THROMBOSIS RESEARCH Printed in Great

l-01.

11,

pp.

Pergamon

Britain

75J-, -63, Press,

of Internal Pfedicine,University Eospitdi "MlheL~ina asthuis", A-esterda~, the Ketheybnds.

Cq7kvtrct

(Received

19.8.1977; Accepted

by

in revised form 28.9.1977. Editor H.C. Godal)

Venous occlusion for 20 min. by a sbhjrgmomanometer cuff ,appliedabove the elbow and maintained at a pressure midway systolic and diastolic, increased the fibrinolytic activity of the blood within the occluded part of the arm as measured by the means of the euglobulin clot lysis time. The plasma obtained after venous occlusion showed a plasmin resistancewhich was much higher than could be explained only by the rise of the antiplasminsdue to hemcconcentration caused by this phenomenon.As the phenomenon could be reproducedby hemoconcentrationin vitro this antiplasmin activity could not be contributedto an tiown antiplasmin liberated from the vascular wall. Fibrinolyticactivity after venous occlusion as measured by the euglobulinclot lysis time affords only an incomplete insight in the real fibrinolyticpotential in vivo.

INIRODJCTION Inhibitionand inactivationof plasmin is ascribed to different antiplastis: a2-macroglobulinCl), al-antitrypsin cl), cl-esterase inhibitor (21, inter-c-inhibitor(11, a2-antiplasti (3,4,5,6,7)and perhaps still unknown inhibitors.Tne nrxt important antiplasmin activity in the blood seems to be the rapid acting a2-antiplasmin (7,8) and the somewhat slawer acting a2 -macroglobulin.After venous occlusion there is usually a shorteningof the euglobulin clot lysis time, produced by the liberationof activator activity (9). 755

19;; Lxd.

7%

WEXPECTED

PLASNIN

RESISTANCE

In this smidy we ?.vestigatedthe fibrin0lyti.m 3CtiVity

vo1.11,so.6

2d

tk

dIltiDhS~in

activity of plasma *beforea?0 after venous occlusion in several r0rrnal individualsusing the antiplasminactivity of whole plasira(10).

Plasmin: (LysofibrinR)purified, activator-freeplasmin produced from porcine blood by :jovoTherapeutiskLaboratoryA/S Copenhagen,was used. This plasmti is obtained by trypsin activation of purified activator-,%e plasminogen from porcine blood. The plasmin is stabilized from porcine blood. One Novo-caseinunit equals approximately6-7 Remmert and Cohen units (11). In this paper all references to units are in tems of Novocasein units. lhe plasmin Novo was dissolved in Wren's buffer pH 7.4. 'Duombin: (TopostasinR)Roche, Basel, Switzeriand,was dissolved in saline (0.9 % NaCl) to a final concentrationof 500 NIH units/ml. Freshly prepared thrombin solution was used. Buffers: Cwren's buffer pH 7.35 500 ml, containing: ------2.9375 g Verona1 Na 3.6675 g NaCl. 107.5 ml 0.1 N.HCl 392.5 ml distilled water; TRIS buffer pH 8.8; _----Versene buffer pH 7.75 (accordingto BrafaMn) 1000 nil,containing: -_----5.85 g NaCl 10.31 g Verona1 Na 725 ml distilled water ml 0.1 N.HCl 250 1 g='TA 2.5 g gelatine (Merck-4070). C&12 solution: 0.025 M. Agarose: (TndubioseR)A 37, 1'IndustrieBiolcque FYancaise S.A., Gennevilliers. Aprotinine: (TrasylolR),100.000 KIU/ml, Bayer, Ieverkusen. and the "plasmin inactivation Sera and plasma, F.D.P., a_2-rnacroglobulin test" was collectedand determinatedas described earlier and the same antisera were used (10).

FiEXPECTED

VOl.11,SO.h

c(

2

PLASMIN

a+*m;-~d by ti;e -an:iplaskr:yeasr’ _____i._i._

Laurel1 (12).

im,o-e,_

RESISTXSCE

1 ~ctmphore3is

methcd of

Antiserum against human a 2-antiplasminwas obtained &from

the Research Cenkxrm Leuven. Antiplasminwas also determined with the S-2251 substrate from Yabi (13,Zi).

The euglobulin fraction of the plasma was isolated by adding 1 ml plasma to 9 ml distilled water and adjusting the p!ito 5.9 with 1 % acetic acid. The precipitated ifraction was separated by centrifugationat 350 g for 3 minutes and redissolved in 1 ml versene buffer. Euglobulinclot lysis time This is the time required for complete lysis of 1 ml euglobulin solution clotted by addition of 0.5 ml CaC12 l/40 mol. and incubated at 37.0°C. Protein determination Protein content was measured by a quantitativebiuret method.

In all subjects the pre-occlusionsample was taken without stasis from an antecubitalvein. A sphygmxnanometercuff was then applied to the upper arm and 3 or 1,blood pressure readings were taken. The cuff was inflated to a value midway the systolic and diastolic arterial pressure as recorded in the arm for 20 min. Blocd was then withdrawn from an antecubitalvein before releasing the occluding cuff. The fibrinolytic activity of each blcxd sample was measured by the euglobulin clot lysis time also. The concentrationsof the antiplatin activity, fibrinogen, total protein content were determined and the plasmin inactivationtest was performed. Plasma concentrationin vitro was effected with the aid of collcdion bags to imitate in vitro the effect of hemoconcentration.From this concentrated plasma we measured the concentrationof proteins and performed the plasmin inactivationtest.

RESULTS

All six healthy persons (five men and one woman) shawed a considerable shorteningof the euglobulin clot lysis time after venous occlusion (table I).

758

LXEXPECTED

PLASMIX

RESISTASCE

v01.11,s0.6

TABLE I

Subject

1. before after 2. before after 3. before after 4. before after 5. before after 6. before after

F.D.?.production(rrg/lSO ml) after addltlon of plasmn T"plasmin inactivationtest")

Euglobulinclot lysis the m mmutes

210

5.0

20

1.0

125

6.3

20

0.8 16

335 13

1.1

145

4.6

21

1.6

120

5.4

28

1.4

133

2.4

12

0.5

TABLE II Subject 1. before after

2. before after

3. before after

4. before after

5. before after

6. before after

%cr,M %a,AT

%l-esterase

inh.

tot.protein g/l

54

83

102

72

82

132

168

118

64

70

90

74

74

95

142

28

97

100

41

155

156

111

86

92

106

69

105

122

81

96

97

116

08 142

98

141

104

66.5

91

80

74 94

81 122

68.5 1OR

LYESPFCTED

rol.11,so.h

i'LAS>fIS RESISTANCE

TABTE III

Subject

3 a2M

%a, -antiplaskn (LaWell)

% antiplasmin F.D.P.prcduction (S-2251)

(~/lo0 inl)after addition of plasmin (plkmin inactivation test)

1. before after

2. before after

aa

84

a4

5.1

128

120

110

0.8

80

a4

88

5.5

110

119

121

0.5

After vemus occlusion we never saw spontaneous F.D.P. generation. There was always a correlationbetween the a2-mcmglobulin concentrationof the serum (detemined immnologically) ard the amount of F.D.P. generated in the pre-occlusionplasm in thirty minutes after addition of plasmin (plasmin inactivationtest)(figure1). This was in agreementwith our earlier findings (10). Tne plasmin inactivationtest wa6 also performed with the plasm obtained after vemus occlusion. In all subjects we observed a very low F.D.P. generation after addition of plasmin (table I, figure 2) also at low concentrationof a2-mcmglobulin fi&gure2).

(table II,

760

UNEXPECTED

PLASMIN

RESISTX?U'CE

. I 104

SO

__c

cone

(**

m.croplob

150

(XnmS)

Figure 1: Correlationbetween a2M concentration and content of F.D.P. generated in plasma after 30 min. of incubation with plasmin (final concentration 0.8 U/ml).

.

*.

.o

5b -

100 Cont.

Ix2

0

150

macro glob. (serum ‘6)

Figme 2: Correlationbetween a M concen-hxtion and the content of F.% .P. genemted in the plasm (obtainedafter venous occlusion) after 30 min. of incubation with plasmin (final concentration 0.8 U/ml).

vo1.11,so.6

LXESPECTED

PLXSXTS

,t?l

RESISTAXE

After concentratingplasm with tne aid of collodion bags to the saie degree as after ve"~us occlusion we observed tinesa;nephenmenon: . a low F.D.?. generationafter addition of plasmin e.g. in one plasm a decrease ,4om more 16 ng% to 1 rg% (a2-M increase fm

2245 4).

DISCUSSION

c activity after venous occlusion as 'Theincrease in fibrinolyti measured by the euglobulinclot lysis time is a well-kmm

phenomxon.

Whether the rise in plasminogenactivator activity finally results in free plasmin is not clear. We performed the "plasmin inactivationtest" (10) before and after venous occlusion. After versus occlusion we never saw spontaneousF.D.P. productionwhich could reflect plasminemia. It is possible that the plasmin formed after verms occlusion is directly bound to the antiplasmirs present in het blood. Therefore we had expected that after venous occlusion: there should be a plamin hypersensitivityof the post-occlusiveplasma, because the plasmin which could have been formed after venous occlusion is already bound to the antiplasminsand the remaing antiplasmins' capacity is therefore diminished. Instead of this plasmin hypersensitivity we found plasmin resistance after venous occlusion. After venous occlusion hemconcentration occurs and this concerns also the different antiplasmins. The rise in the concentrationwas similar to that of the total plasma protein. With the substrate (S-2251) assay we did not see a disproportions? rise in the a2-antiplasminafter venous occlusion (table III). However, the plasmin resistance was much mre

than could be attributedto

hemoconcentration. kfmx

venous occlusion we saw after 30 minutes of incubationof platin

with plasm an mount of generated F.D.P. which was~strongly correlated with the concentrationof ~2-macroglobulinof the plasma (serum)of different normal individuals (figure 1). This was in agreement with our earlier observations (10). The amunt of a2-a.ntiplasmin in nomal individualsis very constant and rapid acting (8). Apparentlya2-antiplamti does not disturb this relationshipbetween a2-mac~globulin and F.D.P. venerationafter incubationof plasm with plasmin for thirty minutes.

762

After

UNEXPECTED

VQMGS

PLASIIIN RESIST.LLliCE

occksion we found a very low F.D.?. production and no

relation with the concentrationof a2-~croglobu1i.n(figure 2). This plasmin resistance after venous occlusion cannot be explained by an inhibitor liberated from the vascular wall as a result of venous occlusion, because we could produce the sac phenomenon in vitro by hanoconcentration of pre-occlusionplasma. The cause of the plasmin resistance after venous occlusion could not be localized at this rroment;whether interactionof proteins or alteration in the confi,wation of proteins takes place remains open. The finding of a considerableplasmin resistance following venous occlusion makes it questionablewhether the shorteningof the euglobulin clot lysis time after venous occlusion represents real enhancement of the fibrinolyticactivity of post-occlusiveblood.

1. HEDNER, U. Fibrinolyticinhibitors.Supplement LIX of Thrombosis et I&thesis Haemorrhagica,p. 281, 1974. 2. SCHREIBER,A.D., KAPLAN, A.P. and AUSTFN, K.F. Plasmin inhibitors of the componentsof the fibrinolyticpathway in man. J.Clin.Invest. 52, 1394, 1973. 3. MULLERTZ, S. Differentmlecular forms of plasminogenand plasmin produced by urokinase in m plasma and their relation to protease inhibitorsand lysis of fibrinogenand fibrin. Biochem.J.143, 273, 1974. 4. MULLERTZ, S. and CLEMMENSEN,I. The primary inhibitor of plasmin in human plasma. Bi0chem.J.159, 545, 1976. 5. MOROI, M. and AOKI, N. Isolationand characterizationof a -plasmin inhibitor from human plasma. J.Biolog.Chem.L251 5956, 1978. 6. COLLEN, D., DE COCK, F. and VERSTRAETE,M. Immunochemicaldistinction between antiplasminsand a -antitrypsin.Thrombosis Res. 1, 245, 1976. 2 7. EDY, J., COD, D. and VERSTRAETE,M. Evidence for a functionally important plasmin inhibitor in plasma, different from a -macroglobulin, a -anti-try&n and antithrambinIII. Vth Congr.Int.Soc.?"nrombosis Hkmostasis, Paris 1975, Abstract 462. 8. TEGER-NILSSON,A.C., G?ZANDER,E., MURWOLD, H., NOPPA, H., OLSSON, R. and WALLMO, L. Determinationof a fast acting plasmin inhibitor in plasma from patients with tendency to thrombosis and increased fibrinolysis.VIth Int.Congr.onThrombosis ard Haemostasis,Philadelphia 1977, Abstract_,Thrxtkosis and Ha-stasis, July 1, p. 145, 1977.

V01.11,4*.5

IXEIPECTED

PLASMIS

RESISTXSCE

3.

1cI.

11.

“+=Ts, Z.J. ) mxsz?;, I]., pIoGaJ’s9;:’ B. and STO??!,0. Infusion of ~mke ?lamin iY‘Frn. Sczmd.J.Clin.LaS.invest. g, 179, 1963.

.“_

_.._L

2. Quantitativeestimtion of protein by electrophoresis1: 12. ~~XxL, '3. az3-"ose~ -el _, containingantibodies.Pnalyt.Biocha?.. z,_45, 1965. 13. z--w ui_ ) J . 35 COCK, F. and COLLL'I,D. Inhibitionof plasmin by ~omal azd mt!Aami> depleted human plasm. %o&osis Res. 8, 513, 1976. 14. FGER-?Z‘sSO;T,AC. Determinationof a new rapid plasm inhibitor in ?xmn bloti by rmzns of a plasmin specific tripeptide substrate. To be published in Scand.J.Clin.Lab.Invest.

Unexpected plasmin resistance after venous occlusion.

THROMBOSIS RESEARCH Printed in Great l-01. 11, pp. Pergamon Britain 75J-, -63, Press, of Internal Pfedicine,University Eospitdi "MlheL~ina asth...
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