von Willebrand Factor Mediates Protection of Factor VI11 from Activated Protein C-catalyzed Inactivation*

Factor VIII, a cofactor of the intrinsic clotting path- way, is proteolytically inactivated by the vitamin K-dependent serine protease, activated protein C in a reaction requiring Ca2+ and a phospholipid surface. Factor VI11 was inactivated 15 times faster than factor VI11 in complex with either von Willebrand (vWf) the large homodimeric fragment, SPIII (vWf Free factor VI11 or factor VI11 in complex with a smaller fragment, SPIII-TI (vWf res- idues 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 VI11 brought about by occupancy of the vWf-binding site. Thrombin cleavage of the factor VI11 light chain to remove the vWf-binding site eliminated the protec- tive effects of vWf. In the absence of phospholipid, high levels of the protease inactivated both free and vWf-bound factor VI11 at equivalent rates. Using the same conditions, isolated heavy chains and the heavy chains of factor VI11 were proteolyzed at similar rates. Taken together, these results suggested that, in the absence of phospholipid, inactivation of factor VI11 is independent of factor VI11 light chain and further

Factor VIII, a cofactor of the intrinsic clotting pathway, is proteolytically inactivated by the vitamin Kdependent serine protease, activated protein C in a reaction requiring Ca2+ and a phospholipid surface. Factor VI11 was inactivated 15 times faster than factor VI11 in complex with either von Willebrand factor (vWf) or the large homodimeric fragment, SPIII (vWf residues 1-1365). Free factor VI11 or factor VI11 in complex with a smaller fragment, SPIII-TI (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 VI11 brought about by occupancy of the vWf-binding site. Thrombin cleavage of the factor VI11 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 vWfbound factor VI11 at equivalent rates. Using the same conditions, isolated heavy chains and the heavy chains of factor VI11 were proteolyzed at similar rates. Taken together, these results suggested that, in the absence of phospholipid, inactivation of factor VI11 is independent of factor VI11 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 VI11 (fluorescence donor) and synthetic phospholipid vesicles labeled with octadecyl rhodamine (fluorescence acceptor) indicated saturable binding and equivalent extents of donor fluorescence quenching for factor VI11 alone or when complexed with SPIII-T4. However, complexing of factor VI11 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 VI11 by inhibiting cofactor-phospholipid interactions.
Factor VIII, a plasma protein absent or defective in individuals with hemophilia A, circulates as a series of Me2+linked heterodimers (1)(2)(3) in noncovalent association with * This work was supported in part by National Institutes of Health Grant HL-38199 (to P. J. F.) and HL-40328 (to F. J. W.) and by the American Heart Association Grant 90-0643 (to P. J. F.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement'' in accordance with 18  vWfl (4, 5 ) . Activation of factor VI11 by thrombin dissociates factor VI11 from vWf, thus allowing its participation as a cofactor in the membrane-dependent activation of factor X by the vitamin K-dependent serine protease factor IXa (see Ref. 6 for review). Protein C, also a vitamin K-dependent plasma protein (7), when activated to a serine protease (activated protein C), becomes a potent inhibitor of blood coagulation (8,9). The basis of this anticoagulant activity is the phospholipid-dependent proteolytic inactivation of factors Va (10)(11)(12) and VIII(a) (12)(13)(14)(15).
Factor VI11 is synthesized as a single-chain precursor represented by the domain structure Al-AZ-B-A3-Cl-C2 with heterodimers formed as a result of proteolysis at the B-A3 junction plus additional cleavages within the B domain (16). The factor VI11 heavy chain is minimally represented by the Al-AZ domains but exhibits significant size heterogeneity resulting from the presence of some or all of the contiguous B domain, whereas the light chain corresponds to the A3-Cl-C2 domains derived from the COOH-terminal end of the precursor. The intact heterodimeric structure is essential for cofactor function in that the subunits of factor VI11 are dissociated by chelating reagents resulting in loss of clotting activity (1,17). While little is known of the role of the heavy chain in factor VI11 function, the light chain has been observed to contain sites for binding of vWf ( l ) , activated protein C (18), and phospholipid (19).
Recent studies have indicated that the association of factor VI11 with vWf protects the cofactor from activated protein C (20,21). In this report we have examined the effects of multimeric vWf and vWf fragments containing the factor VI11 binding site on the interactions of factor VI11 with activated protein C and phospholipid vesicles. Our results are consistent with a mechanism where protection of the cofactor when bound to vWf results from a reduced affinity of the complex for the phospholipid surface.

MATERIALS AND METHODS
Reagents-Human factor VI11 concentrate (KoateTM) was a generous gift from the Cutter Division of Miles Laboratories. ~-1 -T osylamido-2-phenylethyl chloromethyl ketone-treated trypsin (bovine pancreas) was purchased from Sigma and further purified by reverse phase high performance liquid chromatography using a Vydac CIS column (5 pm, 0.45 X 25 cm) developed with a linear gradient from 20-50% acetonitrile in 0.1% trifluoroacetic acid. Staphylococcus aureus V8 protease was purchased from ICN ImmunoBiologicals. Human 01 thrombin was obtained from Enzyme Research Laboratories.

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Proteins-Bovine activated protein C was prepared as described previously (22). Factor VIII, factor VI11 subunits, and vWf were prepared from human factor VI11 concentrates as described previously (23,24). Thrombin-cleaved factor VI11 light chain was prepared from intact factor VI11 heterodimers as follows. Factor VI11 (390 pg) in 5 ml of 0.01 M histidine (pH 6.0), 0.15 M NaCl, and 0.003 M CaClz was treated with human a-thrombin (26 pg, 73 units) for 10 min at 22" C. Clotting assays indicated that factor VI11 had been maximally activated. To the thrombin-cleaved factor VI11 was added 0.5 mM diisopropyl fluorophosphate and 0.03 M EDTA and the reaction mixture incubated overnight. The thrombin-cleaved light chain was isolated following chromatography on a Mono S column (HR5/5) using conditions identical to those described for native factor VI11 light chain (23). SDS-polyacrylamide gel electrophoresis (silver-stained) showed no uncleaved light chain present in the modified light chain preparation. Modified light chain (10.4 pg) was reconstituted with native heavy chains (38 pg; approximately 2-fold molar excess) in 0.44 ml of 0.01 M Hepes (pH 7.2), 0.4 M NaCl, and 0.03 M MnClz for 2 h at 22" C. The activity of the reconstituted factor VI11 prepared from the thrombin-cleaved light chain plus native heavy chains was approximately 140 units/ml. Assuming an upper limit of 66 pg/ml for the concentration of reassociated factor VIII, this level of activity indicated a minimum specific activity of approximately 2 units/pg. Fragment SPIII was prepared from the S. aureus V8 protease digest of vWf and isolated by Mono Q (HR5/5) column chromatography as previously described (24). Fragment SPIII-T4 was prepared from a tryptic digest of SPIII (24). Preparations of SPIII-TI used in the present study effectively blocked fluorescence energy transfer between N-pyrene maleimide-labeled factor VI11 and CPM-SPIII (24), indicating a direct interaction between this fragment and factor VIII.
Protein concentrations were determined by the method of Bradford (25). SDS-polyacrylamide gel electrophoresis was performed as previously described (24). Assays-Factor VI11 activity was measured using a one-stage clotting assay. Inactivation of factor VI11 by activated protein C was carried out as described previously (15). Samples were removed from reaction mixtures at the indicated times and assayed for residual factor VI11 activity. For experiments involving the effects of vWf (derived fragments) on factor VI11 inactivation, factor VI11 was preincubated with a 10-fold molar excess of vWf (fragment) for 1 h a t 22 "C prior to reaction with activated protein C. Fluorescent Labeling of Factor VU-Factor VI11 (90-240 pg/ml) in 0.01 M histidine (pH 6.0), 0.05 M NaC1, 0.005 M CaClZ, 0.01% Tween-20, and 50% (v/v) ethylene glycol was reacted with the sulfhydryl-specific fluorophore CPM. Reactions contained a 20-50-fold molar excess of CPM, assuming a mean molecular mass of 220 kDa for factor VIII heterodimers (17). Prior to reaction, CPM was dissolved in a small volume of dry dimethylformamide and reactions were run overnight at 4 "C in the dark. The unbound fluorophore was separated from the modified protein by gel filtration using a PD-10 column (Pharmacia LKB Biotechnology Inc.) equilibrated in 0.02 M Hepes (pH 7.2), 0.15 M NaCl, and 0.003 M CaCl2. The molar ratio of probe bound to protein ranged from approximately 1.5 to 2.8 for the CPM-factor VIII, which possessed similar clotting activity when compared with the unmodified protein.
Preparation of Phospholipid Vesicles-Vesicles (41 PCPs) were prepared according to the method of Husten et al. (26). OR-PCPs also were prepared as above using approximately 20 pg of OR per 2.5 mg (total) phospholipid. The final concentration of phospholipid was determined by an elemental phosphorous assay (27). The concentration of OR incorporated into PCPs vesicles was determined using an extinction coefficient of 95,400 M" cm" at 564 nm.
Fluorescent Measurements-Fluorescent measurements were made using a SPEX Fluorolog 212 spectrofluorometer. Samples were excited at 387 nm and emission spectra taken using 15-nm slit widths for both excitation and emission monochrometers. Data were collected over the appropriate wavelength ranges at 1-nm increments and a 1-s integration time. Energy transfer was measured for the CPM-factor VI11 (fluorescence donor) and OR-PCPs (fluorescence acceptor) pairing. Determinations for each concentration of acceptor involved spectral analysis for three samples: (i) CPM-factor VI11 plus unlabeled PCPs, (ii) unlabeled factor VI11 plus OR-PCPs, and (iii) CPM-factor VI11 plus OR-PCPs. The corrected emission spectra for the above samples were integrated from 470 to 560 nm. Donor quenching was calculated from the area ratio of sample iii to samples i + ii. All reactions (0.3 ml) were carried out at room temperature in buffer containing 0.02 M Hepes (pH 7.2), 0.15 M NaC1,0.003 M CaCl2, and 0.01 M lysine HCI. For reactions containing vWf or vWf-derived fragments, (CPM-) factor VI11 was first preincubated with the vWf for 1 h at 22" C in the reaction buffer prior to the addition of (OR-) PCPs vesicles.

RESULTS
Effects of vWf on Factor VIII Inactivation-Catalytic amounts of activated protein C inactivated factor VI11 in the presence of Ca2+ and a phospholipid surface. However, prior incubation of factor VI11 with vWf reduced the rate of inactivation (Fig. 1). For this analysis, factor VI11 (mean molecular mass = 220 kDa) was reacted with a 10-fold molar excess of vWf for 1 h prior to reaction with activated protein C. This ratio of vWfifactor VI11 and incubation time were chosen to ensure complete binding of factor VI11 (24). Under these reaction conditions, the time required for enzymatic-catalyzed inactivation of 50% the factor VI11 was increased about 15fold (from -2 min to -30 min).
To determine if protection resulted from either occupancy of the vWf-binding site on factor VI11 or depended upon the mass of the bound substrate, similar analyses were performed using two fragments of vWf, the homodimeric fragment SPIII (vWf residues 1-1365; subunit molecular mass -170 kDa) and the monomeric fragment SPIII-T4 (vWf residues 1-272; -34 kDa) (28). These fragments, which retain the factor VIIIbinding site (29), form stoichiometric complexes with factor VI11 (24). A similar protective effect from activated protein C action to the factor VIII-vWf complex was observed for factor VI11 bound to the SPIII homodimer. Here again, the rate of inactivation of the factor VIII-SPIII complex was approximately 15-fold slower compared with that observed for free factor VIJI. However, complexing of the smaller SPIII-T4 with factor VI11 offered no protection from activated protein C in that essentially similar reaction rates were observed for free factor VI11 and the factor VIII-SPIII-T4 complex. These results suggested that the protection from activated protein C observed for factor VI11 complexed with either intact vWf or SPIII did not result from either occupancy of the vWfbinding site in factor VI11 or potential conformational changes induced in factor VI11 following binding, but instead suggested a mechanism dependent upon the size of the ligand that occupies the vWf-binding site.
Results shown in Fig. 2 indicated that when the vWf- binding site was removed from factor VIII, the protective effect of vWf toward activated protein C-catalyzed inactivation was abolished. For this analysis, factor VI11 was treated with thrombin to effect removal of the NH2-terminal peptide (residues 1649-1689) (30) containing the vWf-binding site from the factor VI11 light chain. Intact factor VI11 rather than factor VI11 light chain was used to prepare the thrombincleaved light chain because proteolysis of the isolated subunit was slow and failed to yield a fully cleaved product. Subsequent reconstitution of native factor VI11 heavy chains with the thrombin-cleaved light chain resulted in the formation of a proteolytically modified factor VI11 with similar (stable) clotting activity (specific activity -2 units/pg) when compared with native factor VI11 but altered only in the light chain so that it lacked the ability to bind vWf. Equivalent rates of activated protein C-catalyzed inactivation of this modified factor VI11 were observed in assays performed in the absence of vWf or following a preincubation with a 10-fold excess of vWf. Thus protection is achieved only when factor VI11 complexes with vWf. Multiple binding domains have been localized within the light chain of factor VI11 and include regions that bind vWf (residues 1670-1684) (31), phospholipid (residues 2303-2332) (32), and activated protein C (residues 2009-2018) (33). The results presented above are suggestive of two possible mechanisms by which the protective effect of vWf is dependent on the size of the binding ligand. The first possibility is that vWf can sterically block access to the activated protein C binding site or mask the cleavage sites located on the heavy chain. The second possibility is that vWf can block the interaction of factor VI11 with membranes and prevent formation of catalytic complex which would include protease, Ca2+, phospholipid, and factor VIII.
The requirement for phospholipid in the formation of the catalytic complex can be overcome by high concentrations of activated protein C (Fig. 3). In the absence of phospholipid, an approximate 15-fold molar excess of enzyme relative to factor VI11 resulted in a rate of inactivation similar to that observed using catalytic levels of enzyme in the presence of phospholipid (see Fig. 1). Further, under these conditions of a high ratio of enzyme to substrate and no phospholipid, the rate of factor VI11 inactivation either in the presence or absence of vWf were equivalent. This experiment suggested that vWf did not mask the cleavage sites in the heavy chain from activated protein C. contained either factor VI11 heavy chains (15 pg/ml), heavy chains (15 pglml) plus 100 pg/ml phospholipid, or intact factor VI11 (20 pg/ ml). Activated protein C (50 pg/ml) was added to each reaction and incubated at 37 "C. Aliquots were removed at the indicated times, denatured, subjected to electrophoresis, and the gel stained with silver nitrate. Lanes 1, 5, and 9 represent heavy chains, heavy chains plus phospholipid, and intact factor VIII, respectively, prior to addition of protease. Lanes 2-4, 6-8, and 10-12 represent 5-, 15-, and 60-min time points following protease addition to reactions shown in lanes 1,5, and 9, respectively. Lane 13 shows activated protein C alone. M , is X io?
In the absence of phospholipid, we observed that cleavage of the heavy chain was also light chain-independent. High concentrations of activated protein C resulted in proteolysis of the isolated factor VI11 heavy chains (Fig. 4). The apparent rate of degradation, as judged by disappearance of heavy chains and appearance of terminal fragments of 48 and 23 kDa, was independent of the presence of phospholipid. Furthermore, for reactions run in the absence of phospholipid, the rate of cleavage of isolated heavy chains was similar to that observed for the heavy chains of intact factor VIII. This result suggested that inactivation of factor VI11 by activated protein C, in the absence of phospholipid, occurs by a mechanism independent of factor VI11 light chain. These experiments suggest that the effect of vWf on activated protein Ccatalyzed inactivation of factor VI11 is to alter a parameter that is dependent upon the presence of the light chain.
Effects of u Wf on Factor VZZI-Phospholipid Znteractions-Fluorescence energy transfer techniques were applied to determine if the results obtained from clotting assays, namely protection of factor VI11 from activated protein C when bound to vWf and SPIII but not to SPIII-T4, were compatible with the effects of these substrates on the interaction between factor VI11 and phospholipid vesicles. For these experiments, factor VI11 was modified with the sulfhydryl-specific fluorophore, CPM (fluorescence donor). We have previously used this probe to modify factor VI11 subunits and have shown incorporation into residues in both the heavy chain (CYS~'~) and the light chain (CYS'~~') (23). Intact factor VI11 incorporated on average about 2 mol of probe per mol of protein, suggesting sites on both subunits were modified. Clotting activity of the CPM-modifiedprotein was similar to the native material (data not shown). Phospholipid vesicles (41 PCPs) were modified with OR at a ratio of approximately 1 mol of probe per 300 mol of phospholipid monomer for use as the fluorescence acceptor. Substitution of OR-phospholipid for unmodified phospholipid used in assays monitoring the activated protein C-catalyzed inactivation of factor VI11 yielded equivalent results (data not shown) indicating that presence of the probe did not alter the interactions of the lipid with factor VI11 or activated protein C. The OR-PCPs had an excitation spectrum that overlapped the emission spectrum of CPM-factor VI11 (Fig. 5). Thus the fluorescence of CPMfactor VI11 should be quenched upon binding to the labeled phospholipid vesicles. However, no donor fluorescence quenching would be observed if prior complexing of CPMfactor VI11 with vWf (or a vWf-derived fragment) prevented its association with the phospholipid surface.
CPM-factor VI11 was titrated with OR-PCPs (Fig. 6). The acceptor quenched the fluorescence of CPM-factor VI11 approximately 12% and this effect was saturable. Incubation of CPM-factor VI11 with vWf or SPIII blocked subsequent donor quenching following addition of OR-PCPs, whereas prior complexing of CPM-factor VI11 with SPIII-T4 yielded a result equivalent to that observed for free factor VIII. These data suggested that prior complexing of factor VI11 with either vWf or SPIII prevented factor VI11 from binding the phospholipid vesicles. The above results, taken together with the results indicating protection of factor VI11 from the protease when the cofactor was complexed with vWf or SPIII but not SPIII-T4, support a model where vWf-mediated protection from activated protein C results from inhibition of the factor VIII-phospholipid interaction.

DISCUSSION
Limited proteolysis of factor VI11 by activated protein C correlates with the observed inactivation of cofactor function (13-15, 30). This proteolysis is restricted to the heavy chain  subunit which is cleaved to several intermediate and terminal digest products (15). However, our earlier observations (18) that (i) heavy chain alone was not a substrate for activated protein C, (ii) isolated light chain inhibited inactivation of factor VIII, and (iii) a (phospholipid-dependent) binding of light chain and the enzyme suggested an integral role for the light chain in this catalytic mechanism. This paper further emphasizes the importance of the light chain in the catalytic mechanism. In this paper we observe that association of the light chain with phospholipid membranes is an essential step in activated protein C-catalyzed inactivation of factor VIII. In addition, we have observed that cleavage of the isolated heavy chain does not appear to be accelerated by these membranes, suggesting that an activated protein C-phospholipid interaction is not sufficient for the rapid cleavage of substrate. Recently we have localized an activated protein C-binding region to light chain residues 2009-2018 (33) located near the COOH-terminal end of the A3 domain.
Also contained within the factor VI11 light chain (A3-C1-C2 domainal structure; residues 1649-2332) (16) are sites for binding vWf and phospholipid. vWf binds very near the NH2 terminus of this subunit in that thrombin cleavage (at residue 1689) dissociates factor VI11 (or light chain) from vWf (34, 35). Results of Foster et al. (31) have further localized the vWf-binding region to residues 1670-1684. Recently, these investigators have suggested that residues 2303-2332 mediate the binding of factor VI11 to phospholipid (32). It is not known how these regions that bind protease, vWf and phospholipid are spatially oriented in the folded protein.
As a result of the multiple macromolecular interactions attributed to the light chain, one can envision several alternative mechanisms for the vWf-dependent protection of factor VI11 from activated protein C-catalyzed inactivation. Protection of factor VI11 was observed when the cofactor was complexed with multimeric vWf or the homodimeric SPIII but not with the smaller monomer, SPIII-T4. This result indicated a size dependence of the binding substrate with respect to protection, thus excluding the possibilities that occupancy of the vWf-binding site would either induce a conformational change or in itself preclude interaction of factor VI11 with protease. Instead, the above result was compatible with the larger vWf substrates either sterically blocking access to the activated protein C-binding sites and/or masking the cleavage sites present in the heavy chain. This latter alternative was suggested by our previous studies which u W f Protection of Factor VIII from Activated Protein C indicated activated protein C initially cleaves factor VI11 a t site(s) within the A2 domain of the heavy ch@n (15) and a close spatial separation of approximately 30 A between the NH2-terminal region of vWf and this domain in the reconstituted factor VIII-vWf complex (24). Our experiments do not allow us to exclude the possibility that vWf sterically blocks the interaction between activated protein C and the light chain. Experimentally, this would be difficult to prove since binding of activated protein C to the light chain is lipiddependent and vWf inhibits the light chain interaction with lipid, it is not possible to set up an assay in which a lipidbound light chain-vWf complex could be used as a ligand for protein C binding. However, vWf does not appear to mask bonds in the factor VI11 heavy chain from cleavage by activated protein C. In the absence of a phospholipid surface and at high protease levels, we observed similar rates of inactivation of factor VI11 and factor VI11 complexed with vWf ( Fig.  3 ) and similar rates of proteolysis of free heavy chain and the heavy chain of intact factor VI11 (Fig. 4). These results suggested a surface-independent mechanism for cofactor inactivation that was also independent of an activated protein C-light chain interaction. These results suggest that the solution phase inactivation of factor VIII, which is slow, is independent of light chain. Factor VI11 binds tightly and reversibly to phospholipid vesicles (36) and platelets (37) and it is when factor VI11 is surface-bound that it is an optimal substrate for activated protein C. Earlier results showed that high concentrations of phospholipid (above 250 pg/ml) dissociated factor VI11 from vWf (38), indicating an antagonistic relationship between vWf and phospholipid for factor VI11 binding. Thus vWf, by interfering with the factor VIII-phospholipid interaction, could potentially reduce the rate of cofactor inactivation by activated protein C.
We have employed fluorescence energy transfer techniques to assess phospholipid binding of free factor VI11 and factor VI11 complexed to vWf, SPIII or SPIII-T4. Fluorescence data indicated saturable binding of both free factor VI11 and factor VI11 complexed with SPIII-T4 to the phospholipid surface. Levels of fluorescence quenching a t saturating levels of phospholipid, an indicator of the distance separating donor and acceptor fluorophores, were equivalent for factor VI11 and factor VIII/SPIII-T4. Thus occupancy of the vWf-binding site by the 34-kDa SPIII-T4 fragment did not perturb the factor VIII-phospholipid interaction, suggesting that the phospholipid and vWf binding sites within the light chain were not juxtaposed so closely that they were mutually exclusive. However, little if any donor fluorescence quenching was observed for factor VI11 in complex with vWf or SPIII, indicating no interaction of these complexes with the phospholipid surface. Thus the effects of vWf, SPIII, and SPIII-T4 on the factor VIII-phospholipid interaction paralleled their effects in the factor VIII-activated protein C system, suggesting that protection from protease resulted from inhibition of cofactor binding to phospholipid.
The protection offered factor VI11 by vWf from activated protein C-catalyzed inactivation has been the subject of two recent reports. Rick et al. (21) showed that vWf modestly decreased the level of factor VI11 inactivation by about 20-30%. These studies used factor VI11 levels of 0.6-0.8 unit/ml and protease concentrations of up to 2.3 pg/ml. Assuming a specific activity for factor VI11 of 5 unitslpg, this would indicate a (weight) ratio of pr0tease:substrate = 16. Thus, the observed lack of significant protection was likely attributed to the high levels of enzyme used and reflected a phospholipidindependent inactivation of the cofactor. Koedam et al. (20) have observed partial protection of factor VI11 (1.2 nM) from the protease (4 nM) by vWf, whereas no protection was observed following thrombin activation of factor VIII, the latter result consistent with protection requiring a physical association between factor VI11 and vWf.
Similar to thrombin-activated factor VI11 (factor VIIIa), the proteolytically modified factor VI11 employed in this study, composed of a thrombin-cleaved light chain bound to a native heavy chain was inactivated by the protease at a rate independent of the presence of vWf. Furthermore, in the absence of vWf, the rate of inactivation of this form of factor VI11 was similar to that observed for native factor VI11 (see Figs. 1 and 2). Although factor VI11 is a good substrate for activated protein C (15), Marlar et al. (12) reported that factor VIIIa was inactivated 30-fold faster by the protease. Since this value was determined from comparison with the inactivation of factor VIII/vWf, the rate enhancement observed for the activated cofactor probably resulted, in part, from its dissociation from vWf. However, preliminary data from our laboratory suggest a significant increase in the rate of factor VIIIa inactivation by the protease. Since thrombin cleaves both factor VI11 subunits during activation, one or both cleavage events must result in increasing the reactivity of the activated cofactor for the enzyme. The above result suggests that it is thrombin cleavage of the heavy chain, not light chain, that disposes factor VIIIa to rapid inactivation by activated protein C.
It is not known what function(s) are impaired in activated protein C-cleaved factor VIII. Since the light chain subunit is not covalently altered it is unlikely that inactivation results from altered phospholipid binding. Activated protein C cleavage of factor Va reduces the affinity of the cofactor for both prothrombin and factor Xa (39). By analogy, inactivated factor VI11 would show reduced affinity for factor X and factor IXa. Studies on the macromolecular interactions among components of the factor Xase enzyme complex and alterations produced by activated protein C are currently in progress.