Journal of 6, 1962...Journal of Clinical Investigation Vol. 41, No. 6, 1962 ACTIVE HAGEMANFACTOR:...

Journal of Clinical InvestigationVol. 41, No. 6, 1962

ACTIVE HAGEMANFACTOR: A PLASMALYSOKINASE OF THEHUMANFIBRINOLYTIC SYSTEM*

By SOTIRIOS G. IATRIDIS ANDJOHN H. FERGUSON

(From the Department of Physiology, School of Medicine, University of North Carolina,Chapel Hill, N. C.)

(Submitted for publication November 13, 1961; accepted February 15, 1962)

Normal human and animal plasmas contain aprecursor globulin, plasminogen, which can be con-verted to plasmin, the active fibrinolytic enzyme,by a variety of activator agents.

The general term "activator" has been used sowidely that it would often seem inadvisable to at-tempt to replace it by a more specific one. Never-theless, this term is inappropriate for the specificcharacterization of agents which convert plasmino-gen into plasmin. Wetherefore propose the spe-cific term plasminoplastin for those agents whichproduce plasmin by "direct" action on human andanimal plasminogen. It is thus implied that agentslike streptokinase, which convert plasminogen toplasmin "indirectly" (in two steps) should not betermed plasminoplastins, but rather lysokinases(1).

Humanplasminogen can be converted to plasmineither by the direct action of tissue, plasma, orurine plasminoplastins, or by the indirect action ofthe lysokinases (e.g., streptokinase). The plasmafactor, which reacts with a lysokinase to form theplasma (or endogenous) plasminoplastin, can betermed proplasminoplastin (proactivator). Thelatter factor has not yet been isolated and char-acterized; therefore, much controversy surroundsits nature and identity.

Fibrinolytic activity can also be developed "spon-taneously," that is, by a mechanism occurring inthe blood itself, as compared with the activity in-duced by the addition of known plasminoplastinsor lysokinases.

Many attempts have been made to elucidate themechanism of the "spontaneous" activation of thefibrinolytic system, but the problem has not yetbeen entirely solved. From a theoretical point ofview, there appear to be at least two possible mech-anisms. The first is the release of plasminoplastin

* Aided by Grant H-1510-C, from the NIH Division ofResearch Grants and Fellowships, USPHS.

from the tissues into the circulation; the second isan increase in the plasma plasminoplastin by someunidentified "activator" mechanism.

Regarding the first possibility, the presence oflarge amounts of plasminoplastin in many tissues(2-4) suggested that local liberation of tissueplasminoplastin from injured tissue is importantin effecting resolution of fibrin deposits locally intissues and, to a lesser degree, in the vascular bed(1). Extending these observations, Kwaan, Loand McFadzean (5-7) presented evidence to sug-gest that the walls of the vessels contain a cholin-ergic effector mechanism, which, when stimulated(e.g., ischemia due to vasoconstriction of vasavasorum; local injection of epinephrine, acetylcho-line, histamine, or serotonin), responds by releas-ing plasminoplastin into the circulation. Astrupand Permin (4) have reported that some tissues,in addition to the large amounts of plasminoplastin,contain measurable quantities of a substance with alysokinase effect. Mullertz (8) extended thisconcept by proposing that the spontaneous increaseof the fibrinolytic activity in blood (which is ac-tually an increase of plasma plasminoplastin) isassociated with increased permeability of the vas-cular endothelium and release of a tissue compo-nent (lysokinase) which combines with pro-plasminoplastin in blood to form plasminoplastin.

Regarding the second possibility, which is herefurther explored, some evidence for a serum lyso-kinase was presented by Lewis and Ferguson (9).They prepared from normal dog serum two frac-tions (fraction K and proplasmin), which re-mained inactive when stored separately, but de-veloped strong fibrinolytic activity after incubationof a mixture of the two for several weeks at 5° C.They concluded that fraction K (serum fibrino-lysokinase) was responsible for the spontaneousappearance of plasmin in whole serum treatedwith chloroform. Presumablv, this serum frac-

1277

SOTIRIOS G. IATRIDIS ANDJOHN H. FERGUSON

tion K was either lysokinase (1) or plasmino-plastin, in the activation of which a serum lysoki-nase was involved.

Further evidence for the existence of a plasmafactor with activity of a lysokinase was given re-cently (10, 11). It was found that normal eu-globulin suspension incubated at 370 C showed anincrease of plasminoplastin activity, while Hage-man-deficient euglobulin suspension did not in-crease its activity tinder the same experimentalconditions. Hence, it was suggested that Hage-man factor may participate in the activation of theproplasminoplastin.

The present study was undertaken in order toinvestigate further the role of Hageman factor inthe generation of the plasma plasminoplastin.The results indicate 1) that the factor which isdeveloped during the incubation of normal eu-globulin suspension is a plasminoplastin; 2) that"active Hageman factor" acts like lysokinase onthe proplasminoplastin to form plasminoplastin;and 3) that human plasmin, devoid of Hageman-like activity, is ineffectual in the generation ofplasma plasminoplastin.

MATERIALS AND METHODS

Xormal human intact plasma. Using a silicone tech-nique, blood from the arm vein of normal donors wascollected in 0.1 vol of 3.2 per cent sodium citrate. Thiswas centrifuged at 40 C for 30 minutes at 3,000 rpm andthe upper two-thirds, transferred to a siliconized tube,were considered as "intact platelet-poor" plasma (12).

Hagenman-deficient plasma. Blood from a well stud-ied patient with Hageman trait (L. C.) (13, 14) wascollected and centrifuged as for the normal.

Chicken intact plasma. Using the silicone technique,jugular vein blood was collected, citrated, and centri-fuged as for human plasma.

Bovine fibrinogen was a 2 per cent stock solution ofArmour's bovine Fraction I (fibrinogen) in borate buf-fer, pH 7.6, from which dilutions in the same buffer weremade as required. In using this fibrinogen to prepare"unheated fibrin plates" (see below), its content ofplasminogen was usually satisfactory for the purposeof testing plasminoplastins.

Thrombin (bovine) was supplied through the courtesyof Dr. J. T. Correll (Upjohn Co.); potency according tomanufacturer. The fibrin, which formed with the abovefibrinogen and this thrombin, did not lyse in the con-trols.

Surface factor (SF) was prepared by a modificationof Waaler's procedure (15). Normal citrated plasmawas adsorbed with concentrated Al (OH) (0.1 ml per

ml) 1 for 5 minutes at 370 C. This plasma is deficientin prothrombin (Factor II), proconvertin (Factor VII),plasma thromboplastin component (PTC; Factor IX) andStuart (Factor X). The adsorption does not decreasethe amount of Hageman factor (XII) appreciably, butit does considerably decrease the amount of plasma throm-boplastin antecedent (PTA; Factor XI) (16). Theadsorbed plasma was exposed to kaolin (10 mg per ml)in a Lusteroid tube for 10 minutes at room temperature.After centrifuging at 3,000 rpm for 15 minutes, the kaolinwas washed three times with 0.85 per cent NaCl solution.The washed kaolin was suspended in a volume of elutingsolution equal to the original plasma volume. Theeluting solution was prepared by dissolving 7 g of NaClin 100 ml of fluid consisting of 1 part imidazole buffer (pH7.4) and 4 parts distilled water. The suspension wasstirred for 30 minutes at room temperature. The kaolinwas then removed by centrifugation at 3,000 rpm for 15minutes and the eluate was dialyzed in Visking casingagainst large amounts of distilled water for 18 hours at40 C. The inactive precipitate, which formed duringdialysis, was removed by centrifugation at 5,000 rpm for20 minutes and discarded. The SF thus prepared wasstable when frozen-stored at - 200 C. One such prepa-ration lost no activity when stored at this temperature for6 months. Repeated thawing and freezing should beavoided, however.

Ratnoff, Davie and Mallett (16) recently used PTA-deficient plasma to prepare "a crude fraction, rich in ac-tivated Hageman factor and apparently deficient in otherknown clotting factors," which "corrected the coagula-tion defect of plasma deficient in Hageman factor, buthad no effect upon the clotting time of plasma deficientin PTA. . . . The preparation did contain plasminogen."Since there was no PTA-deficient subject available to us,we merely prepared from normal plasma the so-called"surface factor" (14) or "activation product" (15), whichwe believe is essentially "activated Hageman factor" plussome active PTA. Other known clotting factors arelacking, however. In particular, all traces of thrombinand prothrombin were eliminated. Our SF always hasa small content of plasminogen, but if this assayed morethan 0.2 caseinolytic units (cas. U) per ml, or if anyactive plasmin or plasminoplastin was demonstrable, thepreparation was considered as unsuitable and discarded.The protein content of our SF preparation averaged 50 ,ugper ml, indicating over 1,000-fold purification in terms of"activity" per unit weight of protein, compared with theoriginal plasma.

Kaolin-SF was prepared by following the same pro-cedure as for SF, except that the washed kaolin-ad-sorbate was simply suspended in distilled water, insteadof eluted.

This material was also free of other clotting factors,as well as of plasmin or plasminoplastin activity, and itsplasminogen content never exceeded 0.4 cas. U per ml.

Bovine plasminogen was prepared by Kline's (17)

1 Cutter Laboratories, Berkeley, Calif.

1278

HAGEMANFACTORAND HUMANFIBRINOLYTIC SYSTEM

procedure from bovine plasma Fraction III, and con-

tained 24 cas. U per ml.Human plasminogen was prepared similarly, and av-

eraged 80 to 100 cas. U per mg N.Human plasmin was prepared by spontaneous activa-

tion of human plasminogen in glycerol (18). After com-

pletion of the activation, the glycerol was removed bydialysis against large quantities of 0.01 N HCO solution.The preparation was adjusted to pH 2 and heated at 1000C for 30 minutes. The heated plasmin solution was al-lowed to cool to room temperature and then was mixedwith an equal volume of 2 M NaCl solution. The mix-ture was centrifuged at 3,000 rpm for 30 minutes. Theprecipitate was dissolved in 0.01 N HCl and kept at - 200C until used. Human plasmin, thus prepared, containedabout 75 cas. U per mg N (see above), and was de-void of any Hageman-like activity.

Urine plasminoplastin (UP) or "urokinase" was a

purified preparation for which we are indebted to Dr.H. 0. Singher (Ortho Laboratories).

Streptokinase (SK) was commercial (Lederle's) Vari-dase. The dry material was readily dissolved in distil-led water to give 10,000 U per ml.

e-Aminocaproic acid (e-aca) was a 4 M stock solution(in distilled water) of a commercially available prepara-

tion (Aldrich Chemical Company).Euiglobulin fraction. The plasma (normal human,

Hageman-deficient, or chicken) was diluted 20-fold withdistilled water and the pH adjusted to 5.3 with 1 per centacetic acid. The suspension was centrifuged at 40 C for15 minutes at 3,000 rpm. The sediment was dissolvedin a volume of borate buffer (pH 7.6) equal to the origi-nal plasma volume.

Euglobulin lysis time. Two-tenths ml of 0.1 per centbovine fibrinogen was pipetted into a glass tube (13 mm

X 100 mm) prewarmed to 370 C. To this was added 0.2ml of the euglobulin preparation (incubated, see below,or otherwise), followed by 0.1 ml of a thrombin-Ca mix-ture (equal parts of 100 U per ml thrombin and 0.025 MCaCl2). The clot was observed every minute and thetime required for complete clot lysis recorded as the eu-

globulin lysis time.Plasminoplastin generation test (PGT) (11). Two ml

of intact, citrated, platelet-poor plasma was pipetted intoa beaker, diluted with 38 ml of distilled water, adjusted topH 5.3 with 1 per cent acetic acid, and incubated at 370 C.At successive time intervals, 4.6-ml samples were re-

moved, centrifuged at 3,000 rpm for 15 minutes at 40 C,the precipitate (euglobulin) dissolved in 0.23 ml of boratebuffer (pH 7.6) and tested for fibrinolytic activity by theeuglobulin lysis time method (see above).

During the incubation there was an increase of thefibrinolytic activity, which was followed for 6 hours.The range and mean of 26 control estimations (from 26normal persons) are shown in Figure 1. The smallgroup studied did not permit any definite correlation ofvariations with age, sex, and so forth. Each curve was

reproducible and characteristic of the individual, pro-

vided that the conditions under which the blood was

Lysis timein minutes

600

500

400

300

200

100

2 3 4 5 6Incubation time in hours

FIG. 1. PGT ON NORMALPLASMAS. The average andthe range of tests with plasmas from 26 subj ects areillustrated.

drawn remained the same. Four separate studies on oneindividual, shown in Figure 2, indicate only minor vari-ations.

Occasionally the above system was slightly modifiedby adding agents such as streptokinase, plasmin, SF, kao-lin (1 mg per ml distilled water) or kaolin-SF. Whenkaolin (1 mg per ml plasma) was added to a normalPGT system the initial lysis time was shortened and themaximal activity was usually reached in 90 to 120 min-utes (11).

Lysis time,n minuwes

400

300 \

200\

100-

2 3 4 5 6

Incubotion time in hours

FIG. 2. PGT ON NORMALPLASMA OF ONE SUBJECT.The four points indicate tests on four separate occasionsover a 1-month period. The curve represents the aver-age of the four determinations.

1279


Instead of lysis time, we may express plasminoplastinactivity in units. By performing the PGT on Hageman-deficient plasma plus SF and incubating until the shortestlysis time was reached in the euglobulin lysis test, maxi-mally activated euglobulin, that is, maximal plasmino-plastin formation was obtained. When the shortest lysistime had been reached, the euglobulin-suspension formingplasminoplastin was centrifuged, the precipitate was re-

dissolved with borate buffer (pH 7.6) and placed in a

water bath of melting ice. Dilutions of this euglobulinwere made (with borate buffer pH 7.6) and tested by theeuglobulin lysis method. A curve relating lysis timesand concentration of plasminoplastin was then drawn.One plasminoplastin unit was defined as that amount ofplasminoplastin corresponding to 7 minutes' lysis time.

Fibrin plate methods. The well known methods ofAstrup and Mullertz (19), as modified by Alkjaersig,Fletcher and Sherry (20), were used.

Proteolytic activity. This activity was determined bya slight modification of the Mullertz (21) caseinolyticassay. The 3 per cent casein solution and the materialto be tested were placed in a water bath at 370 C untiltemperature equilibrium was reached (5 minutes), andequal volumes (2.5 ml) were then mixed in a test tube.Two-ml samples, removed immediately after mixing (zerotime), and after 60 minutes' incubation at 370 C, were

pipetted into test tubes containing 3 ml of 1.7 M perchlo-ric acid. After 45 minutes (at room temperature) theprecipitates were removed by centrifuging and filtering.The absorbance of the supernatants was determined at a

wave length of 275 mAu on the Beckman DU ultravioletspectrophotometer, and the increase per 60 minutes was

calculated. One caseinolytic unit was arbitrarily takenas that amount of enzyme producing an increase of 200/Ag of acid-soluble tyrosine in a medium of 3 per centcasein in 60 minutes at 37° C.

Plasminogen assay. Human plasminogen was con-

verted to plasmin by an optimal concentration of strepto-kinase, and the resulting proteolytic activity was meas-

ured on casein, as above. Bovine plasminogen was acti-vated by optimal urine plasminoplastin and assayed inthe same way.

RESULTS

1. Nature of the fibrinolytic factor developed inthe PGT

By performing the PGT (see Methods) on

normal plasma plus 1 mg per ml kaolin (11 ), andincubating (usually for 2 hours at 370 C) until theshortest lysis time was reached in the euglobulinlysis test, we obtained maximally activated eu-

globulin-K. In the same period, two other prepa-

rations of nonactivated (nonincubated) euglobu-lin were made; i.e., the same plasma (withoutkaolin) was diluted and acidified, and the euglobu-lin recovered by centrifugation, redissolved in

borate buffer (to original plasma volume), andstored in the refrigerator (40 C). After the maxi-mal activity of the incubated euglobulin-K was de-termined, one of the nonactivated euglobulin prep-arations was mixed with urine plasminoplastin(UP) in several amounts, selecting that whichgave a similar euglobulin lysis time (about 13 min-utes) as the maximally activated euglobulin-K;the other was mixed with human plasmin (see Ma-terials), again in an amount yielding a similar ly-sis time. All three active preparations were thenplaced in an ice bath to preserve their similar lyticactivities.

It is a characteristic of the plasminoplastins toinduce more lysis of fibrin when more plasminogenis available (22). On the other hand, the lyticactivity of plasmin is not increased by addingplasminogen. In the first experiment, the un-heated fibrin plate method was employed. Tworows of plates were prepared: the first with nor-mal bovine fibrin (normal amount of plasmino-gen), and the second with bovine fibrin fortifiedwith 2.5 cas. U of bovine plasminogen per plate(per 10 ml of fibrin). The plates thus preparedwere designated "plasminogen-rich."

Table I shows that euglobulin-K behaved likea plasminoplastin (euglobulin-UP) and lysedmore fibrin in the presence of more plasminogen.In the case of euglobulin-plasmin, there was no sig-nificant difference.

The effect of varying amounts of e-aca (22)upon the activity of the three active euglobulinswas next investigated. It can be seen from TableII that the inhibition of the fibrinolytic factor ofeuglobulin-K by the E-aca was analogous to theinhibition of euglobulin-UP, and was quantita-tively different from the inhibition of euglobulin-

TABLE I

Effect of varying plasminogen content uponthe lysis of fibrin *

Lysed area (mm2)

Normal Plaminogen-Preparation tested fibrin rich fibrin

Borate buffer 0 0Euglobulin-K 206 380Euglobulin-UP 480 670Euglobulin-plasmin 365 368

*Fibrin plate method (unheated fibrin containingplasminogen).

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TABLE II

Inhibition of fibrinolytic activity bye-aminocaproic acid *

Per cent inhibition by e-aca

Preparation tested 10-4M 10-3M 10-2M 10-'M

Euglobulin-K 88 99 100 100Euglobulin-UP 74 80 94 100Euglobulin-plasmin 0 56 80 90

* The euglobulin lysis times (in the absence of e-aca) ofthe three preparations were adjusted to the same level andconsidered as 100% activity. The results illustrate theinhibitory effect of e-aca upon the three euglobulins.

plasmin. This fact again tentatively suggests asimilar nature of the fibrinolytic factors in euglobu-lin-K and euglobulin-UP.

The fate of each of the above euglobulins afteracid-heat treatment was also investigated. It isknown that purified preparations of plasmin arestable at low pH (below 4) and that they may bebrought to 1000 C at pH 2, without significantloss of activity (23). Urine plasminoplastin,while stable at temperatures up to 500 C, loses itsstability and is denatured at higher temperatures.Little is known concerning the heat stability ofplasma plasminoplastin. However, it is consideredmore labile than tissue or urine plasminoplastins,at least in the crude materials so far available.

In the present preliminary experiment the la-bility of these factors should not be compared withthat of the purified preparations, mainly becausethe presence of gross impurities in the euglobulinsmay affect their degree of stability.

All three euglobulins were adjusted to pH 2,heated for 30 minutes at 1000 C, brought back toneutrality, and tested for residual activity by theeuglobulin lysis time method. It can be seen(Table III) that the percentage loss in activitydiffered in the three cases. Only in the euglobu-lin-K was there total (100 per cent) loss.

TABLE III

Loss of Jibrinolytic activity by acid-heat treatment *

Per cent loss of activity

Euglobulin-Euglobulin-K Euglobulin-UP plasmin

100 84 78

* All three euglobulins were adjusted to pH 2, heated for30 minutes at 1000 C, brought back to neutrality, andtested.

2. Role of SF (dependent on Hageman factor)in the generation of the endogenous plasmino-plastin

It has been shown (11) that the generation ofendogenous plasminoplastin in Hageman-deficientplasma is defective and that Hageman factor,while not identical with plasminogen and proplas-minoplastin, does participate in the reaction(s) ofplasminoplastin formation. The following ex-periment confirms these facts.

In one beaker (I), 2 ml of Hageman-deficientplasma was mixed with 2 ml of a kaolin-SF prepa-ration (see Materials), then diluted with 36 mlof distilled water, and the pH adjusted to 5.3 with1 per cent acetic acid. In a second beaker (II),2 ml of the same kaolin-SF was diluted 20-foldwith distilled water and the pH adjusted to 5.3 asabove. In a third beaker (III), 2 ml of Hageman-deficient plasma was treated similarly. All threetest systems were incubated at 370 C for 6 hours.At intervals, samples from the first test systemwere processed (see Methods) for the estimationof the euglobulin lysis time. Simultaneously andat the same incubation times, equal volumes of thesecond and third systems (II and III) weremixed in a tube, centrifuged, and the precipitatereconstituted with a volume of borate buffer equalto the original plasma volume (of the third sys-


150 -

100-

50-

lax

\ I~~~~~~~~~~~~~~~.

' I I I I I I

1 2 3 4 5 6Incubation time in hours

FIG. 3. PGTON HAGEMAN-DEFICIENTPLASMA. Curve

I, Hageman-deficient plasma preincubated with SF;curve II and III, Hageman-deficient plasma and SFpreincubated separately and only mixed before centrifu-gation.

1281


Plasminoplostin ^.VW.. "P10SMMOPIUSM7 ~~~~~~~~~INITIAL VELOCITY,wnits/02 m/0.3

0.I,

01.01.0 3.8it to 50 1000.8/"Is S.F. PER CENT

0.86 %%

0.6- w ^ S.FI00%

0.4 ~ '~~''O SF 50%

0.2- |,*' S.F 25%

0~~~ ~ ~ ~ ~ ~ ~ ~S12.52

Incubation time in hoursFIG. 4. GENERATION OF ENDOGENOUSPLASMINOPLASTIN IN HAGEMAN-DE-

FICIENT PLASMAWITH SF. The ordinate gives units of plasminoplastin formed.The abscissa gives incubation periods. The ordinate on the inset figure repre-sents initial reaction velocity. See text for other details.

tem), for the estimation of the euglobulin lysistime.

It can be seen from Figure 3, curve I, that pre-incubation of Hageman-deficient plasma and kao-lin-SF in the same beaker resulted in generationof endogenous plasminoplastin. On the otherhand, when Hageman-deficient plasma and kaolin-SF were preincubated separately (curve II + III)and mixed only at the time of centrifugation, therewas no increase in fibrinolytic activity. This sug-gests that neither Hageman-deficient plasma norkaolin-SF, alone, is a complete and adequate sys-tem for the formation of the endogenous plasmino-plastin, whereas, when preincubated together, eachcontributes to the formation of this endogenousplasminoplastin.

3. Evidence that SF (Hageman-dependent) actsas catalyst in the generation of the endogenousplasminoplastin

Figure 4 shows a PGT study (the values aregiven in units) on the generation of the endoge-nous plasminoplastin by Hageman-deficientplasma with varying additions of SF. The re-lationship (inset figure) observed between SFconcentration and initial reaction velocity may

suggest an enzymatic or catalytic action of SF.However, the present evidence is no more thansuggestive and does not permit an analysis ofkinetics. More work will be needed for the finalanswer.

4. Role of surface

It has been shown (11) that, in the PGT ofHageman-deficient plasma, kaolin causes a sig-nificant shortening of the initial euglobulin lysistime, but this does not progress. The followingexperiment was undertaken to clarify the role of"surface" in the generation of endogenous plas-minoplastin.

It was shown that when the PGTwas performedwith Hageman-deficient plasma and the systemswere incubated in siliconized beakers, a weak SFpreparation, which did not greatly affect the ini-tial euglobulin lysis time, did, however, producea continuously progressive increase of plasmino-plastin (Figure 5, curve 3). While the same weakSF preparation in the presence of kaolin (1 mgper ml) (Figure 5, curve 4) did not affect theinitial euglobulin lysis time obtained with kaolinalone (Figure 5, curve 2), it nevertheless causeda progressive increase of plasminoplastin in the

1282

AaW.&I 21"five


time test 2 was with 1 mg kaolin per ml; test 3 wasFlutes with streptokinase (SK); and test 4 was with

human plasmin. The amounts of SK and plasminwere adjusted so that both gave approximately the

_~"~~%emmmm~m.. 1 same euglobulin lysis times, at zero incubation, asin the kaolin experiment. It was found that, un-der the conditions employed in thes'e experiments,2 cas. U plasmin per ml, 0.2 U streptokinase per

--.. a ml, and 1 mg kaolin per ml had the same effect-....A... J on the initial lysis time (zero incubation).

It can be seen from Figure 6a that test 2 with,-__0_kaolin, and test 3 with SK, gave essentially simi-

lar curves. Onthe other hand, test 4 with plasmin,despite the similar initial value, progressed much

1 2 3 4 5 6 more slowly. The control, test 1, was poor ini-Incubation time in hours tially, but gradually approached the same order of

activity as that in the other systems after about 45. EFFECTS OF KAOLIN (CURVE 2), SF (CURVE 3),husOLIN PLUS SF (CURVE 4) ON PGT OF HAGEMAN-IT PLASMA. The control (curve 1) is a PGT of b) Hageman-deficient plasma test systemsin-deficient plasma without any additive. (Figure 6b). Tests 2, 3, and 4 were run in a

manner analogous to the corresponding tests instages. This leveled off, however, at es- Figure 6a. No control was necessary, since plas-

ly the same end point as was reached, more minoplastin fails to develop in the absence ofby SF alone (Figure 5, curve 3). It can Hageman factor (11) (Figure 5, curve 1). In-

seen from the same Figure 5 (curve 1) that stead, test 5 was run with kaolin-SF (see Ma-the control-namely, Hageman-deficient plasmawithout any additive-did not increase its plas-minoplastin activity over the 6 hours of incubation.

The present experiment tentatively suggests thatthe role of surface, itself, in the generation of en-dogenous plasminoplastin is limited. When SF(Hageman-dependent) is preformed, the surfacedoes not add significantly to the over-all rate ofactivation. Hence, it would appear that in nor-mal plasma the surface contributes to the forma-tion of endogenous plasminoplastin by partici-pating in the activation of Hageman factor.

5. Site of action of SF (Hageman-dependent)

Streptokinase is known to act on a precursor(proplasminoplastin) in the fibrinolytic systemto form a plasminoplastin. Without raising ques-tions regarding the nature or exact mode (s) ofaction, a comparative study was made of strepto-kinase, SF, and plasmin as "activators" in thePGT system with a) normal plasma, b) Hage-man-deficient plasma, and c) chicken plasma.

a) Normal plasma test systems (Figure 6a).Test 1 was a control run without any additive;


300 PGT

(a)NORMAL PLASMA

200

100- >Z, ^

300

200

100

PGT(b) HAGEMAN-DEFICIENT PLASMAAd.... ...........

2 3 4 5Incubation time in hours

6

FIG. 6. EFFECTS OF DIFFERENT ACTIVATOR AGENTS ONPGT OF (a) NORMALPLASMA AND (b) HAGEMAN-DE-FICIENT PLASMA. See text for other details.

Lysisin mi,

5001

400 ~

300

200'

100-

FIG. CAND KADEFICIE?

Hagema

earliersentiallslowly,also be

1283

II I I


terials) to restore SF and the necessary surface,thereby normalizing the initial euglobulin lysistime. Figure 6b, test 2, shows the usual failureof kaolin to develop plasminoplastin activity in theHageman-deficient test system (11). Test 3 withSK, clearly developed plasminoplastin activity.Test 5 with kaolin-SF, behaved like test 3. Onthe other hand, test 4 with plasmin, showed no in-crease in plasminoplastin activity.

The similarity in both systems (Figure 6, a andb) between SK and SF (plus surface) at leastsuggests that both work by forming a plasmino-plastin. Active plasmin is significantly differentin this regard and fails to develop additionalfibrinolytic activity (i.e., plasminoplastin) in theHageman-deficient system.

In the normal plasma system (Figure 6a, test2) kaolin apparently works by surface activation ofthe Hageman factor, since there is no progressionof the initial fibrinolytic activity when kaolin isadded to the Hageman-deficient plasma system(Figure 6b, test 2).

Table IV presents data obtained by testing ini-tial and 5-hour incubates of the previous experi-ment (Figure 6, a and b; tests 3 and 4) by thefibrin plate method. It is evident that in the ab-sence of the Hageman factor (Hageman-deficientsystems) only the system with SK showed in-creased plasminoplastin activity (unheated fibrin)in the 5-hour sample, while that with plasmin notonly failed to develop more activity but lost mostof that possessed at zero time. It can also be seenthat almost identical results were obtained withthe normal euglobulin systems.

c) Chicken plasma test systems (Figure 7).In preliminary experiments, not included in thiscommunication, it was shown that the chicken

TABLE IV

Effect of streptokinase and human plasmin on thegeneration of endogenous plasminoplastin *

Lysed area (mm2)

Unheated Heatedfibrin fibrin

Systems preincubated at -370 C 0 5 hrs 0 5 hrs

Normal euglobulin +SK 221 382 100 100Normal euglobulin +plasmin 144 253 100 85Hag-def. euglobulin+SK 284 371 118 100Hag-def. euglobulin +plasmin 121 63 111 <30

* Fibrin plate method. The preparations were samples obtainedfrom the incubates shown in Figure 6. a and b, tests 3 and 4.

fibrinolytic system, while readily activated withurine plasminoplastin, remained completely in-active in the presence of streptokinase. It wastherefore concluded that chicken plasma has ade-quate plasminogen, but is deficient in proplasmino-plastin. On the basis of these data, and takinginto consideration the additional fact that chickenplasma is Hageman-deficient (24) (also confirmedin our laboratory), we performed the followingPGTexperiment.

Test 1 was a control without any additive; test2 was with 1 mg kaolin per ml; test 3 was with 1ml kaolin-SF per ml. It can be seen (Figure 7)that chicken plasma without additive (test 1) orwith kaolin (test 2) behaved similarly, failing todevelop any plasminoplastin activity. These re-sults were anticipated, since chicken plasma isHageman-deficient. In test 3 it can be seen thatdespite the addition of SF, the chicken test sys-tem did not develop any plasminoplastin activity.The slightly shorter lysis times obtained through-out this test might be attributed to insignificanttraces of human plasminogen (and proplasmino-plastin) in our kaolin-SF preparation (see Ma-terials). It can be concluded that, since kaolin-SFfailed to develop endogenous plasminoplastin in


260-I

220

180

140

100

1 2 3 4 5

Incubation time in hoursFIG. 7. PGT ON CHICKEN PLASMA. Curve 1 is a

control test without any additive; curve 2 is a test withkaolin (1 mg per ml); and curve 3 is a test with kaolin-SF (1 ml per ml).

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a system which is deficient in proplasminoplastin,it only acts when proplasminoplastin is present.Hence, SF is only efficient for the generation ofendogenous plasminoplastin when its precursor(the proplasminoplastin) is available.

DISCUSSION

The present study confirms and extends previ-ous observations on a role of Hageman factor inthe activation of the fibrinolytic system (10, 11,25). It also characterizes as a plasminoplastinthe agent which is developed in the incubationphase of the plasminoplastin generation test(PGT). The elucidation of the role of Hage-man factor in the fibrinolytic system can thereforebe explored in terms of the formation of "endoge-nous plasminoplastin."

The severe defect in the generation of endoge-nous plasminoplastin in Hageman-deficient plasmaindicates a major role for some "activator" factorlacking in such plasma. Our SF preparation (de-pendent upon activation of the Hageman factor)corrects the above defect when it is incubated withthe Hageman-deficient plasma in the PGT.

In a previous study (11) it was found thatHageman factor is not identical with plasmino-gen or proplasminoplastin. It was also establishedthat Hageman-deficient plasma has normal amountsof these factors. There is substantial evidencethat the failure of the Hageman-deficient plasma todevelop plasminoplastin activity is due to the ab-sence of Hageman factor. After restoration ofthe "Hageman clotting factor" defect by additionof SF, the fibrinolytic activity of Hageman-de-ficient system is also normalized, specifically thegeneration of endogenous plasminoplastin.

Our SF preparation is relatively crude and doesnot permit the exact identification of the "activa-tor" agent. It could be the entire "active Hage-man-PTA complex" or "active Hageman" itself(which will be tested when the opportunity to pre-pare PTA-free "active Hageman" occurs). Itdoes not depend upon PTA alone, since this factoris known to be present in the Hageman-deficientsystem. It could conceivably depend upon someas yet unidentified agent, coincidentally absentfrom Hageman-deficient plasma and present inour SF preparation. Future work will be neededfor the final answer.

A minor role of surface, interpreted from itsaction on Hageman-deficient plasma, with andwithout added SF, appears to be limited to someinitial activation of the fibrinolytic system whichdoes not progress unless SF (Hageman-de-pendent) is added. Hence, in normal plasma, thesurface mainly contributes to the activation of thefibrinolytic system by activating Hageman factor.

The observation that Hageman factor is neces-sary for the formation of the endogenous plasmino-plastin raised the question of its site of action.According to our data, the generation of the en-dogenous plasminoplastin is only brought aboutwhen adequate amounts of proplasminoplastin andHageman factor (or SF) are present. Chickenplasma, which is deficient in both factors, does notform endogenous plasminoplastin. It still doesnot do so even after restoration of Hageman fac-tor in the form of kaolin-SF. Presumably, theactive Hageman factor brings about the formationof plasminoplastin by acting on its precursor (theproplasminoplastin). In this respect, Hagemanfactor seems to have the same site, but not neces-sarily the same mode, of action as streptokinase,which is the "type" of a lysokinase.

The suggestion (Figure 4) as to a catalytic orenzymatic mode of action of SF (Hageman-de-pendent) upon proplasminoplastin, is tentativelybased upon the measurement of the initial velocity.It was shown that the progress curves obtainedwith four different amounts of SF are of thegeneral form of most enzyme reactions. By plot-ting the initial velocity against the amount of SF.a curve was obtained, which was slightly deviantfrom the linear curve obtained in pure enzyme-substrate systems. It is known that such deviantcurves are obtained when small amounts of im-purities are present in the substrate. Hardistyand Margolis (26) originally suggested that activeHageman factor acts catalytically to convert PTAinto an "active" form. The supporting evidencecame from Becker's (27) demonstration that di-isopropyl fluorophosphate inhibits the coagulantproperties of active Hageman factor. Similar ob-servations were made by Ratnoff and associates(16). Pending final proof that Hagemran factorhas two natural substrates-namely, proplasmino-plastin and PTA the apparent parallelism may beemphasized.

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Being aware from our previous work (14) thatplasmin preparations are usually contaminatedwith a "Hageman-like" factor, we prepared aplasmin free from such activity (see Materials).With this preparation, we were able to show con-vincingly the inability of plasmin to activate theproplasminoplastin in Hageman-deficient plasma,even when it was used in a concentration of 2cas. U per ml, which corresponds to almost halfthe potential plasmin yield of the plasma plasmino-gen (Figure 6b, test 4). In the correspondingexperiment on normal plasma (Figure 6a, test 4),the reaction takes place in the presence of Hage-man factor, and thus any additional action of plas-min could not be shown. That plasmin is notneeded for plasminoplastin generation indicatesthat this enzyme is unrelated to the factors whichinteract to form endogenous plasminoplastin.

The present evidence, which clearly suggeststhat active Hageman factor participates in endoge-nous plasminoplastin formation, strongly indi-cates that Hageman factor may be the precursorof natural "plasma lysokinase" responsible for theactivation of proplasminoplastin and the increaseof the amount of endogenous plasminoplastin,which is considered to be the main thrombolyticagent in vivo (23). It is implicit in this new hy-pothesis that thrombolytic activity in vivo, a phe-nomenon reflecting the concentration of the plas-minoplastin in the circulation, depends on thelevel of active Hageman factor. Figure 8 is aschematic representation of this hypothesis. As

InactiveHageman factor

S1_ _- cc

INNIBITOR

1%,,ACI

HAGEMANs(PLASMA

Proplasminoplostin

IURFACE p0NTACT- -0..

INHIBITOR

I,rlVEFACTOR R -.

LYSOKINASE)PLASMINOPLASTIN

IN PLASMA IN THROMBUSPlosminogen Plosminooen

,^p INHIBITORSN IPLASMIN PLASMIN

( ? proteolysis) (thrombolysis)

FIG. 8. TENTATIVE SCHEME SHOWING THE POSSIBLE

ROLE OF HAGEMAN FACTOR IN THE THROMBOLYTIC

PROCESS.

can be seen, endogenous plasminoplastin, after itsformation, may convert both plasma plasminogenand plasminogen trapped in a thrombus to plasmin.However, plasma plasmin has an entirely differ-ent fate from plasmin in the thrombus. In theplasma, inhibitory substances rapidly inhibit what-ever plasmin is formed. Consequently, underthese conditions, plasma plasmin is seldom avail-able for effective thrombolysis. On the other hand,plasmin formed within a thrombus is relativelyuninhibited in its action, and thus brings aboutthe dissolution of the fibrin without causing gen-eralized proteolysis. Hence, the concentration ofcirculating plasminoplastin, rather than the amountof plasma plasmin, determines the rate of resolu-tion of fibrin-containing thrombi.

SUMMARY

A simple and sensitive method, the plasmino-plastin generation test (PGT) has been described,which involves determination of the euglobulinlysis time of samples taken from diluted (20-foldwith distilled water) plasma, adjusted to pH 5.3,and incubated at 370 C. It was found that thefibrinolytic factor, which increases during theincubation phase of the PGT, is a plasminoplastin(activator).

The results indicate that the generation of nor-mal endogenous (or plasma) plasminoplastin de-pends on the presence of adequate amounts of"proplasminoplastin" (proactivator) and Hage-man factor. They also suggest that activatedHageman factor brings about the generation ofthe endogenous plasminoplastin by an enzymaticprocess.

These studies are in agreement with the hypothe-sis that plasmin is unrelated to the generation ofendogenous plasminoplastin and to the factorswhich interact to form it.

A tentative scheme is proposed relating thenew hypothesis involving Hageman factor to themain steps in the thrombolytic process. It in-cludes the current concept that thrombolysis typi-cally occurs without significant increase in plasma(circulating) plasmin.

ACKNOWLEDGMENT

We record with pleasure our indebtedness to Miss EveC. Sandlin for technical assistance.

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REFERENCES

1. Astrup, T. Fibrinolysis in the organism. Blood1956, 11, 781.

2. Goldhaber, P., Cornman, I., and Ormsbee, R. A. Ex-perimental alteration of the ability of tumor cellsto lyse plasma clots in vitro. Proc. Soc. exp. Biol.(N. Y.) 1947, 66, 590.

3. Astrup, T., and Sterndorff, I. Fibrinolysokinaseactivity in animal and human tissue. Acta physiol.scand. 1956, 37, 40.

4. Astrup, T., and Permin, P. M. Fibrinolysis in theanimal organism. Nature (Lond.) 1947, 159,681.

5. Kwaan, H. C., Lo, R., and McFadzean, A. J. S.Production of plasma fibrinolytic activity withinveins. Clin. Sci. 1957, 16, 241.

6. Kwaan, H. C., Lo, R., and McFadzean, A. J. S.Lysis of thrombi experimentally produced withinveins. Brit. J. Haemat. 1958, 4, 51.

7. Kwaan, H. C., Lo, R., and McFadzean, A. J. S. In-hibition of plasma fibrinolytic activity by exer-

cised ischaemic muscles. Clin. Sci. 1958, 17, 361.8. Miullertz, S. Activation of plasminogen. Ann. N. Y.

Acad. Sci. 1957, 68, 38.9. Lewis, J. H., and Ferguson, J. H. Studies on a pro-

teolytic enzyme system of the blood. IV. Activa-tion of profibrinolysin by serum fibrinolysokinase.Proc. Soc. exp. Biol. (N. Y.) 1951, 78, 184.

10. Iatridis, S. G. The effects of surface on plasminogenactivation. Fed. Proc. 1961, 20, 58.

11. Iatridis, S. G., and Ferguson, J. H. Effects of sur-

face and Hageman factor on the endogenous or

spontaneous activation of the fibrinolytic system.Thromb. Diath. haemor. 1961, 6, 411.

12. Ferguson, J. H., Wilson, E. G., Iatridis, S. G., Rier-son, H. A., and Johnston, B. R. Enzymes andblood clotting. I. Trypsin as an accessory factor.J. clin. Invest. 1960, 39, 1942.

13. Johnston, C. L., Jr., Ferguson, J. H., and O'Hanlon,F. A. Surface activation of plasma clotting: Afunction of Hageman Factor. Proc. Soc. exp.

Biol. (N. Y.) 1958, 99, 197.14. Iatridis, S. G., Wilson, E. G., Ferguson, J. H., and

Rierson, H. A. Blood clotting activities of throm-bolytic agents with special reference to a Hage-

man-like effect. Thromb. Diath. haemor. 1960, 5,50.

15. Waaler, B. A. Contact activation in the intrinsicblood clotting system. Scand. J. clin. Lab. Invest.1959, 11, suppl. 37.

16. Ratnoff, 0. D., Davie, E. W., and Mallett, D. L.Studies on the action of Hageman factor: Evi-dence that activated Hageman factor in turn acti-vates plasma thromboplastin antecedent. J. clin.Invest. 1961, 40, 803.

17. Kline, D. L. The purification and crystallization ofplasminogen (profibrinolysin). J. biol. Chem.1953, 204, 949.

18. Alkjaersig, N., Fletcher, A. P., and Sherry, S. Theactivation of human plasminogen. I. Spontaneousactivation in glycerol. J. biol. Chem. 1958, 233,81.

19. Astrup, T., and Mullertz, S. The fibrin plate methodfor estimating fibrinolytic activity. Arch. Bio-chem. 1952, 40, 346.

20. Alkjaersig, N., Fletcher, A. P., and Sherry, S.e-Aminocaproic acid: An inhibitor of plasminogenactivation. J. biol. Chem. 1959, 234, 832.

21. Mullertz, S. Formation and properties of the acti-vator of plasminogen and of human and bovineplasmin. Biochem. J. 1955, 61, 424.

22. Sawyer, W. D., Fletcher, A. P., Alkjaersig, N., andSherry, S. Studies on the thrombolytic activity ofhuman plasma. J. clin. Invest. 1960, 39, 426.

23. Sherry, S., Fletcher, A. P., and Alkjaersig, N.Fibrinolysis and fibrinolytic activity in man.Physiol. Rev. 1959, 39, 343.

24. Soulier, J. P., Wartelle, O., and Menache, D.Caracteres differentiels des facteurs Hageman etPTA. R6le du contact dans la phase initiale dela coagulation. Rev. franc. At. clin. biol. 1958, 3,263.

25. Niewiarowski, S., and Prou-Wartelle, 0. R6le dufacteur contact (facteur Hageman) dans la fibrino-lyse. Thromb. Diath. haemor. 1959, 3, 593.

26. Hardisty, R. M., and Margolis, J. The role ofHageman factor in the initiation of blood coagu-lation. Brit. J. Haemat. 1959, 5, 203.

27. Becker, E. L. Inactivation of Hageman factor bydiisopropylfluorophosphate (DFP). J. Lab. clin.Med. 1960, 56, 136.

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