Cross-Linking of Alpha 2-Plasmin Inhibitor to Fibrin by Fibrin- Stabilizing Factor
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Cross-linking of alpha 2-plasmin inhibitor to fibrin by fibrin- stabilizing factor. Y Sakata, N Aoki J Clin Invest. 1980;65(2):290-297. https://doi.org/10.1172/JCI109671. Research Article The concentration of alpha 2-plasmin inhibitor in blood plasma is higher than that in serum obtained from the blood clotted in the presence of calcium ions, but is the same as that in serum obtained in the absence of calcium ions. Radiolabeled alpha2-plasmin inhibitor was covalently bound to fibrin only when calcium ions were present at the time of clotting of plasma or fibrinogen. Whereas, when batroxobin, a snake venom enzyme that lacks the ability to activate fibrin- stabilizing factor, was used for clotting fibrinogen, the binding was not observed. When fibrin-stablizing, factor-deficient plasma was clotted, the specific binding of alpha 2-plasmin inhibitor to fibrin did not occur even in the presence of calcium ions and the concentration of alpha 2-plasmin inhibitor in serum was the same as that in plasma. Monodansyl cadaverine, a fluorescent substrate of the fibrin-stablizing factor, was incorporated into alpha 2-plasmin inhibitor by activated fibrin-stablizing factor. All these findings indicate that alpha 2-plasmin inhibitor is cross-linked to fibrin by activated fibrin-stabilizing factor when blood is clotted. Analysis of alpha 2-plasmin inhibitor-incorporated fibrin by sodium dodecyl sulfate gel electrophoresis showed that the inhibitor was mainly cross-linked to polymerized alpha-chains of cross-linked fibrin. Cross-linking of alpha 2-plasmin inhibitor to fibrin renders fibrin clot less susceptible to fibrinolysis by plasmin. Find the latest version: https://jci.me/109671/pdf Cross-Linking of a2-Plasmin Inhibitor to Fibrin by Fibrin-stabilizing Factor YOICHI SAKATA and NOBuo AoKI, Institute of Hematology and Department of Medicine, Jichi Medical School, Tochigi-Ken, Japan 329-04 A B S TR A C T The concentration of a2-plasmin inhib- solution of fibrin (fibrinolysis) is believed to be checked itor in blood plasma is higher than that in serum ob- or retarded by natural inhibitors of fibrinolysis. tained from the blood clotted in the presence of calcium Natural inhibitors of fibrinolysis inhibit the activa- ions, but is the same as that in serum obtained in the tion of plasminogen to plasmin (inhibitor of plasmino- absence of calcium ions. gen activation or activator inhibitor) or act upon the Radiolabeled a2-plasmin inhibitor was covalently plasmin already formed (plasmin inhibitors). Although bound to fibrin only when calcium ions were present the presence of the former inhibitors in plasma (1) has at the time of clotting of plasma or fibrinogen. Whereas, not been fully substantiated, the presence of plasmin when batroxobin, a snake venom enzyme that lacks the inhibitors has been firmly established (2). ability to activate fibrin-stabilizing factor, was used for Among various proteinase inhibitors capable of in- clotting fibrinogen, the binding was not observed. When hibiting plasmin activity, two inhibitors, a2-plasmin in- fibrin-stabilizing, factor-deficient plasma was clotted, hibitor (a2PI)1 and a2-macroglobulin are considered to the specific binding of a2-plasmin inhibitor to fibrin be physiologically most important (2). Plasmin gener- did not occur even in the presence of calcium ions ated in a plasma milieu is effectively inhibited by these and the concentration of a2-plasmin inhibitor in serum two inhibitors, therefore fibrinogenolysis does not oc- was the same as that in plasma. cur under these conditions. Monodansyl cadaverine, a fluorescent substrate of When fibrin is formed, plasminogen activators and the fibrin-stabilizing factor, was incorporated into a2- plasminogen are adsorbed to fibrin and plasminogen plasmin inhibitor by activated fibrin-stabilizing factor. activation takes place on fibrin molecules to exert an All these findings indicate that a2-plasmin inhibitor effective fibrinolysis. This process of fibrinolysis is ef- is cross-linked to fibrin by activated fibrin-stabilizing ficiently inhibited by a2PI but not by a2-macroglobulin factor when blood is clotted. Analysis of a2-plasmin (2, 3). This hypothesis was substantiated by the dis- inhibitor-incorporated fibrin by sodium dodecyl sulfate covery of congenital deficiency of a2PI (4). The efficient gel electrophoresis showed that the inhibitor was inhibition of fibrinolysis by a2PI has been attributed mainly cross-linked to polymerized a-chains of cross- to the inhibition of binding of plasminogen to fibrin linked fibrin. Cross-linking of a2-plasmin inhibitor to by a2PI (2, 3) in addition to the rapid inactivation of fibrin renders fibrin clot less susceptible to fibrinolysis plasmin formed during plasminogen activation (2,5,6). by plasmin. In a brief, preliminary communication (7), we showed that a2PI was adsorbed to fibrin when plasma was clotted and suggested that the adsorption might be INTRODUCTION another mechanism of the efficient inhibition of fibrinol- The dissolution of fibrin deposits or thrombin in vivo ysis by a2PI. is achieved by plasmin formed from plasminogen by We now present evidence that a2PI is bound to plasminogen activator that has been added extrinsically fibrin by a covalent linkage formed by activated fibrin- for the therapeutic purposes (urokinase and strepto- stabilizing factor (FSF, blood coagulation factor XIII, kinase) or endogenously generated in the vascular trees plasma transglutaminase), and suggest that the covalent (blood activators or vascular activator). Regardless of binding of a2PI to fibrin contributes significantly to the the manner in which plasminogen is activated, dis- stability of fibrin. ' Abbreviations used in this paper: a2PI, a2-plasmin inhibi- Received for publication 9 July 1979 and in revised form tor; FSF, fibrin-stabilizing factor; MDC, monodansyl cadaver- I October 1979. ine; SDS, sodium dodecyl sulfate. 290 J. Clin. Invest. (D The American Society for Clinical Investigation, Inc. 0021-9738/80/02/0290/08 $1.00 Volume 65 February 1980 290-297 METHODS 50 U/ml, CaCl2, 0.375 M). FSF-deficient plasma was kindly supplied by Dr. M. Fujimaki, Department of Clinical Pa- Fibrinogen. Human fraction 1-4 prepared by the method thology, Tokyo Medical College. The blood was obtained from of Blomback and Blomback (8) or human Cohn's fraction I a patient with congenital deficiency of FSF and immediately (Green Cross Corp., Osaka) was used as fibrinogen preparation mixed with 1/10 vol of 3.8% sodium citrate to obtain citrated after removing contaminating plasminogen from the prepara- plasma. When the fibrin formed by the addition of thrombin tion using lysine-Sepharose (Pharmacia Fine Chemicals, Inc., and calcium ions to this plasma was analyzed by sodium do- Piscataway, N. J.) (9). The concentration of FSF present in decyl sulfate gel electrophoresis (14), no formation of y-chain the solution of 250 mg/100 ml of fraction 1-4 or Cohn's fraction dimer of fibrin was observed, indicating the absence of FSF I was 50 or 80% of normal standard plasma, respectively, when in the patient's plasma. Serum was obtained by incubating assayed by an antibody neutralization method (10) using a 1 ml of plasma at 37°C for 2 h with 25 ,ul of 0.5 M CaCl2. Clotting Factor XIII-test kit supplied by Behringwerke AG, a2PI-deficient plasma was obtained from a patient with con- Marburg. Cold-insoluble globulin was not detected in the genital deficiency of a2PI (4). Aprotinin (Trasylol, Bayer fraction 1-4 preparation by double immunodiffusion, single Chemical, Tokyo) was added to the plasma in a ratio of 5 IAI radial immunodiffusion or counter immunoelectrophoresis aprotinin (5,000 U/ml):1 ml plasma to avoid spontaneous using specific antiserum against cold-insoluble globulin (11). fibrinolysis when the plasma was clotted. There was also no characteristic band of subunit cold-in- Radioiodination of protein. Purified a2PI and fibrinogen soluble globulin with mol wt 215,000 when the preparation (fraction 1-4) were radioiodinated by the method of Thorell was analyzed by sodium dodecyl sulfate gel electrophoresis and Johansson (17) or the solid-state lactoperoxidase method with reduction (11). Cohn's fraction I was clearly con- of David (18), respectively, using lactoperoxidase (Calbiochem- taminated with cold-insoluble globulin. Fibrinogen was Behring Corp., American Hoechst Corp., San Diego, Calif.) dissolved in barbital-buffered saline (0.005 M barbital-acetic and 125I-Na (17 Ci/ml) (New England Nuclear, Boston, Mass.). acid-0. 14 M NaCl, pH 7.4) and the concentration was adjusted The labeled a2PI preparation had the radioactivity of 1.1 to 1.5% (wt/vol) clottable protein. x 107 cpm/,ug. Specific activity of a2PI before and after radio- Thrombin. Purified thrombin was prepared from bovine iodination was 1136 U/A280 and 1,050 U/A2s0, respectively. thrombin preparation (Parke, Davis & Co., Detroit, Mich.) a2PI concentration of the labeled a2PI preparation was 60 according to the method of Lundblad (12), and was stored ixg/ml. The labeled fibrinogen preparation had a radioactivity as a 200 U/ml solution in 50% glycerol at -200C. of 6.3 x 104 cpm/Ig. Clottability (97%) was not changed, and Plasmin. Plasminogen was purified from human whole fibrinogen concentration of the labeled preparation was 316 plasma by affinity chromatography using lysine-coupled mg/100 ml clottable protein. Sepharose 4B (13), and was activated with urokinase-coupled Measurement of the binding of a2PI tofibrin. When plasma Sepharose according to the described method (5). Total con- was used, 1 ml of plasma was mixed in a counting vial for version of plasminogen to two-chain plasmin form was as- radioactivity with 5 Al of radiolabeled a2PI and the mixture certained by sodium dodecyl sulfate gel electrophoresis with was clotted by the addition of 40 Iul of thrombin (50 U/ml) reduction (14). Plasmin thus obtained was stored at -20°C as a or a thrombin-calcium mixture (thrombin 50 U/ml, CaCl2 0.375 65 caseinolytic U/ml solution (15) in buffered saline contain- M).