Human blood coagulation Factor VIII (FVIII) ()participates as cofactor for Factor IXa (FIXa) in activation of Factor X (FX) in the intrinsic pathway of blood coagulation(1) . FVIII is synthesized as a single-chain polypeptide displaying a domain structure with the sequence A1-A2-B-A3-C1-C2(2, 3) . The protein circulates in plasma as a heterodimer consisting of FVIII heavy chain (FVIII-HC) and FVIII light chain (FVIII-LC) comprising the domains A1-A2-B and A3-C1-C2, respectively. Because of limited proteolysis in the B-domain, FVIII-HC is heterogeneous (M, 90,000-210,000)(4, 5) . The FVIII heterodimer is metal ion-dependent, since EDTA destroys FVIII activity concomitant with dissociation of FVIII-HC and FVIII-LC(4, 6, 7) . Moreover, the addition of divalent metal ions to inactivated FVIII or to a mixture of isolated subunits results in FVIII reassembly(8, 9) and regain of FVIII activity(8, 9, 10) . Thus, FVIII activity is restricted to the heterodimeric complex of heavy and light chain.

FVIII activation is required for cofactor function in the intrinsic pathway. Activation is accomplished by limited proteolysis of both the heavy and light chain of FVIII(11, 12, 13) . Major cleavage sites are located at or close to the boundaries of the A-domains, in the FVIII-HC at amino acid positions Arg and Arg, and in the FVIII-LC at position Arg(5) . After cleavage of FVIII-HC between the domains A1 and A2 at position Arg, the A2 domain is noncovalently associated with the A1 domain and the A3-C1-C2 comprising light chain(5, 14, 15) . Activated FVIII (FVIIIa) has transient activity, since the A2 domain readily dissociates from the A1/A3-C1-C2 dimer(16) . Both thrombin and Factor Xa (FXa) activate FVIII by limited proteolysis at the three major cleavage sites(5) . However, FXa cleaves some additional sites, one of which is located in the heavy chain at position Arg(5) . The same site is cleaved by activated Protein C and FIXa, which inactivate FVIII(5, 17, 18, 19) . Cleavage at this site, and the release of the region Ser-Arg, promotes FVIIIa dissociation and inactivation(20) . These findings have established the concept that limited proteolysis of FVIII-HC activates FVIII but simultaneously triggers subunit dissociation and concomitant FVIII inactivation.

With respect to FVIII-LC cleavage, its role in FVIII activation and inactivation remains controversial. One problem is that FVIII-LC is susceptible to cleavage at a number of sites, including the positions Arg, Arg, and Arg. It seems evident that FVIII activation by thrombin involves proteolysis at Arg(5, 21) . The same site may be cleaved upon activation by FXa, although this enzyme cleaves at position Arg as well, in a process that coincides with inactivation of FVIII(5, 22) . Inactivation may also be accomplished by FIXa, which cleaves FVIII-LC at position Arg(18, 19) . However, a recent observation suggests that cleavage at this site may be unrelated to FVIII inactivation(22) . Interpretation of these findings is further complicated by the notion that the same enzymes that cleave FVIII-LC also cleave at various positions within FVIII-HC (5, 18, 19, 20, 21, 22) . Thus, the precise role of FVIII-LC cleavage in FVIII function remains unclear. In the present study, we addressed this problem employing FVIII heterodimers that were cleaved exclusively at the FVIII-LC positions Arg or Arg. These were obtained by assembling isolated FVIII-HC with well defined FVIII-LC derivatives. Subsequently, these FVIII species were compared with respect to subunit interaction, FVIII-FIXa complex assembly, and function as cofactor of FIXa in FX activation.

EXPERIMENTAL PROCEDURES

Materials

FVIII and FVIII Subunits

Antibodies and Other Proteins

FVIII Heterodimer Assay

Association of FVIII Subunits

Association Rate Constants

The extent of FVIII assembly at a given time t thus is the resultant of the simultaneously occurring association and dissociation. Since FVIII association is a slow process (see ``Results''), this is most conveniently analyzed in terms of initial rates of association. Under certain experimental conditions, FVIII dissociation is remarkably slow (see ``Results''). In the initial phase, the concentration of dimer formed remains low, whereas the concentrations of free subunits do not differ substantially from their initial values. The reversible association reaction then reflects the simple, bimolecular association process,

When the initial concentration of FVIII-HC (a) is slightly higher than that of FVIII-LC (b), the integrated expression, with the limits t = 0 and t = t, has previously been given as(32) .

Here k represents the association rate constant, and p is the concentration of FVIII dimer formed at time t. Association kinetics were derived from the initial 120 min of FVIII assembly experiments. k was calculated by fitting of data into employing Enzfitter software (Elsevier, Amsterdam, The Netherlands).

Dissociation Rate Constants

Here [FVIII] is the FVIII concentration at t = 0, and k is the first order rate constant of inactivation of FVIII heterodimers.

The apparent K values were calculated from association and dissociation rate constants according to the following equation.

FIXa Binding Assay

Initial Rates of FXa Formation

RESULTS

Regeneration of FVIII Activity from Intact and Cleaved Subunits

Figure 1: Purified FVIII subunit derivatives. FVIII-HC and FVIII-LC derivatives were reduced and subjected to SDS-polyacrylamide gel electrophoresis on a 7.5% (w/v) polyacrylamide gel. Protein was visualized by silver staining. Lanes 1 and 2 contain 1 µg of purified FVIII-HC and 0.25 µg of FVIII-LC, respectively. FVIII-LC was subjected to limited proteolysis by thrombin and FXa, and cleavage products were purified (see ``Experimental Procedures''). Lanes 3 and 4 show 0.25 µg of FVIII-LC and FVIII-LC, respectively. NH-terminal amino acid sequence analysis of FVIII-LC and FVIII-LC was performed to verify the positions of cleavage by thrombin and FXa. The derived sequences of amino acids are shown together with the NH-terminal sequence of intact FVIII-LC(5) .

In preliminary experiments, the effect of pH and divalent cations was examined with respect to regeneration of FVIII activity from isolated subunits. The results were essentially similar to those described previously by Fay(8) , with the exception that Ca proved more effective than Mn with respect to regain of FVIII activity (results not shown). Conditions were identified that promoted FVIII regeneration from intact FVIII-LC and an excess of FVIII-HC, yielding a specific activity of 1.9 ± 0.2 units/µg (mean ± S.D.). In terms of effectively assembled protein, thus excluding the excess of FVIII-HC, the specific activity was 4 units/µg. This value is similar to that of the FVIII starting material used for the preparation of the isolated subunits. These conditions, thus, are appropriate for studies on the role of FVIII-LC cleavage in regeneration of FVIII activity. FVIII-LC was found to be similar to intact FVIII-LC with respect to the specific activity of regenerated FVIII (1.8 ± 0.2 units/µg). In contrast, FVIII-LC restored FVIII activity to a lower specific activity (0.9 ± 0.1 units/µg). This suggests that either FVIII-LC containing heterodimers have reduced activity, or that the process of heterodimer formation is less efficient when the sequence Ser-Arg is lacking from FVIII-LC.

Assembly of FVIII Heterodimers from Intact and Cleaved Subunits

Figure 2: Reconstitution of FVIII-LC derivatives with FVIII-HC. Varying concentrations of FVIII-LC (), FVIII-LC (), and FVIII-LC () were tested for complex assembly with FVIII-HC (415 nM) into active FVIII heterodimers. Subunits were incubated in 400 mM NaCl, 40 mM CaCl, 1% (w/v) HSA, 20 mM Hepes (pH 7.2). After 30 min of incubation at 22 °C, FVIII activity (panel A) and FVIII heterodimer formation (panel B) were assessed as described under ``Experimental Procedures''.

Association and Dissociation Kinetics of FVIII Subunits

Figure 3: Ionic strength dependence of FVIII subunit interaction. Association (panel A) of FVIII-HC (415 nM) with intact FVIII-LC (125 nM) was performed in the presence of varying concentrations of NaCl in 1% (w/v) HSA, 20 mM Hepes (pH 7.2), and either 10 mM () or 40 mM () CaCl. After 1 h of incubation at 22 °C, FVIII activity was determined as described under ``Experimental Procedures.'' The same incubation conditions were used for dissociation of reassociated FVIII (panel B). FVIII subunits were associated for 20 h in 150 mM NaCl, 1% (w/v) HSA, 20 mM Hepes (pH 7.2), and either 10 mM () or 40 mM () CaCl prior to dilution to 0.4 nM FVIII activity. Residual FVIII activity was measured after 3 h of dissociation. Data represent the average ± S.D. of three experiments.

Association (Fig. 4A) of FVIII-HC with various FVIII-LC derivatives as well as dissociation (Fig. 4B) of various reassociated FVIII species was investigated time-dependently. Association and dissociation data were fitted into models of noncatalytic interaction and single exponential decay, respectively (see ``Experimental Procedures''), in order to derive association (k) and dissociation (k) rate constants and apparent K values (Table 1). Comparison between the various FVIII-LC derivatives demonstrates that FVIII-LC differs from FVIII-LC and intact FVIII-LC in that it displays a 2-3-fold lower association rate constant (Table 1). For FVIII-LC and FVIII-LC, the dissociation rate constants were found to be equal, and slightly higher than the dissociation rate constant of heterodimers containing intact FVIII-LC. Similarly, no differences between FVIII-LC species were apparent under much more stringent dissociating conditions (Fig. 4B). As the association process, and thus k, is particularly ionic strength-dependent, the same holds for the apparent K values derived from these data. K values may be 2-3-fold lower at lower NaCl concentrations (cf. Fig. 3).

Figure 4: Interaction between FVIII subunit derivatives. FVIII-HC (415 nM) was reconstituted with 125 nM of intact FVIII-LC (), FVIII-LC (), and FVIII-LC () in a buffer consisting of 400 mM NaCl, 40 mM CaCl, 1% (w/v) HSA, 20 mM Hepes (pH 7.2) (panel A). At various time points FVIII activity was determined, and association rate constants were calculated from data between 0 and 120 min of association employing under ``Experimental Procedures.'' For inactivation studies (panel B), FVIII first was assembled in 400 mM NaCl, 40 mM CaCl, 1% (w/v) HSA, 20 mM Hepes (pH 7.2) and subsequently diluted to 0.4 nM FVIII activity in 1% (w/v) HSA, 20 mM Hepes (pH 7.2) containing 400 mM NaCl and 40 mM CaCl (solid line) or 7.5 mM NaCl and 10 mM CaCl (dashed line), and decay of FVIII activity was determined. Data were fitted into under ``Experimental Procedures,'' and the resulting curves are shown. Data are given as the average ± S.D. of three experiments.

Interaction of FVIII-LC Derivatives with FIXa

Figure 5: FIXa binding to immobilized FVIII-LC variants. Intact FVIII-LC (), FVIII-LC (), and FVIII-LC () were immobilized to the anti-FVIII-LC monoclonal antibody CLB-CAg 12 (1 µg/well). The amount of FVIII-LC derivatives bound to the antibody was quantified employing an immunological assay (see ``Experimental Procedures'') and was found to be equivalent (1 pmol/well) for the FVIII-LC derivatives. Subsequently, FIXa was added, and after 4 h of incubation FIXa binding was assessed as described elsewhere(23) . Data represent the average ± S.D. of three experiments.

Activation of FVIII by FXa and Thrombin

Figure 6: Initial rates of FXa formation. FX (0.56 µM) was activated in a mixture containing FVIII (0.4 nM), FIXa (0.3 nM), phospholipids (100 µM), and Ca (10 mM). FX activation was started by the addition of various concentrations of thrombin () or FXa (). Progress curves of FX activation served to derive the initial rates of FXa formation. Each data point represents the average ± S.D. of at least three experiments. In order to detect cleavage fragments, FVIII (32 nM) was activated for 3 min with thrombin (FIIa) or FXa (both 5 nM) under the same conditions, except that FIXa and FX were omitted. Subsequently, activated FVIII was subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting. FVIII-LC fragments were visualized employing a polyclonal antibody against FVIII-LC. The positions of the FVIII-LC cleavage products (73 and 67 kDa) upon incubation with thrombin or FXa, respectively, are indicated in the inset.

Effect of FVIII-LC Cleavage on FX Activation

Figure 7: FXa formation in the presence of reconstituted FVIII. Intact FVIII-LC (), FVIII-LC (), or FVIII-LC () (125 nM) were reconstituted with FVIII-HC (415 nM) in 400 mM NaCl, 40 mM CaCl, 1% (w/v) HSA, 20 mM Hepes (pH 7.2) for 24 h at 22 °C. Reconstituted FVIII (0.08 nM) was added to FIXa (0.3 nM), phospholipids (100 µM), CaCl (10 mM), and acetylated FX (0.2 µM), and FXa formation was determined (panel A). In panel B, the same experiment was performed, employing native FX instead of chemically modified FX. Data are given as the average ± S.D. of three experiments. The inset shows an immunoblot analysis of FVIII-HC reassociated for 24 h with intact FVIII-LC (lane 1), FVIII-LC (lane 2), and FVIII-LC (lane 3). FVIII-HC was visualized employing a chicken antibody (see ``Experimental Procedures'') directed against the A1 domain of FVIII-HC.

Previous studies have established that, in the absence of exogeneous FVIII activating enzymes such as FXa or thrombin, FVIII activation is fully dependent on FXa formed during the initial phase of FX activation (11, 12) . To assess the contribution of feedback activation caused by FVIII-HC cleavage by FXa, the experiments of Fig. 7A were also performed using native, unmodified FX as the substrate. As shown in Fig. 7B, FVIII containing FVIII-LC or FVIII-LC displayed a 4-fold higher rate of FXa formation than observed in the presence of chemically modified FX (Fig. 7A). This implies that under the conditions of Fig. 7, feedback cleavage of FVIII-HC by FXa contributes to the activity of fully activated FVIII. Moreover, the same rate of FXa formation was observed in the presence of heterodimers containing FVIII-LC or FVIII-LC, demonstrating that these two FVIII-LC derivatives are functionally equivalent. Fig. 7B further shows that the initial rate of FXa formation (<3 min) was more than 10-fold reduced when the reconstituted FVIII heterodimer contained intact instead of cleaved FVIII-LC. Apparently, FVIII-LC cleavage, either by thrombin or by FXa, is essential for developing the full cofactor potential of FVIII-HC.

DISCUSSION

The objective of the present study was to assess the role of FVIII-LC cleavage by thrombin and FXa, with special reference to FVIII subunit interaction and FVIII activation and inactivation. Our experimental approach employed FVIII-LC derivatives obtained by limited proteolysis using thrombin and FXa (Fig. 1). Subsequently, these cleavage products were reassembled with FVIII-HC in order to obtain FVIII heterodimers that were cleaved in one subunit only. Isolated FVIII subunits could be effectively reassociated into active heterodimers (Fig. 2). For intact FVIII-LC, our results are similar to those of previous studies of FVIII assembly(8, 16) . To describe subunit interaction in more detail, we have assessed association and dissociation kinetics. Low ionic strength was found to promote FVIII subunit association but simultaneously to destabilize the heterodimer (Fig. 3). The same ionic strength dependence has recently been reported for the subunit association of activated FV (FVa)(37) , which is similar to FVIII in that it also comprises a heterodimer of the domains A1-A2 and A3-C1-C2(37, 38) . For FVa subunit interaction, K values have been reported (38) that are at least one order of magnitude lower than for the interaction between FVIII-HC and intact FVIII-LC (Table 1). Since K values for FVIII subunit interaction represent apparent equilibrium constants that are highly dependent on ionic strength (Fig. 3), nonequilibrium rate constants may be considered as more appropriately reflecting subunit interaction. Comparison between the association rate constants demonstrates that assembly of FVIII heterodimer is about 50-fold slower than of FVa (Table 1, (39) ). However, the dissociation rate constants of associated FVIII and FVa subunits indicate that, except at relatively low ionic strength, dissociation is negligibly slow for both FVIII and FVa (Fig. 4B, (39) ). It may seem contradictory that subunit association rates are lower at the higher ionic strength (Fig. 3), whereas optimal regain of FVIII activity from isolated subunits requires the same condition (Table 1; cf. (8) ). Apparently, the extent of heterodimer formation is fully controlled by the dissociation process.

The dissociation rate constant of uncleaved FVIII-HC and FVIII-LC (Table 1) is 1000-fold lower than that of the interaction between the A2 domain and the A1/A3-C1-C2 dimer(16) . This supports the concept that limited proteolysis of FVIII-HC, besides activating FVIII, also triggers subunit dissociation and concomitant FVIII inactivation. We have considered the possibility that, in analogy with FVIII-HC, FVIII-LC cleavage also affects subunit interaction. Although FVIII-LC proved to be less efficient than intact FVIII-LC and FVIII-LC in heterodimer assembly and regain of FVIII activity from isolated subunits (Fig. 2), heterodimer dissociation rates were strikingly similar (Table 1, Fig. 4B). Therefore, we conclude that the FVIII-LC fragment Ser-Arg, although promoting subunit association, has no effect on dissociation of FVIII into its constituent units.

In the current model of FVIII regulation, FXa is believed to cleave FVIII-LC at position Arg prior to Arg(5) . The latter cleavage thus may be a secondary event that is associated with FVIII inactivation and as such could serve the same function as FVIII-HC cleavage at Arg(21, 22) . Our data, however, do not support this view. First, heterodimers containing FVIII-LC do have FVIII activity (Fig. 2). Second, FVIII containing FVIII-LC displays the same subunit dissociation rate constant as heterodimers containing FVIII-LC or uncleaved FVIII-LC (Table 1, Fig. 4B). Moreover, FVIII-LC and FVIII-LC have the same affinity for FIXa (Fig. 5) and contribute to FVIIIa activity to the same extent (Fig. 7). Collectively, these data suggest that thrombin and FXa follow distinct pathways in cleaving FVIII, which yield different but functionally indistinguishable FVIIIa species. Whereas this view could be confirmed in a system of purified components (Fig. 6), the physiological mechanism of FVIII activation seems to be more complex. The notion that a point mutation at the thrombin cleavage site Arg leads to FVIII dysfunction and hemophilia A (21, 40, 41, 42) implies that cleavage at Arg by FXa does not compensate for the lack of cleavage at Arg by thrombin. This raises the question of whether Arg is susceptible to proteolysis by FXa when FVIII is in complex with its physiological carrier protein von Willebrand Factor (vWF). Several studies have reported that vWF indeed interferes with events involving the amino-terminal portion of FVIII-LC, such as the activation of FVIII by FXa (43) and the interaction of FIXa with the FVIII-LC region Gln-Asp(23) . It seems conceivable that cleavage of FVIII-LC at Arg provides a relatively insignificant pathway of FVIII activation, since it may be restricted to situations where FVIII is dissociated from vWF.

Its tight association with vWF (K 10M) (44) prevents FVIII from interacting with components that bind with lower affinity, such as FIXa (23) and phospholipids(45) . Thus, disruption of the FVIII-vWF complex is required for cofactor function. It has been well established that dissociation is accomplished by FVIII-LC cleavage at position Arg(13, 22, 46, 47) . Our rigorous approach of FVIII subunit reassembly and cofactor function analysis allowed us to establish a second role for FVIII-LC cleavage. Employing vWF-free conditions, we found that cleavage of FVIII-LC alone at either Arg or Arg activates FVIII to a substantial extent (Fig. 7). This implies that cleavage of FVIII-HC, although determining the final extent of FVIIIa activity (Fig. 7B), is not absolutely required for FVIII activation. Moreover, this finding provides an explanation for the observation that dysfunctional FVIII with a substitution at the Arg-Ser cleavage site in FVIII-HC has some residual FVIII activity and is associated with mild hemophilia A (47, 48) .

One conclusion of our study is that FVIII activity is completely lacking when FVIII is composed of uncleaved subunits (Fig. 7A). Apparently, the amino-terminal portion Glu-Arg of the light chain of FVIII is an activation peptide that needs to be cleaved off for exposure of cofactor activity. Within this fragment, the sequence Glu-Arg serves a dual role in regulating FVIII activity. First, this sulfated, acidic sequence promotes the high affinity interaction between FVIII and vWF(28, 36, 49) . On the other hand, the same region seems to inhibit some interaction involved in FX activation. Since our data seem to exclude the possibility that FVIII-LC cleavage affects FIXa binding (Fig. 5), we propose that the region Glu-Arg interferes in binding of FVIII to other components of the FX-activating complex, such as FX or phospholipids. In contrast, the region Ser-Arg has no apparent role in the cofactor function of FVIII. Although this region does contribute to FVIII heterodimer assembly (Table 1), the physiological implications of this phenomenon remain unclear.

FOOTNOTES

*

This study was financially supported by the Netherlands Organization for Scientific Research (NWO) (Grant 900-526-191). 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. Tel.: 31-20-512-3120; Fax: 31-20-512-3332.

()

The abbreviations used are: FVIII, Factor VIII; FVIII-LC, Factor VIII light chain; FVIII-LC, thrombin-cleaved Factor VIII light chain; FVIII-LC, Factor Xa-cleaved Factor VIII light chain; FVIII-HC, Factor VIII heavy chain; FV, Factor V; FXa, Factor Xa; FIXa, Factor IXa; FVIIIa, Factor VIIIa; vWF, von Willebrand Factor; PPACK, D-phenylalanyl-L-prolyl-L-arginine chloromethyl ketone; EGR-CK, L-glutamyl-glycyl-L-arginine chloromethyl ketone; HSA, human serum albumin.

ACKNOWLEDGEMENTS

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