©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cleavage of Factor VIII Light Chain Is Required for Maximal Generation of Factor VIIIa Activity (*)

Lisa M. Regan (1), Philip J. Fay (1) (2)(§)

From the (1) Department of Biochemistry and the (2) Hematology Unit, Department of Medicine, University of Rochester School of Medicine and Dentistry, Rochester, New York 14642

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Thrombin-catalyzed activation of heterodimeric factor VIII occurs by limited proteolysis, yielding subunits A1 and A2 derived from the heavy chain (HC) and A3-C1-C2 derived from the light chain (LC). The roles of these cleavages in the function of procoagulant activity are poorly understood. To determine whether LC cleavage contributes to the potentiation of factor VIII activity, factor VIII heterodimers were reconstituted from native HC and either thrombin-cleaved LC (A3-C1-C2) or intact LC and purified by Mono S chromatography. The reconstituted factor VIII form containing the A3-C1-C2 subunit had a specific activity (2 units/µg) that was 3-fold greater than that of the reconstituted factor VIII form containing native LC (0.6 units/µg). Factor Xa generation assays using the hybrid heterodimer showed an initial rate that was unaffected by the presence of von Willebrand factor and a reduced lag time when compared with the native heterodimer. The A1/A3-C1-C2 dimer was dissociated by chelation, and the purified A1 subunit was reacted with either the A3-C1-C2 subunit or the LC in the presence of Mnto reconstitute the dimer. Factor VIIIa heterotrimers were reconstituted from either A1/A3-C1-C2 or A1/LC plus the A2 subunit. The authentic factor VIIIa heterotrimer (A1/A3-C1-C2/A2) had 3-fold greater activity than the form containing the LC. However, upon reaction with thrombin, the activity of the latter form was increased to that of the factor VIIIa form containing native subunits. The incremental increase in fluorescence anisotropy of fluorescein-Phe-Phe-Arg chloromethyl ketone-modified factor IXa was markedly greater in the presence of HC/A3-C1-C2 ( r = 0.037) compared with HC/LC ( r = 0.011) and approached the value obtained with factor VIIIa ( r = 0.051). These results suggest that cleavage of factor VIII LC directly contributes to the potentiation of coagulant activity by modulating the conformation of the factor IXa active site.


INTRODUCTION

Factor VIII, the component of blood coagulation deficient or defective in individuals with hemophilia A, is synthesized as a 300-kDa precursor protein (1, 2) with the domain structure A1-A2-B-A3-C1-C2 (3) . It is processed to a series of Me-linked heterodimers (4, 5, 6) produced by cleavage at the B-A3 junction, generating a light chain (LC)() (A3-C1-C2 domains) and a heavy chain (HC) (A1-A2-B domains). Additional cleavage sites within the B domain result in variable sized heavy chains (3) minimally represented by the contiguous A1-A2 domains. The two chains can be separated by chelating reagents (4, 6) and isolated following ion-exchange chromatography. The separated chains have no activity, but factor VIII activity can be reconstituted by combining the subunits in the presence of divalent metal ion (6, 7) .

Factor VIII circulates in plasma as a procofactor associated with a carrier protein, von Willebrand factor (vWf) (8) . Binding is noncovalent and involves both electrostatic and hydrophobic interactions (9) . A primary vWf-binding site is contained within the NH-terminal portion of factor VIII LC (10) , and this association prevents factor VIII from binding to phospholipid surfaces (11, 12, 13) .

Factor VIII functions in the intrinsic Xase complex as a cofactor for factor IXa in the surface-dependent conversion of factor X to factor Xa. This activity is dependent upon conversion of factor VIII to the active cofactor form, factor VIIIa.() Thrombin, the principal activator of factor VIII, cleaves factor VIII HC at Arg, which liberates the B domain fragments, and at Arg, which splits the HC into the A1 and A2 subunits (14) . Thrombin also cleaves factor VIII LC at Arg(14) , liberating an acidic rich region and creating a new NHterminus. Thus, factor VIIIa is a heterotrimer of subunits designated A1, A2, and A3-C1-C2. The A1 and A3-C1-C2 subunits retain the Melinkage and can be isolated as a stable A1/A3-C1-C2 dimer. The A2 subunit is only weakly associated with the dimer. Factor VIIIa activity can be reconstituted from the isolated A1/A3-C1-C2 dimer and the A2 subunit (15) .

The functions of the thrombin-catalyzed cleavages in factor VIII are not well understood. Cleavage of the LC is believed to be responsible for dissociation of factor VIIIa from vWf (16) . This cleavage may also add to the cofactor activity of factor VIIIa (17, 18, 19) , although this hypothesis is controversial (20) . In this study, reconstitutions of factors VIII and VIIIa from purified subunits were utilized to determine the contribution of LC cleavage to factor VIIIa activity. Results indicate that the thrombin cleavage of factor VIII LC contributes to the potentiation of factor VIIIa activity by directly affecting the conformation of the factor IXa active site.


MATERIALS AND METHODS

Reagents

Human factor VIII concentrates (Koateand Koate-HP) were generously provided by the Cutter Division of Miles Inc. Recombinant factor VIII was a generous gift from Debra Pittman (Genetics Institute). The reagents -thrombin and factor IXa(Enzyme Research Labs), DEGR-CK and PPACK (Calbiochem), fluorescein-Phe-Phe-Arg chloromethyl ketone-modified factor IXa (Fl-FFR-factor IXa) (Molecular Innovations), and the chromogenic substrates S-2238 ( H- D-phenylalanyl- L-piperyl- L-arginyl- p-nitroanilide) and S-2765 ( N-benzyloxycarbonyl- D-arginyl- L-glycyl- L-arginyl- p-nitroanilide dihydrochloride) (Kabi Pharmacia/Chromogenix) were purchased from the indicated vendors. Phospholipid vesicles were prepared as described previously (21) .

Proteins

Factor IXa was inactivated with the active-site inhibitor DEGR-CK as described previously (22) . Factor VIII (23) , factor VIII subunits (23) , factor VIIIa subunits (15) and vWf (24) were prepared as described previously. Recombinant factor VIII was used as the starting material for some of the subunit preparations, where it behaved equivalently compared with the plasma-derived material. The A3-C1-C2 subunit was prepared by reacting factor VIII LC (3.78 µ M) with thrombin (70 n M) for 12 h in buffer A (20 m M Hepes, pH 7.2, 5 m M CaCl, and 0.01% Tween 20) containing 100 m M NaCl. These conditions resulted in complete conversion of the LC to A3-C1-C2 as determined by SDS-PAGE and silver staining. Thrombin was subsequently inactivated by adding PPACK in half-molar equivalents until no amidolytic activity was detected using the chromogenic substrate S-2238. Following inactivation of thrombin, the reaction was dialyzed for 4 h against buffer A containing 100 m M NaCl. A control reaction, which did not contain thrombin, was performed in parallel with the complete reaction.

Reconstitution of Factor VIII(a) Forms

Factor VIII activity was reconstituted from A3-C1-C2 (700 n M) or native LC (700 n M) by incubation with native HC (920 n M) at room temperature in buffer A containing 30 m M MnCl. Reconstituted factor VIII was diluted 1:3 in buffer B (10 m M histidine, pH 6.0, 5 m M CaCl, and 0.01% Tween 20) and applied to a Mono S column equilibrated with buffer B containing 100 m M NaCl. Factor VIII was eluted with a 30-ml linear gradient of 100 m M to 1 M NaCl in buffer B, and 1-ml fractions were collected. Factor VIII activity was measured by a one-stage clotting assay using plasma that had been chemically depleted of factor VIII activity (25) . Protein concentrations were determined by the Coomassie Blue dye binding method of Bradford (26) .

Factor VIIIa was reconstituted in a two-step process. First, the A1 subunit (300 n M) was reacted at room temperature with either A3-C1-C2 (400 n M) or the LC (400 n M) in buffer A containing 10 m M MnCl. Progress of the Me-dependent linkage was monitored by reacting an aliquot of the reaction mixture with the A2 subunit for 10 min at room temperature and assaying for clotting activity.

In the second step, the A1/A3-C1-C2 or A1/LC dimer was diluted at least 3-fold in buffer A containing 0.5 mg/ml bovine serum albumin and incubated with the A2 subunit. Dilution of the dimer was performed to decrease the concentration of Mnsince divalent metal ions interfere with the association of the A2 subunit with the dimer (15) . Factor VIIIa activity was determined by clotting assay, and optimal activity was achieved within 10 min.

Factor Xa Generation Assays

The rate of conversion of factor X to factor Xa was monitored in a purified system. Assays contained reconstituted factor VIII(a) (2 n M), factor IXa (1 n M), factor X (200 n M), and 100 µg/ml phospholipid vesicles in buffer A containing 100 m M NaCl. Selected reactions also contained vWf. Aliquots were removed at the indicated times, and EDTA (50 m M final concentration) was added to stop the reaction. Initial rates of factor Xa generation were determined by the addition of the chromogenic substrate S-2765 (460 µ M). Reactions were read continuously at 405 nm for 2 min using a Beckman DU650 spectrometer.

Fluorescence Anisotropy Measurements

All fluorescence anisotropy measurements were made using a Spex Fluorolog 212 spectrophotometer operated in the L format. Excitation and emission wavelengths were 495 and 520 nm, respectively, and polarizers were manually rotated. Fluorescence measurements were made using a microcell (200 µl) initially containing Fl-FFR-factor IXa (20 n M) and phospholipid vesicles (10 µg/ml) in buffer A containing 100 m M NaCl. The anisotropy was determined by acquiring data at each polarizer position for 30 s. Three measurements were made over an 3-min time period and averaged for each determination. Either HC/LC or HC/A3-C1-C2 (40-50 n M) was added, and the anisotropy was determined as described above. Due to the concentration of the reconstituted factor VIII forms, the total volume in the microcell increased 25% after the addition of factor VIII. Control reactions indicated that this dilution factor did not affect the values determined for anisotropy. Activation of the factor VIII forms was achieved following the addition of thrombin (4 n M). Reactions were run for 1 min, and the anisotropy was again determined as described above.


RESULTS

Reconstitution of Factor VIII from Isolated Subunits

Active factor VIII heterodimers can be reconstituted from isolated subunits in the presence of divalent metal ion (27) , and this property was used to determine whether the thrombin-catalyzed cleavage of the LC affected factor VIII activity. Factor VIII was reconstituted from either A3-C1-C2 or the LC plus native HC in the presence of Mn(Fig. 1). Using these reaction conditions, reconstitution of factor VIII forms was complete by 20 min as judged by a leveling of regenerated cofactor activity. The factor VIII form reconstituted with A3-C1-C2 (HC/A3-C1-C2) exhibited 3-fold greater activity than reconstituted factor VIII derived from native LC (HC/LC).


Figure 1: Reconstitution of factor VIII activity from cleaved or native LC and native HC. Reconstitution of factor VIII from native HC (920 n M) and A3-C1-C2 (700 n M; ) or LC (700 n M; ) was carried out as described under ``Materials and Methods.'' Aliquots were removed at the indicated time points and assayed for factor VIII activity.



Purification of Reconstituted Factor VIII

The enhanced activity observed for the HC/A3-C1-C2 heterodimer compared with the HC/LC heterodimer could result from either an increased extent of reconstitution or a higher intrinsic specific activity. To differentiate between these alternatives, HC/A3-C1-C2 and HC/LC were run separately on a Mono S column to purify the reconstituted heterodimers. In each case, two protein peaks were eluted from the column (Fig. 2). SDS-PAGE analysis of the two peaks showed that the first one consisted of free HC, while the second contained both the HC and LC (or A3-C1-C2) (data not shown). Densitometric scans of the stained gels showed that the HC and LC (or A3-C1-C2) in the latter peak were present at equivalent stoichiometries (data not shown), suggesting that these subunits are linked to form factor VIII heterodimers. Column fractions were assayed for factor VIII activity, which was present only in the heterodimer fraction. The peak HC/A3-C1-C2 activity was observed to elute one fraction later (fraction 17) than the peak HC/LC activity (fraction 16). This result likely reflects the reduction in acidic character of HC/A3-C1-C2 following loss of residues 1649-1689. Furthermore, the peak activity of this fraction was 3-fold greater than the HC/LC activity. Protein determinations across both sets of fractions from the column revealed similar concentrations. The specific activity of HC/A3-C1-C2 was determined to be 2 units/µg, while that of HC/LC was 0.6 units/µg. This result suggests that the difference in activity is not due to a variance in the extent of factor VIII reconstitution, but rather reflects a higher intrinsic specific activity for the heterodimer containing thrombin-cleaved LC.


Figure 2: Mono S chromatography of reconstituted factor VIII. Factor VIII was reconstituted from native HC and native LC ( top panel) or A3-C1-C2 ( bottom panel) and purified as described under ``Materials and Methods.'' Absorbance was measured at 280 nm (). Factor VIII activity () was determined in a one-stage clotting assay.



The elevated specific activity of HC/A3-C1-C2 compared with HC/LC suggested a partially activated factor VIII molecule. To further examine the activation state of this factor VIII form, the rates and extents of cofactor activation were assessed. Equivalent concentrations of the two factor VIII forms were reacted with thrombin. Both HC/A3-C1-C2 and HC/LC reached peak activation within 2 min (data not shown). But, while HC/LC was activated 10-fold, HC/A3-C1-C2 was activated 3-fold. The final potentiated activity levels of the two forms were equivalent. This result was expected since once HC/A3-C1-C2 and HC/LC are activated by thrombin, both forms consist of an A1/A3-C1-C2/A2 subunit structure. Furthermore, the similarity in rates of activation (data not shown) suggested that the increased specific activity of the HC/A3-C1-C2 heterodimer did not result from it possessing the capacity for enhanced reactivity with thrombin.

Activity of HC/LC and HC/A3-C1-C2 in a Purified System

An alternative explanation for the enhanced activity of the hybrid factor VIII form could reflect a sequestering of native factor VIII by endogenous vWf present in the plasma used for assay. Thus, the higher apparent activity of HC/A3-C1-C2 could result from its inability to bind vWf. To eliminate this variable, a series of experiments were performed using a factor Xa generation assay composed of purified reagents. In these assays, factor VIII derived from native HC and intact or thrombin-cleaved LC was used to assemble the intrinsic Xase complex. Reactions were run in the absence of exogenous thrombin, thereby relying on feedback activation of the factor VIII forms by endogenously formed factor Xa. Both the HC/LC- and HC/A3-C1-C2-containing reactions showed a lag time preceding the initial rate of product formation (Fig. 3). This lag reflects the structure of factor VIII and illustrates the requirement for activation of the cofactor by limited proteolysis (28) . However, the observed lag time for the hybrid form was about one-third that observed for HC/LC. After 15 min, similar rates of factor Xa generation were observed, suggesting that both factor VIII forms had been fully cleaved following feedback activation by generated factor Xa. The enhanced activity of the hybrid factor VIII form did not result from altered reactivity during proteolytic activation since both factor VIII forms were activated at the same rates and to the same extent by factor Xa (data not shown).


Figure 3: Factor Xa generation assay using factor VIII forms in the presence and absence of vWf. HC/A3-C1-C2 (2 n M; and ) or HC/LC (2 n M; and ) was preincubated with either vWf (100 n M; and ) or the equivalent amount of bovine serum albumin ( and ) for 2 h prior to the addition of factor IXa (1 n M), factor X (200 n M), and 100 µg/ml phospholipid vesicles in buffer A containing 100 m M NaCl. Reactions were performed as described under ``Materials and Methods.''



Inclusion of vWf (Fig. 3) did not affect the factor Xa-generating activity of the HC/A3-C1-C2 form, whereas its presence markedly reduced the amount of factor Xa generated in the reaction containing the native factor VIII subunits. This result was consistent with the requirement for dissociation of factor VIII from vWf in the expression of cofactor activity and the observation that factor Xa is a poor activator of vWf-bound factor VIII (29) . That low levels of factor Xa generation are observed in this latter reaction may reflect the presence (and subsequent activation) of unbound factor VIII, possibly resulting from competition for vWf binding by the phospholipid surface (12) .

Reconstitution of Factor VIIIa Activity

To determine the effect of LC structure on the activity of heterotrimeric factor VIIIa, the following experiments were performed. Heterotrimeric molecules were reconstituted from isolated A1 and A2 subunits derived from factor VIII HC with either native LC or A3-C1-C2. Reconstitution of the factor VIIIa heterotrimer involved a two-step reaction whereby the divalent metal ion linkage was formed between A1 and either the LC or A3-C1-C2, creating a stable dimer, followed by the metal ion-independent association of the A2 subunit with the dimer to yield the heterotrimer. The presence of all three subunits is required for factor VIIIa activity (15) .

For these experiments, the A1 subunit was reacted with a slight excess (1.3-fold) of either the LC or A3-C1-C2 in the presence of 10 m M MnCland at moderate ionic strength (Fig. 4). Reaction conditions were chosen to help ensure saturation of the A1 subunit since this subunit contains the primary interactive site for the A2 subunit (30) as well as to mimic our previously defined conditions that promote factor VIII reconstitution from the HC and LC (27) .

A time course of factor VIIIa reconstitution was performed by incubating either A1/LC or A1/A3-C1-C2 with the A2 subunit. Results from this experiment are shown in Fig. 4. Reconstitutions of A2 with either A1/LC or A1/A3-C1-C2 yielded saturable levels of factor VIIIa activity. However, the activity associated with the heterotrimer composed of A1/LC/A2 (20 units/ml) was 3-fold less than the activity associated with the authentic factor VIIIa heterotrimer (60 units/ml). This result was consistent with the above observation using heterodimeric factor VIII that suggested that native LC possessed inherently lower specific activity than the cleaved form.


Figure 4: Reconstitution of factor VIIIa activity. A1/A3-C1-C2 (100 n M; ) or A1/LC (100 n M; ) was incubated with A2 (200 n M) as described under ``Materials and Methods''. At the indicated time points, an aliquot was removed and assayed for factor VIIIa activity. After 90 min at room temperature, an aliquot of each reaction was reacted with thrombin (10 n M) and, at the indicated time points, assayed for factor VIIIa activity.



To ensure that this observation did not reflect a less efficient reconstitution of the A1/LC/A2 heterotrimer and hence a lower concentration of active material, the two factor VIIIa forms were reacted with thrombin (Fig. 4). Exposure of the heterotrimer formed from A1/A3-C1-C2 and A2 to thrombin had no effect on its activity, consistent with its subunit structure being derived from this protease. However, reaction with thrombin enhanced the activity of the A1/LC/A2 heterotrimer such that its potentiated value was equivalent to that of native factor VIIIa. Since the LC was the only subunit now cleaved in this form of the molecule, this result directly indicates that its cleavage is required for maximal expression of factor VIIIa activity.

Factor Xa Generation Assay with Factor VIIIa Forms

Factor Xa generation assays similar to those described for the factor VIII forms were performed using factor VIIIa forms derived from the native HC components (A1 and A2 subunits) and thrombin-cleaved or intact LC (Fig. 5). Linear regression analysis of the data points for the reaction containing A1/A3-C1-C2/A2 had a correlation value of 0.995, indicating the absence of a lag time for the generation of factor Xa in this reaction. A lag time was observed for the reaction containing A1/LC/A2, suggesting that this form of the cofactor is less than fully activated. However, this lag time appeared somewhat shorter than that for the reaction containing HC/LC, consistent with A1/LC/A2 possessing an intermediate level of cofactor activity.

Stability of Reconstituted Factor VIIIa

Factor VIIIa activity is highly labile at low concentration and physiologic pH as a result of dissociation of the A2 subunit from the dimer (15, 31) . To determine whether the NH-terminal region of the LC influenced factor VIIIa stability, factor VIIIa was reconstituted from either A1/A3-C1-C2 or A1/LC plus the A2 subunit at pH 7.2, and the loss of activity upon dilution was monitored. In addition, a similar set of assays was performed in the presence of DEGR-factor IXa, which has been shown to stabilize factor VIIIa under similar reaction conditions (22, 32) . Inclusion of the latter experiment was to evaluate whether the NH-terminal region of the LC affected the interaction of factor VIIIa with factor IXa. The results of these experiments are shown in Fig. 6. The loss of factor VIIIa activity upon dilution was similar with both factor VIIIa forms, suggesting that the NH-terminal region of the LC does not contribute to the association with the A2 subunit. Furthermore, the equivalent loss of activity of the two factor VIIIa forms in the presence of DEGR-factor IXa suggested that this region of the LC did not affect the ability of factor VIIIa to bind to the protease.


Figure 6: Stability of factor VIIIa in the presence and absence of DEGR-factor IXa. Factor VIIIa was reconstituted from either A1/A3-C1-C2 (100 n M) or A1/LC (100 n M) and the A2 subunit (100 n M) in buffer A containing 500 µg/ml bovine serum albumin for 10 min. Factor VIIIa derived from A3-C1-C2 ( and ) or LC ( and ) was incubated in the presence ( and ) or absence ( and ) of DEGR-factor IXa in buffer A and assayed for factor VIIIa activity. Reactions were then diluted 50% with buffer A containing 180 m M NaCl and 500 µg/ml bovine serum albumin, incubated for 5 min, and reassayed. This procedure was repeated until factor VIIIa was diluted 12.5-fold. The 100% levels of activity were A1/A3-C1-C2/A2 (26.2 units/ml), A1/A3-C1-C2/A2 + DEGR-factor IXa (29.3 units/ml), A1/LC/A2 (6.9 units/ml), and A1/LC/A2 + DEGR-factor IXa (9.1 units/ml).



Effects of Factor VIII Forms on Fluorescence Anisotropy of Fl-FFR-factor IXa

Recently, Duffy et al. (33) observed an incremental increase in fluorescence anisotropy following association of factor VIII with factor IXa modified at its active site with a fluorescent probe. This anisotropy value was further increased following activation of factor VIII by thrombin. Thus, this parameter may be used as an indicator of the activation state of the cofactor. Results presented in indicate the fluorescence anisotropy values for Fl-FFR-factor IXa alone, in the presence of either HC/LC or HC/A3-C1-C2, and following activation of the heterodimer forms by thrombin. The fluorescence anisotropy value of Fl-FFR-factor IXa was increased 0.01 upon binding HC/LC. A further increase was observed following activation by thrombin, resulting in an overall incremental increase of 0.05. These effects were similar to the original observations in the prior study (33) . In contrast, the fluorescence anisotropy value for Fl-FFR-factor IXa was increased 0.037 upon HC/A3-C1-C2 binding and was followed by a relatively small increase upon activation by thrombin such that the final anisotropy value was equivalent to that obtained following activation of the native heterodimer. Thus, the observation that interaction with the hybrid dimer form results in an initial anisotropy increase that markedly exceeds that for the native dimer and approaches the magnitude of that for factor VIIIa suggests that this factor VIII form may be factor VIIIa-like in its effect on the conformation of the factor IXa active site.


DISCUSSION

Activation of factor VIII is a prerequisite for its role as the cofactor for factor IXa in the intrinsic Xase complex. Thrombin, the physiologic activator of factor VIII (34) , cleaves this substrate at Argand Argin the HC and Argin the LC, converting the inactive procofactor to the active form. Little is known about the role(s) of these cleavages and their contribution to the potentiated activity of factor VIIIa. In the factor Xase complex, the cofactor binds both the (phospholipid) surface and factor IXa. However, these activities are present in the unactivated molecule. A high affinity interaction of factor VIII with phospholipid vesicles ( K 2 n M (35) ) is mediated through residues located at the COOH-terminal end of the LC (C2 domain residues 2300-2332 (36) ), and this interaction is likely unaffected by thrombin cleavage. Recent results of Duffy et al. (33) indicate a Kof 2 n M for the factor VIIIa-factor IXa interaction. A similar high affinity interaction was observed by these investigators when factor VIII was substituted for factor VIIIa.

This study focuses on the potential contribution to cofactor activity following LC cleavage. Independent of any effect on activity, cleavage at this site is required to dissociate factor VIII from its physiologic carrier protein, vWf, thus allowing for interaction with a thrombogenic surface. A primary binding site for vWf is contained within the acidic region defined by LC residues 1649-1689 (10, 16) . Cleavage at this site results in a marked reduction in the affinity of factor VIII for vWf (37, 38) . However, recent observations of the failure of this cleavage fragment to bind vWf (39) as well as the capacity for factor VIII C2 domain fragments to inhibit the factor VIII-vWf interaction (40, 41) suggest that this interaction is more complex than originally perceived.

In this study, we present data that indicate that the LC cleavage of factor VIII contributes to the potentiation of factor VIIIa activity. We have done this utilizing purified factor VIII(a) subunits and taking advantage of the capacity for these isolated subunits to be reconstituted to produce active material. The result of reconstitution of native HC and A3-C1-C2 is a factor VIII molecule with 3-fold higher specific activity than factor VIII reconstituted from native HC and native LC (2 versus 0.6 units/µg). It must be noted that the specific activity of purified factor VIII (either plasma-derived or recombinant) is 2-4 units/µg. The reason for the lower specific activity of reconstituted factor VIII (0.6 units/µg) is not known, but may result from partial denaturation of the subunits during the metal ion chelation and/or subsequent subunit purification steps. The hybrid material showed a markedly reduced lag time compared with factor VIII reconstituted from native subunits, when assayed without prior activation in a purified factor Xa generation system. The lag observed in this assay is attributed to the time required for the low levels of factor Xa generated to feedback activate factor VIII, yielding the active cofactor (28) . Both factor VIII forms followed similar rates of activation by factor Xa (data not shown), suggesting that the more factor VIIIa-like activity of the hybrid form does not result from differential rates of activation. In addition, the activity of the hybrid form was unaffected by the presence of vWf, consistent with it lacking the primary vWf interactive site. On the other hand, authentic factor VIII showed a reduced catalytic rate in the presence of vWf, consistent with factor Xa being a poor activator of vWf-bound factor VIII (29) .

A factor VIIIa molecule derived from A1/LC/A2 possessed 3-fold less activity than factor VIIIa derived from A1/A3-C1-C2/A2. However, reaction of either the hybrid heterodimer (HC/A3-C1-C2) or the heterotrimer (A1/LC/A2) with thrombin resulted in a further potentiation of activity, yielding values equivalent to those of native factor VIIIa. Therefore, we conclude that cleavage of the LC by thrombin contributes to the potentiation of factor VIIIa activity, and cleavage at both chains is required to yield the maximally activated molecule.

Several earlier reports have attempted to determine whether LC cleavage contributed to cofactor activity. Shima et al. (19) isolated DNA from a patient with mild hemophilia A and found an Arg Cys mutation, creating a thrombin-resistant HC. Partially purified factor VIII from this patient had 3% that of normal plasma factor VIII activity, which could still be activated 5-fold with thrombin. Activation correlated with cleavage of the LC, and since there was no vWf present in these preparations, the increase in factor VIII activity did not result from cofactor release of vWf. Although only limited regions of the factor VIII gene from this patient were examined and therefore other mutations may have gone undetected, the results presented in this study support the notion that cleavage of factor VIII LC contributed to the increase in factor VIIIa activity.

Using site-directed mutagenesis, Pittman and Kaufman (18) demonstrated a requirement for thrombin cleavage at both the HC and LC sites for factor VIII activation. Thrombin-resistant factor VIII molecules were created by making conservative changes at either Arg(in the LC) or Arg(in the HC). These factor VIII molecules were resistant to activation by thrombin at the altered site, while thrombin cleavage at the unmodified site was not affected. These investigators concluded that both cleavages were required for factor VIII activation. However, a general concern of studies involving site-directed mutagenesis is that even conservative substitutions may affect protein characteristics such as folding and/or stability as well as interprotein interactions (42) .

Studies using the snake venom protease from Bothrops jararacussu (20) , which produces a thrombin-like cleavage of factor VIII HC, but does not cleave the LC, show that cleavage of the HC alone leads to a factor VIIIa activity that is 60% that of factor VIII cleaved by thrombin. Although it was concluded that LC cleavage was not necessary for potentiation of activity, this observation is in agreement with our results, indicating that factor VIII requires LC cleavage for maximal activation.

Bihoureau et al. (17) attempted to dissect the relative contributions of each thrombin cleavage to changes in factor VIII activity. In these studies, factor VIII concentrates were activated with thrombin, and at various time points, the thrombin was inactivated by the addition of hirudin. Factor VIII was then purified by ion-exchange chromatography and analyzed by clotting assay and by SDS-PAGE. Initial reaction with thrombin yielded partially (5-fold) activated factor VIII molecules possessing cleaved LC but intact HC as judged by silver staining of the gels. Further thrombin activation of these factor VIII molecules resulted in a 15-fold (overall) activated factor VIIIa and generation of polypeptides corresponding to the A1, A2, and A3-C1-C2 subunits. These results suggested that LC cleavage by thrombin potentiated factor VIII activity. However, the potential for low levels of HC cleavage contributing to the enhanced activity could not be excluded.

Results from this study exclude several possible mechanisms by which LC cleavage might potentiate factor VIIIa activity. First, both the HC/LC and HC/A3-C1-C2 heterodimers were activated at apparently similar rates by thrombin and factor Xa, suggesting that initial cleavage of the LC does not improve subsequent cleavage of the HC. This result is consistent with the finding that the presence of vWf, which binds to this portion of the LC, does not affect the catalytic efficiency of thrombin cleavage of the HC (43) . Second, the intrinsic stability of the A1/LC/A2 heterotrimer, as judged by activity measurements following dilution of the sample, was similar to that observed for the native factor VIIIa heterotrimer. Thus, the affinity of the A2 subunit for either dimer form appeared equivalent. This result is interesting in that a primary binding site for A2 is defined by the COOH-terminal acidic region of the A1 subunit (30) . Thus, the presence of an additional acidic residue-rich region located in the NH-terminal region of intact LC does not appear to influence the association of the A2 subunit. Finally, results on the capacity of active site-modified factor IXa to enhance the association of A2 with either A1/LC or A1/A3-C1-C2 suggest that this region of the LC is not involved and/or does not interfere with the association of factor VIIIa with factor IXa.

Other laboratories have shown that cofactor binding involves changes in the active site of coagulant enzymes. Ye et al. (44) have shown that the binding of thrombomodulin derivatives to DEGR-thrombin causes a change in the emission intensity of the dansyl probe bound in the thrombin active site. This apparent conformational change in the thrombin active site is consistent with the change in substrate specificity of the thrombin such that it is now able to activate the anticoagulant protein C. The cofactor factor Va has been shown to cause conformational changes in the factor Xa active site as well as to affect the distance of the active site above the membrane surface (45) . Armstrong et al. (46) have recently shown that factor Va also can cause a moderate shift in the active site of the activation intermediate meizothrombin. Therefore, factor Va may function to alter the conformation and/or orientation of both enzyme and substrate.

Using similar types of fluorescence studies, Mutucumarana et al. (47) determined that the active site of DEGR-factor IXa is far above the membrane surface (>70 Å) and that the binding of factor VIIIa does not alter this distance, although a change in fluorescence intensity and in the anisotropy of the probe was observed. Duffy et al. (33) showed that the binding of factor VIII to an active site-labeled factor IXa produces a small anisotropy change, whereas a significantly larger change in anisotropy is produced by activating factor VIII with thrombin. This result appears to correlate with the role of factor VIIIa in increasing kfor the factor IXa-dependent conversion of factor X by several orders of magnitude.

We have utilized the sensitivity of fluorescence anisotropy of an active site-labeled factor IXa to factor VIII structure to evaluate the role of LC cleavage. The anisotropy of Fl-FFR-factor IXa was increased incrementally in the presence of HC/LC ( r = 0.011) and was further increased following activation to factor VIIIa ( r = 0.051), consistent with the earlier results of Duffy et al. (33) . However, in the presence of HC/A3-C1-C2, the initial incremental increase in anisotropy ( r = 0.037) was 4-fold greater than that observed with HC/LC and approached the value observed with the fully activated cofactor. Subsequent conversion of the hybrid factor VIII form to factor VIIIa yielded an anisotropy value equivalent to that obtained following activation of HC/LC. These results suggest that HC/A3-C1-C2 is more factor VIIIa-like than the native heterodimer with respect to its effect on the conformation in and around the factor IXa active site and hence its effect on the catalytic efficiency of factor IXa in the tenase complex. We propose that this alteration in conformation is dependent upon LC cleavage and explains mechanistically the observed higher specific activity and more efficient factor Xa-generating properties of the hybrid factor VIII heterodimer as well as the requirement for this cleavage to yield maximally activated cofactor.

  
Table: Effect of factor VIII forms on the fluorescence anisotropy of Fl-FFR-factor IXa



FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL38199 and HL30616 and by an established investigatorship (to P. J. F.) from the American Heart Association. A portion of this work was presented at the 67th Scientific Sessions of the American Heart Association, Dallas, TX, November 14-17, 1994. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Hematology Unit, P. O. Box 610, University of Rochester Medical Center, 601 Elmwood Ave., Rochester, NY 14642. Tel.: 716-275-6576; Fax: 716-473-4314.

The abbreviations used are: LC, factor VIII light chain; HC, factor VIII heavy chain; vWf, von Willebrand factor; dansyl, 5-dimethylaminonaphthalene-1-sulfonyl; DEGR-CK, dansylglutamyl- L-glycyl- L-arginyl chloromethyl ketone; DEGR-factor IXa, factor IXa modified with DEGR-CK; Fl-FFR-factor IXa, factor IXa modified with fluorescein- D-phenylalanyl- D-phenylalanyl- L-arginyl chloromethyl ketone; PPACK, D-phenylalanyl- L-prolyl- L-arginyl chloromethyl ketone; PAGE, polyacrylamide gel electrophoresis.

Factor VIIIa, thrombin-activated factor VIII, and factor VIIIa subunits are designated relative to the domain sequences as follows (3): A1, residues 1-372; A2, residues 373-740; and A3-C1-C2, residues 1690-2332. Noncovalent subunit interactions are denoted by slashes, and covalent associations are denoted by hyphens.


ACKNOWLEDGEMENTS

We thank Drs. James Brown and George Mitra and the Cutter Division of Miles Inc. for the therapeutic concentrates used to prepare factor VIII subunits and Debra Pittman and the Genetics Institute for the gift of recombinant factor VIII. We also thank Tammy L. Beattie for excellent technical assistance.


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