Definition of the affinity of binding between human von Willebrand factor and coagulation factor VIII

Factor VIII and von Willebrand factor are two plasma proteins essential for effective hemostasis. In vivo, they form a non-covalent complex whose association appears to be metal ion dependent. However, a precise definition of the nature of the molecular forces governing their association remains to be defined, as does their binding affinity. In this paper we have determined the dissociation constant and stoichiometry for Factor VIII binding to immobilized von Willebrand factor. The data demonstrate that these proteins interact saturably and with relatively high affinity. Computer assisted analyses of the Scatchard data favour a two site binding model. The higher affinity site was found to have a Kd of 62 (+/- 13) x 10(-12) M while that of the lower affinity site was 380 (+/- 92) x 10(-12) M. The density of Factor VIII binding sites (Bmax) present on von Willebrand factor was 31 (+/- 3) pM for the high affinity binding site and 46 (+/- 6) pM for the lower site, corresponding to a calculated Factor VIII: von Willebrand factor binding ratio of 1:33 and 1:23, respectively.     

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.     

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.     

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.     

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.     

Sulfation of Tyr1680 of human blood coagulation factor VIII is essential for the interaction of factor VIII with von Willebrand factor

The acidic region of the Factor VIII light chain was studied with regard to structural requirements for the formation of a functional von Willebrand factor (vWF)-binding site. Factor VIII mutants lacking the B domain, with additional deletions and an amino acid replacement within the sequence 1649-1689 were constructed using site-directed mutagenesis and expressed in Cos-1 cells. These mutants, which were recovered as single-chain molecules with similar specific activities, were compared in their binding to immobilized vWF. Deletion of amino acids 741-1648 or 741-1668 did not affect the binding of Factor VIII to vWF. However, a mutant with a deletion of residues 741-1689 was no longer capable of interacting with vWF. This indicates a role for residues within the sequence 1669-1689 in the formation of a vWF-binding site. When recombinant Factor VIII was expressed in the presence of chlorate, an inhibitor of protein sulfation, the resulting Factor VIII displayed strongly reduced binding to vWF. vWF binding was completely abolished when within the sequence 1669-1689 the tyrosine residue Tyr1680, which is part of a consensus tyrosine sulfation sequence, was replaced by phenylalanine. The Factor VIII sequence 1673-1689 was identified as a high affinity substrate for tyrosylprotein sulfotransferase (Km = 57 microM) in cell-free sulfation studies. It is concluded that sulfation of Tyr1680 is required for the interaction of Factor VIII with vWF. Two synthetic peptides that represent the sequence 1673-1689, but differ with respect to sulfation of Tyr1680 are shown to have vWF binding affinity that is considerably lower than the Factor VIII protein. Several models to accommodate our findings are discussed.     

Localization and characterization of a heparin binding domain peptide of human von Willebrand factor.

Human von Willebrand factor, a plasma glycoprotein which plays a critical role in regulating hemostasis, binds heparin, but the physiological importance and mode of this interaction is poorly understood. Using the motif of an amino acid sequence of a consensus heparin binding synthetic peptide, a 23-residue sequence (Tyr565-Ala587) of human von Willebrand factor was identified that retains the consensus motif and binds heparin with affinity comparable with native von Willebrand factor and the consensus peptide. In a fluid phase binding assay, the Tyr565-Ala587 peptide competed effectively with von Willebrand factor for binding heparin. Synthesis and testing of peptides overlapping Tyr565-Ala587, as well as adjacent cationic regions, showed this core sequence to be the optimal linear binding domain. Far ultraviolet circular dichroism spectrometry of the Tyr565-Ala587 peptide suggested that the peptide undergoes conformational change upon binding heparin. The Tyr565-Ala587 peptide thus encompasses part (or all) of a functionally important heparin binding domain of von Willebrand factor. Further study of this and related peptides may be useful for exploring how heparin may influence von Willebrand factor-mediated platelet hemostasis.     

Construction and characterization of an active factor VIII variant lacking the central one-third of the molecule

The primary structure of factor VIII consists of 2332 amino acids that exhibit 3 distinct structural domains, including a triplicated region (A domains), a unique region of 909 amino acids (B domain), and a carboxy-terminal duplicated region (C domains), that are arranged in the order A1-A2-B-A3-C1-C2. The B domain (residues 741-1648) of factor VIII is lost when factor VIII is activated by thrombin, which proteolytically processes factor VIII to active subunits of Mr 50,000 (domain A1), 43,000 (domain A2), and 73,000 (domains A3-C1-C2). To determine if the B domain is required for factor VIII coagulant activity, a variant was constructed by using recombinant DNA techniques in which residues 797-1562 were eliminated. This shortened the B domain from 909 to 142 amino acids. This variant factor VIIIdes-797-1652 was expressed in mammalian cells and was found to be functional. The factor VIIIdes-797-1562 protein was purified and shown to be processed by thrombin in the same manner as full-length factor VIII. The factor VIIIdes-797-1562 variant also bound to von Willebrand factor (vWF) immobilized on Sepharose. These results indicate that most of the highly glycosylated B domain of factor VIII is not required for the expression of factor VIII coagulant activity and its interaction with vWF.     

Thrombin-activated and factor Xa-activated human factor VIII: differences in cofactor activity and decay rate.

The decay of human coagulation factor VIIIa has been studied by kinetic methods that ensure no interference through proteolytic feedback. The rate of decay of factor VIIIa activity was found to vary with the activator used to activate factor VIII. Thrombin-activated factor VIII-von Willebrand factor complex (fVIII-vWf) decayed at a rate of 0.31 min-1, whereas factor Xa-activated fVIII-vWf decayed at 0.11 min-1 under the same conditions. Factor VIII free of von Willebrand factor (factor VIII: C), although decaying at a generally slower rate after activation, still showed a dependence of decay rate on activator: thrombin-activated factor VIII:C decaying at a rate of 0.06 min-1, and factor Xa-activated factor VIII: C at 0.01 min-1. Readdition of von Willebrand factor (18 micrograms/ml) to factor VIII:C did not alter the observed activity or decay rate. The decay of the two species of factor VIIIa was studied, using the fVIIIa-vWf complex, in the presence of varying levels of factor IXa. Plots of reciprocal decay rates vs factor IXa concentration were linear, and nearly parallel for the two factor VIIIa species, with a mean slope of 0.56 min.nM-1. In addition to these studies, we have confirmed previous studies showing that the two forms of factor VIIIa differ in cofactor activity, but they do so in the same ratio as in their decay rates. We suggest that this difference and that observed in decay rate have a common cause, and incorporate this into a potential kinetic model of factor VIII activation and decay.     

Collagen-bound von Willebrand factor has reduced affinity for factor VIII

von Willebrand factor (vWf) is a multimeric adhesive glycoprotein that serves as a carrier for factor VIII in plasma. Although each vWf subunit displays a high affinity binding site for factor VIII in vitro, in plasma, only 2% of the vWf sites for factor VIII are occupied. We investigated whether interaction of plasma proteins with vWf or adhesion of vWf to collagen may alter the affinity or availability of factor VIII-binding sites on vWf. When vWf was immobilized on agarose-linked monoclonal antibody, factor VIII bound to vWf with high affinity, and neither the affinity nor binding site availability was influenced by the presence of 50% plasma. Therefore, plasma proteins do not alter the affinity or availability of factor VIII-binding sites. In contrast, when vWf was immobilized on agarose-linked collagen, its affinity for factor VIII was reduced 4-fold, with KD increasing from 0.9 to 3.8 nM. However, one factor VIII-binding site remained available on each vWf subunit. A comparable reduction in affinity for factor VIII was observed when vWf was a constituent of the subendothelial cell matrix and when it was bound to purified type VI collagen. In parallel with the decreased affinity for factor VIII, collagen-bound vWf displayed a 6-fold lower affinity for monoclonal antibody W5-6A, with an epitope composed of residues 78-96 within the factor VIII-binding motif of vWf. We conclude that collagen induces a conformational change within the factor VIII-binding motif of vWf that lowers the affinity for factor VIII.     

Ontogeny of reactivity to endothelial cell markers during development of the embryonic and fetal rat lung.

The reactivity of endothelial cells to putative endothelial cell-specific markers varies with species, with vessel size and with the organ studied. To determine their value in studies of fetal rat lung, and whether organ immaturity would also influence reactivity, we studied endothelial cell immunoreactivity to antibodies against Factor VIII/von Willebrand factor (VIII/vWF), and binding reactivity to Bandeiraea (Griffonia) simplicifolia 1 lectin (BSL 1) during rat fetal lung development. Using an indirect immunofluorescent technique to detect Factor VIII/von Willebrand factor (VIII/vWF), endothelial cells lining the aortic arches were identified as early as day 11 of gestation (term = 22 days), prior to lung development. Immunoreactivity to VIII/vWF was subsequently localized to intrapulmonary endothelial cells and was not dependent on vessel size. In contrast, binding reactivity of FITC-conjugated BSL 1 was observed to both endothelial cells and to the basement membrane of developing airways, thus limiting its value as endothelial cell marker. During very early lung development solitary angioblasts could not be identified by reactivity to either VIII/vWF antibodies or to BSL 1, and neither marker appears to be of value for studies of early angiogenic events.     

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.     

Inheritance of a new bleeding disease in a herd of swine with Willebrand's disease

A herd of swine affected by Willebrand's disease was begun in 1967 at the Mayo Clinic in order to study the inherited hemostatic abnormality in swine as a model for the human disease. Affected individuals have bleeding times in excess of 15 minutes, extremely low levels of Willebrand factor (less than or equal to 0.25 percent of normal), and decreased levels of VIII coagulant activity. Individuals with long bleeding times, higher levels of Willebrand factor and normal levels of VIII coagulant activity began to appear in the colony. It is hypothesized that this new (N) condition is inherited as a simple autosomal recessive (N/n) at a locus separate and independent of the similarly autosomal recessive (A/a) von Willebrand locus. In addition, the Willebrand locus is epistatic to the N locus, i.e., individuals will only express the new condition provided there is at least one normal allele at the von Willebrand locus. Therefore, individuals with genotype aa--are all von Willebrand phenotypically, and A-nn individuals have the new disease.     

Specificity of phosphatidylserine-containing membrane binding sites for factor VIII. Studies with model membranes supported by glass microspheres (lipospheres).

Factor VIII functions in an enzyme complex upon the activated platelet membrane where phosphatidylserine exposure correlates with expression of receptors for factor VIII. To evaluate the specificity of phosphatidylserine-containing membrane binding sites for factor VIII, we have developed a novel membrane model in which phospholipid bilayers are supported by glass microspheres (lipospheres). The binding of fluorescein-labeled factor VIII to lipospheres with membranes of 15% phosphatidylserine was equivalent to binding to phospholipid vesicles (KD = 4.8 nM). Purified von Willebrand factor (vWf), a carrier protein for factor VIII, decreased membrane binding of factor VIII with a Ki of 10 micrograms/ml. Likewise, normal plasma decreased bound factor VIII by more than 90% whereas plasma lacking vWf decreased the binding of factor VIII by only 20%. Proteolytic activation of factor VIII by thrombin, which releases factor VIII from vWf, increased liposphere binding in the presence of vWf and in the presence of normal plasma. Although factor V is homologous to factor VIII and binds to lipospheres with the same affinity, purified factor V was not an efficient competitor for the membrane binding sites of factor VIII. These results indicate that phosphatidylserine-containing membrane sites have sufficient specificity to select thrombin-activated factor VIII from the range of phospholipid-binding proteins in plasma.     

The role of Willebrand factor in platelet - blood vessel interaction, including a discussion of resistance to atherosclerosis in pigs with von Willebrand's disease

Von Willebrand pigs have all the manifestations of the severe human disease. The role of Willebrand antigen (VIII R:AG) and ristocetin cofactor (VIII: RWF) was assessed in these pigs by (1) transfusion and (2) "in vitro" bleeding time assay. The skin bleeding time became normal when the level of transfused Willebrand factor (VIII R:AG/RWF) was raised in the plasma above 30 U/dl. After single or repeated transfusions, skin capillary endothelium and platelets were still distinguished from normal by VIII R:AG deficiency. When incisions in excised porcine skin ("in vitro" bleeding time) were perfused with blood and plasma fractions, haemostasis occurred when plasmatic Willebrand factor exceeded 30 U/dl whether the skin or platelets came from normal or from von Willebrand pigs. The platelet plug occluding the skin incision contained VIII R:AG by immunofluorescence. Willebrand factor appears to coat surfaces and to serve as a platelet attachment protein. These bleeder pigs are resistant to atherosclerosis. If platelets are involved in early atherosclerotic lesions, the role of Willebrand factor in platelet - blood vessel interaction may be important.     

Reconstitution of human factor VIII from isolated subunits

Human factor VII heterodimers were fractionated into component heavy and light chains using an anti-light chain specific monoclonal antibody immunosorbant. Neither the light chain nor the heavy chain alone possessed activity. Factor VII activity was reconstituted by recombining the subunits in the presence of Mn2+ or Ca2+. Reconstitution of activity also showed ionic strength dependence suggesting the importance of hydrophobic and electrostatic interactions. All factor VIII heavy chains (93 to 210 kDa) recombined with the 83 kDa light chain as judged by retention of all reconstituted heterodimeric forms by the monoclonal immunosorbant. Maximum specific activity (3 units/micrograms) was obtained at a 1:1 molar ratio of light chain:heavy chain. The presence of von Willebrand factor enhanced the rate of factor VIII reconstitution as much as 5-fold. This effect was both ionic strength-dependent and dose-dependent up to a 25-fold weight excess of von Willebrand factor over factor VIII.     

Effects of amidination and chemical cross-linking on human factor VIII (antihemophilic factor).

The bifunctional reagent dimethyl suberimidate, reacting with primary amino groups of proteins, was used to cross-link highly purified human factor VIII. Reaction products were reduced with beta-mercaptoethanol or treated with Rhizopus arrhizus triglyceride lipase. The proportions of the dissociated subunits and their oligomers were calculated from the relative staining intensities of individual bands following polyacrylamide electrophoresis in the presence of sodium dodecyl sulfate. Low concentrations of dimethyl suberimidate (up to 0.5 mM) produced covalently linked dimers which retained full functional (coagulant and von Willebrand factor) activities. Treatment with increasing concentrations of dimethyl suberimidate resulted in an almost simultaneous appearance of both trimeric and tetrameric species, suggesting the existence of specific intra-dimer contacts. A parallel decrease of functional activities was observed at higher concentrations of dimethyl suberimidate. A monofunctional reagent (ethyl acetimidate), reacting similarly with primary amino groups, amidinated factor VIII at rates similar to dimethyl suberimidate. Up to 40% amidinated factor VIII retained full biological activities. We conclude that the most reactive lysine residues are not involved in the active sites responsible for either coagulant or von Willebrand activity.     

Portal vein thrombosis and high factor VIII in Turner syndrome

BACKGROUNDS/AIMS: Turner syndrome is not usually associated with thrombotic events. The aim of this study is to report 3 Turner syndrome patients with portal vein thrombosis and, in 2 of them, high factor VIII. These findings are compared to values in Turner syndrome patients without thrombosis and controls. METHODS: In different years, 3 patients with Turner syndrome were initially seen at the Gastroenterology Clinic of Hospital de Clínicas de Porto Alegre, Brazil, for portal vein thrombosis. After the most common causes of portal vein thrombosis and thrombophilias had been excluded, the 2 surviving patients were studied for clotting factors VIII, IX and von Willebrand factor. The same factors were also assessed in 25 Turner syndrome patients without thrombosis and 25 normal girls. RESULTS: One of the patients with portal vein thrombosis died before the study. In the 2 surviving patients, factors VIII and von Willebrand levels were >150 IU/dl, which is considered to be high. In Turner syndrome patients without thrombosis, the mean factor VIII level was 127.2 +/- 41.1 IU/dl and for von Willebrand factor 101.2 +/- 26.9 IU/dl, while in control girls these were 116.0 +/- 27.6 and 94.28 +/- 27.5 IU/dl, respectively. Factor VIII and von Willebrand factor were not different between these 2 groups. When non-O blood group Turner syndrome patients and normal girls were compared, the former had significantly higher levels of factor VIII. CONCLUSIONS: This is the first report on the unusual finding of portal thrombosis in patients with Turner syndrome in whom high levels of factor VIII and von Willebrand factor were found. Factor VIII is higher in the non-O blood group Turner syndrome patients without thrombosis when compared to normal girls.     

Proteolytic activity of alpha 2-macroglobulin-enzyme complexes toward human factor VIII/von Willebrand factor

The low level of enzymatic activity of certain alpha 2-macroglobulin-proteinase complexes could be important to the function of factor VIII/von Willebrand glycoprotein since it is especially sensitive to proteolytic cleavage. To test this possibility, complexes of alpha 2-macroglobulin with plasmin, trypsin, and thrombin were formed in at least a 2:1 molar ratio of alpha 2-macroglobulin:proteinase and tested for effects on the factor VIII procoagulant activity of the factor VIII/von Willebrand glycoprotein. Neither the alpha 2-macroglobulin-trypsin complex nor the alpha 2-macroglobulin-plasmin complex affected factor VIII procoagulant activity. The behavior of the alpha 2-macroglobulin-thrombin complex was different. When alpha 2-macroglobulin and thrombin were incubated in a mole ratio of 3:1 or less, factor VIII procoagulant activity was enhanced to about the same extent as with free thrombin. Even at a 24:1 mole ratio, the mixture could produce 45% of the increase in factor VIII activity obtained with free thrombin. The isolated alpha 2-macroglobulin-thrombin complex could also activate the factor VIII procoagulant function to about 45% of the level obtained with an identical amount of uncomplexed thrombin. Analysis of the alpha 2-macroglobulin-125I-labeled thrombin complexes by rechromatography or by polyacrylamide gel electrophoresis in sodium dodecyl sulfate indicated that this activation was not due to free thrombin. We conclude that the alpha 2-macroglobulin-thrombin complex retains sufficient proteolytic activity to activate the procoagulant function of factor VIII/von Willebrand glycoprotein despite the latter being a very large substrate, having an estimated molecular weight of 1-20 million.     

Identification of discontinuous von Willebrand factor sequences involved in complex formation with botrocetin. A model for the regulation of von Willebrand factor binding to platelet glycoprotein Ib

We have used proteolytic fragments and overlapping synthetic peptides to define the domain of von Willebrand factor (vWF) that forms a complex with botrocetin and modulates binding to platelet glycoprotein (GP) Ib. Both functions were inhibited by the dimeric 116-kDa tryptic fragment and by its constituent 52/48-kDa subunit, comprising residues 449-728 of mature vWF, but not by the dimeric fragment III-T2 which lacks amino acid residues 512-673. Three synthetic peptides, representing discrete discontinuous sequences within the region lacking in fragment III-T2, inhibited vWF-botrocetin complex formation; they corresponded to residues 539-553, 569-583, and 629-643. The 116-kDa domain, with intact disulfide bonds, exhibited greater affinity for botrocetin than did the reduced and alkylated 52/48-kDa molecule, and both fragments had significantly greater affinity than any of the inhibitory peptides. Thus, conformational attributes, though not strictly required for the interaction, contribute to the optimal functional assembly of the botrocetin-binding site. Accordingly, 125I-labeled botrocetin bound to vWF and to the 116-kDa fragment immobilized onto nitrocellulose but not to equivalent amounts of the reduced and alkylated 52/48-kDa fragment; it also bound to the peptide 539-553, but only when the peptide was immobilized onto nitrocellulose at a much greater concentration than vWF or the proteolytic fragments. These studies demonstrate that vWF interaction with GP Ib may be modulated by botrocetin binding to a discontinuous site located within residues 539-643. The finding that single point mutations in Type IIB von Willebrand disease are located in the same region of the molecule supports the concept that this domain may contain regulatory elements that modulate vWF affinity for platelets at sites of vascular injury.