Seperation of plasminogen activators from normal and lytic human plasma

Life Sciences Yol . 5, pp . 731-741, 1966 . Printed in Great Britain.

Pergamon Press Ltd .

3~Aß~TIOA OF PLl81LIH00SA ACPIVATOHB FHOI( HOEII~L ~D L2TIC HOII~Z PL~9L IIlla Haaberg Departoent of Hioohsmistry, IIaiversity of Hslsinti, Finland. (Received 22 February 1966) The plaemiaogsn eotivators oooupy a oeatral position is the theories oonoernlng the meahaniem of fibrinolysis (1,2) .

Laoording to aooeptsd

visxe the oonasntration of the b1oAd aotivator rsflsate the physiologiasl thrombolytia eotivity, removing fibrin olots inside the blood vessels.

Of

particular interest is the mechanism of formation of the natural blood aotivator, xhiah is essential for the maintenance of thrombolytio activity in circulating blood .

lfaturally occurring activators is tissues, blood

end urine (3,4) have been eztensively studied, horever comparatively little ie lrnorn about the origin and moleauls o! the natural blood aotivator .

ltfillsrts (5,6) presented evidence far the presence of tree

plaemiaogsn aotivator in plasma from oases of sudden aaozaemio death .

It

is generally thought that the blood aotivator, through diffusion or adsorption into fibrin dissooistee from s hypothetical aotivator-antiaotivator oomplez, aarmally present in plasma and similar to the plasmiaantiplaemia aomplez (2) .

This primary fibrinolytio meahaaism i~olves the

activation of intrinsic plot plaemiaogsn, and leads to the dissolution of the clot through the protsolytia notion of free plaemin (q) "

Ths

aotivator once formed sots directly on the prosnsyms, plaemiaogsn, activating the protsolytio sn$yms plasmia, xhioh oleo autooatalyaes the traneformatioa . The formatioa of plaemiaogsn aotivatare has also Dean studied xith an aotivator of bacterial origin, obtained in a etoichiometrio reaction xith streptoünass (3~) and the proaotivatas in human plasma (1,8) . 731

The

pT"!~T~OGEN ACTIYATOES

732

Yol . 5, No . 8

szistsnoe of proaotivator se a unique protein is still a utter of controversy, although lately evidence has been brought in favour of the hypoths. see that ~ interacts with huaan plas=inogen ( 9, 10) or plasma (11,12,13) to fore an activator in blood . In the present xork a natural plasainogea activator has been separated f~oa spontaneously lytic huean plaeaa by gel filtration and ehoun to be of different aoleoular sire ae the 3~-activator .

Both ooa

pounds produced, after isolation, strong activation of bovine plaerinogsn as detsrdnsd by the lyeie of fibrin, xhile having none or negligible protsolytio aotivitT directly oa plaeeiaogen-free, heated fibrin .

The

~-activator xas forasd both with norwal huaaa plaeaa and the lytic plasma obtained poet mortem from oases of sudden death .

The possibility that the

natural aotivstar isolated from lytio blood may be identical with some of the knoxa fibrinolytio components ie discussed . Material 'and Msthode Blood plasma .

llaa" mal human mizsd blood bank plasma xae used, con-

taining acid citrate deztrose solution according to the II .S .P . Formula A . The plasma was separated fresh xith pressure and iroasn at -20° Cz ) . Blood was drawn post martsm at different periods of time following cases of sudden death s ) .

It was taken from the femoral vein xith

silioonised equipment, and was immediately osatrif~gsd at 900 z g for 20 minutes and +5 ° C .

111 plasma samples ware stored at -20°C in polythene

containers until used in ezpsrimente . Fibrinolytio âotivity .

Ths fibrin plate methods (14,15) xers used

in a comparative xay for the demonstration of activator effect and free protease activity (plsemin) . z

Platse xers prepared in petri dishes 9 z 9ôm

obtained fron the Finnish Bed Cross Blood Transfusion 9ervioe, sad a) from the IIniversity Department of Forensic Medicine

Yol . 5, No . 8

PLASYINOGEN aCTIYaTORB

733

by clotting 12 ml 0 .2 ~ bovine fibrinogen (Hsbi, grads H 1, lyophilised, purity 85-95 ~ of total protein content) xith 20 H .I .H . units of thrombin (Topoetasine, Hofitian-La 8oohs, Hassl, 9xitaerland) . forced for 16 hours at +37 ° C .

Incubation xas psr-

The unheated plats xas calibrated for

activator response xith purified uroünase~ ) .

Averagely 40 ~ higher

activities xere obtained xith urokinase (0 .01-1 .0 Leo unite par spot ~unhsated~) compared rith the activities obtained xith purified human plsemin (Hnbi, 0 .005-1 .0 casein unite per spot ~hsatsd~) .

Ths dose

reepones curves xsre parallell (log-log scale) in~the range 55-700 mm 2 . $iaos human plsemin causes the activation of bovine plasmiaogen, the plate reepones xas also tested xith unheated fibrin .

The does response curves

for plsemin xith heated and unheated fibrin xsrs not parallell .

An

increasingly higher (> 50 ~) difference in nativity xas obtained above the asdium range (300 u2 ~unheated), apparently due to plaeminogen content in the human plsemin preparation . Plasma samples xere tested directly on the plates (Table 1) .

Plasma

protein factions could be tested directly xithout the removal of salt (16), xhile xhen using the plot lysis method (1~) salt xas fir et removed by ultrafiltration (18) and dialysis against 0 .15 11 HaCl at +5 ° C (osllulose tubing, Arthur H . Thomas, Philadelphia, II .S .A ., 832 inches), until R ~ 0 .13-0 .15 (intsrpolnted from n curve made xith 0 .003 lI HaCl using the

Philiphe conductivity measurement bridge PB 95~) " The buffer used xith the fibrin plate and clot lyais iethode xas 0 .05 K Tria-HC1 pH 7 .8 containing sodium chloride to R ~ 0 .15 .

A linear

dose response xas obtained xith 2 .5-100 units of urokinase xhen measuring the slot lyeie time at +3~aC .

A clot xas made by first mining the ice

cold solutions of 0 .1 ml 0 .2 °~ bovine fibrinogen, 0 .2 ml plasma sample, 0 .2 ml buffer, and by rapid addition, 10 unite of thrombin (100 H .LH . zzz)

fom Lso Pharmaceutical Produate, Denmark

~34

Yol. 5, No . 8

PLASYIAOGEA ACTIYATOE3

units per ml in saline) .

Time xa~ rsoorded for a glass bead to resoh the

bottom of the lysis tube (0 .8 z 10 am) (lî) " FibriaoAren ooaoentration and ooairulation xers tested aooording to llorrisson s syneresis method as modified by Eergstrôm st al . (20) .

For

mioroeoals determinations 0.2 ml lytio plasma xaa mi :ed with 0.5 al phosphdte buffer pH 5 .9 (R + 0.15) to give

pH

6.3 .

At asro time 0.13 ml

of 0.2 lI lysine methylester dihydroohloride (Mann Hseearoh Laboratories Bex 7ork, II .S .A,i xas added.

üith this method a mean value 0.25-0.26

fibrinogen xas obtained xith the normal human plasma, in agreement xith reported values (20) .

ühen human fibrinogen xas added to lytio plasma,

the recovery xas about 80 ~ of the mean .

X11 the lytio plasma samples

ahoxn in Table 1 xers inooagulsbls under these oonditiona xith 5

H.I .H .

units of thrombin after 2 hours at room temperature . Preparation of 9g-activator .

~a 8 ml saapls of fresh human or lytio

plasma xas incubated xith 8000 units of a highly purifled strsptokinase (gabikinaae, 250.000 units per vial) for 15 minutes at +3q° C and

~.8 .

pH

ühen activity xae.msasursd on fibrin plates, the highest protsolytic sffeot xas obtained with 100 and 250 units of SR, xhile the plasminogen activating sffeot xns highest xith 1000 units per ml plasma . sample xaa submitted to gel filtration through 3ephadez 0-200.

The incubated The 5g-

activator xas recovered in the first protein peak immediately after the Vo elution volume (130-136 ml ) . Oel filtration .

A gel bed of approzimately 500 ml (column diameter

3 .8 om) xas packed xith Ssphadsz 0-200 (40-120 R, Pharmaoia, IIppsala, 9seden) aooasding to Oelotte st al . (21) .

Ths hydrostatic pressure during

the elution xas 25-23 om, xith a flox rats 7-7 "5 ml per half hour . eluant buffer xas 0.05 X Trie-HC1 pH 8.0 in 1 )I NaCl . in a cold room at +5 ° C .

The

The rune xers made

The elution profile xas standardi$sd xith normal

human plasma by measuring the abaorbanoy st 280 mu in 1120 diluted

vol .

5, No . 8

eluatse .

PLAS1üxOGEx ACTIVATORS

735

Three peaks rere obtained rich normal (22) and lytio plates .

l5rsotioas xsre oonoeatrated by ultrafiltration (18) and dislyaed . Proteins iron normal aad lytia plnesa rare identified Eros the oonoentrsted frsotioas by paper elsotrophoresie (24) using sasplss oorresponding to 0 .006-0 .012 et original plasma . Hioasea~ .

Lytio plasea sasplee rers tested for ssooth susols oon-

traoting aotivity on the isolated guinea pig dens (25) .

~ . Palmer

(London) kysograi xae used for registration of oontraotions .

The deus

xae suspended from a frontal rriting lever in a 6 .5 ml bath of Tyrode (aerated) solution at +3T-38 ° C .

3yathstio vasoaotive peptide (bradykinin,

BE9-640, 3andos, Baesl, 9ritaerland) raa used ss a standard . Eeeults and Disouseion The saterisl used in this investigation is presented in Table 1, and represents 13 sssples of inooagulable plassa Eros individual oases of sudden death .

The fibrinogen-i~se saeples rare strongly lytio, and in

the eajarity of oases s free protease (plassin) oould be deteoted .

The

high ratios betreen unhssted~heatsd fibriaolytio sotivity iadioated the preeeaoe of an sotivntor in the blood .

Plaesa rich protease also oon-

tainsd plassinogen, as shorn by iaoubatioa rith for oonoentrations of Sâ (Table 1) (26) .

Ths relatively higher sotivity on unheated plates rich

the protease containing eaaplee, xae therefore presusably a result of the ooebinsd effects of plassin and plsssinogsn (see sethods) .

Ia other

sasplse the diaappearanoe of plassin and plsesinogen was follored by the Bole appearance of activator sotivity, particularly in loss sasplss tested after a longer tine period (Table 1) . greater lability of plassin .

This could bs ezplainsd by the

PLdSYINOGEN ACTIVATORS

736

Yol .

5,

No .

Tsble 1 FI8HIa0Lr1'IC ACTIPIT7 1DI 2 iIITH IHCOlOÜLIBLS HOII~H PL~S1U B7 THS PL~TS 101THOD8

Post mortal time 5 " 5 hours 44 .5 3Z .5

IInheated fibrin

Heated fibrin

+31C~hsatsd fibrin

256

98

103

453

0

0

218

0

0

"

312

62

102

150

0

" "

524 240

0 ±

0 +

4 "5

1T9

51

98

T

"

355 150

6T .

TO _

"

4T7 258

36 T8

408

±

_

0

113

13 6 .5 18 6 .5-14 .5

9 7 8 .5 7

llarmal plaama

0

T2

Yalubs refer to sotivit~ obtained xith 0 .06 ml samples of plasma diluted 1 :2 xith buffer pH T .B, or buffer containing 100 H . .LH . unite of 3[ per et plasma . Before measuring the f[-aotivatin~ effect plasma samples xere preinoubatsd for 10 minutes at +3T C . Bovine fibrin substrate in all oases . Plasma fibrinogen content bslox T ~ of the normal mean value . Time indicates estimated hours after sudden death xhsn blood xae drmrn .

8

Yol . 5, No . 8

FIO . 1

PLASè~INOGEN ACTIYATOR4

737

Oel filtration on Ssphadez 0-200

o--ß elution profile of plasma proteins by abaorbancy from eluates

" --" 3g-activator from an aotivation mizture xith normal plasma (A) ~" plaeminogen activating Emotions from lytic plsema (H) ( " ) fibrinolytic sotivity on unheated fibrin platen using 0 .03 (A) and 0 .06 (H) ml eluate . Ho sotivity oocurred rich heated fibrin over the entire elution range . On the basin of these results, the aotivator oompound xae isolated from the lytio plsema choosing a sample xhioh contained no demonstrable amounts of free plaemin (408 ~ 2 unheated) .

The elution of plsema protein

in correlation to aotivator sotivity ie shoxn in Fig . 1, H .

As found by

plate analyses directly iron the sluatea, and xith concentrated fractions (by ultrafiltration), the natural aotivator xae eluated betxeea the second and third plasma protein peaks, xith more sotivity in the third peak area . According to Oelotte et al . (21 ), the plasma proteina are roughly retarded on Sephadez G-200 in the order of decreasing sedimentation ooeffioisate . The fir et peak contains mainly (22) a2 - and ß - macroglobulias, lipo2 proteins and T-g~obulin (19 9), the aeaond T-globuline and some a- and

~38

PLASIQINOGEN ACTIYATORS

Yol . 5, No . 8

~-globuline (7 3, 4-5 S), the thlrd, albumine, al - and a2 globuline (4 S s 3 .5 3) "

~ similar eleotro-phorstio distribution of the lytio plasms

proteins ras found, rhea oonosntratsd plasma fractions rare oospared by paper eleotro-phoresis xith noraal huaaa plasma .

Comparison xith the

0-200 filtrated 3[-sotivstor ooaplez (!`ig . 1, A), shore that the natural sotivstor is s e~aller sins protein .

1s reported by Baoh~aan st al . (3)

the notivator isolated Eros pig heart is s 3 .36-2 .77 3 protein .

Lexie

(23) found that elution of plassdaogsn ooourre~d is the eeoond peak area . If judged by the reported ~oleaular xeights (89 .000) for plsaainogen sad plaerin (12), these fibrinolytio oomponsnte ~ interfer is the sotive elution range (ees ~ethode) . eluates (Fig . 1, B) .

Hoxevsr, free plasma xas not found in the

Yhea oo~paring the lysis ti~ss rich the aonwntrated

sluates (p ~ 0 .15) of the aotivators, a muoh stronger lysis ooourrsd xith the Sâ-sotivstor, shoxing that the oonoentration of the natural oospouad was relatively lox . To investigate the possibility that si~ilar prsaursore may have been involved 1n the for~atioa of both aotivatore, lytic plae~a xas inoubatsd xith s high ooaoeatration of Sb (1000 unite per ~1) and gel filtrated . Results shored that plsa~a oontsiaing preforaed notivator (same as Fig . 1, B), ae rell as a plasma sample xith ao ds~onstrable aaount of notivator, horever fibrinogen-free (apt listed), yielded 3â-sotivstor in the first elution peak (oompars !'ig . 1, ~) .

Thus general lysis as indicated by lox

fibrinogen (0 .018 ~) or activator content in the plasma did not ezhaust the prosotivator ooapound neoeseary to form the Sg-a°tivstor complez . 9lsilarity xith tissue notivator xas sustained by the comparatively high stability of the natural sotivstor oospound .

A lyti° plasma sample

oontainlng sotivstor and plasmla (355 mm 2 unheated~67 mm2 heated, Table 1) retained 60 ~ of the plasmiaogen sotivating activity after 30 days at +5 ° C, xhile plaemin disappeared completely (37 ~ left after 4 days) .

Repent

results by Holemans and Johnston (27) suggest that vasoactive peptides

Yol . 5, No . 8

PLABHINOGEN ACTIYATOES

739

suoh as eledoisin and bradyldain rhea pcrfused in s blood-lee systea prodnoe the release of plssainogsn aotivstos t~os the vessel wells .

31aos

bradykinin is nasally bound to a globulin in plasra and may bs released by protease notion, the eventual influenos of spontaneous lysis ran i~sstigatsd .

Ia a series of ezperineate lytio plseaa res incubated rith

bradyünin releaslag agents (29), and in other series, synthetic peptide ras added to the lytio plasma .

The release of the peptide could be

effected is the lytia plssaa as cell ss its normal destruction, rhea oo~pared rich normal Mann plae~a in parallsll ezperi=ents .

A notable

c:ocptlon occurred rich one of the plae~a samples tested, rhioh yielded lees bradykinin-line activity, ae judged by the contraction of smooth ~usols, apparently due to a oo~parativcly high oontent of tree protca~c (98 ia2 heated, Table 1) .

In the oases tested, in spite of wtivstar

oontent, the peptide appeared to regain boned to its preoursas in plasaa . Trhsn the lytia plana rare tested directly on the guinea pig ileua, ao contraction occurred rich 0 .1 ~1 sseples in the scnsitivity range betreca 8-80 aNg bradyüain per ~1 . lihile ~uoh of the evidsnw obtained in this ror]c appears to suggest s closer siailarity betrecn the natural gad tissue activator, the aaterial is still too limited, and nesda further elucidation particularly oonocraing the iaterrslationehips betrscn the various fibrinolytio plaeaa oosponents, rhioh seriously liait the interpretation of sotivlty uasurcaents . Loinorledg ent This rescaroh has been supported by grants f~oa the gational Hcsearah Council for Soisnass, the 81gns end Ans Oylleaberg foundation, and by generous supplice of research ~atcrial iron AB nabi, 3toohhola, 8seden .

~40

PL98è~A0GEN ACTIYATORS

Yol. 5, No . 8

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T . Aetrup, Thro~bolytio Activity and Rslsted Phenonsna, Friedrioh-âarl-$oTis s~ e~rlag~îu~~gâr ,i~~lZJb~l)

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T, Astrup and 3. 16111srts, Arah . Hioohem . Biophps . , 40, 346 (1952)

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A. P, Flstohsr, Hioohso. J . , ~, 677 (1954)

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19 .

3. lßillsrts, Bioohse. J. , 61, 424 (1955)

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H, Oelotte, P, Flodin and J, %illander, Aroh Bioohs~ . Hiophys . euDDl . 1. 319 (1962)

PLéSI[INOGEN ACTIYAT08S

Yol . 5, No . 8

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24 .

Bsolman Technical Bulletin 6095 A (1961)

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II . Hauberg, Ann . Acad . Soient . Fenn . , A 11 , p . 16 (1962)

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D . L . buns, Proo . p . 1312 (1963

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E . Holemane and J . 0 . Johneton, Proo . lOth Co Haeoat . , 3traebourg, p . 49 (1965

h Co

. euro . 300 . Hasuat ., Lisbon, . euro . 300 .

741