the journal of blood - semantic scholar...the procoagulant property of normal plasma that is...

Blood, Vol. 58, No. 1 (July), 1981

BLOOD The American

The Journal of

Society of Hematology

VOL. 58, NO. 1 JULY 1981

REVIEW

The Factor VIII Complex: Structure and Function

By Leon W. Hoyer

Normal human plasma contains a complex of two proteins

that are important in hemostasis and coagulation. The

factor VIII procoagulant protein (antihemophilic factor) and

the factor VIll-related protein (von Willebrand factor) are

under separate genetic control, have distinct biochemical

and immunologic properties, and have unique and essential

T HE IMPORTANCE of factor VIII in hemostasis

and blood coagulation is obvious from the clini-

cal problems in the factor VIII deficiency diseases,

classic hemophilia, and von Willebrand’s disease.

During the past decade there has been intense

research interest in these diseases, the two most

common hereditary bleeding disorders, and in the

properties of factor VIII. These studies have led to an

evolving understanding of factor VIII structure and

function.

The concept that factor VIII has two distinct

biologic functions-coagulant activity and a role in

primary hemostasis-was first suggested as an expla-

nation for the dual defect in von Willebrand’s disease.’

The logical inference that factor VIII is a bifunctional

molecule was strengthened by reports that proteins

purified from human and bovine plasmas had both

factor VIII procoagulant activity and the capacity to

interact with platelets in a way that might reflect an in

vivo role in primary hemostasis.25 Subsequent studies

have suggested an alternative interpretation, and it is

now generally accepted that plasma factor VIII is a

complex of two components that have distinct func-

tions, biochemical and immunologic properties, and

genetic control. The properties of these components

are summarized in Table I and the relationship is

illustrated in Fig. I.

One component of the factor VIII complex has

antihemophilic factor procoagulant activity and is now

usually designated VlII:C. It is inactivated by human

antibodies and can be measured (as VllI:CAg) when

these reagents are used for immunoassays. The other,

physiologic functions. While the nature of their interaction

and the details of the biochemical structures remain to be

determined, the information now available permits a

preliminary understanding of the molecular defects in

hemophilia and von Willebrand’s disease.

larger component comprises the majority of the

protein mass, interacts with platelets in a way that

promotes primary hemostasis, and can be immunopre-

cipitated by heterologous antisera. It is usually desig-

nated factor Vill-related protein (VIIIR) or von

Willebrand factor since it is reduced in quantity or is

qualitatively abnormal in von Willebrand’s disease.

Although it has been suggested that the two compo-

nents are properties of a single macromolecule,6’7

several kinds of data demonstrate the essential differ-

ences of the two proteins.

(1) The factor VIII procoagulant protein and the

factor Vill-related protein are controlled by different

genes. Isolated VlII:C deficiency is characteristic of

hemophilia, a disease transmitted by X-chromosomal

inheritance. In contrast, reduced or abnormal VIIIR is

found in von Willebrand’s disease, and the inheritance

pattern is that of an autosomal gene.

(2) The two proteins can be separated by chroma-

tography or centrifugation in high ionic strength

buffers (1 M NaCI or 0.24 M CaCI2).8” In most

studies, the inclusion of protease inhibitors in the

buffers does not affect the separation.’2”3

From the Hematology Division, Department of Medicine,

University of Connecticut School of Medicine, Farmington, Conn.

Supported in part by USPHS Research Grants HL 16626 and

HL 16872.

Submitted November 19, 1980, accepted February 12, 1981.

Address reprint requests to Leon W. Hoyer, M.D., University of

Connecticut Health Center, Farmington, Conn. 06032.

(C I 981 by Grune & Stratton, Inc.

0006-4971/81/5801-000l$02.00/0

For personal use only.on November 9, 2017. by guest www.bloodjournal.orgFrom

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- ? CELL - ENDOTHELIAL CELL

- MEGAKARYOCYTE

�ThVIII:C VIIIR SUBUNIT

/VIIIR POLYMER

(VIII:C) (VIIIRPOLYMER)

VIII:C

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VI IIRPOLYMER

HIGH IONICSTRENGTH REDUCTION VIII:C

+

2 LEON W. HOVER

THE FACTOR VIII COI4�LEX IN PLASMA

Table 1 . The Components of the Factor VIII Complex

Vlll:C The factor VIII procoagulant protein: the antihemo-

philic factor.

Identified as:

Factor VIII procoagulant activity (VlIl:C)

The procoagulant property of normal plasma

that is measured in standard coagulation

assays.

Factor VIII procoagulant antigen (Vlll:CAg)

Antigenic determinants closely associated

with VIII:C. Measured by immunoassays

carried out with human antibodies.

VIIIR The factor VIII-related protein: the von Willebrand factor.

A large polymeric protein that is necessary

for normal platelet adhesion and bleeding

time in vivo.

Identified as:

Factor-VllI-related antigen (VIIIR:Ag)

Antigenic determinants on VIIIR that are

detected by heterologous antibodies.

Ristocetin cofactor (VIIIR:RC)

The property of normal plasma VIIIR that

supports ristocetin-induced agglutination of

washed normal platelets.

(3) The plasma concentration of the two proteins,

measured by both function and immunoassays, vary

independently under certain conditions, the most strik-

ing being the posttransfusion period in patients with

von Willebrand’s disease.’

(4) The two proteins have different antigenic deter-

minants. Factor VIII procoagulant activity is macti-

vated by human antibodies from multitransfused

hemophiliacs and patients with spontaneous inhibitors;

ristocetin cofactor activity (VIIIR:RC) and the bleed-

ing time are characteristically normal in these

patients. Each protein can be measured by an immu-

noassay that is independent of the other protein.’4”5

Human anti-VIII:C coupled to Sepharose remove

x�-GIRc11L&PE

VIIl:C (and VIII:CAg) from plasma, but there is no

change in VIIIR:Ag levels.’6

(5) The biologic properties of the two proteins are

also independent. The procoagulant protein retains

full VIII:C activity in the virtual absence of VIIIR

(i.e., an VIII:C/VIIIR:Ag ratio of 1 2,600: 1 ),17 and the

factor-Vill-related protein can have VIIIR:RC activ-

ity in the absence ofdetectable VIII:C.”

The two components of the factor VIII complex do

interact, however. Their concentrations vary together

under most normal, stressful, and pathologic situa-

tions,’9 and standard purification methods separate

the intact (two component) factor VIII complex from

other plasma proteins. This interaction will be consid-

ered in detail after a description of the properties of

the two components.

FACTOR VIII PROCOAGULANT PROTEIN:

ANTIHEMOPHILIC FACTOR

Biochemical Properties

At the present time, there is little published infor-

mation about the biochemical properties of the factor

VIII procoagulant protein, per se. With few excep-

tions, studies of VIII:C function have been carried out

with the intact factor VIII complex, and it is only

recently that the factor VIII procoagulant protein has

been characterized after separation from VIIIR as

well as from the other plasma pr��jfl5#{149}l72O2�

Bovine VIII:C has been purified approximately

300,000-fold from plasma by Vehar and Davie.2#{176}

Although the G-200 gel filtration properties of the

protein with VIII:C activity suggested a molecular

weight of 250,000-300,000, analysis in sodium dode-

cyl sulfate/urea-polyacrylamide gel electrophoresis

identified a triplet of protein-staining bands with

Fig. 1 . The factor VIII com-plex. This schematic interpreta-tion indicates the interaction of

the two components and theirgenetic control. The effects of

VIIIR high ionic strength and reductionSUBUNITS are also noted.




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60 80 100 120

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Fig. 2. The Sephadex G-200 gel filtration properties of VIII:C

that has been separated from VIIIR. Vlll:C procoagulant andVlll:CAg measurements are indicated. For reference, the void

volume (V0) is noted, as are the elution volumes of lgG (G) andalbumin (A). (Modified from Hoyer and Trabold.2’ ) (A) VIII:C free ofdetectable VIllA. (B) Thrombin-activated Vlll:C. A part of thepreparation characterized in panel A was incubated with 5 x104U/ml human thrombin for 4 hr at 37’C prior to gel filtration.(C) Thrombin-inactivated VIII:C. A part of the preparation charac-terized in panel A was incubated with 102U/mI human thrombinfor 4 hr at 37’C prior to gel filtration.

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THE FACTOR VIII COMPLEX 3

molecular weights of 85,000, 88,000, and 93,000. The

electrophoretic properties did not change when pun-

fled VIlI:C was reduced with 2-mercaptoethanol, but

proteolytic cleavage to smaller proteins could be

accomplished by incubation with thrombin, purified

factor Xa, or activated protein C.2#{176}The purified bovine

VIII:C had no platelet-aggregating activity. Antibod-

ies to the VIII:C protein raised in rabbits inhibited

bovine VIII:C procoagulant activity but had no effect

on the platelet-aggregating activity of bovine plasma.

This observation strongly suggests that the coagulant

protein is separate from the protein responsible for

platelet-aggregating activity.

Human factor VIII procoagulant protein has not

been purified to homogeneity and it may be difficult to

obtain the necessary volume of human plasma that has

been collected in a way that reduces the likelihood of

VIII:C modification in vitro. A major unresolved

problem in VllI:C purification is the poor yield

obtained with all standard methods. For example, only

0.4 mg of bovine VIII:C protein was obtained from

each 125-liter batch of specially collected bovine plas-

ma, an overall VlII:C recovery that was estimated to

be l%.20

We have recently completed studies of VlII:C sepa-

rated from VIIIR and other human plasma proteins by

an immunoadsorbent technique.’7 While this VIII:C is

not stable in the absence of added bovine serum

albumin or similar proteins that prevent loss of VIII:C

from very dilute solutions, the preparation can be

studied by both VIII:C functional assays and

VlII:CAg measurements to determine properties of

VIIt:C when it is separate from VIIIR. Although it is

difficult to exclude the possibility that the protein was

modified during purification, protease inhibitors were

present during the procedure, and the purified VIII:C

had the same ratio of functional to immunologic

activity, as did the plasma from which it was

prepared.

The estimated molecular weight of the human

factor VIII procoagulant protein separated in this

manner is 285,000.21 This value has been calculated

from the properties of VIII:C on Sephadex G-200 gel

filtration (Fig. 2A), from which Stokes’ radius can be

estimated by comparison with standards, and sucrose

density gradient centrifugation (8.25). These proper-

ties of unactivated VIII:C are similar to those recently

obtained for bovine factor V by Nesheim and cowork-

ens.22 The correspondence is not surprising since the

cofactor role of factor V in prothrombin activation is

like that which VIII:C plays in factor X activation.

Both proteins are highly asymmetric molecules that

are activated by thrombin proteolysis.

Factor VIII procoagulant activity is not inactivated

60 80 100 120

Elution Volume (ml)

0.0005 U/mI Thrornbin‘4,

when the intact factor VIII complex is incubated with

reducing agents, e.g., 0.05 M 2-mercaptoethanol.7

This stability is striking when compared to the rapid

loss of nistocetin cofactor activity in these experiments.

Intact thiol groups do appear to have an important role

in VIII:C function, however, and a variety of thiol

inhibitors, including the specific reagent, p-chloromer-




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Fig. 3. The relationship of factor VIII procoagulant activity

and VIII:CAg in normal individuals and in patients with hemophiliaand von Willebrand’s disease. In the hemophilia panel, the largecircle at the origin represents 43 plasma samples with no detect-able Vlll:C or VIll:CAg. The large circle with an asterisk represents18 plasmas that had <0.01 U/mI VllI:C and 0.01-0.06 U/mIVlll:CAg.

4 LEON W. HOVER

curibenzoic acid, inactivate VIII:C.23 Factor VIII

procoagulant activity is also affected by pH and

calcium concentration. VlIl:C is most stable between

pH 6.9 and 7.2, and a marked loss occurs below pH 6

and above pH 8. The very low VllI:C content of

EDTA and ion-exchange resin-treated plasmas mdi-

cate that plasma cation concentration is also impor-

tant.24

Although neither human nor bovine VIll:C has

been purified in sufficient quantity for carbohydrate

analysis, there is indirect evidence in both cases that

the molecule contains carbohydrate residues. Concon-

avalin-A-agarose binds the purified VIII:C in these

preparations, and VIIl:C procoagulant activity is

eluted by the sugar, a-D glucopyranoside.’7’2#{176}

The limited success of VIll:C purification has made

it necessary to express the plasma content of this

protein by reference to standardized pools of human

plasma collected and stored in a way that reduces the

likelihood of VIlI:C activation or loss. Thus, all

VIII:C and VIII:CAg values are arbitrary measures

that express a ratio. They do not have any molecular

interpretation at the present time. One can, of course,

estimate the amount of protein that corresponds to a

“normal plasma level” of I U/ml. From measure-

ments ofthe intact human factor VIII complex’8’25 and

the proportion of protein that is VIlI:C,6 the value is

approximately 50 ng/U. A similar value can be

obtained from the specific activity of apparently

homogeneous bovine VIII:C (4500 U/mg).2#{176} It must

be recognized, however, that the estimated plasma

concentration, 222 ng/ml, is incorrect if the purified

VIll:C includes protein that has been either activated

or inactivated.

Immunologic Properties

Human anti-VIlI:C, obtained from multitransfused

hemophilic patients that develop inhibitors and from

rare individuals who form autoantibodies that macti-

vate VIIl:C, do not form detectable immunoprecipi-

tates with VIII:C or with the factor VIII complex.

Nevertheless, these sera can be used to detect VIII:C

antigen determinants by antibody neutralization

assays26 and by more sensitive immunoradiometric

assays for VIII:CAg.’5’2729

VIlI:CAg immunoassays require radiolabeled hu-

man anti-VIlI:C that has been purified by preparation

of immune complexes followed by their separation and

dissolution at low pH. The antibodies have been

obtained from high-titer sera (greater than 1000

Bethesda units/ml), and there does not appear to be

any consistent difference between hemophilic and

spontaneous anti-VIII:C. To date, similar results have

been obtained with two different assay methods: fluid

phase and two-site solid-phase separations.’5’2729 The

sensitivity of VlIl:CAg assays is 0.01-0.03 U/mI in

most laboratories, and the coefficient of variation for

these assays is ca. l0%.’�

In general, there is excellent correlation between

plasma factor VIII procoagulant activity and

VIII:CAg content (Fig. 3). VIII:CAg determinants

are more stable than VIII:C activity, however, and this

is striking in the case ofserum in which the VIII:CAg

values are 6O9’o-8O% of those in the corresponding

1.5

I t t U

0 0.5 1.0 1.5





plasma, even though there is no residual VIII:C activi-

ty.2729 Careful studies carried out with purified

VIII:C and human ct-thrombin demonstrated a dose-

dependent loss of VIII:CAg reactivity at thrombin

concentrations below 0.1 NIH U of thrombin/mI-to

60% of the original value-and qualitative changes in

VIlI:CAg determinants upon exposure to higher

thrombin concentrations.2’ These experiments proba-

bly overestimated the effect of thrombin on VIll:C,

since they were carried out in purified systems and the

usual plasma protein inhibitors were not present. It is

clear that thrombin has a detectable effect on

VIII:CAg, but that this measure is much more stable

than procoagulant activity.

Synthesis

Despite many investigations, the site of VIII:C

synthesis is not known. Both transplantation and

perfusion studies strongly suggest that VIII:C is

released by the liver under some conditions.3#{176}33 These

studies, carried out using standard procoagulant

assays, are not definitive, however, and it will be

important to verify these observations by immunoas-

say data and protein synthesis studies. Even though

there is strong suspicion that the liver plays an impor-

tant role in VIlI:C production, the normal or increased

VIII:C values in severe hepatic disease strongly

support the concept that there are extrahepatic

sources of this protein. In any case, it is not yet known

what cell type is responsible for VIII:C synthesis.

Function

It is generally agreed that VIIl:C accelerates blood

coagulation by its cofactor role in the enzymatic

activation of factor X by factor IXa. In the presence of

phospholipid and calcium, VIII:C markedly enhances

this reaction; in the absence of factor IX,, it does not

have any intrinsic capacity to activate factor X.3436

Native VIII:C may not participate in this reaction,

however, and VIlI:C activity is enhanced when plasma

or factor VIII concentrates are incubated with dilute

thrombin.37’38 It is likely, in fact, that thrombin activa-

tion is essential for VIII:C activity.36

It has been presumed that thrombin activates

VIII:C by a proteolytic modification, and this effect

has now been demonstrated.20’2’ Incubation with

thrombin appears to cause a cleavage in each of the

protein chains of bovine VIII:C,2#{176} and thrombin-

activated human VIII:C has gel filtration and ultra-

centrifugation properties of a 1 1 6,000-dalton protein,

in contrast to the 285,000 value calculated for the

unactivated molecule (Fig. 2B).2’ Factor VIII proco-

agulant activity is inactivated by higher concentra-

tions of thrombin-or more prolonged exposure to the

enzyme-and a further shift is noted in the gel filtra-

tion properties of the (nonfunctional) protein

measured by VIII:CAg (Fig. 2C).2’ Thus, thrombin-

mediated activation and inactivation are associated

with proteolytic modifications of VIII:C structure.

FACTOR VIll-RELATED PROTEIN:

VON WILLEBRAND FACTOR

Biochemical Properties

As the bulk of the factor VIII complex consists of

factor VI I I-related protein (V I I I R, von Willebrand

factor), data obtained for bifunctional factor VIII

reflect VIIIR properties.39�’ In all of these studies, the

most striking property of purified factor VIII is its

very large size. Agarose gel filtration separations

suggested a molecular weight greater than I 06,3940 and

the molecular weight obtained by sedimentation equi-

librium studies carried out in 6 M guanidine was

1.12 x 106.41 Although highly purified human factor

VIII is not dissociated by 6 Mguanidine or 1% sodium

dodecyl sulfate (SDS), subunits can be detected when

VIIIR is reduced with 2-mercaptoethanol or dithio-

threitol. A single band is detected on SDS poly-

acrylamide gel electrophoresis, and the subunits have

an estimated molecular weight of 195,000-

240,000.� � 8,39-41

Studies carried out with highly purified factor VIII

may be misleading, however, for they examine only

the small fraction of the molecules (ca. 5%-10%) that

are not lost during the series of separations. It is now

apparent that factor VIII-related protein is, in fact, a

heterogeneous population of multimers that have a

range of molecular weights from ca. 850,000 to over

12 x 106. This property first became apparent when

the technique of crossed immunoelectrophoresis iden-

tified VIIIR heterogeneity.42 Zone electrophoresis in

agarose did not resolve the different forms, however,

and the population of multimers was not recognized

until VIIIR was examined in agarose or agarose/

acrylamide gels in the presence of sodium dodecyl

sulfate.7’43 These studies of porcine and human VIIIR

were carried out with purified materials, and it was

possible that the apparent multimeric pattern could

have been caused by aggregation that occurred during

purification. Subsequent analysis of unmodified

normal human plasma has demonstrated that VIIIR

circulates as a population of very large multimers and

that the size distribution is not an artifact induced by

purification methods, freezing, or calcium chela-

tion”45 (Fig. 4).

The smallest polymers detected in normal human

plasma have an apparent Mr of 0.85 x I �6#{149}This would

appear to be a disulfide-bonded tetramer of the basic

ca. 200,000 subunit. The larger forms have M� indicat-




6 LEON W. HOVER

2.8 x i06-1.9 x 106-

0.95 x iO6�__

1gM NJ HEM VWD VWDI It

Fig. 4. The polymer pattern of human plasma VIIR analyzed by SDS-agarose electrophoresis. The migration of 1gM and 1gM polymers

is indicated on the left and their molecular weights are noted. The Vlll:R in plasma samples was identified by autoradiography afterincubation with tThl�Iabeled rabbit anti-VIIIR:Ag according to the method of Hoyer and Shainoff.” From the left. these plasmas are from anormal individual and patients with severe von Willebrand’s disease (VIIIR:Ag < 0.01 U/mI). severe hemophilia. type I von Willebrand’sdisease. and type II von Willebrand’s disease.

ing that they are composed of an integral number of

these tetramers, i.e., they have M� of 1 .7 x 1 06, 2.5 x

106, 3.4 x 106, etc. (Fig. 4). Serum obtained from

blood clotted in glass tubes at 37#{176}Chas the same

population of VIIIR.44 As many as 8 separate bands

can be detected in most normal plasmas, and there is

in addition a population of poorly resolved VIIIR with

Mr of ca. 8-1 2 x 106.

Purified human VIIIR has also been analyzed by

standard biochemical methods. It is a glycoprotein

containing 5%-6% carbohydrate, and hexose, hexos-

amine, and sialic acid have been specifically identi-

fled.39’4’ The amino acid composition has also been

determined in these studies: methionine, tyrosine, and

tryptophane values are relatively low and there are no

free sulfhydryl groups.39’

The amount of VIIIR protein in plasma has been

calculated from the specific activity of highly purified

factor VIII’8’39’ and from immunoradiometric assays

of plasma in which purified VIIIR was used as a

standard.25 The estimated values for VIIIR protein in

normal human plasma have been between 5 and 10

sg/ml. This is approximately 100 times greater than

the concentration of VIII:C protein.

Immunologic Properties

Rabbits immunized with purified human factor

VIII form useful immunoprecipitating antibodies.�

Although the sera vary in their ability to inactivate

VIII:C, they all form immunoprecipitates with VIIIR

and they inactivate plasma ristocetin cofactor activity

as well as other measures of VIIIR-platelet interac-

tion.2’47’48 They are monospecific in immunoprecipitin

assays if prepared with sufficiently purified factor

VIII, but absorption with VIIIR-depleted plasma

fractions is often necessary. A number of different

assays have been used to detect and quantify this

factor Vill-related antigen. Laurell electroimmunoas-

say, counter immunoelectrophoresis, and radioimmu-

noassay all give similar results,”4’46’49 and antisera

prepared in different laboratories have had generally

consistent properties in these immunochemical assays.

These rabbit antisera vary in their effect on human

VIII:C activity.50 The properties of the material used

for immunization appear to be very important in this

regard, and the VIII:CAg content depends on the

purification method. Although small amounts of anti-

VIII:CAg may be present in some sera (in addition to

the anti-VIIIR:Ag) they do not affect the immuno-chemical measurements. The interaction of anti-

VIIIR:Ag with the intact factor VIII complex will also

inactivate VIII:C, and anti-VIIIR:Ag coupled to

agarose removes both VIIIR and VIII:C from plas-

ma.’6’7

In general, there is a good correlation between the

factor VIII procoagulant activity and the factor VIII-

related antigen concentration in normal human plas-

mas (Fig. 5). Parallel increases in VIII:C and

VIIIR:Ag have been noted in plasmas from patients

with a wide range of nonhematologic diseases and

from normal individuals subjected to physiologic stim-

uli.”�

von Willebrand Factor Activity

Factor-VIII-related protein has a central role in

normal platelet function, a property that has been

designated “von Willebrand factor” activity because it

is deficient in patients with von Willebrand’s disease.’

The prolonged bleeding time in these patients is

presumed to be due to the reduced plasma VIIIR

content and it is corrected by transfusion of VIIIR-

rich cryoprecipitate. Two in vitro platelet assays are

usually abnormal in von Willebrand’s disease: ristoce-

tin-induced platelet agglutination and retention of




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� VON WILLEBRAND’S DISEASE (23)0 HEMOPHILIA (42)

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FACTOR yin-RELATED ANTIGEN (Units/mI)

Fig. 5. The relationship of factor VIII procoagulant activity and factor VIll-related antigen in the plasmas of normal individuals andpatients with hemophilia or von Willebrand’s disease.

THE FACTOR VIII COMPLEX

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platelets in glass bead columns.’ Both are corrected

when purified normal factor VIII is added to blood of

a patient with severe von Willebrand’s disease.

Although the way in which a prolonged bleeding time

is a consequence of the reduced level of factor VIII

protein is not yet known, there is a good correlation

between this abnormality and reduced levels of plasma

VIIIR:Ag and ristocetin cofactor measurements.5’

The use of ristocetin for in vitro assessment of

VIIIR function has become widely adopted in a rather

short period of time. The initial observation-reduced

or absent platelet aggregation when ristocetin was

added to platelet-rich plasma (PRP) from patients

with von Willebrand’s disease52-provided a simple

measure that many laboratories have incorporated

into the routine evaluation of patients with bleeding

disorders. Unfortunately, the assessment of ristocetin-

induced aggregation in patient PRP has limitations as

a diagnostic technique. In addition to its qualitative

nature, normal or reduced aggregation at one or more

ristocetin concentrations, it may be falsely positive in

some patients with primary platelet disorders and it is

not sufficiently sensitive to detect mild or moderate

von Willebrand’s disease.53 While abnormal aggrega-

tion in von Willebrand’s disease is the consequence of

a plasma deficiency and can be corrected by addition

of normal VIIIR, the phenomenon also requires the

presence of a normal platelet surface protein that is

deficient in Bernard-Soulier syndrome.54

Ristocetin-induced platelet aggregation is more

critically examined by assays in which dilutions of

plasma are tested with washed normal platelets and a

fixed concentration of ristocetin. These ristocetin

cofactor assays can be done with freshly washed plate-

lets or with formaldehyde-fixed platelets that remain

satisfactory as test reagents for several weeks if kept in

the refrigerator. The rate of ristocetin-induced aggre-

gation is related to the amount of plasma that is

added, and the value can be obtained from the aggre-

gometer tracing or by measurement of the time

required by detectable platelet agglutination.55 The

different methods give similar results under most

conditions.

Although there is a good correlation of ristocetin

cofactor activity in vitro and presumed von Willebrand

factor activity in vivo, as judged by freedom from

abnormal bleeding and by bleeding time measure-

ments,5’ there are exceptions. For example, the

VIIIR:RC value may become normal in von Wille-

brand’s disease during pregnancy and in inflammatory

states, or after transfusion with factor VIII, even

though the bleeding time remains prolonged.56’57 In

addition, patients with a variant form of von Wille-

brand’s disease have long bleeding times in spite of

low-normal ristocetin cofactor values and increased

reactivity when ristocetin is added to their platelet-

rich plasma.45’58 Thus, the value of ristocetin cofactor

assays as in vitro measures of VIIIR function must not




8 LEON W. HOVER

obscure the fact that they do not always reflect in vivo

biologic function. In this regard, it should also be

emphasized that VIIIR:Ag and ristocetin cofactor

assays measure different properties of the VIIIR

protein. While immunoassays detect all VIIIR mole-

cules with specific antigenic determinants, the protein

does not always have biologic activity. Moreover, arti-

factually increased immunoassay values are obtained

for the smaller VIIIR polymers when they are

compared to whole plasma standards by the Laurell

electroimmunoassay method. In contrast, ristocetin

cofactor measurements only identify VIIIR protein

that can interact with platelets, and this capacity is

limited to the larger polymers.59 Thus, plasmas or

purified proteins that lack the larger VIIIR polymers

will have a very low ratio of VIIIR:RC to VIIIR:Ag,

and, conversely, material that is relatively enriched in

large forms will have higher values for ristocetin

cofactor activity than VIIIR:Ag, when compared to

the whole plasma standard.

Radiolabeled purified VIIIR binds specifically to

platelet membranes, and it has been suggested that

discrete receptor sites are present.6#{176}62 Ristocetin,

which also binds to the platelet membrane,63 appar-

ently makes this platelet receptor available to VIIIR

and enhances its binding. In fact, it has been shown

that increasing concentrations of ristocetin cause more

VIIIR molecules to bind to the platelets.60’6’ A correla-

tion between VIIIR binding to platelets and ristocetin-

induced platelet aggregation has also been demon-

strated.62 VII1R polymer heterogeneity complicates

these analyses, however, for it is now recognized that

only large VIIIR bind to platelets in the presence of

ristoceti n �

Several kinds of data suggest that VIIIR carbohy-

drate is important in the factor-VIII-platelet interac-

tion and in ristocetin-induced aggregation. Initial

studies indicated that sialic acid removal reduced

ristocetin-induced platelet aggregation by 65%;64 other

studies found no change in reactivity even though the

sialic acid was removed.65 The latter studies suggested

that oxidation of the penultimate galactose modified

ristocetin-induced platelet aggregation, and there was

a 90% reduction of VIIIR:RC function when these

residues were oxidized. Subsequent reversal of the

reaction by galactose reduction caused full regenera-

tion of VllIR:RC function. No change in VIII:C

activity was noted when intact factor VIII complexes

were modified in a way that removed or oxidized these

carbohydrate groups.64’65

Carbohydrate residues are also important in VIIIR

intravascular survival. The asialo-derivative is cleared

by the rabbit liver with a T’/2 of 5 mm; the value for

normal VIIIR is 240 mm.64 Other studies have demon-

strated that the rabbit liver lectin that specifically

binds asialoglycoproteins binds to the asialo-deriva-

tive, not the native or asialo-agalacto-VlIlR.66

Synthesis

Immunofluorescent studies have identified

VIIIR:Ag in endothelial cells of arteries, arterioles,

capillaries, and veins throughout the body, as well as

in megakaryocytes and platelets.67’68 In addition, both

VIIIR:Ag and VIIIR:RC have been identified in the

medium from cultured human umbilical cord endothe-

hal cells.69’70 Direct evidence of VIIIR synthesis by

endothelial cells has also been obtained in tissue

culture.69 Factor VIII coagulant activity was not

detected in these culture media, however. While it is

possible that VIII:C might have been inactivated by a

protease present in the media, VIII:CAg was also

undetectable.7’ Thus, either the culture conditions

and/or cell source (umbilical cord veins) are unsatis-

factory for VIlI:C synthesis or VIIl:C is synthesized

by a different cell type.

THE INTERACTION OF VIII:C and VIIIR IN THE

FACTOR VIII COMPLEX

Although VIll:C and VIIIR have very distinct

properties, it would be an oversimplification to suggest

that they have no relationship and that they are simply

two proteins that happen to copurify. Several observa-

tions indicate that in plasma they interact through

noncovalent bonds to form a complex. For example,

the concentrations of the two proteins are closely

correlated in normal plasma and in most (nonhemato-

logic) disease states (Fig. 5)#{149}l.14.46.72 In contrast, there

is no correlation of either protein with factor V activi-

ty.72 The interaction between the components remains

intact when VIIIR interacts with heterologous anti-

bodies (e.g., rabbit anti-(whole) factor VIII) and

VIII:C is included in the immune complex. Immuno-

precipitates obtained with rabbit anti-VIIIR have

coagulant activity73 and elicit anti-VIll:C when

injected into other rabbits.74 Moreover, anti-VIIIR

coupled to agarose removes both VIII:C and VIIIR

from plasma.16”7 IfVIIl:C and VIIIR did not interact,

one would not expect the VIIJ:C to remain with the

immunoadsorbent. An alternative reason for a loss of

VIIl:C from the plasma, direct binding to unrecog-

nized anti-VIII:C in the rabbit antiserum, can be

discarded since the VIII:C can be recovered from the

beads by a modest increase in the ionic strength that

does not affect immunologic reactions.’7

Human antibodies to factor VIII have a different

effect. Although agarose-bound antibodies remove

VIII:C procoagulant activity (and VIIl:CAg) from

plasma, plasma VIIIR:Ag and ristocetin cofactor

activity are unaffected.’6 The difference between the

human and the heterologous antibodies in these exper-





iments is likely to be due to the very different concen-

trations of VIII:C and VIIIR protein in plasma. Since

less than 1% of the protein in the factor VIII complex

is VIII:C, the amount of VIIIR removed with VIII:C

may be so small that the assays cannot detect a change

in VIIIR concentration. In addition, the factor VIII

complex appears to be “destabilized” when VIII:C

interacts with human antibodies. The basis for this

suggestion is found in studies of soluble complexes

obtained by incubating human anti-VIII:C with

normal plasma. Immune complexes were detected in

late-eluting fractions, as well as at the void volume,

even though VIII:C was limited to the void volume

fractions when the antibody was not present.75

A relatively high-affinity interaction between

VIII:C and VIIIR can also be inferred from the effect

of reducing agents on plasma VIII:C. After plasma is

exposed to low concentrations of dithiothreitol or 2-

mercaptoethanol, VIII:C has the properties of a rela-

tively small protein on sucrose density centrifugation,

agarose gel filtration, and ethanol precipitation.76

While this change could be due to a direct effect of the

reducing agents on VIII:C properties, they return to

“normal” if hemophilic plasma (VIIIR free of VIII:C

activity) is added. This sequence strongly suggests

that the presence of intact VIIIR modifies the proper-

ties of the VIIl:C protein in standard separation tech-

niques.

The data summarized above suggest that VIII:C

and VIIIR interact in some way, but we have very

little information about the way in which this complex

is formed. Nevertheless, the susceptibility to dissocia-

tion by high salt buffers is indirect evidence that

electrostatic forces are important. The biologic impor-

tance of the plasma interaction is also uncertain, but

VIII:C instability in the absence of VIIIR (or in

plasmas that have relatively reduced VIIIR) suggests

that complex formation protects VlII:C from proteo-

lytic inactivation.77

THE FACTOR VIII DEFICIENCY DISEASES

Hemophilia A

Although the low factor VIII concentration in

plasma has prevented biochemical studies in classic

hemophilia (hemophilia A), immunologic techniques

have begun to define the molecular defect. These

studies have attempted to distinguish diminished

production of normal factor VIII from synthesis of

nonfunctional protein.

Two kinds of immunologic studies must be clearly

differentiated as this question is considered. The initial

work, done with human antibodies by the technique of

antibody neutralization, identified “nonfunctional but

antigenically cross-reacting AHF-like protein” in 10%

of hemophilic plasmas.26’78’79 The plasmas were desig-

nated cross-reacting material positive (CRM + ), and

they are now known to have normal levels of V I I I :CAg

even though VIII:C is very low (2%-lO% of normal).’5

Shortly thereafter, quantitative immunoassays were

carried out with rabbit antisera to human factor VIII.

These studies identified (by quantitative immunopre-

cipitin measurements) normal levels of “factor-VIlI-

like protein” in all hemophilic plasmas.46 Moreover, all

hemophilic plasmas neutralized the VIII:C inactivat-

ing properties of this rabbit antiserum. Plasmas from

patients with severe von Willebrand’s disease did not

form immunoprecipitates nor did they neutralize the

anti-VIII:C activity.46 These studies, and others

carried out with heterologous antisera, led to the

concept that nonfunctional but immunologically cross-

reactive material is present in all hemophilic plas-

mas.’4’46 It is now recognized that immunochemical

assays using heterologous antisera-whether done by

immunoprecipitation, hemagglutination, or radioim-

munoassay-detect VIIIR:Ag, not antigens related to

VIII:C function. Thus, they do not demonstrate an

intact factor VIII complex in hemophilia A, only

normal VIIIR:Ag synthesis as one might expect from

the normal bleeding time and the normal values of in

vitro assays of von Willebrand factor activity. It was

subsequently shown that VIIIR purified from hemo-

philic plasmas cannot be distinguished from normal

VIIIR by standard biochemical methods.�’#{176} Thus,

VIIIR:Ag measurements can be used as an assay that

distinguishes hemophilia from most forms of von

Willebrand’s disease, but they do not provide any

information about the nature of hemophilia. They

measure the product of a different (autosomal) gene.

Immunologic study of hemophilia has become possi-

ble, however, as human anti-VIII:C have been used in

quantitative immunoradiometric assays for VIII:C

antigenic determinants.’5’27 This technique has permit-

ted both qualitative and quantitative evaluation of

VIIIC:Ag and a number of studies have been

published during the past 2 yr.’5’27�29’8#{176}They have

identified several different patterns of VlII:C and

VIII:CAg in hemophilic plasmas (Fig. 3).

There is no detectable VIII:CAg in most plasmas

from patients with severe hemophilia (VIll:C <1% of

normal). In one-fourth of these plasmas (33 of I 34)

there are low VIII:CAg levels-usually I%-l0% of

normal, but rarely as much as 28%-even though

there is no detectable VIII:C coagulant activity.’5’27

29,80,81 Variable VIII:CAg levels are present in plasmas

of patients with mild or moderate hemophilia; usually

there is slightly more immunoreactive material than

VIII:C. A more extreme difference is observed for the

small group of hemophilic plasmas that have normal

VIII:CAg even though the coagulant activity is low.

These plasmas are from the same patients in which




10 LEON W. HOVER

CRM+ hemophilia can be identified by antibody

neutralization assays.26

Thus, patients with hemophilia have VIII:C defi-

ciency transmitted by X-chromosome inheritance and

they have normal VIIIR synthesis and function.

Nonfunctional VIII:C-like molecules are synthesized

by some hemophilic patients, and plasma concentra-

tions of immunoreactive protein are normal in rare

instances. These patients have an X-chromosome

mutation that modifies VIII:C structure. The absence

ofdetectable VIII:C protein in the remaining patients

may reflect a structural defect that is so severe that

antigenic reactivity is lost (as well as coagulant func-

tion) or it may indicate that there is no protein in the

plasma.

The evolving understanding of the molecular defect

in hemophilia has been based on new methods

(VIII:CAg and VIIIR:Ag measurements) that have

supplemented standard coagulation assays. This has

also improved our ability to provide informed genetic

counseling for families affected by this disease. It is

now widely recognized that hemophilia carrier detec-

tion has been facilitated by the combined measure-

ments of VIII:C and VIIIR:Ag, and most (>85%)

hemophilia carriers can be identified when the two

assays are done in laboratories that have excellent

assay quality control and sufficient experience with

reference populations of normal and genetically obli-

gate carriers.82’83 Carrier women have normal

VIIIR:Ag levels; VIII:C is reduced since only half of

their X-chromosomes (on the average) direct normal

VIII:C synthesis. The maintenance of an abnormal

ratio suggests that the physiologic influences that

affect the factor VIII complex act by modifying the

production, release, and metabolism of the two

proteins in a consistent way.

VlII:CAg stability in the presence ofamniotic fluid

had led to a further advance in genetic counseling, and

prenatal diagnosis of hemophilia is feasible by immu-

noassay analysis of fetal blood samples obtained by

fetoscopy at I 8-20 wk of gestation.84’85 In collabora-

tive studies carried out with Dr. M. J. Mahoney of the

Department of Human Genetics of Yale University

School of Medicine, immunoradiometric assay for

VIII:CAg (and VIIIR:Ag as a control protein) has

excluded-or accurately identified-hemophilia in

utero for 35 fetuses tested through October 1 , I 980.86

von Willebrand�s Disease

Immunologic and biochemical assays have also clar-

ified our understanding ofvon Willebrand’s disease. In

this case, the hemostatic disorder is transmitted by an

autosomal locus that affects VIIIR structure and

concentration. Qualitative and quantitative VIIIR

defects have been identified in this disease; the

reduced VIII:C levels appear to be secondary.

In its most frequent form, von Willebrand’s disease

is a mild or moderate bleeding disorder in which all of

the components of the factor VIII complex are

reduced in quantity and the bleeding time is

prolonged.”87 Plasma VIII:C and VIII:CAg may be

slightly higher than VIIIR:Ag and VIIIR:RC, but the

values are usually similar (Figs. 3 and 5). The VIIIR

multimer pattern appears to be normal since all of the

VIIIR polyers are reduced in quantity.45 The bleeding

time prolongation is variable, but it is usually asso-

ciated with reduced ristocetin cofactor activity.5’

While these patients all have genetic defects that

affect VIIIR, the hemostatic defect may be quite

inconsistent and the results of plasma assays and

bleeding time measurements may vary considerably

from time to time.88

Severe von Willebrand’s disease is a distinct (and

unusual) condition in which individuals have very low

levels of both factor VIII components, a markedly

prolonged bleeding time, and a major bleeding diathe-

sis. Family studies often demonstrate that these

patients are homozygous offspring of parents with

mild, or asymptomatic, von Willebrand’s disease.

Although VIIIR:Ag and VIIIR:RC levels are usually

less than 1% of normal in severe von Willebrand’s

disease (Fig. 5), some VIIIR:Ag can usually be

detected by sensitive assay methods.89

It is not certain why VIIl:C is low in von Wille-

brand’s disease, for these patients have the genetic

capacity to synthesize this protein. Recent studies

suggest that normal VIIIR protects VIII:C from mac-

tivation, and it is possible that VIIIR deficiency

permits accelerated VIII:C inactivation in vivo.77 It is

also possible that plasma VIIIR levels may, in some

poorly understood way, have an effect on VIII:C

synthesis or its release into the plasma.33 It is likely

that an understanding of the low baseline VIII:C level

will clarify the mechanism of the delayed rise and

prolonged survival of VIII:C (and VIII:CAg) after

transfusion in von Willebrand’s disease.’ At the pres-

ent time, one can simply suggest that normal (trans-

fused) VIIIR may stabilize VIlI:C or it may directly

influence VIII:C synthesis or release.

Most patients with von Willebrand’s disease have

similar levels of the different factor VIII properties-

this has been designated the “classical” pattern. Other

patients have been found to have nonfunctional VIIIR,

and these individuals have normal or slightly reduced

VIII:C and VIIIR:Ag, very low VIIIR:RC, and a

prolonged bleeding time. The VIIIR:Ag pattern is

abnormal on crossed immunoelectrophoresis, and this





has suggested that there is an abnormal and nonfunc-

tional VIIIR.90’9’ It is now apparent that the more

rapid VIIIR migration reflects an increased propor-

tion of small VIIIR and an absence of the largest

multimers (Fig. 4)#{149}45.92 The shift in multimer distribu-

tion changes the VIIIR electrophoretic mobility, since

agarose electrophoresis separates large molecules

according to size as well as charge. The important

consequence of the abnormal polymer distribution is

impaired hemostasis in the variant form of von Wille-

brand’s disease. Platelet binding, ristocetin cofactor

activity, and bleeding time correction all require the

large VIIIR forms and their deficiency causes a bleed-

ing diathesis.45’59’92 This variant form of von Wille-

brand’s disease has been designated “type II” by some

investigators to distinguish it from the classical “type

I” pattern in which there is a normal polymer pattern

and a similar reduction in all components of the factor

VIII complex.

A distinction has recently been made between two

subtypes of type II von Willebrand’s disease and the

abnormal VIIIR polymer patterns are slightly

different. In addition, PRP from patients with the

“type IIB” disorder has increased reactivity when

tested for ristocetin-induced platelet aggregation and

plasma VIIIR:RC is normal or minimally reduced.45’58

In contrast, patients with the more common “type

lIA” disease have markedly reduced ristocetin cofac-

tor activity and there is no aggregation when ristocetin

is added to their PRP.

The importance of carbohydrate groups in VIIIR

function has been considered in a previous section. It

is, therefore, not surprising that reduced VIIIR carbo-

hydrate has been identified in some patients with von

Willebrand’s disease. VIIIR subunits prepared from

these plasmas do not have detectable carbohydrate

when tested after SDS-polyacrylamide gel electropho-

resis, and the VIIIR sialic acid content is reduced.93’94

While a carbohydrate abnormality may be responsible

for some instances of nonfunctional VIIIR, another

series suggests that it is an uncommon form of von

Willebrand’s disease.95

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73. Bird P. Rizza CR: A method for detecting factor VIII

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1981 58: 1-13

LW Hoyer The factor VIII complex: structure and function

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