Synthesis, processing, and secretion of recombinant human factor VIII expressed in mammalian cells

The synthesis, processing, and secretion of factor VIII expressed from heterologous genes introduced into Chinese hamster ovary cells has been studied. The results show factor VIII to be synthesized as a primary translation product of approximately 230 kDa that can be detected in the lumen of the endoplasmic reticulum. In this compartment, the majority of the factor VIII is in a complex with a resident protein of the endoplasmic reticulum, binding protein, and may never appear in the medium. Some factor VIII transits the endoplasmic reticulum to the Golgi apparatus, where it is cleaved to generate the mature heavy and light chains. In the absence of von Willebrand factor in the medium, the secreted heavy and light chains are unassociated and subsequently degraded. In the presence of von Willebrand factor in the medium, the heavy and light chains are secreted as a stable complex and activity accumulates linearly with time. The utilization and complexity of asparagine-linked carbohydrate present on the secreted recombinant-derived factor VIII and human plasma-derived factor VIII were compared and found to be very similar. In both cases, the asparagine-linked carbohydrate moieties on the heavy chain are primarily of the hybrid or complex-type. In contrast, the factor VIII from both sources contains a high-mannose type of asparagine-linked carbohydrate on the light chain.     

Inactivation of human factor VIII by activated protein C: evidence that the factor VIII light chain contains the activated protein C binding site

Factor VIII is represented as a series of heterodimers composed of an 83(81) kDa light chain noncovalently bound to a variable size (93 to 210 kDa) heavy chain. Activated protein C inactivates factor VIII causing several cleavages of the factor VIII heavy chain(s). When factor VIII subunits were dissociated and component heavy and light chains isolated, the heavy chains were no longer a substrate for proteolysis by activated protein C. However, when factor VIII heavy chains were recombined with light chain, the reconstituted factor VIII activity was inactivated by activated protein C. The rate of factor VIII inactivation catalyzed by activated protein C was reduced by the presence of free light chain. The extent of this inhibition was dependent upon the concentration of light chain. Control experiments indicated that this protective effect of free light chain was not the result of inhibition of the activated protein C - lipid interaction. Fluorescence analysis demonstrated binding between the factor VIII light chain, chemically modified with eosin maleimide, and activated protein C, modified at its active site by dansyl-Glu-Gly-Arg chloromethyl ketone. Similar to proteolysis of factor VIII by activated protein C, this binding was dependent upon a lipid surface. Based upon the degree of fluorescence quenching, a spatial distance of 26 A was calculated separating the two fluorophores. These results demonstrate direct binding of activated protein C to the factor VIII light chain and suggest that this binding is an obligate step for activated protein C-catalyzed inactivation of factor VIII.     

A monoclonal antibody to factor IX that inhibits the factor VIII:Ca potentiation of factor X activation

A murine monoclonal antibody (IgG1k, Kd approximately 10(-8) M) specific for an epitope located on the heavy chain of human factor IXa was used to study structure-function relationships of factor IX. The antibody inhibited factor IX clotting activity but did not impair activation of factor IX either by factor XIa/calcium or by factor VIIa/tissue factor/calcium. The antibody also did not impair the binding of factor IXa to antithrombin III. Moreover, the antibody did not prevent calcium and phospholipid (PL) from inhibiting the binding of factor IXa to antithrombin III. The antibody also failed to impair activation of factor VII by factor IXa/calcium/PL. Furthermore, the antibody did not interfere with the very slow activation of factor X by factor IXa/calcium/PL. In contrast, the antibody did interfere with factor X activation when reaction mixtures also contained factor VIII:Ca/von Willebrand factor. The marked acceleration of factor X activation observed in control mixtures was not observed in mixtures containing the antibody. Similar results were obtained in reaction mixtures containing the Fab portion of the antibody and factor VIII:Ca free of von Willebrand factor. In additional experiments, factor VIII:Ca/von Willebrand factor was found to inhibit the binding of the antibody to 125I-factor IXa as determined using an immunosorbent assay. Moreover, the antibody displaced factor VIII:Ca from the factor X activator complex (IXa/calcium/PL/VIII:Ca) as evidenced by an altered elution pattern on gel filtration chromatography. From these observations, we conclude that the antibody impairs the clotting activity of factor IXa through interference with its binding of factor VIII:Ca. This suggests a significant role for the heavy chain (residues of 181-415) of factor IXa in binding factor VIII:Ca.     

Topography of the human factor VIII-von Willebrand factor complex

Factor VIII circulates in noncovalent complex with von Willebrand factor (vWf). The topography of this complex was evaluated by fluorescence energy transfer using factor VIII subunits modified with N-(1-pyrenyl)maleimide (NPM; fluorescence donor) and vWf-derived fragments modified with 7-diethylamino-3-[4'-maleimidylphenyl]-4-methyl coumarin (CPM; fluorescence acceptor). Results from a previous study indicated an interfactor VIII subunit distance of 20 A separating Cys528 and Cys1858 in the factor VIII heavy and light chains, respectively (Fay, P.J., and Smudzin, T. M. (1989) J. Biol. Chem. 264, 14005-14010). Fluorophore modification of the vWf SPIII homodimer (residues 1-1365) indicated multiple attachment sites at Cys126/135/1360 as determined from sequence analysis of fluorescent tryptic peptides derived from the modified protein. Based upon donor quenching data, an interfluorophore distance of approximately 28 A was calculated separating NPM-factor VIII light chain or factor VIII reconstituted from NPM-light chain plus unmodified heavy chain, from CPM-SPIII. A similar value (29 A) was obtained for NPM-light chain paired with CPM-SPIII-T4 (vWf residues 1-272), suggesting that donor quenching resulted primarily from modified residue(s) Cys126/135 in the acceptor. No energy transfer was observed for the NPM-heavy chain/CPM-SPIII pairing. However, when NPM-heavy chain was reassociated with unmodified light chain prior to reaction with CPM-SPIII or CPM-SPIII-T4, energy transfer was observed with calculated interfluorophore distances of approximately 31 and 34 A, respectively. Levels of acceptor resulting in maximal donor quenching suggested an equimolar stoichiometry of factor VIII (light chain)/vWf fragment in the reconstituted complexes. These results indicate a close spatial arrangement among the A3 domain of factor VIII light chain, the A2 domain of factor VIII heavy chain, and the NH2 terminus region of vWf in the factor VIII-vWf complex.     

von Willebrand factor is a cofactor for thrombin-catalyzed cleavage of the factor VIII light chain

The proteolytic activation of highly purified, heterodimeric porcine factor VIII and factor VIII-von Willebrand factor complex by thrombin was compared at I 0.17, pH 7.0, 22 degrees C. During the activation of factor VIII, heavy-chain cleavage is necessary to activate the procoagulant function, whereas light-chain cleavage is required to dissociate factor VIII from von Willebrand factor. The kinetics of activation of free factor VIII and factor VIII-von Willebrand factor complex were identical. The steady-state kinetics of thrombin-catalyzed heavy-chain cleavages and light-chain cleavage of factor VIII either free or in complex with von Willebrand factor were studied using sodium dodecyl sulfate-polyacrylamide gel radioelectrophoresis and scanning densitometry of fragments derived from 125I-labeled factor VIII. Association of factor VIII with von Willebrand factor resulted in an 8-fold increase in the catalytic efficiency (kcat/Km) of light-chain cleavage (from 7 x 10(6) to 54 x 10(6) M-1 s-1). The catalytic efficiencies of heavy-chain cleavage at position 372 (approximately 6 x 10(6) M-1 s-1) and position 740 (approximately 100 x 10(6) M-1 s-1) were not affected by von Willebrand factor. We conclude that von Willebrand factor promotes cleavage of the factor VIII light chain by thrombin which is followed by rapid dissociation of the complex, so that the rate-limiting step becomes heavy-chain cleavage at position 372. This accounts for the observation that von Willebrand factor has no effect on the kinetics of activation of factor VIII by thrombin.     

Mechanisms of plasmin-catalyzed inactivation of factor VIII: a crucial role for proteolytic cleavage at Arg336 responsible for plasmin-catalyzed factor VIII inactivation

Plasmin not only functions as a key enzyme in the fibrinolytic system but also directly inactivates factor VIII and other clotting factors such as factor V. However, the mechanisms of plasmin-catalyzed factor VIII inactivation are poorly understood. In this study, levels of factor VIII activity increased approximately 2-fold within 3 min in the presence of plasmin, and subsequently decreased to undetectable levels within 45 min. This time-dependent reaction was not affected by von Willebrand factor and phospholipid. The rate constant of plasmin-catalyzed factor VIIIa inactivation was approximately 12- and approximately 3.7-fold greater than those mediated by factor Xa and activated protein C, respectively. SDS-PAGE analysis showed that plasmin cleaved the heavy chain of factor VIII into two terminal products, A1(37-336) and A2 subunits, by limited proteolysis at Lys(36), Arg(336), Arg(372), and Arg(740). The 80-kDa light chain was converted into a 67-kDa subunit by cleavage at Arg(1689) and Arg(1721), identical to the pattern induced by factor Xa. Plasmin-catalyzed cleavage at Arg(336) proceeded faster than that at Arg(372), in contrast to proteolysis by factor Xa. Furthermore, breakdown was faster than that in the presence of activated protein C, consistent with rapid inactivation of factor VIII. The cleavages at Arg(336) and Lys(36) occurred rapidly in the presence of A2 and A3-C1-C2 subunits, respectively. These results strongly indicated that cleavage at Arg(336) was a central mechanism of plasmin-catalyzed factor VIII inactivation. Furthermore, the cleavages at Arg(336) and Lys(36) appeared to be selectively regulated by the A2 and A3-C1-C2 domains, respectively, interacting with plasmin.     

The size of human factor VIII heterodimers and the effects produced by thrombin

The heterodimeric structure of factor VIII was demonstrated by two approaches. First, the native molecular weights of several partially purified fractions of factor VIII were determined by measurement of Stokes radii and sedimentation coefficients to be approx. 237 500, 201 000 and 141 000. These measured molecular weights correlated with those derived from polypeptide chain composition, in which each molecule would consist of a doublet polypeptide of Mr 83 000/81 000 plus one predominant high-Mr polypeptide of either 146 000, 120 000 or 93 000. In addition, immunoadsorption using a monoclonal antibody specific for the light-chain doublet removed all of the heavy chains. Separation of the heavy chains from the light chain by EDTA further illustrated the non-covalent nature of the heterodimers. All forms had coagulant activity which was potentiated 13-15-fold by an equimolar amount of human alpha-thrombin. Thrombin converted the Mr 83 000/81 000 doublet to one of Mr 73 000/71 000, and cleaved the largest polypeptides to a transient intermediate form of Mr 93 000 which was further cleaved to polypeptides of Mr 51 000 and 43 000. Potentiation of coagulant activity was correlated with proteolytic cleavage of either or both the doublet and the Mr 93 000 polypeptides. These data indicate that human factor VIII purified from plasma consists of a group of heterodimers, composed of a light chain of Mr 83 000 (81 000) and a heavy chain which varies in size between Mr 170 000 and 93 000, each form of which is similarly potentiated and cleaved by thrombin.     

Characterization of human factor VIII and interaction with von Willebrand factor. An electron microscopic study

Blood coagulation factor VIII is a large glycoprotein that circulates in plasma at relative low concentration (0.1 microgram/ml). It consists of a heterogeneous mixture of a series heavy-chain peptides (90-200 kDa), each associated with a light chain of 80 kDa. To gain insight into the physical properties of the protein, we have characterized purified human factor VIII by electron microscopy and rotary shadowing. Electron microscopy of rotary shadowed factor VIII molecules showed predominantly a single globular domain structure, with a somewhat asymmetric shape, while two-domain structures were also encountered. The overall dimensions of the globular domains ranged from 4 x 6 nm to 8 x 12 nm. EDTA treatment of factor VIII reduced the overall dimensions (2.5 x 5 nm to 6 x 10 nm) while treatment with thrombin reduced the dimensions to a small extent. In complexes with von Willebrand factor, factor VIII appeared localized at the globular domains of von Willebrand factor multimers. In addition, incubation of factor VIII with Staphylococcus aureus V8 protease fragments SpII and SpIII revealed only binding to the globular domains of SpIII. In this study, the first morphological characterization of human factor VIII is presented, together with its direct localization on von Willebrand factor multimers.     

Intersubunit fluorescence energy transfer in human factor VIII

Human factor VIII circulates as a series of active heterodimers composed of a light chain (83 kDa) linked by divalent metal ion(s) to a variable sized heavy chain (93-210 kDa). Purified factor VIII subunits were modified with sulfhydryl-specific fluorophores. Probe selection was based upon the limited number of free cysteine residues in each subunit. Levels of probe incorporation suggested the presence of a single reactive cysteine residue per subunit. Amino-terminal sequence analysis of fluorescent tryptic peptides derived from the modified subunits indicated fluorophore attachment sites at Cys528 of the heavy chain (A2 domain) and Cys1858 of the light chain (A3 domain). Subunit reassociation was measured by fluorescence energy transfer using light chain modified with N-[1-pyrenyl] maleimide (fluorescence donor) and heavy chain modified with 7-diethylamino-3-[4'-maleimidophenyl]-4-methylcoumarin (fluorescence acceptor). Donor fluorescence quenching paralleled the formation of factor VIII clotting activity, and both effects were saturable with respect to added heavy chain. Based upon the degree of donor quenching, a distance of 20 A was calculated separating the two fluorophores. These results indicate a close spatial relationship between the A2 domain of heavy chain and the A3 domain of light chain in the factor VIII heterodimer.     

von Willebrand factor mediates protection of factor VIII from activated protein C-catalyzed inactivation.

Factor VIII, a cofactor of the intrinsic clotting pathway, is proteolytically inactivated by the vitamin K-dependent serine protease, activated protein C in a reaction requiring Ca2+ and a phospholipid surface. Factor VIII was inactivated 15 times faster than factor VIII in complex with either von Willebrand factor (vWf) or the large homodimeric fragment, SPIII (vWf residues 1-1365). Free factor VIII or factor VIII in complex with a smaller fragment, SPIII-T4 (vWf residues 1-272), were inactivated at the same rate, suggesting that this effect was dependent upon the size of factor VIII-vWf complex rather than changes in factor VIII brought about by occupancy of the vWf-binding site. Thrombin cleavage of the factor VIII light chain to remove the vWf-binding site eliminated the protective effects of vWf. In the absence of phospholipid, high levels of the protease inactivated both free and vWf-bound factor VIII at equivalent rates. Using the same conditions, isolated heavy chains and the heavy chains of factor VIII were proteolyzed at similar rates. Taken together, these results suggested that, in the absence of phospholipid, inactivation of factor VIII is independent of factor VIII light chain and further suggest that vWf did not mask susceptible cleavage sites in the cofactor. Solution studies employing fluorescence energy transfer using coumarin-labeled factor VIII (fluorescence donor) and synthetic phospholipid vesicles labeled with octadecyl rhodamine (fluorescence acceptor) indicated saturable binding and equivalent extents of donor fluorescence quenching for factor VIII alone or when complexed with SPIII-T4. However, complexing of factor VIII with either vWf or SPIII eliminated its binding to the phospholipid. Since a phospholipid surface is required for efficient catalysis by the protease, these results suggest that vWf protects factor VIII by inhibiting cofactor-phospholipid interactions.     

pH-dependent association of factor VIII chains: enhancement of affinity at physiological pH by Cu2+

Reconstitution of factor VIII from isolated heavy chain (HC) and light chain (LC) shows pH-dependence. In the presence of Ca2+, up to 80% of native factor VIII activity was recovered over a wide range of pH. In contrast, affinity of HC and LC was maximal at pH 6.5-6.75 (Kd approximately 4 nM), whereas a Kd approximately 20 nM was observed at physiological pH (7.25). The effect of Cu2+ (0.5 microM total Cu2+) on maximal activity regenerated was negligible at pH 6.25-8.0. However, this level of Cu2+ increased the inter-chain affinity by approximately 5-fold at pH 7.25. This effect resulted from an approximately 1.5-fold increased association rate constant (k(on)) and an approximately 3-fold reduced dissociation rate constant (k(off)). High affinity (Kd=5.3 fM) of the factor VIII heterodimer for Cu2+ was estimated by increases in cofactor activity. No significant increase in inter-chain affinity was observed when either isolated chain was reacted with Cu2+ followed by addition of the complementary chain. Together, these results suggest that the protonation state of specific residues modulates inter-chain affinity. Furthermore, copper ion contributes to the maintenance of the heterodimer at physiologic pH by a mechanism consistent with bridging the two chains.     

A critical evaluation of the available methods for the determination of factor VIII von Willebrand

Von Willebrand factor (vWf) is the major component of the circulating factor VIII complex. The von Willebrand molecule includes factor VIII related antigen (VIIIR: Ag) which represents the molecular substrate of the von Willebrand activity expressed as Ristocetin cofactor (VIIIR:RCoF) activity. Several methods have been developed for VIIIR: Ag evaluation, among the first being the rocket-immunoelectrophoresis method of LAURELL. Radial immunodiffusion (MANCINI's method) was also used. Subsequently, radioimmunological assays, either as radioimmunoassay (RIA) or immunoradiometric assay (IRMA), were developed with improvements in sensitivity, so that levels of VIIIR: Ag lower than 0.1% of normal can be detected. More recently, an enzyme-linked immunosorbent assay (ELISA), characterized by the use of enzyme-conjugated antibody was proposed. This method shows a sensitivity similar to immunoradiometric methods but without using any dangerous reagent. Finally, a nephelometric method was proposed for factor VIII antigen evaluation. For a qualitative evaluation of von Willebrand factor crossed-immunoelectrophoresis and multimeric analysis can be used. In the first case, the use of precipiting antibodies against von Willebrand factor may demonstrate a peak with different characteristics related to the biochemical property of von Willebrand. Multimeric analysis in SDS-agarose gel electrophoresis followed by staining with labelled antifactor VIII antibodies gives information about different polymeric forms of circulating VIII/vW factor. Von Willebrand factor activity, expressed as its ability to induce platelet aggregation in the presence of the antibiotic Ristocetin, can be carried out using normal formalin fixed platelets, either with aggregometer or visual methods (glass slide test or tubes test and microtritation plate). The corrected evaluation of factor VIII complex by all these techniques together with the clotting activity assay allows a satisfactory study of factor VIII properties.     

Myosin from human erythrocytes

We have purified myosin from human erythrocytes using methods similar to that for other cytoplasmic myosins with a yield of about 500 micrograms/100 ml of packed cells. It consists of a 200-kDa heavy chain and light chains of 26- and 19.5 kDa and therefore differs from the isozyme in platelets which has light chains of 20- and 15 kDa. At low ionic strength, the myosin forms short bipolar filaments like those of platelet myosin. Eight of eight monoclonal antibodies to platelet myosin also bind to erythrocyte myosin. Like most myosins, it has a high ATPase activity in the presence of Ca2+ or EDTA, but is inhibited by Mg2+. Myosin light-chain kinase transfers 1 phosphate from ATP to the 20-kDa light chain, and this stimulates the actin-activated ATPase. Thus, myosin may play a role in shape changes in the erythrocytes.     

Purification of myosin from Ehrlich ascites tumour cells (phosphorylation of its light chain and heavy chain)

1. The myosin molecule from Ehrlich ascites tumour cells consists of heavy chains of about 200 kDa and three species of light chains of 20, 19 and 15 kDa. 2. The heavy chain can be phosphorylated in vitro either by endogenous Ca2+-independent kinase or by casein kinase II. 3. The 20 and 19 kDa light chains can be phosphorylated either by an endogenous kinase or by myosin light chain kinase from chicken gizzard. 4. The Ca2+-ATPase activity of the purified myosin was 0.3 mumol/min mg protein. The Mg2+-ATPase activity was activated 14-fold by actin upon the light chain phosphorylation.     

Inactivation of factor VIII by activated protein C and protein S

Factor VIII was inactivated by activated protein C in the presence of calcium and phospholipids. Analysis of the activated protein C-catalyzed cleavage products of factor VIII indicated that inactivation resulted from the cleavage of the heavy chains. The heavy chains appeared to be converted into 93- and 53-kDa peptides. Inactivation of factor VIII that was only composed of the 93-kDa heavy chain and 83-kDa light chain indicated that the 93-kDa polypeptide could be degraded into a 68-kDa peptide that could be subsequently cleaved into 48- and 23-kDa polypeptides. Thus, activated protein C catalyzed a minimum of four cleavages in the heavy chain. Activated protein C did not appear to alter the factor VIII light chain. The addition of protein S accelerated the rate of inactivation and the rate of all of the cleavages. The effect of protein S could be observed on the cleavage of the heavy chains and on secondary cleavages of the smaller products, including the 93-, 68-, and 53-kDa polypeptides. The addition of factor IX to the factor VIII-activated protein C reaction mixture resulted in the inhibition of factor VIII inactivation. The effect of factor IX was dose dependent. Factor VIII was observed to compete with factor Va for activated protein C. The concentration dependence of factor VIII inhibition of factor Va inactivation suggested that factor VIII and factor Va were equivalent substrates for activated protein C.     

Factor VIII/von Willebrand Factor has potent lectin activity

Highly-purified plasma and platelet Factor VIII/von Willebrand Factor had potent lectin activity when measured in a haemagglutination assay. This lectin activity was inhibited by monoclonal and heterologous antibodies to Factor VIII/von Willebrand Factor as well as by hexosamines, mannose and net-positively charged amino acids.     

Cryoprecipitate of intermediate purity produced in a closed thaw-siphon system from DDAVP stimulated blood donor plasma.

The effects of intravenous administration of DDAVP to blood donors and the use of DDAVP plasma for the production of cryoprecipitate in the closed thaw-siphon system were evaluated. DDAVP treatment produced on the average a 3.2-fold rise in plasma levels of factor VIII. Von Willebrand factor antigen increased to a lesser extent. Cryoprecipitate prepared from 220-280 ml aliquots of DDAVP stimulated donor plasma contained 472 +/- 210 units of factor VIII and 276 +/- 130 units of von Willebrand factor antigen. The average yield of factor VIII was 57% of that in the prefrozen plasma. The specific activity of factor VIII in cryoprecipitate was 0.77 +/- 0.44 U/mg protein, comparable to that for intermediate purity concentrates. Thus, by the use of DDAVP and the thaw-siphon technique it is possible to produce cryoprecipitate 4-7 times as potent as conventionally manufactured preparations.     

Peptide affinity chromatography of human clotting factor VIII: Screening of the vWF-binding domain

The region of von Willebrand factor, which is involved in the complex formation with factor VIII, was used to generate a panel of octapeptides. A peptide ladder was generated from the von Willebrand factor region aa40 to aa100 and was synthesized on cellulose membranes by spot technology. Four peptides with affinity for factor VIII were identified by incubation with plasma derived factor VIII and recombinant factor VIII. The peptides denoted as 010 (LCPPGMVRHE), 011 (RCPCFHQGK), 014 (CFHQGKEYA) and 015 (RDRKWNCTDHVC) were further characterized by real-time interaction analysis and small scale affinity chromatography. Biotinylated peptides were used for blotting assays. These experiments showed that the peptides are directed against the light chain of FVIII. We consider these peptides as valuable tools for in situ labeling and also as ligands suitable for affinity chromatography.     

Enhanced secretory ability for the human factor VIII light chain produced in baculovirus-infected insect cells

The light and truncated heavy chains of human factor VIII, expressed separately in baculovirus-infected insect cells, exhibited different secretory behaviour when compared with each other and with a biologically active fusion molecule of the truncated heavy and light chains.The light chain was very efficiently secreted into culture medium, as judged by high extracellular protein levels and the absence of evidence for light chain retention within cells.Alternatively, proteins containing the heavy chain sequence were poorly secreted and appeared to be sequestered within cells, suggesting that regions within the heavy chain are responsible for the low levels of secreted protein which have generally been observed for recombinant factor VIII.     

Carbohydrate on human factor VIII/von Willebrand factor. Impairment of function by removal of specific galactose residues.

Human factor VIII/von Willebrand factor protein containing 120 +/- 12 nmol of sialic acid and 135 +/- 13 nmol of galactose/mg of protein was digested with neuraminidase. The affinity of native factor VIII/von Willebrand factor and its asialo form for the hepatic lectin that specifically binds asialoglycoproteins was assessed from in vitro binding experiments. Native factor VIII/von Willebrand factor exhibited negligible affinity while binding of the asialo derivative was comparable to that observed for asialo-alpha1-acid glycoprotein. Incubation of asialo-factor VIII/von Willebrand factor with Streptococcus pneumoniae beta-galactosidase removed only 62% of the galactose but abolished binding to the purified hepatic lectin. When the asialo derivative was incubated with purified beta-D-galactoside alpha2 leads to 6 sialyltransferase and CMP-[14C]NeuAc, only 61% of the galactose incorporated [14C]NeuAc. From the known specificites of these enzymes, it is concluded that galactose residues important in lectin binding are present in a terminal Gal/beta1 leads to 4GlcNAc sequence on asialo-factor VIII/von Willebrand factor. The relative ristocetin-induced platelet aggregating activity of native, asialo-, and agalacto-factor VIII/von Willebrand factor was 100:38:12, respectively, while procoagulant activity was 100:100:103.