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Changes in the Electrophoretic Patterns of Euglobulin Fractions Following Activation of the Fibrinolytic System by Exercise Department of Physiology, University of Toronto, Toronto, Ontario Received February 5, 1971 MCALPPNE, V., MPLOJEVIC, S., and MONKHOUSE, F. C. 1971. Changes in the electrophoretic patterns of euglobulin fractions following activation of the fibrinolytic system by exercise. Can. J. Physiol. Pharmacol. 49, 672-677. A study was made of changes in blood fibrinolytic activity in a group of young male university students following varying levels of exercise. The subjects were divided into two groups. Those partaking in regular athletic programs were classified as athletes and those not as nonathletes. There was an increased fibrinolytic activity with increased severity of exercise in all subjects. Several of the nonathletic group showed a much greater maximum effect than their athletic counterparts, and all nonathletes showed the effect at much lower levels of exercise. Increase in fibrinolytic activity correlated well with increase in pulse rate. When euglobulin fractions from the plasma samples were subjected to microzone electrophoresis, there was a band change in the region of the beta globulins in many of the samples following exercise. This band change is apparent only when fibrinogen is present in the euglobulins and appears to be due to fibrinogenolysis as a result of the activation of the fibrinolytic system. These band changes were observed when the specific activity was as little as 0.058 unit of plasmin per milligram protein. Our results show that electrophoretic pattern changes of euglobulin fractions is a relatively sensitive and rapid method for detecting activation of the fibrinolytic system. MCALPPNE,V., MILOJEVIC,S., et MONKHOUSE, F. C. 1971. Changes in the electrophoretic patterns of euglobulin fractions following activation of the fibrinolytic system by exercise. Can. J. Physiol. Pharmacol. 49, 672-677. SUP des Ctudiants de l'UniversitC, nous avons ktudiC changements de l'activitb fibrinolytique du sang ii la suite d'exercices de diffkrentes intensitks. Les sujets sont rhpartis en deux groupes. Le groupe des athlktes incluent les sujets participant rCgulibrement 8 des exercices d'athlktisme. Le groupe des non-athlktes comprenant tous les autres sujets. Chez tous les sujets nous avons not6 une augmentation de I'activitC fibrinolytique proportionnellement 8 l'intensit6 de lkxercice fourni. Chez plusieurs sujets non-athl&tes, l'effet maximum atteint Ctait bien plus ClevC que chez les athlktes. De plus, chez les non-athlbtes, nous notons une augmentation de I'activitC fibrinolytique pour des intensitks d'exercices ne donnant pas d'effets chez les athlbtes. L'augmentation de I'activitC fibrinolytique est en bonne correlation avec l'augmentation de la frCquence cardiaque. L'Clectrophorkse de la zone euglobuline du plasma montre aprhs l'exercice chez plusieurs sujets une modification au voisinage de la rtgion des p-globulines. Le changement n'apparait que lorsque la fraction englobuline contient du fibrinogbne et serait dQ 8' la fibrinogenolyse aprbs activation du syst2me fibrinolytique. Les modifications sont observCes pour une activitC spCcifique aussi basse que 0.058 unitCs de plasmine par milligramme de protiines. Les risultats indiquent les modifications de l'Clectrophor6gramme de la fraction euglobuline est une mCthode relativement sensible et rapide pour dktecter l'activation du syst&mefibrinolytique.

Introduction Activation of the fibrinolytic system by exercise has been observed by numerous investigators over the past two decades (Biggs et al. 1947; Fearnley and Lackner 1955; Cash and A4cGill 1969). Such studies include exercise sf walking, running, and bicycling. It has been reported also that the degree of activation of the fibrinolytic system is proportional to the severity of the exercise (Menon 1967). Our investiga-

tions confirm the above finding but show that the degree of activation of the fibrinolytic system of any individual shows a better correlation with the increase in pulse rate than with the amount sf work done. Those who are in good physical condition have to do more work to show detectable fibrinolytic changes than do those in poorer physical condition. Furthermore, it was discovered that this change in the fibrinolytic system during exercise was detect-

McALPINE ET AL. : EUCiLOBULIN ELECTROPHORESIS

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able in electrophoretic patterns of the euglobulin fraction. It is the latter finding which forms the main justification for this report.

Materials and Methods Euglobulin Prepuration Plasma was collected after centrifugation of citrated blood at 2006 r.p.m. for 10 min, and diluted 1 to 19 with distilled water slightly acidified with acetic acid. Sufficient 1% acetic acid was then added to lower the pH to 6.0. After standing 15 rnin to allow precipitation, the samples were centrifuged at 3000 r.p.m. for 15 min. All procedures were carried out in a cold room ( 4 "C) . The precipitate was dissolved in 0.1 114 potassium phosphate buffer (pH 7.6) in a volume equal to that of the original plasma. All samples were frozen and kept at -20 " C until just prior to assay. Potassium Phosphate Buffer This was prepared as 0.1 M solution from KzHP84 with the pH adjusted to 7.6 with orthophosphoric acid. Thrombin-Bufler Solution A 1 ml aliquot of thrombin solution (100 units/ml) was mixed with 90 mI of phosphate buffer solution as prepared by Fearnley ( 1957). Fibrinogen Bovine fraction I (Sigma) was purified and made deficient in plasminogen following the method of Johnson et al. (1964). The stock solution was adjusted to contain 2.0% protein which was 85-90% clottable. Concentrations used in the euglobulin lysis test were either 200 mg % o r 500 mg %, made up in potassium phosphate buffer (pH 7.6). Thrombin Thrombin Topical (Parke Davis) was made up as a stock soIution in 0.85% NaCl to contain 100 units/ ml. It was stored at -20 "C and diluted in 0.85% NaCl soIution to 10 units/ml just prior to use. Urokinase (Calbiochem Grade B ) A stock solution was made up in potassium phosphate buffer (pH 7.6) to contain 200 Ploug units/ml, and stored at -20 "C. Electrophoresis Samples were applied to cellulose acetate strips and subjected to a current from a 250 V source for 35 rnin in a Beckman microzone cell, mode1 R-101. Barbitone buffer (pH 8.7) was used. At the completion of the run, the strips were first stained in Ponceau S and then restained, if necessary, in 0.001% Nigrosin in 2% acetic acid. Assays for Fibainoly tic Activity Euglobulin Eysis Time Test A two-phase system was used in which the plasminogen was activated by addition of urokinase, and the activated material then added to a standard fibrinogen and clotted with thrombin. Phase I-Tubes containing 0.25 ml of euglobulin, 0.2 ml of potassium phosphate buffer (pH 7.6), and

673

0.05 ml (10 Ploug units) of urokinase were incubated at 37 " C for 4 min. At the same time, tubes containing 0.25 ml of euglobulin and 0.25 ml of buffer were incubated. Two controls were used, one containing 0.45 ml of buffer plus 0.05 ml of urokinase, the other containing 0.50 ml of buffer. Phase IZ-Tubes ( 10 X 75 mm) each containing 0.2 ml of fibrinogen were placed in an ice bath and 0.2 ml of the incubation mixture from phase I was added to each and mixed well before and after the addition of 0.2 ml of thrombin topical (10 units/ml). The tubes were kept in the ice bath for 10 min to allow clot formation. The rack of tubes was then transferred to a water bath at 37 " C and the time for lysis observed. The results of samples activated with urokinase were a measure of total plasmin and plasminogen content. Those of the unactivated samples were a measure of the free plasmin. One euglobulin plasmin unit was defined as that amount of enzyme which would lyse a standard fibrinogen clot (fibrinogen of 500 mg % ) in 10 rnin (velocity 0.1 min-') . Dilute Whole Blood Clot Lysis Time A modification of the Fearnley (1957) procedure was used. A 0.2 ml aliquot of fresh citrated blood was added to each of two tubes containing 1.8 ml of thrombin-buffer solution, and mixed well. The mixture was allowed to clot for 15 rnin at 4 "C. The tubes were then rimmed and rotated briskly between the palms of the hands to promote clot retraction, then incubated at 37 " C and observed at intervals for lysis. Results were expressed in velocity ( 1 /lysis time). Exercise Procedures All subjects were healthy male students (average age 21.9 years). Each subject rested in a supine position for 30 rnin prior to exercising. After this time a control pulse rate and blood sample were taken. Blood was taken into l/loth the volume of 3.8% trisodium citrate. Exercise was done on a treadmill for 8 rnin using a 5" angle of elevation with speeds varying from 3.5 to 8.5 miles per hour. The pulse rates and blood samples were taken immediately on completion of exercise and 15 rnin later. Syringes and test tubes were kept at 4 "C and blood samples were processed immediately in a cold room (4 "C).

In Fig. 1, a summary of the results of both euglobulin and whole blood dilution lysis tests following exercises of varying severity is shown. Only three people, 5. F., L. H., and P. T., were not able to continue all exercise levels for the full 8 min. The assays shown here were done on the samples taken immediately after cessation of the exercise. The results of the euglobd i n lysis test are shown as specific activity (plasmin units per milligram of protein). The dilute whole blood 8ysis test results are shown

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SPEED ( m.p.h.1

FIG. 1. Increase in fibrinolytic activity with increasing severity s f exercise. Exercise was performed on a treadmill for 8 rnh at the rates (m.p.h.) shawn. The treadmill was set at an angle sf 5 " . Initials refer to individual subjects. The subjects J.F.*, E.M.*, and P.T.* were not able . are shown in terms of percentage to continue for the full 8 min at the rate s h o ~ ~ nValues increase in velocity over control for the dilute whole blood test and in specific activities (units per milligram protein) for the euglobulim assay where the control values were zero.

as increase in velocity from the control value (velocity being the reciprocal of lysis time). Subjects were divided into two goups. Those partaking regularly in athletic programs were classified as athletes and those not as nonathletes. It is apparent that there is an increase in activity with increasing severity of exercise in all subjects. Several of the nonathletic goug show a muck greater maximum effect than. their athletic counterparts, and all nonatlaletes show the effect at much lower rates of exercise. Pt was also found that the increase in pulse rate correlated very well with the increase in fibrinolytic activity (Fig. 2). The eraglobulins were subjected to electrophoresis in a Beckman microzone apparatus and it was immediately apparent that there was a band shift in the region of the beta globulins in many of the samples following exercise and that this band shift correlated with detectable fibrinolytic activity. In Fig. 3, typical results are shown. These results pose the question as to what the shift represented and whether or not it codd be used as a rapid method far detection of

PULSE RATE IBeats/rnrn )

FIG. 2. The relation between increase in fibrinolytic activity of ebsglsbulins and pulse rate following exercise. ( @ ) Athlete, (0) nonathlete.

fibrinolytic activity. Consequently, a series of experiments was set up to examine the changes in electrophoretic patterns sf euglsbulins when human blood or euglobullin fractions were activated with varying concentrations of urokin-

MCALPINE ET

AL.: EUGLOBULIN

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SAMPLE

475

ELECTROPHORESIS

PATTERN ,

SPECIFIC ACTIVITY

HUMAN PLASMA a

L.H.

J.M.

M.M.

b

0

I .26

c

0.3 1

Q

0

t>

0.1I

C

0

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HUMAN P L A S M A FIG. 3. Euglobulin electrophoretic patterns of three subjects before and after exercise, showing typical response to: ( I ) light exercise (M.M.), (2) heavy exercise (J.M.), and ( 3 ) exhaustive exercise (L.H.) . (a) Sample taken before exercise, ( b ) immediately after exercise, and ( c ) 15 min after exercise. Specific activity refers to units of plasmin per milligram protein.

ase. Figure 4 shows typical results of such experiments. In the one experiment, varying amounts of urokinase were incubated with blood for 10 min at 37 "C. The blood was then centrifuged and the euglobulins prepared. In the other experiment, the euglobulins were prepared first and then activated with urokinase. As can be seen in both experiments, with increasing concentrations of urokinase the beta 2 band moved toward the beta 1 band. These band changes therefore appeared to be related to the conversion of plasminogen to plasmin. The influence of the fibrinogen in blood upon the band changes was then investigated. Fresh human plasma was prepared and half of it was activated with 2 units/ml of urokinase for 10

min at 37 "C. Defibrinated and nondefibrinated plasma samples were made from both the activated and nonactivated blood (defibrination was done by heating the plasma to 54 "C for 4 min). The euglobulins were prepared and electrophoresis was performed. The results are shown in Fig. 5 , Plasma defibrination results in the loss of the beta 2 band. It is therefore apparent that the changes in the euglobulins observed following exercise or activation of the fibrinolytic system were the result of fibrinogenolysis by free plasmin rather than a shift from plasminogen to plasmin. Any darkening seen in the alpha bands under exercise conditions may be attributed to fibrinogen breakdown products or other polypeptides, or to the general plasma

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CANADIAN JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY.

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1971

H U M A N PLASMA

0-0

0'0

HUMAN P L A S M A FIG. 4. Electrophoretic patterns s f euglobulins prepared from fresh citrated blood, incubated with varying amounts of urokinase and urskinase-activated euglobulins. Note band shift with increasing activity.

prepared from human plasma reflect @asrninogen activation. These changes were observed when the specific activity (units per millDiscussion limarn protein) was as little as 0.058. The wale Our in vitrta and in vivo results show that level reached during exercise by any of the electroplmoretic pattern changes of euglc~bulins subjects was 1.26. It can be seen that zone

protein concentration which accompanies exer-

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McALPINE ET AL. : EUGLOBULIN ELECTROPHORESIS

677

FIG. 5. Electrophoretic patterns of euglobulins prepared from defibrinated and nondefibrinated plasmas from normal and urokinase-activated fresh blood. Note that only in the euglobulins prepared from nondefibrinated plasma was the second band present and thus the shift is only visible with nondefibrinated plasma. Two units per milliliter of urokinase were used to activate the blood.

R., MACFARLANE, R. G., and PILLING,J. 1947. electrophoresis of plasma euglobulin fractions BIGGS, Observations on fibrinolysis. Experimental acis a relatively sensitive and rapid method for tivity produced by exercise or adrenaline. Lancet, detecting activation of the fibrinolytic system. 1, 402-405. Our results confirm those of other workers that CASH,9. D,, and MCGILL,R. C . 1969. Fibrinolytic response to moderate exercise in young male exercise is a physiological means of activating diabetics and nondiabetics. J. Clin. Pathol. 22, the fibrinolytic system. The results also indicate 32-35. that in order for exercise to measurably activate PEARNLEY, G . R., and LACKNEW, R. 1955. The fibrinthe system, such exercise must be sufficient to olytic activity of normal blood. Brit. J. Haematol. cause a substantial increase in pulse rate. Pres1,189-198. G . R., BALMFORTH, G., and FEARNLEY, E. ent work in our laboratory indicates that over- FEARNLEY, 1957. Evidence of a diurnal fibrinolytic rhythm; ventilation also c~ntributesto the activation of with a simple method of measuring natural the system. If the activation of the fibrinolytic fibrinolysis. Clin. Sci. 16, 645. system is a physiological mechanism for pre- JOHNSON, A. J., TSB, A. Q., and NEWMAN,J. 1964. Preparation of fibrinogen deficient in plasmioventing the build up of fibrin deposits on the ogen and determination of clottable protein. In endotheliunl of vessels and by this means preBlood coagulation hemorrhage and thrombosis. venting atherosclerosis, then exercise can obEdited by Tocantins, L. M., and Kazal, L. A. pp. viously be beneficial. However, to be beneficial 462-464. in this way the exercise must be a challenge, MENON,I. S . 1967. Effect of strenuous and graded exercise on fibrinolytic activity. Lancet, 1, 700though not necessarily exhausting or distressing 702. in nature.

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This article has been cited by: 1. Nikolaos D. Hamouratidis, Theophilos E. Pertsinidis, George P. Bacharoudis, George S. Papazachariou. 1988. Effects of exercise on plasma fibrinolytic activity in patients with ischaemic heart disease. International Journal of Cardiology 19:1, 39-45. [CrossRef] 2. D.H Osmond, E.K Lo, A.Y Loh, E.A Zingg, A.H Hedlin, J.A Millar, B.H Clappison, C.I Johnston. 1978. KALLIKREIN AND PLASMIN AS ACTIVATORS OF INACTIVE RENIN. The Lancet 312:8104, 1375-1376. [CrossRef] 3. L. BERRENS, E. JANKOWSKI, I. JANKOWSKI-BERNTSEN. 1976. Complement component profiles in urticaria, dermatitis herpetiformis, and alopecia areata. British Journal of Dermatology 95:2, 145-152. [CrossRef]