Interaction of Hageman factor, prekallikrein activator and plasmin

Pergamon Press

Life Sciences Vol. 13, pp . 1201-1210, 1973 . Printed in Great Britain

INTERACTION OF HAGEMAN FACTOR, PRSKALLIRREIN ACTIVATOR AND PLASMIN Pierce M .E . Webster, V .H . Beaven, Y . Nagai, S . Oh-iehi and J .V . Ezperimental Therapeutics Branch National Heart and Lung Institute National Institutes of Health Bethesda, Maryland 20014

(Received 29 June 1973 ; in final form 28 August 1973) When plasmin was incubated with active Hageman factor prepared from acetone-activated human plasma by adsorption and elution from pre-treated supercel, no change in prekallikrein activator (PRA) activity was found, even though by polyacrylamide gel electrophoresis and by supercel adsorption it was shown that the active Hageman factor had been converted to the 30,000 molecular weight fragment . These data are in agreement with the concept that PKA is derived from Hageman factor, but do not support the concept that the conversion of plasminogen to plasmin is necessary for maximal generation of PKA activity in human plasma . Also reported is a radiochemical method for the measurement of PKA (active Hageman factor) activity which is 300 times more sensitive than the guinea pig ileum bioassay and 10 times more sensitive than the clotting tests . In our initial studies of the mechanism by which prekallikrein is activated in human plasma (1), we showed that the chromatography of acetoneactivated human plasma on DEAE-cellulose columns resulted in the separation of several peaks, each of which were capable of forming kinins from intact plasma .

We suggested that these active peaks might represent a series of

enzymes, each enzyme in turn activating the next .

However, more recent

evidence by Nagasawa et al . (2), Raplan and Auaten (3) and Cochrane and Wuepper (4) have clearly demonstrated that active Hageman factor itself can activate prekallikrein and that the multiple peaks of activity are due to partial digestion of active Hageman factor, presumably by plasmin .

One of

these fragments, a 30,000 molecular weight molecule, is thought to be five times more active than active Hageman factor in its ability to convert prekallikrein to kallikrein and thus it has been postulated that the conversion of plasminogen to plaemin is necessary for maximal generation of bradykinin when human plasma is activated (3) .

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Hagemin Factor and Plasmin

In the present studies, partially purified active Hageman factor was prepared from acetone-activated human plasma by adsorption and elution from pre-treated supercel as described by Speer et al . (5) . factor existed in multiple forms .

This active Hageman

However, conversion of this mixture to

the 30,000 molecular weight form by incubation with plasmin occurred with no change in prekallikrein activator (PKA) activity .

Also reported is a

sensitive radiochemical method for the measurement of PKA activity . Methods Clotting teats were conducted with plasma congenitally deficient in Factors XII, %I and LX as previously described (6), except that 0 .1 M Tris buffer, pH 8 .0, containing 0 .15 M NaCl and celite 512 (50 mg/ml) were used . The incubation time was shortened to 1 .0 min since more prolonged incubation inhibited the clotting time of active Hageman factor .

Active factors were

determined in the absence of celite, while in the presence of celite both active and inactive factors were measured .

The authors are indebted to

Dr . P . G . Iatridis, Indiana University School of Medicine, for the human Factor XII deficient plasma ; to Dr . C . J . Kociba, Ohio State University, for the Factor XI deficient bovine plasma ; and to Dr . M . Wicherhauser, American National Red Cross, for the Factor IX deficient human plasma . Prekallikrein activator (PKA) activity was determined by measuring the amount of kallikrein formed from purified prekallikrein .

The kallikrein so

generated was determined by a radiochemical method employing p-tosyl-L arginine [~]-methyl ester ([ 3H]-TAMe) which was originally devised by Beavers et al . (7) for the measurement of human urinary kallikrein and modified by Imanari et al . (8) for the determination of prekallikrein .

In a final volume

of 50 ul, the enzyme solution iB added to a mixture of 10 ul 0 .5 M Tris buffer, pH 8 .0, and 20-30 ul prekallikrein (containing 0 .015 EU (esterase units) where 1 EU is defined as that amount which will hydrolyse 1 umole

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Vol . 13, No. 9

TAMe per min at 25° and pH 8.0) in a plastic mictotube and incubated at room temperature for 10 min .

j3H]-TAMe (10 N1 containing 0.14 uc, 280

me/mmole) was added and the reaction mizture was incubated for an additional 30 min at room temperature,

During this latter incubation the plastic

microtube was floated in a closed counting vial . containing 10 ml tolueneliquiflor counting solution and 50

ul

atop solution (1 volume glacial acetic

acid added to 10 volumes 0.02 H TAMe) .

The reaction was terminated by

mizing the contents of the plastic microtube with the counting solution by v shaking for 10 sec .

This technique thus

takes advantage of the preferential

solubility of the tritiated methanol in the toluene-liquiflor solution and the almost complete insolubility of the [3H]-TAMe (9) .

All samples are run

in duplicate with blank determinations in the absence of prekallikrein . Prekallikrein,

freed of inhibitors to plasmin and plasma kallikrein,

waa prepared by a modification of the method of Kaplan and Austen (10) .

Hexa-

dimethrine bromide (100 ug/ml, Abbott Laboratorien) was added to fresh ACD human plasma (containing approximately 1 .5 EU/ml)

stored at room temperature

and the plasma dialyzed overnight at room temperature against several changes of 0 .01 H Tris buffer, pH 7 .0, containing 100 ug/ml hexadimethrine bromide. The dialyzed plasma was added to a DEAg-cellulose column (Whatman D$-23)

(2,0 ml

column volume/1 .0 ml dialyzed plasma) which had been previously equilibrated with the same buffer and the column developed at room temperature with about two column volumes of buffer . and frozen in liquid nitrogen .

Active fractions are pooled, placed in tubes The prekallikrein preparations had no

detectable esterase activity . Acetone-activated and dialyzed human plasma was prepared as previously described (1) .

Supercel, coated with plasma protein and air-dried ae described

earlier (5) (pre-treated supercel), was washed once with tap water .

To this

was added acetone-activated dialyzed plasma to a final concentration of 150 mg supercel per

ml .

The suspension was stirred for 10 min at room

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Hagemin Factor and Plasmin

temperature and centrifuged .

The adsorbed supercel was washed three times

with tap water and eluted with one-half to one-third the original plasma volume of 0 .007 M ammonia by stirring for 10 min at room temperature .

The

elutee was adjusted to pH 7 .4 and frozen in liquid nitrogen . Plasminogen was purified from human plasma by adsorption to a lyeineagarose column (11) and elution with 0 .2 M 1-lysine in 0 .02 M Tris buffer, pH 7 .4 .

The plaeminogen solution (8 .8 CTA units/ml) was diluted 4-fold with

a 50X solution of glycerol in 0 .1 M Tris, pH 8 .0, and activated with 25 unite atreptoldnase per ml diluted solution by standing at room temperature for 24 hr .

Plasmin (0 .2 ml) thus obtained was added to the supercel elutee (0 .5

ml) and the solution was incubated for 2 hr at room temperature . Gel electrophoresis using 7X polyacrylamide gel was conducted in Trisglycine buffer, pH 8 .3, according to the directions of Davia (12) .

Percent

adsorbance of the gel was measured at 280 nm in a Gilford spectrophotometer 240 equipped with a gel scanner .

The gel was cut into 1 .5 mm slices and

divided into two pieces so that it could be assayed in the presence or absence of prekallikrein (20 ul) in the radiochemical method .

Each piece was placed

in an assay tube containing 10 ul 0 .5 M Tris buffer, pH 8 .0, and crushed with a microhomogenizer .

The homogenizer was rinsed with 20 ul water .

The direct bioassay employing the guinea pig ileum has been described elsewhere (13) .

For kallikrein determination, 0 .4 ml of human plasma heated

to 60° was used se substrate .

For PKA determinations, the kallikrein was

removed from the samples by filtration through columns of SBTI-agarose and 0 .4 ml of untreated human plasma was used as substrate . Ia all of the ezperiments silicone-coated or plastic glassware was used . The plasma and preparations for assay were stored in liquid nitrogen and thawed only once .

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Results The PKA activity of the solutions could be measured either by the bioassay or by the radiochemical technique described under Hethods.

In the

latter procedure (Fig . 1), a linear dose-response curve was obtained with concentration of activator up to 3 mU (milliunite)/ml where 1 unit is defined es that mount of activator Found in 1 .0 ml of acetone-activated plasma .

r s ô

s

s

wu . ...

~....~ sern~ae

FIG. 1 Radiochemical technique for measurement of prekallikrein activator activity . mU (milliunite) . Inasmuch as pre-treated supercel had previously been shown to bind Hageman factor from human plasma (5), we attempted to prepare inactive Hagemaa factor by a modification of this

technique-the addition of

heaadimethrine bromide (100 ug/ml) to the plasma to prevent activation . However,

as shown in Table 1,

this ezperimant was unsuccessful in that

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TABLE 1 Simultaneous Activation of Nageman Factor and Prekallilcrein Activator on Adsorbed Supercel Active Hageman factor unite/ml

Fraction

Prekallikrein 'Activator unite/ml

Supercel elutee

22

22

Ammonium sulfate precipitate

42

47

the preparations eluted with dilute ammonia contained appreciable amounts of both active Hageman factor, as measured by its clotting activity, and PKA, as measured in the bioassay .

Furthermore, when these fractions were

further purified by ammonium sulfate precipitation (5), both the active Hageman factor and PKA activities increased concomitantly .

These results

suggested that both activities might reside within the same molecule . We then examined the ability of the PKA activity of acetone activated plasma to be adsorbed to supercel .

As shown in Table 2 with one individual

plasma, only trace amounts of human plasma kallikrein were found in the supercel eluates, the major portion of the activity being in the supernatant . PKA activity, oa the other hand, waA only partially adsorbed to supercel . The relative amounts found in the supernatant and in the elutee varied from one plasma to another ; sometimes the activity was equally divided between the two fractions .

Clotting teats conducted in the presence of celite 512

indicated that approximately 40X of the äageman factor, 18X of Factor %I (PTA) and 6X of Factor IX activity could be recovered from the supercel . These supercel eluates also shortened the clotting time of the three deficient plasma in the absence of celite, suggesting that the clotting factors in the eluates were present in their active forms .

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TABLE 2 Separation by Pre-treated Supercel of the Prekallikrein Activator Activity Found in Acetone-Activated Human Plasma Fraction

Rallilcrein units/ml*

Prekallikrein Activator unite/ml* Bioassay Esterase

Acetone-activated plasma

.75

.40

.37

Supercel eluate

.03

.14

.18

Supercel supernatant

.80

.44

.31

* A unit ie that amount of activity found in 1 .0 ml of a standard acetone-activated human plasma . Prekallikrein activator activity was freed of kallikrein by SBTI-,agarose affinity chromatography . Polyacrylamide gel electrophoresic analyses of the aupercel supernatant (Fig . 2A) showed that the PKA activity was predominantly in the prealbumin region, indicating that the prekallikrein activator found in the supernatant was the 30,000 molecular weight fragment . natant eluate

The PKA activity found in the supet-

(Fig . 2B), on the other hand, was found distributed in a number

of peaks throughout the gel .

No activity was recovered in the prealbumia region

Polyacrylamide gel electrophoresis of eupercel supernatant (A) and ). supercel eluate (B) . Percent adsorbante of the gel at 280 nm ( Prekallikrein activator activity (o -o) .

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and these data are in agreement with Kaplan and Auaten (3) who also found multiple forms of active Hageman factor . We then investigated the role of plaemin in converting active Hageman factor into the 30,000 molecular weight fragment .

As mentioned earlier,

Kaplan and Auaten (3) had found that dige~ tion of their active Hageman factor with plaemin caused a five-fold increase in prekallikrein activator activity .

However, when plaemin was incubated with the active Hageman

factor found in the aupercel eluates (Table 3), no or only a slight increase in PKA activity was found .

Plasmin by itself had no PKA activity .

Readsorption

of the aupercel eluate with pre-treated aupercel (150 mg/ml) removed all of the PKA activity .

However, after the aupercel eluate was digested with

plaemin, readsorption with aupercel no longer adsorbed this activity .

Poly-

acrylamide gel electrophoretic analysis of the plaemin-treated active Hageman factor showed that the PKA activity now migrated in the albumin region . TABLE 3 The Action of Plasmin on Active Hageman Factor Prekallikrein Activator munits/ml

Treatment Incubated for 2 hr at 25 ° Active Hageman Factor Plasmin Active Hageman factor

+ plaemin

210 < 5 230

Recovery after aupercel treatment Active Hageman factor* Active Hageman factor + plaemin

<10 210

Supercel eluate . Discussion These data are in agreement with the concept that the prekallikrein activator activity is derived from Hageman factor .

However, they do not

support the hypothesis that the conversion of plasminogen to plaemin is

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Hagemin Factor and Plasmin

necessary for maximal generation of bradykinin in han plasma (3) .

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Rather,

the present data would suggest that active Hageman factor itself has full capacity for activating prekallikrein,

since active Aagemaa factor and PRA

activities were concomitantly increased by ammonium sulfate treatment of supercel eluates of human plasma and since plasmin could completely comrert the partially purified active Bageman factor prepared from acetone-activated plasma to the 30,000 molecular weight fragment without increasing its PRA activity .

It may be that earlier workers (3) had a mixture of active and

inactive Aageman factor and that activation of inactive Hageman factor by plasmin caused the five-fold increase in PRA activity . than one form of active Hageman factor may exist, to ue

(Kaplan, personal communication)

Alternatively, more

since it has been reported

that some preparations of active Bageman

factor have full clot-promoting and PRA activities while others have full clot-promoting activity but only partial PKA activity . The radiochemical method for measuring kallikrein ie a technique which was originally devised by Beaven et al . (7) for the measurement of human urinary kallikrein and modified and simplified by Imanari et al . (8) in their sassy of human plasma prekallikrein.

The present procedure is a simple,

sensitive, and accurate method which can be used not only for the measurement of prekallikrein (B) but for prekallikrein activator (active Hageman factor) as well .

This method is approximately 300 times more sensitive than the

bioassay employing the guinea pig ileum,

50 times more sensitive than that

with the rat uterus and 10 times more sensitive than the clotting tests . Values for prekallikrein activator (Table 2)

were similar whether measured

by the direct bioassay employing the guinea pig ileum (13) or by the radiochemical technique.

Even crude acetone activated and dialyzed plasma can be

assayed by this method and, although the kallikrein must be removed from these solutions by filtration through SThepharose for the bioassay, this step is not necessary for the esterase method as the values can be corrected

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by blank determinations in the absence of prekallikrein. Adsorption of active Hageman factor preparations with pre-treated aupercel may also be a useful method for the separation of the 30,000 molecular weight form from the higher molecular weight forms .

Complete

recovery of the small molecular weight fragment was obtained in the aupercel supernatants, in contrast to recoveries from polyacrylamide gale which in our hands never exceeded 30X .

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