THROMBOSIS RESEARCH 67; 613-618, 1992 0049-3848/92 $5.00 + .OOPrinted in the USA. Copyright (c) 1992 Pergamon Press Ltd. All rights reserved.
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CALCIUM BINDING TO ABNORMALFIBRINOGENS WITH A SINGLE AMINO ACID REPLACEMENTIN THE NHP-TERMINAL REGION OF FIBRIN o:- OR F-CHAIN
Nobuhiko Yoshida’, Michio
Matsudal,
2, Hajime Hideo
Hirata3, Shinji Asakural, Wada4, Shigeru Shirakawa4,
Kensuke Yamazumi ‘, and Minoru Okuma5
’ Institute of Hematology, Jichi Medical School, Tochigi 2 Department of Internal Medicine, Toshiba General Hospital, Tokyo 9 Department of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo 4 Second Department of Internal Medicine, Faculty of Medicine, Mie University, Mie 5 First Division, Department of Internal Medicine, Faculty of Medicine, Kyoto University, Kyoto, Japan
(Received 6.5.1992; accepted in revised form 17.7.1992 by Editor A. Takada)
INTRODUCTION
Human fibrinogen has three high affinity calcium binding sites (l-5). Two of them are located in the two D-domains (6-g), especially in residues 311-336 of the y -chain (4). Calcium bound to two high affinity binding sites in D domains has a proqective effect in the plasmic digestion of fibrinogen which results in the formation of fragments D 1 and E (4, 10). The third site is located in the NH2-terminal disulfide knot (NDSK) of fibrintogen) (11, 121, but the precise regions or structures required for calcium binding are not known yet. The role of calcium bound to NDSK region is not known at all. Calcium binding studies of abnormal fibrinogens with structural defect in NDSK have not yet been reported, but should contribute to provide such informations. In this report we describe calcium binding studies of fibrinogens Kyoto II (13) and Ise (14) with an amino acid replacement in the NHz-terminal region of fibrin a -- and B -chain, respectively. - -------
-._-------
Correspondence should be Internal Medicine, Toshiba Tokyo 140, Japan.
Key Words:
Dysfibrinogenemia,
addressed to Nobuhiko Yoshida, M.D., Department General Hospital, 6-3-22 Higashi-Oi, Shinagawa-ku,
Calcium
binding
613
to fibrinogen,
NDSK
of
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MATERIALSANDMETHODS Fibrinogen was purified as previously described Calcium binding to fibrinogen (9). Heterozygous abnormal fibrinogen Kyoto II has the replacement of A a prolineby leucine (131, and heterozygous abnormal fibrinogen Ise has the replacement of BBglycine-15 by cysteine (14). Equiiibrium dialysis to characterize the calcium binding properties of fibrinogen was performed as described (51. The buffer used (50 al4 Tris-HCl, 0.135 M NaCl, pH 7.61 was filtered through a Chelex 100 column (Bio-Radl. Fibrinogen (3 &ml) was added with 5 nM EGTA and dialyzed against the above buffer with 3 mMEGTA, followed by extensive dialysis against the above buffer without EGTA at 4 “C. 0.1 ml of EGTA-treated fibrinogen (2.4 u&ml) was dialyzed against 20 al of buffer containing calcium at concentrations from 2 to 40 ,u M at 25°C for 48 h. Each ves(Du Pont-New England Nuclear). After dialysis, sel contained 2 ,u Ci of rsCaa+ 50-rl aliquots of the materials inside and outside the dialysis bag were Biofluor (Du Pont-New England mixed with 20 ml of scintillation fluid, Nuclear), and counted in an LX-700 liquid scintillation system (Aloka, Tokyo, Japan). Scatchard analysis was perforMed assuming Mr 340,000 for fibrinogen. Plaslic Fragment D I was prepared as described (51. Fibrinogen (2.5 mg/nl) in 50 EM Tris-HCl, 0.135 M NaCl, PH 7.4, was incubated with 5 mMCaCl* at 37 “C for 30 min and treated with 0.1 me/ml human plasminogen and 3000 units/r1 streptokinase for 18 h at 37 “C. Plasmic digests were analyzed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis according to the method of Laemmli (15). RESULTSANDDISCUSSION A Scatchard analysis of equilibrium dialysis data (Fig. 1Nl showed that the number of high affinity calcium binding sites for the normal fibrinogen is 2.97 (about 31 with a dissociation constant of 2.9,uM. 12-
r Fig.
1 Calcium binding to fibrinogen. r, moles of Ca*’ bound per mole of fibrinogen: C, free Ca2’ concentration in moles/liter. N, normal control; K, fibrinogen Kyoto II; I, fibrinogen Ise.
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The number of calcium binding sites for fibrinogens Kyoto II and Ise showed the normal value of about 3 (3.22 and 2.95, respectively) (Fig. 1K and dissociation constants of high affinity binding (3.1 and 111, with comparable 2.9 p M, respectively).
The dissociation constant obtained (2.9-3.1 rM1 is in the same range as those (2-3.7,~ Ml for human (4, 51, rat (2) and bovine (16) fibrinogen but at variance with those (8.7-26rM) found by other groups (l-3, 171 for human fibrinogen, the reason for which remained unknown. Nieuwenhuizen et al. (121 suggested that residues A a 17-19, BB 15-53 and/or ~54-78 are involved in the third high affinity calciua binding from the comparison of the formula of NDSK with that of fragment E3 which does not bind calcium. Abnormal fibrinogens with structural defects in these residues are very rare (18-231 and calcium binding studies have not been performed. Calcium binding studies have been reported only for fibrinogens with abnormal Y chains (5, 9, 17, 24, 251. Liu et al. proposed that residues BB 15-53 are not required for calcium binding since fibrinogen New York I with a deletion of BB 9-72 can polymerize with thrombin in the presence of calcium (20). However, it will be difficult to draw such a conclusion, because the role of calcium bound to NDSK region is not known at all, and enhancement of fibrin polymerization will be due to calcium bound not to the third high affinity site but to some low affinity sites (4, 26). Prolonged thrombin clotting time in fibrinogens Kyoto II and Isa was partially corrected in the presence of physiological concentration of calcium (13, 141, which does not necessarily mean the normal calciun binding to the third high affinity site. Plasmic digestion of fibrinogens Kyoto II and Ise in the presence of calcium resulted in the generation of the normal fragment D 1 and E (figures not shown), suggesting the normal calcium binding to two high affinity sites in D domains. Abnormal fibrinogens with defective calcium binding to D domains lack the ability to be protected by calcium against further attack by plasnin (5, 17). In conclusion, the normal calcium binding to fibrinogans Kyoto will mean that a single amino acid replacement of A a prolineby BBglycine-15 by cysteine does not affect high affinity calcium NDSK. Calcium binding studies for another abnormal fibrinogens with defects in these regions will provide further insights into the function relationship in the calcium binding to the central fibrinogen.
II and leucine binding structural structuredomain
Ise or to
of
ACKNOWLEDGMENTS This work was supported in part by a Scientific Grant-in-Aid from the Ministry of Education of the Government of Japan, a Research Grant for Intractable Diseases from the Ministry of Health and Welfare of the Government of Japan, and by a grant-in-aid from the Japan Private School Promotion Foundation. We are discussions for excellent
grateful to Drs. Masaaki Moroi and Stephanie M. Jung for helpful during the course of this work. We also wish to thank Yuko Muto technical assistance.
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