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Release of alpha 2-plasmin inhibitor from plasma fibrin clots by activated coagulation factor XIII. Its effect on fibrinolysis. J Mimuro, … , S Kimura, N Aoki J Clin Invest. 1986;77(3):1006-1013. https://doi.org/10.1172/JCI112352. Research Article When blood coagulation takes place in the presence of calcium ions, alpha 2-plasmin inhibitor (alpha 2PI) is cross-linked to fibrin by activated coagulation Factor XIII (XIIIa) and thereby contributes to the resistance of fibrin to fibrinolysis. It was previously shown that the cross-linking reaction is a reversible one, since the alpha 2PI-fibrinogen cross-linked complex could be dissociated. In the present study we have shown that the alpha 2PI-fibrin cross-linking reaction is also a reversible reaction and alpha 2PI which had been cross-linked to fibrin can be released from fibrin by disrupting the equilibrium, resulting in a decrease of its resistance to fibrinolysis. When the fibrin clot formed from normal plasma in the presence of calcium ions was suspended in alpha 2PI-deficient plasma of buffered saline, alpha 2PI was gradually released from fibrin on incubation. When alpha 2PI was present in the suspending milieu, the release was decreased inversely to the concentrations of alpha 2PI in the suspending milieu. The release was accelerated by supplementing XIIIa or the presence of a high concentration of the NH2-terminal 12-residue peptide of alpha 2PI (N-peptide) which is cross-linked to fibrin in exchange for the release of alpha 2PI. When the release of alpha 2PI from fibrin was accelerated by XIIIa or N-peptide, the fibrin became less resistant to the fibrinolytic process, resulting in an […] Find the latest version: https://jci.me/112352/pdf Release of a2-Plasmin Inhibitor from Plasma Fibrin Clots by Activated Coagulation Factor XIII Its Effect on Fibrinolysis Jun Mimuro, Shigeru Kimura, and Nobuo Aoki Institute ofHematology and Department ofMedicine, Jichi Medical School, Tochigi-Ken 329-04, Japan Abstract glutaminase, activated blood coagulation Factor XIII (XIIIa) (2-4). The a2PI thus cross-linked renders the fibrin clot more When blood coagulation takes place in the presence of calcium resistant to fibrinolysis. Particularly, the naturally occurring fi- ions, a2-plasmin inhibitor (a2PI) is cross-linked to fibrin by ac- brinolytic process that occurs subsequently to fibrin formation tivated coagulation Factor XIII (XIIIa) and thereby contributes and is caused by fibrin-associated plasminogen activation (5) is to the resistance of fibrin to fibrinolysis. It was previously shown mainly inhibited by a2PI cross-linked to fibrin (6). that the cross-linking reaction is a reversible one, since the a2PI- a2PI serves only as a glutamine substrate for XIIIa in the fibrinogen cross-linked complex could be dissociated. In the cross-linking reaction (4), and the cross-linking occurs between present study we have shown that the a2PI-fibrin cross-linking lysine residues of fibrin a-chains and a glutamine residue of the reaction is also a reversible reaction and a2PI which had been a2PI molecule that is the second residue from the NH2-termi- cross-linked to fibrin can be released from fibrin by disrupting nal (4). the equilibrium, resulting in a decrease of its resistance to fi- We have previously suggested that the a2PI cross-linking brinolysis. When the fibrin clot formed from normal plasma in reaction may be a reversible equilibrium reaction that is common the presence of calcium ions was suspended in a2PI-deficient to the general cross-linking reaction catalyzed by transglutam- plasma of buffered saline, a2PI was gradually released from fibrin inases (7, 8), since the a2PI-fibrinogen cross-linked complex could on incubation. When a2PI was present in the suspending milieu, be dissociated to each of its components to reach a new equilib- the release was decreased inversely to the concentrations of a2PI rium state when the isolated complex was incubated with XIIIa in the suspending milieu. The release was accelerated by sup- (9). It is important to know if the a2PI cross-linked with fibrin plementing XIIIa or the presence of a high concentration of the could be released physiologically, because such a release of a2PI NH2-terminal 12-residue peptide of a2PI (N-peptide) which is from fibrin would make the fibrin less resistant to the fibrinolytic cross-linked to fibrin in exchange for the release of a2PI. When process, thereby accelerating fibrinolysis. the release of a2PI from fibrin was accelerated by XIIIa or N- In the present study we have demonstrated that a2PI once peptide, the fibrin became less resistant to the fibrinolytic pro- cross-linked with fibrin could be released by the function of XIIIa cess, resulting in an acceleration of fibrinolysis which was pro- and the release could result in an acceleration of fibrinolysis. portional to the degree of the release of a2PI. These results suggest the possiblity that a2PI could be released from fibrin in Methods vivo by disrupting the equilibrium of the a2PI-fibrin cross-linking reaction, and that the release would result in accelerated throm- Plasma. Blood was withdrawn from a normal individual, a patient with bolysis. congenital deficiency of a2PI (10), or a patient with congenital deficiency of Factor XIII, into 0.1 vol of 3.8% sodium citrate or into 0.1 vol of 100 Introduction U/ml heparin. The blood was then centrifuged to prepare platelet-rich or -poor plasma. The concentration of a2PI in plasma was immunolog- When blood coagulation takes place '20% of the a2-plasmin ically determined (2). inhibitor (a2PI),' a major inhibitor of fibrinolysis (1) present in Purified proteins. a2PI was purified from human plasma by the pre- plasma, is cross-linked to the a-chain of fibrin by plasma trans- viously described method (1 1). The concentration was determined spec- trophotometrically using El' = 7.03 at 280 nm (11). The protein was dissolved in Tris-buffered saline (0.05 M Tris-HCI, 0.15 M NaCl, pH This work was presented in part at the International Congress on Fibri- 7.4). Human fraction 1-4, prepared according to the method of Blomback nolysis, 29 March 1984. and Blomback (12), was used as the fibrinogen preparation after removing Dr. Aoki's present address is First Department of Medicine, Tokyo contaminating plasminogen and plasma fibronectin from the preparation Medical and Dental University, Yushima 1-5-45, Bunkyo-Ku, Tokyo with lysine-Sepharose (13) and gelatin-Sepharose (14), respectively. The 113, Japan. concentration was determined spectrophotometrically using El' = 15.1 Receivedfor publication 27 August 1984 and in revisedform 28 Oc- at 280 nm (12) and the coagulable protein was >95%. Factor XIII was tober 1985. purified from human plasma by the method described by Curtis and Lorand (15). Factor XIII activity was assayed by the dansylcadaverine 1. Abbreviations used in this paper: a2PI, a2-plasmin inhibitor; '251-FDP, incorporation method as described by Lorand et al. (16). 1 U of Factor radiolabeled fibrin degradation products; N-peptide, the NH2-terminal XIII is defined as the activity of Factor XIII present in 1 ml normal 12-residue peptide of a2PI; t-PA, tissue plasminogen activator; XIIIa, pooled standard plasma. Thrombin-modified Factor XIII was prepared activated coagulation Factor XIII. as described previously (4) and was converted to XIIIa by calcium ions added at the time of the experiments. Purified thrombin was prepared J. Clin. Invest. from a bovine thrombin preparation (Mochida Pharmaceuticals, Tokyo, © The American Society for Clinical Investigation, Inc. Japan) according to the method of Lundblad (17). The hirudin (a throm- 0021-9738/86/03/1006/08 $1.00 bin inhibitor obtained from leeches) used was a grade IV preparation Volume 77, March 1986, 1006-1013 from the Sigma Chemical Co., St. Louis, MO, and was shown to have 1006 J. Mimuro, S. Kimura, and N. Aoki no plasmin inhibitor activity when tested by the chromogenic ( 18) and fying screen; E. I. Du Pont de Nemours & Co.) for 24 h at -70'C. The clot lysis (19) methods. Tissue plasminogen activator (t-PA) was purified a2PI was also identified by cutting the disc gel transversely into slices of from the culture media of a human melanoma cell line according to the 3 mm thickness and counting each slice for radioactivity. method of Rijken and Collen (20). The t-PA activity was assayed by the a2PI released was examined for its antiplasmin activity. For that clot lysis method using a calibration curve constructed with the WHO purpose, 2 ml of normal plasma containing radiolabeled a2PI was clotted, standard preparation kindly supplied by Dr. P. J. Gaffney, National In- washed, suspended, and incubated in 2 ml of buffered saline containing stitute for Biological Standards and Control, London, England. Native 0.6 U/ml XIIIa in the same way as described above, except for the omis- Glu-plasminogen, whose amino-terminal group is glutamic acid, was sion of aprotinin. At 20 and 60 min of incubation, aliquots of 800 or 2 prepared from fresh plasma in the presence of aprotinin (10 kallikrein M1 were removed from the suspending media for antiplasmin activity or inhibitor units/ml) (Mochida Pharmaceuticals) by affinity chromatog- radioactive counting, respectively. For antiplasmin activity an aliquot raphy with lysine-Sepharose followed by DEAE-Sephadex chromatog- of 800 Ml was mixed at 370C with 200 Al of 5 mM S-2251 and then with raphy (21). Plasmin was prepared by activating plasminogen with uro- 100 ul of plasmin solution (4 nmol of substrate hydrolyzed/second per kinase-coupled Sepharose 4B (1 1). Plasmin activity was determined by ml). Increase of absorbance at 405 nm was monitored by a DU-8 spec- the amidolytic method (22) using the chromogenic substrate, H-D-Val- trophotometer (Beckman Instruments, Inc., Fullerton, CA), and the im- Leu-Lys-p-nitroanilide (S-2251; Daiichi Pure Chemicals Co., Tokyo, mediate-type antiplasmin activity was calculated as described previously Japan).

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