Kinetics of factor VIII light-chain cleavage by thrombin and factor Xa. A regulatory role of the factor VIII heavy-chain region Lys713-Arg740.

Activation and limited proteolysis of factor VIII have been investigated with respect to the role of the heavy-chain region Lys713-Arg740. The kinetics of factor VIII activation have been analyzed in a system consisting of human factor VIII, factor IXa, factor X phospholipids, and thrombin or factor Xa. Plasma-derived factor VIII is activated by thrombin with a second-order rate constant of 3.3 +/- 0.3 x 10(6) M-1 s-1, which proved to be slightly higher than for activation by factor Xa. The second-order rate constant of activation by thrombin of plasma-derived factor VIII in the presence of a monoclonal antibody against the sequence Lys713-Arg740 is markedly reduced. The same result was obtained for activation by thrombin and factor Xa of factor VIII with a deletion including the sequence Lys713-Arg740, des-(713-1637)-factor VIII. This suggests that the region Lys713-Arg740 promotes factor VIII activation by both thrombin and factor Xa. Since factor VIII activation is associated with proteolysis, cleavage of factor VIII heavy and light chains was analyzed quantitatively. These studies indicated that heavy-chain cleavage of des-(713-1637)-factor VIII is similar to that of plasma-derived factor VIII. In contrast, cleavage of the light chain of des-(713-1637)-factor VIII is clearly reduced. Furthermore, the secondorder rate constant (0.2 +/- 0.1 x 10(6) M-1 s-1) of des-(713-1637)-factor VIII light-chain cleavage by thrombin was reduced tenfold compared with that of plasma-derived factor VIII. Proteolysis by factor Xa yielded similar results. The rate of des-(713-1637)-factor VIII light-chain cleavage by thrombin is similar to that of isolated light-chain, but isolated light-chain is cleaved by factor Xa 20-fold more efficiently than the light chain in des-(713-1637)-factor VIII. We conclude that activation of factor VIII by both thrombin and factor Xa is closely associated with light-chain cleavage. Furthermore, within the factor VIII heterodimer, the heavy-chain sequence Lys713-Arg740 promotes both activation and light-chain proteolysis.

Enhanced thrombin sensitivity of a factor VIII-heparin cofactor II hybrid.

Generation of thrombin at a site of vascular injury is a key event in the arrest of bleeding. In addition to the conversion of fibrinogen into the insoluble fibrin, thrombin can initiate a number of positive and negative feedback mechanisms that either sustain or down-regulate clot formation. We have modulated the thrombin sensitivity of human blood coagulation factor VIII, an essential cofactor in the intrinsic pathway of blood coagulation. We have substituted an acidic region of factor VIII corresponding to amino acid sequence Asp712-Ala736 by amino acid sequence Ile51-Leu80 of the thrombin inhibitor heparin cofactor II. Functional analysis of the resulting factor VIII-heparin cofactor II hybrid, termed des-(868-1562)-factor VIII-HCII, revealed an increase in procoagulant activity as measured in a one-stage clotting assay. Incubation of purified des-(868-1562)-factor VIII-HCII with different amounts of thrombin showed that this protein was more readily activated by thrombin when compared with des-(868-1562)-factor VIII, a control protein lacking amino acid sequence Ile51-Leu80 of heparin cofactor II. This was manifested by an increase in the second order rate constant of activation by thrombin for des-(868-1562)-factor VIII-HCII (12.0 +/- 0.48 x 10(6) M-1 s-1) compared with des-(868-1562)-factor VIII (1.77 +/- 0.21 x 10(6) M-1 s-1). Our data suggest that amino acid sequence Ile51-Leu80 of heparin cofactor II endows factor VIII with increased sensitivity towards thrombin which results in accelerated clot formation.

The sequence Glu1811-Lys1818 of human blood coagulation factor VIII comprises a binding site for activated factor IX.

In previous studies have shown that the interaction between factor IXa and VIII involves the light chain of factor VIII and that this interaction inhibited by the monoclonal antibody CLB-CAg A against the factor VIII region Gln1778-Asp1840 (Lenting, P.J., Donath, M.J.S.H., van Mourik, J.A., and Mertens, K. (1994) J. Biol. Chem. 269, 7150-7155). Employing distinct recombinant factor VIII fragments, we now have localized the epitope of this antibody more precisely between the A3 domain residues Glu1801 and Met1823. Hydropathy analysis indicated that this region is part of a major hydrophilic exosite within the A3 domain. The interaction of factor IXa with this exosite was studied by employing overlapping synthetic peptides encompassing the factor VII region Tyr1786-Ala1834. Factor IXa binding was found to be particularly efficient to peptide corresponding to the factor VIII sequences Lys1804-Lys1818 and Glu1811-Gln1820. The same peptides proved effective in binding antibody CLB-CAg A. Further analysis revealed that peptides Lys1804-Lys1818 and Glu1811-Gln1820 interfere with binding of factor IXa to immobilized factor VIII light chain (Ki approximately 0.2 mM and 0.3 mM, respectively). Moreover, these peptides inhibit factor X activation by factor IXa in the presence of factor VIIIa (Ki approximately 0.2 mM and 0.3 mM, respectively) but not in its absence. Equilibrium binding studies revealed that these two peptides bind to the factor IX zymogen and its activated form, factor IXa, with the same affinity (apparent Kd approximately 0.2 mM), whereas the complete factor VIII light chain displays preferential binding to factor IXa. In conclusion, our results demonstrate that peptides consisting of the factor VIII light chain residues Lys1804-Lys1818 and Glu1811-Gln1820 share a factor IXa binding site that is essential for the assembly of the factor X-activating factor IXa-factor VIIIa complex. We propose that the overlapping sequence Glu1811-Lys1818 comprises the minimal requirements for binding to activated factor IX.

Characterization of des-(741-1668)-factor VIII, a single-chain factor VIII variant with a fusion site susceptible to proteolysis by thrombin and factor Xa.

A factor VIII variant has been characterized in which the heavy chain is directly fused to the light chain. Des-(741-1668)-factor VIII lacks the processing site at Arg1648, as Arg740 of the heavy chain is fused to Ser1669 of the light chain. The sequence of the fusion site is similar to that of other cleavage sites in factor VIII. The fusion site of des-(741-1668)-factor VIII was readily cleaved by both thrombin and factor Xa, and the same result was obtained for heavy chain cleavage. In contrast, des-(741-1668)-factor VIII cleavage by thrombin at position Arg1689 proceeded at a lower rate than the analogous cleavage by factor Xa, which presumably takes place at position Arg1721. The rate of cleavage at position Arg1689 by thrombin was also lower than that at the other processing sites. When des-(741-1668)-factor VIII was activated by thrombin, initial rates of factor Xa formation were similar to the rates obtained when plasma-derived factor VIII was activated by thrombin or factor Xa. Remarkably, activation of des-(741-1668)-factor VIII proceeded at a higher rate by factor Xa than by thrombin. These results indicate that factor VIII activation is strongly associated with cleavage at position Arg1689 or Arg1721. For the interaction between des-(741-1668)-factor VIII and von Willebrand factor, a Kd value of (0.8 +/- 0.3) x 10(-10) M was determined, which is similar to that of heterodimeric factor VIII. The affinity of single-chain des-(741-1668)-factor VIII for factor IXa was found to be 27 +/- 6 nM. The in vivo recovery and half-life of des-(741-1668)-factor VIII were assessed in guinea pigs. Upon infusion of des-(741-1668)-factor VIII at a dosage of 50 units/kg body weight, a rise of 1.0 +/- 0.3 unit/ml in factor VIII activity was obtained. The same recovery was determined for wild-type factor VIII. The half-life of des-(741-1668)-factor VIII was found to be 3 +/- 1 h, compared with 4 +/- 2 h for heterodimeric recombinant factor VIII. In conclusion, des-(741-1668)-factor VIII displays normal activity, is readily cleaved by thrombin and factor Xa at its fusion site, binds with high affinity to von Willebrand factor and factor IXa, and behaves like heterodimeric recombinant factor VIII in guinea pigs. By virtue of these properties, des-(741-1668)-factor VIII may prove useful for the treatment of bleeding episodes in patients with haemophilia A.

Cleavage at arginine 145 in human blood coagulation factor IX converts the zymogen into a factor VIII binding enzyme.

The transition of the factor IX zymogen into the enzyme factor IXa beta was investigated. For this purpose, the activation intermediate factors IX alpha and IXa alpha were purified after cleavage of the Arg145-Ala146 and Arg180-Val181 bonds, respectively. These intermediates were compared for a number of functional properties with factor IXa beta, which is cleaved at both positions. Factor IXa alpha was equal to factor IXa beta in hydrolyzing the synthetic substrate CH3SO2-Leu-Gly-Arg-p-nitroanilide (kcat/Km approximately 120 s-1 M-1) but was less efficient in factor X activation. Factor IX alpha was incapable of generating factor Xa but displayed reactivity toward p-nitrophenol p-guanidinobenzoate and the peptide substrate. The catalytic efficiency, however, was 4-fold lower compared with factor IXa alpha and factor IXa beta. Factor IX alpha and factor IXa beta had similar affinity for the inhibitor benzamidine (Ki approximately 2.5 mM), and amidolytic activity of both species was inhibited by Glu-Gly-Arg-chloromethyl ketone and antithrombin III. Unlike factor IXa beta, factor IX alpha was unable to form SDS stable complexes with antithrombin III. Moreover, inhibition of factor IXa beta and factor IX alpha by Glu-Gly-Arg-chloromethyl ketone followed distinct pathways, because factor IX alpha was inhibited in a nonirreversible manner and displayed only minor incorporation of the dansylated inhibitor into its catalytic site. These data demonstrate that the catalytic site of factor IX alpha differs from that of the fully activated factor IXa beta. Factor IX and its derivatives were also compared with regard to complex assembly with factor VIII in direct binding studies employing the immobilized factor VIII light chain. Factor IX alpha and factor IXa beta displayed a 30-fold higher affinity for the factor VIII light chain (Kd approximately 12 nM) than the factor IX zymogen. Factor IXa alpha showed lower affinity (Kd approximately 50 nM) than factor IX alpha and factor IXa beta, which may explain the lower efficiency of factor X activation by factor IXa alpha. Collectively, our data indicate that cleavage of the Arg180-Val181 bond develops full amidolytic activity but results in suboptimal binding to the factor VIII light chain. With regard to cleavage of the Arg145-Ala146 bond, we have demonstrated that this results in the transition of the factor IX zymogen into an enzyme that lacks proteolytic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a binding site for blood coagulation factor IXa on the light chain of human factor VIII.

The interaction between human factor IXa and factor VIII or its constituent units was investigated. Equilibrium binding studies were performed employing factor VIII light chain that was immobilized on a monoclonal antibody. Factor VIII light chain was observed to bind factor IXa with high affinity (Kd = 14.8 +/- 3.2 nM) and approximately 1:1 stoichiometry. Optimal interaction required NaCl concentrations below 0.2 M and the presence of Ca2+ ions. Factor VIII light chain in solution effectively inhibited binding of factor IXa to the immobilized light chain (Ki = 10.9 +/- 1.9 nM). The isolated factor VIII light chain and the factor VIII heterodimer were equally effective in factor IXa binding, demonstrating that this interaction did not require the factor VIII heavy chain. Factor Xa and activated Protein C were found to be inefficient (Ki > or = 1.2 microM) in competing with factor IXa, indicating that the high affinity for factor VIII light chain was unique for factor IXa. The factor IXa-factor VIII light chain interaction was inhibited by von Willebrand factor, but this effect was abolished by cleavage of the factor VIII light chain by thrombin. An antibody that inhibits von Willebrand factor-factor VIII complex formation did not compete for factor IXa binding. In contrast, association of factor IXa with the factor VIII light chain was inhibited by an antibody directed against the factor VIII region Gln1778-Asp1840. We propose that this sequence provides a factor IXa binding site and that its exposure requires dissociation of the factor VIII-von Willebrand factor complex.

Biological activity of recombinant factor VIII variants lacking the central B-domain and the heavy-chain sequence Lys713-Arg740: discordant in vitro and in vivo activity.

Recombinant factor VIII variants with overlapping deletions spanning the region Lys713-Ile1668 have been expressed in mammalian cells, and analysed for biological activity both in vitro and in vivo. Two distinct assay systems were used to measure the activity in vitro. The one-stage coagulation assay served to assess factor VIII procoagulant activity while a spectrophotometric assay was used for the quantification of factor VIII cofactor activity in factor IXa-dependent factor X activation. Deletion of the entire B-domain (Ser741-Arg1648) resulted in a protein with similar procoagulant and cofactor activity. In contrast, factor VIII-del(713-1637), which has a deletion that also comprises the heavy-chain sequence Lys713-Arg740, had lost factor VIII procoagulant activity while factor VIII cofactor activity was retained. This functional inconsistency was further addressed by comparing purified factor VIII-del(713-1637) with factor VIII-del(868-1562), a mutant with normal in vitro activity. Kinetic studies of factor Xa formation revealed that higher concentrations of thrombin were required to develop the cofactor activity from factor VIII-del(713-1637) than needed for factor VIII-del(868-1562) or plasma factor VIII. The physiological significance of this finding was assessed in dogs with haemophilia A. Both deletion mutants were similar to plasma factor VIII with regard to binding to von Willebrand factor and half-life and recovery. Employing the cuticle bleeding time model, factor VIII-del(868-1562) was found to be indistinguishable from plasma factor VIII, whereas factor VIII-del(713-1637) was less effective. The increased thrombin-resistance of factor VIII-del(713-1637) thus limits both procoagulant activity and haemostatic efficacy in cuticle bleeding. These observations suggest that the heavy-chain sequence Lys713-Arg740, although dispensable for factor VIII cofactor function per se, is involved in the proteolytic activation of factor VIII both in vitro and in vivo.

Transcriptional regulation of retinoic acid receptor beta in retinoic acid-sensitive and -resistant P19 embryocarcinoma cells.

As in other embryocarcinoma (EC) cell lines retinoic acid (RA) rapidly induces expression of the nuclear retinoic acid receptor (RAR) beta in murine P19 EC cells, while RAR alpha is expressed constitutively. In the RA-resistant P19 EC-derived RAC65 cells, however, there is no such induction and an aberrant (smaller) RAR alpha transcript is expressed. RAR gamma 1 is expressed at low levels in both cell lines. To study the regulation of the RAR beta gene and the possible involvement of RAR alpha protein in transcriptional activation of the RAR beta gene we transfected these cells with a construct containing a 1.6 kb promoter fragment of the human RAR beta gene fused to the CAT gene. Upon transient assays in P19 EC cells CAT activity is enhanced rapidly by RA, to more than 100-fold in a concentration-dependent fashion. On the contrary no activity can be observed in the RA-resistant RAC65 cells; however, co-transfection of hRAR alpha, hRAR beta or hRAR gamma 1 restores the RA-dependent induction of CAT activity. These results clearly show that RAR alpha and RAR gamma 1 can transactivate the RAR beta gene; that RAR beta can stimulate its own expression and that resistance to RA in RAC65 cells is probably due to the altered RAR alpha transcript present in these cells.