Further evidence for the presence of two plasma inhibitors of fibrinolysis distinct from α2-macroglobulin

THROMBOSIS RESEARCH Printed in Great Britain

':ol .

11, pp . 267-273, 1977 Peraamon Press, Ltd .

B R I E F C 0 M M U N T i - A T I 0 N FURTHER EVIDENCE FOR THE PRESENCE OF TWO PLASMA INHIBITORS OF FIBRINOLYSIS DISTINCT FROM a 2 -MACROGLOBULIN Michael J . Gallimore and Ulla Hedner Institute for Thrombosis Research, Rikshospitalet, Oslo,Norway, and Coagulation Laboratory, Allmanna Sjukhuset, Malmo, Sweden . (Received 20 .4 .1977 ; in revised form 16 .6 .1977 . Accepted by Editor H .C . Godal) INTRODUCTION Gallimore and Shaw (2) briefly described a human plasma fraction distinct from a 2 -macroglobulin and a 1 -antitrypsin which possessed both antiplasmin and antiactivator activities . Hedner (7) isolated an a 2 -globulin with a molecular weight of 75,000 from human plasma which inhibited the activation

of

plasminogen by urokinase and streptokinase and was a weak inhibitor of plasmin .

Gallimore (4) separated several anti-

proteases from human serum and the isolation and purification of one of these with both antiplasmin and antiactivator activities has recently been reported

(5) .

This inhibitor had a

molecular weight of approximately 80,000 and was named "intera-antiplasmin" because it migrated as an inter-a-globulin on agarose gel electrophoresis . Both Mullertz (10) and Collen and co-workers (1) have published evidence indicating that an antiplasmin distinct from the other antiproteases is present in human plasma and Hedner and Collen (8) have reported the immunochemical distinction between the two inhibitors which they are studying .

Recently

Moroi and Aoki (9) have described the isolation of an a 2 globulin with both antiplasmin and antiactivator activities and a molecular weight of 63-67,000 . There is therefore evidence for the presence of more than 267



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FIBRLNOLYSIS :2 PLASMA INHIBITORS

Vol .ll,No .2

one fibrinolytic inhibitor distinct from, a 2 -macroglobulin and antithrombin 111 in the inter-n-globulin region of human plasma . In the present communication further information is presented on this subject .

MATERIALSANDMETHODS "Antiplasmin 11"solution was prepared as described elsewhere (4) .

This preparation inhibited both plasmin and urokinase-

induced fibrin clot lysis . Antiserumagainsttheinhibitorofplasminogen activation was raised in rabbits as described elsewhere Antiserumagainstantiplasmin Dr . D .

(1)

(7) .

was a generous gift from

Collen, Laboratory of Blood Coagulation, Dept . of

Medical Research, University of Leuven, Leuven, Belgium . Antiserum againstthefibrinolytic inhibitordescribedbyMoroi andAoki

(9)

was kindly supplied by Prof . N . Aoki, Jichi Medical

School, Minamikawachi-Machi, Tochigi-ken, Japan 329-04 . Immunophoresis was performed at pH 8 .6 in 1% agarose electrophoresis plates . Crossedimmunophoresis was performed according to the method of Ganrot (6) . RESULTS

Fig .1 is a diagramatic representation of the results obtained when human serum and a serum fraction exhibiting both antiplasmin and antiactivator activities

("antiplasmin 11")

were tested by immunophoresis against the three antisera . Single precipitin arcs were obtained with the individual antisera with both serum and "antiplasmin 11" solution .

The

protein precipitating with antiactivator antiserum migrated slightly more towards the anode than the proteins reacting with antiplasmin antisera and the antiserum supplied by Professor Aoki, which had the same mobilities .

Whem mixtures of anti-

activator antiserum and either the antiserum provided by Dr . Collen or that provided by Professor Aoki were tested



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269

against "antiplasmin 11" solution only two precipitin arcs were obtained,

both of which had the same migration rates .

The arc

corresponding to antiplasmin was nearer to the antisera wells than that corresponding to antiactivator .

FIG . 1 Diagramatic representation of immunophoresis of serum and antiplasmin 11 solution against the various antisera . 1 p1 volumes of serum or antiplasmin it solution (5 mg/ml) in sample slots, 15 ul antisera in wells .

A= Antiplasmin 11 B= Serum ANTISERA 1= Antiplasmin (1) 2= Antiplasmin (9) 3= Antiactivator (7)

FIG . 2 Radial immunodiffusion of the three antisera against antiplasmin 11 solution and human serum .



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Fig .

Vo1 .11,N0 .2

2 shows the results obtained when human serum and

"antiplasmin 11" solution were tested by radial immunodiffusion against the three antisera .

These results clearly show that

the two antiplasmin antisera cross-react with a common antigen and that antiactivator is immunologically distinct from this protein . Fig . 3 shows that the "antiplasmin 11" preparation gives two precipitin rockets when tested by crossed immunophoresis against a mixture of both the antiplasmin and antiactivator antisera .

FIG . 3 Crossed immunoelectrophoresis of 7 ul antiplasmin 11 solution using agarose gel containing antiserum against antiactivator (7) and antiplasmin (1, 9) .

Peak A = antiactivator, Peak B =

antiplasmin . Note : The precipitin peaks corresponding to the two antigens were identified in gels containing the individual antisera . DISCUSSION The results described in this communication confirm the observations of Hedner and Collen (B) that two distinct plasma proteins exist which cross-react immunochemically with either



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F7BRINOLYSIS :'' PLASMA INHIBITORS

antiactivator antiserum

271

(7) or antiplasmin antiserum

(1) .

They also indicate that the protein reacting with antiplasmin antiserum is the same as that isolated from human plasma by Moroi and Aoki

(9) .

Both of these proteins are present in the

serum fraction named "antiplasmin 11" by Gallimore (4) and have been detected in samples in the late stages of antiactivator purification . Gallimore

(3)

separated two proteins from "antiplasmin 11"

solution by isoelectric focusing and preparative agarose-gel electrophoresis, both of which inhibited fibrin clot lysis . The isolation procedure for the most acidic of these proteins together with some of its properties has been published (5), this protein was named "inter-a-antiplasmin" .

The other

protein was slightly less acidic and was obtained in much smaller quantities .

It exhibited weaker antiplasmin and anti-

activator activities and as it had very similar properties to "inter-a-antiplasmin" was regarded as being a modified form of the more acidic protein

(some properties of the two proteins

together with those of antiactivator and antiplasmin are given in Table I) . TABLE I Some Properties of the low Molecular Weight Fibrinolytic Inhibitors .

Enzymes inhibited

Plasmin

Inhibitor 1

+

Urokinase Trypsin

+

Inhibitor 2

Anti-

Anti-

activator

plasmin

+

+

+

+

+

ND

Elastase

ND

ND

Thrombin

ND

ND



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TABLEI .cont'd .

Enzymes inhibited

Inhibitor 1

* Inhibitor 2

** Anti-

*** Anti-

activator plasmin Physical characteristics Sedimentation coefficient

3 .5

Molecular weight

75-80,000 a

3 .5 75-80,000 a

ND 75,000 a

3 .43

63,000 b 67,000 c

Isoelectric point pH 4 .4-4 .8

4 .6-5 .1

NE

ND

Electrophoretic mobility

Inter-a-globulins a 2 -globulin a 2 -globulin

+ Marked inhibition, + weak inhibition, - no inhibition . ND - Not determined .

a Determined by gel filtration ;

b determined by ultra centrifugation ;

c determined by sodium

dodecyl sulphate gel electrophoresis . *Gallimore 1973

(3) ; **Hedner 1973

(7) ; ***Moroi and Aoki 1976

(9) . As it is possible that these two proteins correspond to antiplasmin (1, 9) and antiactivator (7) experiments are in progress whereby further samples of these proteins are being fractionated from human plasma both to identify them and to investigate their functional activities . REFERENCES 1 . COLLEN, D ., DE COCK, F ., and VERSTRAETE, M . Immunochemical distinction between antiplasmin and a 1 -antitrypsin . Thromb .

Res .

7, 245, 1975 .



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273

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Ed . Peeters, pp . 151-156, 1975 . 6 . GANROT, P .O . Crossed i mmunoelectrophoresis . clin . Lab .

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30, 414,

1973 . 8 . HEDNER, U ., and COLLEN, D . Immunochemical distinction between the inhibitors of plasminogen activation and antiplasmin in human plasma .

Thromb . Res .

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251, 5956, 1976 .

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