATA, Y. and KOHAKURA, M. Congenital deficiency of a*-plasmin inhibitor associated with severe hemorrhagic tendency. J. Clin. Invest. 63:877-884, 1979.
5)
MATSUDA, M., IWANAGA, S. and NAKAMURA, S. A simple, large scale method for preparation of plasminogen-free fibrinogen. Thromb. Res. 1:619-630, 1972.
6)
LLJNDBLAD,R. L. A rapid method for the purification of bovine thrombin and the inhibition of the purified enzyme with phenylmethylsulfonyl fluoride. Biochemistry 10:2501-2506, 1971.
7)
AOKI, N. and YAMANAKA, T. The crz-plasmin inhibitor levels in liver diseases. Clin. Chim. Acta 84:99-105, 1978.
8)
THORELL, 3. 1. and JOHANSSON, B. G. Enzymatic iodination of polypeptides with "'1 to high specific activity. Biochim. Biophys. Acta 251:363-369, 1971.
9)
LORAND, L. The fibrin-stabilizing factor system of blood plasma. Ann. N.Y. Acad. Sci. 202:6-30, 1972.