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.     

The effect of plasma von Willebrand factor on the binding of human factor VIII to thrombin-activated human platelets

The binding of 35S-labeled recombinant human Factor VIII to activated human platelets was studied in the presence and absence of exogenous plasma von Willebrand factor. In the absence of added von Willebrand Factor, platelets bound 210 molecules of Factor VIII/platelet when the unbound Factor VIII concentration was 2.0 nM (Kd = 2.9 nM). As the von Willebrand factor concentration was increased, the number of Factor VIII molecules bound/platelet decreased to 10 molecules of Factor VIII bound/platelet at 24 micrograms/ml of added vWF. Addition of an anti-vWF monoclonal antibody that inhibits the vWF-Factor VIII interaction attenuated the ability of vWF to inhibit binding of Factor VIII to platelets. In contrast, addition of a control anti-vWF antibody that does not block the vWF-Factor VIII interaction did not affect the ability of vWF to inhibit Factor VIII binding to platelets. From the vWF concentration dependence of inhibition of Factor VIII-platelet binding, a dissociation constant for the Factor VIII-vWF interaction was calculated (Kd = 0.44 nM). To further elucidate the role that vWF may play in preventing the interaction of Factor VIII with platelets, the platelet binding properties of a Factor VIII deletion mutant (90-73) which lacks the primary vWF-binding site was studied. The binding of this mutant was unaffected by added exogenous vWF. These observations demonstrate that Factor VIII can interact with platelets in a manner independent of vWF but that excess vWF in plasma can effectively compete with platelets for the binding of Factor VIII. In addition, since cleavage of Factor VIII by thrombin separates a vWF-binding domain from Factor VIIIa, we propose that activation of Factor VIII by thrombin may elicit release of activated Factor VIII from vWF and thereby make it fully available for platelet binding.     

A major factor VIII binding domain resides within the amino-terminal 272 amino acid residues of von Willebrand factor

We have identified a Factor VIII (FVIII) binding domain residing within the amino-terminal 272 amino acid residues of the mature von Willebrand Factor (vWF) subunit. Two-dimensional crossed immunoelectrophoresis showed direct binding of purified human FVIII to purified human vWF. After proteolytic digestion of vWF with Staphylococcus aureus V8 protease (SP), FVIII binding was seen only with the amino-terminal SP fragment III and not with the carboxyl-terminal SP fragment II. A monoclonal anti-vWF antibody (C3) partially blocked FVIII binding to vWF and SP fragment III. FVIII also bound to vWF which had been adsorbed to polystyrene beads. This binding was inhibited in a dose-dependent manner by whole vWF, SP fragment III, and by monoclonal antibody C3. Binding could not be inhibited by SP fragment I, which contains the middle portion of the vWF molecule, or by reduced and alkylated whole vWF. SP fragment II caused only minimal inhibition. Trypsin cleavage of SP fragment III produced a monomeric 35-kDa fragment containing the amino-terminal 272 amino acid residues of vWF. This fragment reacted with monoclonal antibody C3 and inhibited the binding of FVIII to vWF in a dose-dependent manner. These studies demonstrate that a major FVIII binding site resides within the amino-terminal 272 amino acid residues of vWF.     

The binding of factor IXa to cultured bovine aortic endothelial cells. Induction of a specific site in the presence of factors VIII and X

Previous studies have demonstrated a Factor IX and IXa binding site on the endothelial cell surface for which both the zymogen and enzyme compete with equal affinity. In this report, we demonstrate that the affinity of Factor IXa, but not Factor IX, for the cell surface is increased in the presence of both Factors VIII and X. When Factor Xa formation was studied in the presence of saturating concentrations of Factors VIII and X, the half-maximal rate was observed at a Factor IXa concentration of 151 +/- 12 pM. Active site-blocked Factor IXa, 5-dimethylaminonaphthalene-1-sulfonyl-Glu-Gly-Arg-Factor IXa, was a more effective inhibitor of Factor X activation (Ki = 124 pM) than was Factor IX (Ki = 3.0 nM). Radioligand binding studies carried out in the presence of Factors VIII and X confirmed the presence of a selective endothelial cell Factor IXa binding site with Kd = 127 +/- 27 pM. In contrast, when Factor IXa binding was studied in the absence of other coagulation factors, or in the presence of Factor VIII (thrombin-activated or unactivated) alone, this new high affinity site was not observed. Competitive binding studies indicated that Factor IXa was 12 times more effective as an inhibitor of Factor IX-endothelial cell binding in the presence of Factors VIII and X. Consistent with the increased affinity of Factor IXa binding in the presence of factors VIII and X, cell-associated Factor IXa coagulant activity decayed 7 times more slowly in the presence of these coagulation factors. These results demonstrate selective Factor IXa-endothelial cell binding in the presence of Factors VIII and X, suggesting this interaction could be a physiologic occurrence.     

Localization of the binding site for a factor VIII activity neutralizing antibody to amino acid residues Asp1663-Ser1669

We have identified the Factor VIII amino acid sequence Asp-Tyr-Asp-Asp-Thr-Ile-Ser (1663-1669) as the binding site of a Factor VIII activity neutralizing antibody (28 Bethesda units/mg). The binding site of another neutralizing antibody (10 Bethesda units/mg) overlapped only at Asp1663 and Tyr1664, whereas an antibody with minimal neutralizing activity (0.2 Bethesda units/mg) bound only at Asp1665-Ser1669. Residues comprising antibody binding sites were determined by blocking Factor VIII neutralization and/or binding to insolubilized Factor VIII with overlapping peptides, or with variant peptides in which a single amino acid was deleted or replaced with glycine. Eight additional antibodies to flanking sequences, and with similar affinities for Factor VIII, had little or no neutralizing activity (0-3.0 Bethesda units/mg). These studies suggest that Asp1663 and Tyr1664 may be structural features important to Factor VIII function.     

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.     

Platelet-derived microparticles express high affinity receptors for factor VIII.

Factor VIII is a cofactor in the tenase enzyme complex which assembles on the membrane of activated platelets. A critical step in tenase assembly is membrane binding of factor VIII. Platelet membrane factor VIII-binding sites were characterized by flow cytometry using either fluorescein maleimide-labeled recombinant factor VIII or a fluorescein-labeled monoclonal antibody against factor VIII. Following activation by thrombin, most platelets bound factor VIII within 90 s. In addition, over the course of several minutes, membranous vesicles (microparticles) were shed from the platelet plasma membrane and each microparticle bound as much factor VIII as a stimulated platelet. Over 30 min, stimulated platelets (but not microparticles) lost the capacity to bind factor VIII. Factor VIII bound saturably to microparticles from platelets stimulated with thrombin, thrombin plus collagen, or the complement proteins C5b-9. The binding of factor VIII was compared to factor V, a structurally homologous coagulation cofactor. Analysis of microparticle binding kinetics yielded similar on and off rates for factor VIII and factor Va and KD values of 2-10 nM. In the presence of 20 nM factor Va, the binding of factor VIII to microparticles was increased, and there was a comparable increase in platelet tenase activity. At higher factor Va concentrations, factor VIII binding and tenase activity were inhibited. Conversely, factor VIII had a similar dose-dependent effect on factor Va binding and platelet prothrombinase activity. Synthetic phospholipid vesicles containing phosphatidylserine competed with microparticles for binding of factor VIII and factor Va. These studies indicate that activated platelets express a transient increase in high affinity receptors for factor VIII, whereas platelet-derived microparticles express a sustained increase in receptors. The binding characteristics of platelet membrane receptors for factor VIII are similar to those for factor Va.     

Molecular characterization of a unique von Willebrand disease variant. A novel mutation affecting von Willebrand factor/factor VIII interaction

von Willebrand factor (vWF) plays a central role in blood coagulation, mediating the adhesion of the initial platelet plug to the subendothelium, and serving as the carrier for factor VIII (FVIII) in the circulation. In previous studies, we have mapped the epitope for an anti-vWF monoclonal antibody which inhibits the interaction between FVIII and vWF to a region spanning Thr78 to Thr96 of the mature protein (Bahou, W.F., Ginsburg, D., Sikkink, R., Litwiller, R., and Fass, D. N. (1989) J. Clin. Invest. 84, 56-61). We now report the identification of a mutation within this region of vWF that results in decreased FVIII binding. Sequence analysis of polymerase chain reaction amplified platelet vWF mRNA from a von Willebrand disease (vWD) patient with a disproportionately low FVIII level identified a single nucleotide substitution (G----A), resulting in the conversion of Arg91----Gln. Recombinant vWF carrying this substitution showed decreased binding to FVIII compared with wild-type vWF or vWF carrying a polymorphic substitution in the same region (Arg89----Gln). These observations suggest a critical role for Arg91 in the interaction of vWF with FVIII and identify the molecular mechanism for a variant of vWD associated with unusually low FVIII levels.     

The light chain of factor VIII comprises a binding site for low density lipoprotein receptor-related protein.

In the present study, the interaction between the endocytic receptor low density lipoprotein receptor-related protein (LRP) and coagulation factor VIII (FVIII) was investigated. Using purified components, FVIII was found to bind to LRP in a reversible and dose-dependent manner (K(d) approximately 60 nM). The interaction appeared to be specific because the LRP antagonist receptor-associated protein readily inhibited binding of FVIII to LRP (IC(50) approximately 1 nM). In addition, a 12-fold molar excess of the physiological carrier of FVIII, i.e. von Willebrand factor (vWF), reduced the binding of FVIII to LRP by over 90%. Cellular degradation of (125)I-labeled FVIII by LRP-expressing cells ( approximately 8 fmol/10(5) cells after a 4.5-h incubation) was reduced by approximately 70% in the presence of receptor-associated protein. LRP-directed antibodies inhibited degradation to a similar extent, indicating that LRP indeed contributes to binding and transport of FVIII to the intracellular degradation pathway. Degradation of FVIII was completely inhibited by vWF. Because vWF binding by FVIII involves its light chain, LRP binding to this subunit was studied. In ligand blotting experiments, binding of FVIII light chain to LRP could be visualized. More detailed analysis revealed that FVIII light chain interacts with LRP with moderate affinity (k(on) approximately 5 x 10(4) M(-1) s(-1); k(off) approximately 2.5 x 10(-3) s(-1); K(d) approximately 50 nM). Furthermore, experiments using recombinant FVIII C2 domain showed that this domain contributes to the interaction with LRP. In contrast, no association of FVIII heavy chain to LRP could be detected under the same experimental conditions. Collectively, our data demonstrate that in vitro LRP is able to bind FVIII at the cell surface and to mediate its transport to the intracellular degradation pathway. FVIII-LRP interaction involves the FVIII light chain, and FVIII-vWF complex formation plays a regulatory role in LRP binding. Our findings may explain the beneficial effect of vWF on the in vivo survival of FVIII.     

The role of von Willebrand factor multimers and propeptide cleavage in binding and stabilization of factor VIII

von Willebrand factor (vWF) is a multimeric glycoprotein that promotes platelet aggregation and stabilizes coagulation factor VIII in the plasma. vWF is also required for the stable accumulation of recombinant factor VIII secreted from cells in a heterologous expression system. In this report, we show that vWF can promote the in vitro reconstitution of factor VIII activity from dissociated heavy and light chains of factor VIII, suggesting that vWF may act to promote stable assembly of factor VIII subunits at the site of secretion. The structural requirements for vWF propeptide cleavage and for vWF multimerization in its binding and stabilization of factor VIII was examined using specifically altered recombinant vWF. The mutant vWF molecules were also assayed for their function in ristocetin-induced platelet agglutination mediated through the platelet receptor GPIb. Deletion of the vWF propeptide produced a dimeric vWF molecule that failed to mediate platelet agglutination, suggesting that multimerization is required for vWF to attain functional GPIb binding. This mature dimeric form of vWF, however, was fully capable of binding to and supporting stable secretion of factor VIII. A vWF mutant with an altered propeptide cleavage site formed large multimers of uncleaved pro-vWF that functioned in platelet agglutination. However, this noncleavage mutant neither bound to or supported stable accumulation of factor VIII. Analysis of the vWF propeptide, expressed independently, demonstrated that it could not bind factor VIII or stabilize its secretion. These results show that the dimeric mature vWF subunit is sufficient to bind and stabilize factor VIII and that the presence of uncleaved vWF propeptide inhibits both factor VIII binding and stabilization.     

The binding of 35S-labeled recombinant factor VIII to activated and unactivated human platelets

Recombinant-derived human Factor VIII was labeled intrinsically with [35S]methionine, and its binding to washed human platelets was studied. Binding measurements were performed by incubating Factor VIII and platelets for 15 min at room temperature in Tyrode's solution supplemented with Ca2+ (5.0 mM), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (5.0 mM), 0.50% bovine serum albumin, and the Factor Xa and thrombin inhibitors 5-dimethylaminonaphthalene-1-sulfonylglutamylglycinylarginyl chloromethyl ketone and 5-dimethylaminonaphthalene-1-sulfonyl-arginine-N-(3-ethyl-1, 5-pentanediyl)amide. Separation of free from bound Factor VIII was accomplished by centrifugation through oil, and nonspecific binding was determined with excess unlabeled Factor VIII. Binding was saturable, reversible, and stimulated 20-fold after platelet activation with thrombin. Furthermore, binding was specific in that bound labeled Factor VIII could be displaced by excess unlabeled Factor VIII, but not by Factor V. Scatchard analysis indicated a single class of binding sites with Kd = 2.9 nM and 450 sites/activated platelet. The time course of displacement indicated a t1/2 of bound Factor VIII of approximately 5 min. When platelets were incubated in Ca2+, both the heavy and light chains of Factor VIII were bound, whereas exposure to EDTA resulted in the binding of the light chain only. These results demonstrate the specific reversible binding of Factor VIII to human platelets, likely mediated through the light chain.     

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.     

Stoichiometry of the porcine factor VIII-von Willebrand factor association

Factor VIII and von Willebrand factor (vWF) are glycoproteins that form a tightly bound complex in plasma. The interaction of porcine factor VIII with porcine vWF was studied by analytical velocity sedimentation. A single approximately 240-kDa species of factor VIII was isolated for use in the analysis. In contrast, when analyzed by agarose/sodium dodecyl sulfate-polyacrylamide gel electrophoresis, vWF consisted of a population of greater than 10 multimers derived from a 270-kDa monomer. A single boundary (So20,w = 7.2 S) was observed during velocity sedimentation of factor VIII at 260,000 x g. A single boundary also was observed for vWF (weight-average So20,w = 21 S) at 42,000 x g. Under condition of excess factor VIII, the weight-average So20,w of the factor VIII-vWF complex was 40 S at 42,000 x g. At 260,000 x g, the factor VIII-vWF complex had sedimented completely, leaving only free factor VIII. The height of the plateau region of the factor VIII sedimentation velocity curve at 260,000 x g was studied as a function of several starting concentrations of vWF. The experiments were done under conditions in which the effect of radial dilution was negligible so that the plateau height was a measure of the concentration of free factor VIII. The plateau height decreased linearly as the concentration of vWF was increased, indicating that the association was essentially irreversible under the conditions used. A stoichiometry of 1.2 vWF monomers/factor VIII molecule was calculated from the slope of the line. Assuming one factor VIII-binding site/vWF monomer, these results indicate that all factor VIII-binding sites are accessible in the vWF multimer.     

The interaction of the factor VIII/von Willebrand factor complex (VIII/vWf), with guanidinium-derivatized matrices.

Five different guanidinium (Gu)-derivatized agarose matrices were investigated for their potential in chromatographically resolving the Factor VIII/von Willebrand complex, VIII/vWf, fibrinogen, Fg, and fibronectin, Fn, from cryoprecipitate. Using conventional NaCl gradient methodology it was found that the order of elution of specific plasma proteins, and the yield of VIII/vWf, varied with the methods used to derivatize the agarose beads. Good yields of VIII:C (generally 30-45%) were obtained with Gu-matrices prepared by bis-oxirane coupling procedures. Cryoprecipitate binding studies showed that the capacity of Gu-Sepharose 4B, prepared by isourea modification of amino-Sepharose 4B, was 36 units VIII/vWf per ml matrix. The product, depleted of both Fg and Fn, had a specific activity of 2 units VIII:C per mg total protein, (yield 100% vWf:Ag and 47% VIII:C).     

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.     

Binding of human factor VIII to phospholipid vesicles

Factor VIII, a protein cofactor involved in blood coagulation, functions in vitro on a phospholipid membrane surface to greatly increase the rate of factor X activation by factor IXa. Using gel filtration, rapid sedimentation, and resonance energy transfer we have studied the interaction of recombinant-derived human factor VIII with small and large unilamellar phospholipid vesicles composed of phosphatidylserine and phosphatidylcholine. Resonance energy transfer, from intrinsic fluorophores in factor VIII to dansyl-phosphatidylethanolamine incorporated into vesicles, has been adapted for quantitative equilibrium measurements. Factor VIII binds rapidly and reversibly to small and large vesicles. At 8 degrees C the interaction of factor VIII with small vesicles fits a simple bimolecular model with a KD of 2 nM and a phospholipid binding site defined by 180 phospholipid monomers. At 25 degrees C the binding of factor VIII to small vesicles containing 20% phosphatidylserine can be described by an apparent KD of 4 nM; the phospholipid/protein ratio at saturation was 170. Binding to large vesicles was demonstrated with a KD of 2 nM and a phospholipid/protein ratio at saturation of 385. Binding was dependent upon the phosphatidylserine mole fraction and was nonlinear from 0 to 30% phosphatidylserine content. A direct comparison of factor VIII and factor V binding indicated that the affinity of factor V to phospholipid vesicles was equivalent to that of factor VIII and that the phosphatidylserine requirement was lower. A model is proposed to explain the nonlinear phosphatidylserine dependence of binding for factor VIII.     

Crystal structure of human factor VIII: implications for the formation of the factor IXa-factor VIIIa complex

Factor VIII is a procofactor that plays a critical role in blood coagulation, and is missing or defective in hemophilia A. We determined the X-ray crystal structure of B domain-deleted human factor VIII. This protein is composed of five globular domains and contains one Ca(2+) and two Cu(2+) ions. The three homologous A domains form a triangular heterotrimer where the A1 and A3 domains serve as the base and interact with the C2 and C1 domains, respectively. The structurally homologous C1 and C2 domains reveal membrane binding features. Based on biochemical studies, a model of the factor IXa-factor VIIIa complex was constructed by in silico docking. Factor IXa wraps across the side of factor VIII, and an extended interface spans the factor VIII heavy and light chains. This model provides insight into the activation of factor VIII and the interaction of factor VIIIa with factor IXa on the membrane surface.     

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.