VWF

THROMBOSISRESEARCH21; 295-309, 1981 0049-3848/81/030295-15$02.00/O Printed in the USA. Copyright (c) 1981 Pergamon Press Ltd. All rights reserved.

PARALLEL ACTIVITY

DESTRUCTION OF FACTOR VIII PROCOAGULANT AND AN 85,000 DALTON PROTEIN IN HIGHLY PURIFIED

FACTOR

C.G Cockburn, Rosemary Wilson and R.M.Hardisty

VIII/VWF

J. de Beaufre-Apps,

Julia

Department of Haematology, Institute of Child Health and Hospital for Sick Children, London, W.C.l. England

(Received28.8.1980;in revised form 19.12.1980. Acceptedby Editor A.L. Bloom) ABSTRACT Concentrated, highly purified human factor VIII/van Willebrand factor (FVIII/vWF) (produced by two different methods) in O.lM ammonium bicarbonate, pH 7.4 was digested with human plasmin (0.004 cu/ml) or human or bovine were assayed for thrombin (0.1 U/m), and the digests procoagulant (VIIIC) activity. Digestion samples were-lyophilized and then subjected to two different new electrophoretic methods to examine trace proteins, which would not be detectable by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoreThe results show that both thrombin and plasmin sis. a single-chain protein with an apparent proteolyse molecular weight of 85,000 daltons, but at different sites. The initial thrombin-induced proteolysis of this protein, which comprised about 2% of highly purified FVIII/vWF, was largely complete by the time VIIIC was maximally activated as judged by the l-stage assay. The loss of VIIIC induced by both thrombin and as measured by the Z-stage assay (which shows plasmin, no activation) paralleled the rate of proteolysis of Since no other proteolytic the protein by both enzymes. paralleled the events were observed which even remotely activity loss of VIIIC, we conclude that the biological by the 85,000 defective in haemophilia may be expressed dalton protein reported in this study.

Key

words:

Factor

VIII,

plasmin,

295

thrombin

296

FVIII/vWF DIGESTS & VIIIC ASSAYS

Vo1.21, No.3

INTRODUCTION It has been shown that plasmin rapidly destroys FVIII procoagulant activity (VIIIC), as measured by both the land a-stage assays (l-41, and that thrombin activates VIIIC before destroying it (2,3,5), although certain groups of workers suggest that the activation effect is peculiar to the l-stage assay (6). Atichartakarn et al (1978) (3) showed that VIIIC inhibitor binding was not reduced by the action of plasmin but was reduced by thrombin. This suggests that thrombin and plasmin exert their actions at different sites on the moiety responsible for the expression of VIIIC. There would seem to be no general agreement as to what is responsible for the full expression of VIIIC. The most likely reason that thrombin and plasmin have not been reported to proteolyse factor VIII/van Willebrand factor (FVIII/vWF) during the VIIIC activation or inactivation process is that only a small proportion of the FVIII/vWF protein is actually cleaved. By developing two simple electrophoretic methods which will handle much larger amounts of protein than a single polyacrylamide gel, we have been able to observe proteolytic events during VIIIC inactivation by plasmin and by implication those attributes of and thrombin, FVIII/vWF which are necessary for the full expression of procoagulant activity. MATERIALS

AND

METHODS

Materials All chemicals were Analar grade unless otherwise Materials for the production of polyacrylamidegels stated. and benzamidine hydrochloride were obtained from Eastman Chemical Company and sodium dodecylsulphate (SDS) specially pure) from BDH Chemicals Ltd. Epsilon aminocaproic acid (EACA), hirudin (highly purified) and other protein reagents were obtained from Sigma Chemical Company. Plasmin was supplied by AB Kabi, Stockholm, Sweden and was stored at a concentration of 12.5 casein units (cu)/ml at Bovine thrombin (highly purified) was -2OOC in 20% glycerol. obtained from Sigma Chemical Company and was stored at a concentration of 100 U/ml in a O.lM ammonium bicarbonate buffer, pH 7.4, under liquid nitrogen. Human thrombin was obtained from the Bureau of Biologics, FDA, Bethesda, Maryland, U.S.A. and was stored in a similar manner. Agaroee Ltd.

ITCHYwas

obtained

from

Piiarmacia

Fine

Chemicals

Vo1.21, No.3

297

FVIII/vWF DIGESTS 6 VIIIC ASSAYS

purification of FVIII/vWF was performed by the method of Hershgold et al (7) employing the modifications described by Guisasola et al (4); diisopropylfluorophosphate (DFP) was also added to the protein solution to a final concentration of 5mM immediately after the ammonium sulphate precipitation step. The FVIII/vWF had approximately 150 U ristocetin cofactor (VIIIR:VWF), procoagulant (VIIIC) and antigenic activity (VIIIR:AG) per ?g? protein and the properties desOnce purified, it was either cribed by Guisasola et al (4). used immediately or snap-frozen and stored at -2O'C for a few days until use. FVIII/vWF was also purified by an alternative method from lyophilized intermediate or high-purity FVIII concentrates, based on the method of Newman et al (8) and outlined by modifications:Switzer and McKee (91, with the following a) the FVIII/vWF concentrate was dissolved in 0.2M benzamidine hydrochloride and heparin (2 U/ml),

EACA

+ O.OlM

b) the protein from the 12% (w/v) polyethylene glycol precipitation was dissolved in 0.014 M trisodium citrate, 0.13M sodium chloride, were added 0.2M EACA, pH 6.5, to which DFP and hirudin to final concentrations of 5mM and 2 U/ml respectively, c) this material was then subjected to precipitation (35% saturation at 2OC), d) the precipitate was redissolved buffer and subjected to Sepharose same buffer.

an ammonium

sulphate

in the citrate saline EACA 4B gel chromatography in the

Methods The FVIII/vWF at a protein FVIII/vWF-digestion by plasmin concentration of 0.3 - 0.6 g/l was desalted rapidly in small (approximately 25 x 1 cm) Sepharose 4B columns with O.lM During this process only about ammonium bicarbonate, pH 7.4. 5% of the VIIIC was lost as judged by the 2-stage method, Plasmin was then added to a final concentration of 0.04 or The digestions were performed at room temperature 0.004 cu/ml. Once the plasmin (21-24OC) in sterile polystyrene test tubes. had been added the digestion mixture was divided into two to enable digestion aliquots to be removed from each by two laboratory personnel within a couple-of seconds of each other. The following digestion aliquots were taken at regular intervals: 50 ~1 for VIIIC assay; 50 ~1 for reduced SDS polyacrylamide up to 500 ~1 for unreduced SDS gel electrophoresis (PAGE); agarose gel electrophoresis and 1.0 ml for unreduced SDS PAGE. Each digestion aliquot was taken into l/20 volume of saturated EACA in a precooled tube at O'C, then snap-frozen The samples for VIIIC assay were taken into within one minute. l/10 volume of saturated EACA and stored at O°C until assay within an hour of collection.

298

FVIII/vWF DIGESTS & VIIIC ASSAYS

Vo1.21, No.3

FVIII/vWF-digestion by thrombin This was carried out in exactly the same way as the plasmin digestions except that bovine or human thrombin was used at a concentration of O.lU/ii.L. Each digestion aliquot was taken into a precooled tube contain6 times the amount of thrombin ing enough hirudin toneutralise present, then snap-frozen within 1 min. The samples for VIIIC assay were taken into precooled tubes containing enough hirudin to neutralise 3 times the amount of thrombin present in the sample and stored at O°C until assay within an hour of collection. FVIIIC

assays

The l-stage assay was performed acoording to the A.lethod of Hardisty and Macphernon (10) except that the haemophilic substrate plasma with U% normal VIIIC activity was activated by a 6-minute incubation with kaolin at 37OC. For the P-stage assay the method of Denson (11) was used. Reduced

SDS

polyacrylamide

gel

electrophoresis

(SDS

PAGE)

Proteins were reduced and denatured by the addition of t volume 6% (v/v) 2-mercaptoethanol, 6% (w/v) SDS, 0.06M ethylene diaminetetra-acetic acid (EDTA) and 8M urea, then by heating to 100°C for 2 minutes. The samples were then electrophoresed through 8% polyacrylamide gels with 1% of crosslinking O.OlM EDTA, 0.1% SDS using a continuous O.lM sodium phosphate, gel and running buffer with a pH of 7.2. The samples were electrophoresed at a current of 4 mA/5 mm diameter gel until the bromophenol blue tracking dye had migrated 80% of the length of the gel. SDS

Agarose

gel

electrophoresis/SDS

PAGE

The samples for this assay were lyophilized over phosthen dissolved in 120 ~1 of a solution of phorous pentoxide, 0.06M sodium phosphate, O.OlM EDTA, 1% SDS, 5M urea, + bromophenol blue tracking dye at a pH of 7.2 and heated to 100°C for 2 minutes. The agarose gels were made by pouring a 1.25% agarose C solution in O.lM sodium phosphate, O.OlM EDTA, 0.1% SDS buffer, pH 7.2, into 8mm-diameter capped glass tubes. After solidification the upper surfaces of the gels were cut flat with a razor blade and the capped bases of the tubes were replaced by a perforated membrane. The tubes were mounted in an electrophoretic apparatus in which the level of the lower buffer reservoir matched that of the upper one so that there wae no nett hydrostatic pressure on the gels. Both upper and lower running buffer reservoirs contained the same buffer as the The gels and they were 'pre-electrophoresed' before use. FVIII/vWF samples were then electrophoresed at a current of 23 mA/gel until the bromophenol blue band had penetrated the to 6 mA/gel until gels by 1 cm, then the current was increased the electrophoresis was complete, i.e. when the bromophenol blue tracking dye had migrated 80% of the length of the gel.

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299

FVIII/vWF DIGESTS & VIIIC ASSAYS

After the electrophoresis the gels were either removed on sterile unscratched plastic from their tubes, placed surfaces and transversely sectioned into 3 mm discs, or snapfrozen in liquid nitrogen and then stored in small airtight tubes at -2O'C until they could be sectioned. Each section was placed in a small numbered plastic tube, and 40 pl of the SDS denaturing and reducing agent was added to each, together with a microspatula load of urea crystals The tubes were then all heated to 100°C and (30 + 6 ?g). ? violently agitated for 2 min, then, while still molten, centrifuged at 1000 g for a further 2 min. Then each section was reheated in turn to 100°C, taken up in a 250 ~1 Hamilton syringe and layered onto the surface of an 8% SDS polyacrylamide gel in which electrophoresis was performed as described above. 8% Unreduced SDS PAGE present in FVIII/vWF

of

the

low-molecular-weight

proteins

If one attempts to electrophorese large amounts of unreduced FVIII/vWF into an SDS gel, the high-molecular-weight components block the surface of the gel, trapping any lowmolecular-weight components and only allowing them to enter We therethe gel slowly, so they do not resolve into bands. for electrophoresed the FVIII/vWF through a separating mixture above the gel surface, which allowed all the protein to enter but which significantly reiarded the electrophoresis of the FVIII/vWF oligomers, so that all the low-molecular-weight components of the FVIII/vWF mixture had entered the polyacrylamide gel by the time the larger components reached the top of the gel. The separating gel consisted of a 2 cm layer of Sepharose 2B that had been washed 10 times in a large excess of the polyacrylamide gel running buffer plus 5M urea (high The protein samples were lyophilized, then dissolvpurity). ed in a minimal volume (usually 120 ~1) of 1% SDS, O.OlM EDTA, blue and 0.06M sodium phosphate , plus a little bromophenol 4.7 M urea, all at a pH of 7.2. Just before the electrophoresis the residual urea solution above the Sepharose layer was removed with a Hamilton syringe and the protein samples were carefully layered onto the Sepharose so as to form a flat interface. Initially the samples were electrophoresed at 2 mA/gel until the bromophenol blue marker had penetrated the The current was then increased polyacrylamide gels by 1 cm. Gels incorporating the buffer system described to 4 mA/gel. by Laemmli (12) were also used for the procedure. Staining

and

calibration

of

gels

All gels were stained for over 4 hours at room temperature in a filtered near-saturated solution of coomassie brilliant blue 6250 in 40% methanol, 10% acetic acid, and The destained by diffusion in filtered 7.5% acetic acid. gels were scanned at 550 nM in a Gilford InsCrument 240 scanning spectrophotometer and the stain uptake of each band

300

was

FVIII/vWFDIGESTS & VIIIC ASSAYS

calculated

by

Vo1.21, No.3

triangulation.

The gels were calibrated with the following proteins: glyceraldehyde-3-phosphate dehydrogenaae, human haemoglobin chains cross-linked by the method of Davies and Stark (131, yeast alcohol dehydrogenaae, human serum albumin and crosalinked human serum albumin and human IgG. RESULTS The

effect

of

Plaamin

on VIIIC

Control experiments show that when EACA is added to FVIII/vWF in an ammonium bicarbonate solution at pH 7.4 to l/10 saturation before the addition of plasmin, no change in VIIIC can be detected by the Z-stage assay for over 1 hour. In the absence of EACA plaamin destroys VIIIC as judged by the Z-stage VIIIC assay. The,reaulta of seven experiments show a highly discontinuous loss of VIIIC (Fig.1): 0.004 cu of plasmin per ml destroys 20 - 40% of the VIIIC in the first then no further loss of VIIIC can usually be detected minute, for a period of up to several minutes; this is followed by a series of further rapid losses of VIIIC. FIGURE

1

Destruction of VIIIC by 0.004 cu plasmin/ml as judged by the Z-stage clotting assay in three separate experiments.

la PO

,..,. ..

Q

‘;.,..

m

‘.., .,

I i

u Z"

‘b,

..

“‘... ,, ‘..,

!

CTO-O~O

!-”

‘..,

:

,

‘0.. \

.

,.,

\ ,

‘.. ..

I .,..

:---._

,___.

\

Q-0

..,.

0

The

effect

of Thrombin

on VIIIC

Control experiments show that when hirudin is added to FVIII/vWF in ammonium bicarbonate solution at pH 7.4 before the addition of thrombin, a negligible change in VIIIC detected by the Z-stage assay occurs during a period of 1 hour. The results obtained by the Z-stage assay (Fig.21 always show that thrombin destroys VIIIC. No hint of activation of VIIIC has ever been observed and it is always found that during the first 4 minutes of the thrombin digestions, the loss of VIIIC resembles a zero-order process, in that it is directly proportional to the time of digestion.

301

FVIII/vWF DIGESTS & VIIIC ASSAYS

Vo1.21, No.3

On the other hand the l-stage assays (Fig.21 invariably followed by a decline which more or less show a rise in VIIIC, parallels the loss of VIIIC as judged by the 2-stage method. The degree of activation of VIIIC can be quite variable; Fig.2 shows a sample that was minimally activated whereas Before thrombin other samples were activated up to 13-fold. activation the l-stage assay gives a much lower level of VIIIC than the a-stage method and during the later phases of the digestion more VIIIC remains as judged by the l-stage method. FIGURE

2

D

Changes in VIIIC induced by O.lU thrombin as measured by the l-stage O---O and the 2-stage LO clotting assays

)6 Y 28

;I

8% Reduced Plasmin

SDS

PAGE

digestion

,&;_, I

of

to

I5 1D ?5 wNI*YlNuX*

)I

FVIII/vWF

By the time plasmin had destroyed 90% of the VIIIC, nochange could be seen in the polyacrylamide gels as judged by eye, and scanning the gels at high sensitivity also failed to reveal significant differences (Fig.Ja). Gel scanning of subsequent digestion aliquots revealed subtle changes in the gels, but showed (Fig.3b) that when FVIII/vWF was digested with 0.04 cu of plasmin per ml, only one 200,000 dalton subunit in approximately six was proteolysed in a period of 100 min (Fig.Jb), although 9% of the VIIIC had been destroyed by 10 minutes. The slight but reproducible increase in the proportion of protein in the 200,000 dalton band during the first 8 min appeared to be due to a transitory decrease in the variable amount of protein at the top of This effect is not the polyacrylamide gels during this period. co-relatable with the loss of VIIIC. Thrombin

digestion

of

FVIII/vWF

Thrombin had no effect on FVIII/vWF as judged by reduced The gel series were also scanned at high SDS PAGE (Fig.4). again without significant changes being observed. sensitivity,

302

FVIII/vWF DIGESTS & VIIIC ASSAYS

j

‘-,.

1

26

4-a

lw firm

lsb~2fPj26

7ojQQ

Vo1.21, No.3

1.

‘c:r ??

.

0.

TYMYM

tia

IminI

b

a

FIGURE Digestion gels.

1 .

.

of FVIII

by

0.04

3

cu plasmin/ml.

Reduced

a) The position of the 200,000 dalton subunit 9096 of the VIIIC was lost within the first

SDS PAGE

is indicated. 10 minutes

in

8%

Over

b) The 200,000 dalton subunit of FVIII expressed as a percentage of the total protein, estimated by Cooma#sie brilliant blue staining, followed by gel scanning at high sensitivity. FIGURE

4

Digestion of FVIII by O.lU thrombin/ml. Reduced SDS PAGE in 8% gels. The position of the 2d0,OOO dalton band is indicated.

303

FVIII/vWF DIGESTS & VIIIC ASSAYS

Vo1.21, No.3

&arose

gel

electrophoresis

Plasmin

digestion

of

followed

by

reduced

SDS

PAGE

FVIII/vWF

In order to find out if one particular molecular weight fraction of the whole FVIII/vWF was attacked by plasmin this electrophoretic method was used since 3-4 times the quantity of FVIII could be fractionated on a single SDS agarose gel as on an acrylamide gel and since the method can resolve numerous components,each of which is characterized by electrophoretic mobilities in two gel systems. During the time taken by plasmin to destroy 90% of the MIIC activity, three highly repeatable changes could be observed by The first was a reduction in the this electrophoretic system. amount of material excluded from the 1.25% SDS agarose gels (Fig.5a). The second change was that two bands with apparent and 155,000 daltons, which electromolecular weights of 165,000 phorese in the SDS gels in parallel to the bulk of the FVIII/vWF, are produced by plasmin (Fig.5b) at a rate which does not parallel In the case illustrated, this material comthe loss of VIIIC. prised about 4% of the protein both in the starting material and after 4 minutes of digestion (67% loss of VIIIC), but after 16 minutes of digestion it comprised 10% of the total protein. The low electrophoretic mobility of this protein in the agarose gels shows that the bulk of it at least is disulphide-linked to the The third and most striking change is 200,000 dalton oligomers. that a protein with an apparent molecular weight of 85,000 daltons is destroyed by plasmin at a rate which parallels theloss of VIIIC. The results of an experiment are shown in Fig.gc where at least 90% of the 85,000 dalton protein was destroyed by 16 minutes of incubation of FVIII/vWF with 0.004 cu of plasmin per ml. During this time the level of VIIIC had fallen to 7.6% of its The 85,000 dalton protein has a characteristic initial value. high mobility in the SDS agarose gels, showing that it is not disulphide-linked to any significantly large proteins. This protein represents about 2% of the total purified FVIII/vWF by weight. Thrombin

digestion

of

FVIII/vWF

Thrombin rapidly caused the destruction of the 85,000 dalton but for an unknown reason much of the high molecularprotein, weight protein consistently failed to penetrate the initial agarose These components were never sufficiently gels by more than 3 mm. 'spread-out' to determine whether thrombin exerts a proteolytic The results presented in the next section, action on any of them. suggest that thrombin is unlikely to proteolyse a componhowever, ent of the high molecular-weight protein.

8% Unreduced the

SDS

PAGE

of the

of

FVIII/vWF

low

molecular-weight

comnonents

in

FVIII/vWF

Plasmin

digestion

When a large quantity of FVIII/vWF (over 0.5 mg) is subjected several low molecular-weight bands to this form of electrophoresis, We consistently find that most can be seen (Fig.6a - first gel). of the bands increase in staining intensity slightly when the FVIII by 0.004 cu/ml of /vWF has been proteolysed for 1 - 2 minutes plasmin (about 25% loss of VIIIC and increase little or no more There is no overall correlation between the increase thereafter. in staining intensity of any or all of these bands and#elossofVIIIC

Vo1.21, No.3

VIII/vWF DIGESTS & VIIIC ASSAYS

304

a b

- - _. -

.

*r

FIGURE

5

Digestion of FVIII by 0.004 cu plasmin/ml. Analysis of protein from 1.25% SDS agarose gel sections by reduced SDS PAGE in 8% gels

a)

Distribution

b)

Distribution 165,000 and

of the

200,000

dalton

subunit

of bands with apparent 150,000 daltons

of

FVIII

molecular-weights

of

of the 85,000 dalton protein. c) Distribution The parallel destruction of VIIIC (2-stage assay) and Inset the5,OOO dalton protein (estimated by summing all the areas of the peaks of this protein from successive gel scana). U area

VIIIC (mm2/2)

(U/m.L x 31,

o--4

85,000

dalton

protein

peak

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FVIII/vWF DIGESTS & VIIIC ASSAYS

i) fail to demonstrate Gel scans ii) fail to demonstrate bands and mobility of any of the bands. Thrombin

digestion

of

305

the destruction of any of these a shift in electrophoretic

FVIII/vWF

In the first minute of the digestion with human or bovine thrombin (when VIIIC has usually become maximally activated as judged by the l-stage method and has lost about 20% of its activity as judged by the 2-stage method) the 85,000 dalton band is largely destroyed and appears to contribute to the band with an apparent molecular-weight of 77,000 daltons. Thereafter the 77,000 dalton band is destroyed by thrombin at a slower rate and a band at the front of the 62,000 dalton band and another with an apparent molecular-weight of 50,000 daltons appear after 1 minute of thrombin digestion. Significant changes in the other bands are not observed; there are no major shifts in electrophoretic mobility or relative staining intensity of any of them. The same results were obtained whether bovine or human thrombin purified by either method. was used, and with FVIII/vWF The two unreduced gel systems gave similar results, although the resolution in the iaemmli system was better. FIGURE

6

a)

Digestion of FVIII by O.lU/ml thrombin. Unreduced SDS PAGE of FVIII subjected to electrophoresis through a layer of Sepharose 2B.

b)

Scans of the gels shown above, the VIIIC levels in Units/ml as determined by the l-stage assay and apparent molecular-weights of the bands are indicated.

8 E

7 --._

TT:,Z .

;;;14F pz _k-. JCL _L ._ L _i

306

FVIII/vW?J DIGESTS & VIIIC ASSAYS

The proposed mechanism of proteolysis of the 85,000 dalton protein by plasmin and thrombin

/

Vo1.21, No.3

IX

PLASMIN 1

DISCUSSION These results show that both plasmin and human and bovine thrombin destroy a protein with an apparent molecularpresent at a level of about 2% in weight of 85,000 daltons, highly-purified FVIII/vWF prepared by two different methods, at a rate that parallels the loss of VIIIC as assayed by a P-stage On the basis of these results we conclude that the method. 85,000 dalton protein is likely to be necessary for the full expression of VIIIC as both plasmin and thrombin proteolyse it, but at different sites. As shown in Fig.7, plasmin must cleave this protein, which copurifies with FVIII/vWF, within a difor its destruction in the reduced sulphide loop, thus accounting but not the unreduced SDS gels, and thrombin must exert its action outside the disulphide loop, accounting for the destruction of the band in both gel systems. Comparison of apparent molecularweights of the purified protein by reduced and unreduced SDS PAGE provides additional evidence for the existence of one or more disulphide loops (C.G.Cockburn and P.A.McKee - in preparation).

1. 85,000 daltons a-stage assay,

high procoagulant activity as measured by the little activity as measured by the l-stageassay.

2. 77,000 daltons procoagulant l-stage and 2-stage assays. 3. No procoagulant

activity

measured

by both

the

activity.

Our findings might well explain the VIIIC inhibitor binding results obtained by Atichartakarn et al (3). Vehar and Davie (14) have described a protein with a molecular-weight of 70,000 daltons which they believe to be VIIIC, and which is also highly susceptible to the action of thrombin. The overall purification of this protein from bovine plasma was about 500,000 fold, which would also agree with our findings as the 85,000 dalton protein

Vo1.21, No.3

E'VIII/vWF DIGESTS & VIIIC ASSAYS

represents about 2% of our whole FVIII/vWF purified 10,000 fold with respect to human assay).

307

which has been plasma (2-stage

In common with many other workers (2, 3, 5, 14, 15) we have observed that thrombin induces an increase in VIIIC as FVIII measured by a l-stage method. The l- and P-stage obviously measure different attributes of assays, however, VIIIC. Switzer et al (16) have recently found that when FVIII/vWF is purified in the presence of protease inhibitors the FVIII has little procoagulant activity as judged by the They conclude that VIIIC circulates in vivo as l-stage method. a precursive form which is activated by a_thrombin-like enzyme. The major difference between the two VIIIC assays is that the a-stage assay incorporates a 15-minute incubation in which FXa Davie and Fujikawa (17) reported that is allowed to build up. It is likely that the l-stage method only FXa activates VIIIC. measures llactivated VIIIC" and the 2-stage method tends to assay all the VIIIC (the unactivated portion being activated during the incubation). * Pick and Hoyer (18) and Pick et al (19) have shown that VIIIC inhibitor IgG neutralises relatively low-molecular-weight which would support our conclusions. Also procoagulant protein, Lane et al (20) have isolated material .wkich binds to an antiVIIIC affinity column and found that the protein recovered from the column by dissociation with ammonium thiocyanate had apparent daltons by reduced SDSPAGE molecular-weights of 35,000 and 62,000 In the same series of experiments the 200,000 dalton subunit of FVIII was found to be extensively degraded after elution from an anti-whole FVIII affinity column and bore a striking resemblance to our plasmin-digested FVIII (4). The 35,000 and 62,000 dalton protein subunits might well be proteolytic degradation products as Atichartakarn et al (3) have of the 85,000 dalton protein, shown that VIIIC inhibitor neutralizing capacity is not readily It is well known that many proteases destroyed by plasmin. in contrast to the other biological attrirapidly destroy VIIIC, Our own urnpublished observations butes of FVIII/vWF (l-4, 21,221. of protease inhibitors must be used show that a whole 'cocktail' in the FVIII purification to produce FVIII/vWF that has a high specific activity as measured by the 2-stage clotting assay, as In spite well as being largely homogeneous by reduced SDS PAGE. of all our endeavours to minimise proteolysis during the FVIII purifications our results suggest that a proportion of the 85,000 dalton protein has invariably been thrombin activated to The 85,000 dalton protein might ultithe 77,000 dalton protein. mately prove to be a proteolytic derivative of a larger protein As the whole FVIII we purify has a ratio of which occurs in vivo. VIIIC (a-stage assay):VIII related antigen:ristocetin cofactor events which occur during activity of 1:l:l + 0.1, any proteolytic the purification procedure to produce the 85,000 dalton protein are unlikely to decrease its biological from a 'parent' molecule activity as measured by the 2-stage assay. Our Proposals concerning the 85,000 dalton protein which we believe to be required for the full expression of VIIIC are summarized in Fig.7. The methods developed in this study might facilitate its isolation and characterization.

FVIII/vWF DIGESTS & VIIIC ASSAYS

308

vo1.21, No.3

ACKNOWLEDGEMENTS

for

This work was supported by a grant Research into Crippling Diseases.

from

the

National

Fund

REFERENCES

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on human

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4. GUISASOLA,

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5. HULTIN,

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7. HERSHGOLD,

8. NEWMAN,

J., Methods, for factor VIII

M.H. and PUSZKIN, S. JOHNSON, A.J., KARPATKIN, the production of intermediateand high-purity concentrates. Br.J.Haematol. 2, l-20, 1971.

9. SWITZER,

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