The effect of von Willebrand factor on activation of factor VIII by factor Xa
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1 Eur. J. Biochem. 189, (1990) 0 FEBS 1990 The effect of von Willebrand factor on activation of factor VIII by factor Xa Johannes A. KOEDAM, Rob J. HAMER, Nel H. BEESER-VISSER, Bonno N. BOUMA and Jan J. SIXMA Department of Haematology, University Hospital Utrecht, The Netherlands (Received August 31/December 15, 1989) - EJB Factor VIII has to be activated before it can serve efficiently as a cofactor in the intrinsic pathway of blood coagulation. This activation occurs through specific proteolytic cleavages in the molecule by either thrombin or factor Xa. In this study, we show that von Willebrand factor inhibits the activation of factor VIII by factor Xa. Incubation of factor VIII (30 Ujml) with 0.1 pg/ml factor Xa resulted in a 1.6-fold activation followed by a decay of coagulant activity. In the presence of 10 pg/ml von Willebrand factor, activation and inactivation of factor VIII was completely inhibited. In contrast, the activation of factor VIII by thrombin was not influenced by von Willebrand factor. At high concentrations of factor Xa (10 pg/ml), von-willebrand-factor-bound factor VIII could be cleaved and activated. The generated proteolytic fragments were identical to the fragments produced in the absence of von Willebrand factor and all fragments were released from von Willebrand factor. The major products were light-chain-derived fragments of molecular mass 66/68 kda and 60 kda and heavy-chain-derived fragments of 40 and 42 kda. Also minor products of 12,20/21,23,27 and 30 kda were observed, most of which were specific for cleavage of factor VIII by factor Xa. Factor VIII is an important plasma protein which acts as a cofactor in the intrinsic pathway of coagulation. It does so by dramatically increasing the V,,, for the activation of factor X by the enzyme factor IXa in the presence of calcium and a phospholipid surface [I]. The critical role of factor VIII in hemostasis is illustrated by the severe bleeding tendency (known as hemophilia A) in its absence [2]. It is a key protein in the regulation of the coagulation cascade, since its cofactor activity can be enhanced through feed-back mechanisms by thrombin and factor Xa. Factor VIII activity is subsequently destroyed by the regulatory-enzyme-activated protein C. Both activation and inactivation of factor VIII occur through specific proteolytic cleavages within the molecule [3-51. Factor VIII is synthesized as a single-chain precursor molecule of 2332 amino acids [6, 71 and purified from plasma as a carboxy-terminal light chain of 80 kda which is associated with an amino-terminal heavy chain ranging in molecular mass between kda [4, In many studies, the kinetics of factor-viii and factor-x activation [I, , as well as the changes that occur within the factor VIII molecule upon activation by thrombin or factor Xa [3, 4, 17, 191, have been described. These studies have ignored, however, that factor VIII circulates as a non-covalent complex with von Willebrand factor [20]. Formation of this complex is of great physiological importance for factor VIII, since it prolongs its half-life in the circulation [21,22]. Since von Willebrand factor is a very large glycoprotein which represents 99% of the mass of the factor-viii - von-willebrand-factor complex, it is conceivable that it modulates the activation and inactivation of factor VIII. In a previous paper [23], we followed the proteolytic steps involved in activation of factor VIII by thrombin and the Correspondence to J. J. Sixma Department of Haematology, University Hospital Utrecht, P.O. Box 85500, NL-3508 GA Utrecht, The Netherlands Abbreviation. BSA, bovine serum albumin. release of activated factor VIII (factor VIIIa) from von Willebrand factor. The present study was initiated to investigate the effect of von Willebrand factor on the activation of factor VIII by factor Xa and thrombin and the release of factor VIII proteolytic products from von Willebrand factor. MATERIALS AND METHODS Materials Thrombin (T-6759), hirudin (grade IV), and bovine serum albumin were from Sigma Chemical Co. St Louis, MO). Triton X-100 and Tween-20 were obtained from E. Merck AG (Darmstadt, FRG). Cephalin from an APTT kit (Boehringer Mannheim GmbH, Mannheim, FRG) was used as a source of phospholipids. Its phospholipid content was determined according to Fiske and Subbarow [24]. The chromogenic substrate N-benzoyl-L-isoleucyl-L-glutamylglycyl-L-arginine p-nitroanilide (S2222) was purchased from KabiVitrum (Stockholm, Sweden). All other chemicals were of the highest grade available. Proteins Factor VIII was purified from commercial human factor VIII concentrate (Hyland Hemofil HT) and radiolabeled with Na' 251 (Amersham, Buckinghamshire, UK) as described previously [Ill. The resulting factor VIII had a specific activity of 8000 U/mg and a specific radioactivity of 10 pci/pg. The preparation was stored at -70 C in 50 mm Mes buffer ph6.5, containing 150 mm NaCl, 50 mm CaC12, 1 mm benzamidine and 0.005% Triton X-100. In addition, the radiolabeled factor VIII contained 10 mm 6-aminohexanoic acid, 10 pg/ml soybean trypsin inhibitor and 0.1 % bovine serum albumin (BSA). Factor VIII activity was measured with a one-stage clotting assay [25,26] using kaolin/cephalin from
2 230 Boehringer Mannheim GmbH (Mannheim, FRG) and factor- VIII-deficient plasma from a severe hemophilia A patient. von Willebrand factor was obtained during the purification of factor VIII as described elsewhere [ll, 271. The preparation had a specific activity of 100 U von Willebrand factor antigen/mg and no detectable Factor VIII (< 1 mu/ml); 1 U factor VIII or von Willebrand factor is defined as the amount present in 1 ml of a fresh frozen pool from 40 healthy donors. Purified human factor Xa was a generous gift of Dr J. C. M. Meijers from our laboratory [28]. This material contained no detectable prothrombin or thrombin as judged by gel electrophoresis. Activation of,factor VIII by factor Xa Factor VIII (final concentration 30 Ujml) was incubated at 37'C with factor Xa (0.1 pg/ml, 2 nm) in a final volume of 100 pl Michaelis buffer containing 8 pg/ml phospholipid, 10 mm CaCl, and 0.3% BSA. von Willebrand factor was included in the reaction mixture at concentrations varying between 0-30 pg/ml. At various times, 5-p1 aliquots were removed and diluted 1000-fold in ice-cold Michaelis buffer and tested for factor VIII coagulant activity. Control experiments indicated that the amount of factor Xa still present in the diluted samples did not influence the factor VIII activity during the coagulation assay. Neither did the factor Xa in the sample significantly shorten the clotting time of hemophilic plasma. In a parallel set of experiments, factor VIII activation by thrombin was tested. For these incubations, final concentrations of 0.5 U/ml thrombin and 5 mm CaC1, were used, while phospholipids were omitted from the mixture. At various times, aliquots from the incubation mixture were diluted in ice-cold Michaelis buffer containing 0.05 U/ml hirudin. Otherwise, the conditions were as described for the activation with factor Xa. Proteolysis of factor VIII To study the degradation of factor VIII by factor Xa, factor VIII (0.5 U/ml, cpm) was incubated at 37 C with factor Xa (10 pg/ml, 200 nm) in 100 p1 Michaelis buffer (28.5 mm sodium barbital, 28.5 mm sodium acetate, 116 mm NaCI, ph 7.35) containing 8 pg/ml phospholipid, 5 mm CaCI, and 0.01% BSA in the absence or presence of 25 pg/ml von Willebrand factor. At the indicated times, 8-pl aliquots were removed, added to 50 pl non-reducing electrophoresis sample buffer and immediately boiled for 5 min. The samples were analyzed on SDS gels (see below). Binding of factor VIII to von Willebrand factor and elution qf the bound factor VIII Polystyrene microtiter wells (Removawell strips, Greiner B.V., Alphen a/d Rijn, The Netherlands) were coated with von Willebrand factor (100 pl, 1 pg/ml in Michaelis buffer) for 2 h at 37T, followed by blocking of the wells with 3% BSA/O.l% Tween-20 [29] factor VIII (I0000 cpm/well) was bound to the immobilized von Willebrand factor for 2 h at 37 C and the wells were washed with 0.3% BSA/O.l% Tween-20. This binding system has been described and characterized before [29]. To study the release of proteolytic products of factor VIII from von Willebrand factor, the wells were incubated at 37 C with factor Xa (10 pg/ml, 200 nm) in a Fig. 1. Proteolysis offactor VIIZ. lz5i-factor VIII (30000 cpm, 0.5 U/ ml) was incubated with thrombin (10 Ujml) or factor Xa (10 pg/ml) for 5 min at room temperature. In the case of factor Xa, cephalin (0.025 vol.) and CaCI, (5 mm) were included. The proteins were analyzed by electrophoresis on SDS 5-15% gradient polyacrylamide slab gels followed by autoradiography. The molecular mass (kda) of the bands is indicated. Lane 1, factor VIII; lane 2, factor VIII cleaved by thrombin; lane 3, factor VIII cleaved by factor Xa volume of 100 yl containing 8 pg/ml phospholipids, 10 mm CaC1, and 0.01 YO BSA. At different times, 50-p1 aliquots were removed and added to 30 pl non-reducing electrophoresis sample buffer. The wells were washed five times and the remaining radioactivity was eluted with 80 pl sample buffer. In a parallel experiment, thrombin (0.5 Ujml) was used instead of factor Xa. In this case, the CaCI, concentration was 5 mm and phospholipid was omitted. Control incubations contained no enzyme to determine nonspecific (spontaneous) release or 250 mm CaCI, to determine the reversibility of factor VIII binding. The amount of radioactivity released from the wells and the amount remaining bound was counted in a Packard Autogamma 5210 gamma counter (Packard Instrument Co., Downers Grove, IL). Gel analysis SDS/polyacrylamide slab gel electrophoresis [30] was carried out with 5-15% polyacrylamide gradient gels. A mixture of high-molecular-mass proteins (Bio-Rad Laboratories, Richmond, CA) and low-molecular-mass proteins (Pharmacia Biotechnology, Uppsala, Sweden) was run on each gel as molecular mass markers. Gels were stained with Coomassie blue R-250 and dried between cellophane sheets (Bio-Rad). Autoradiography was performed using Kodak X-Omat AR5 film (Eastman Kodak Co., Rochester, NY) and DuPont Cronex Lightening Plus intensifying screens (DuPont Co., Wilmington, DE). The intensity of the bands on autoradiograms was quantified by scanning with a Beckman model CDS-200 densitometer (Beckman Instruments Inc., Fullerton, CA).
3 23 1 -vwf + vwf A 60/ 20 ' \ time (min) A B time (mid Fig. 2. Effect of von Willebrandfactor on factor- VIIIactivation. Factor VIII (30 Ujml) was activated with (A) factor Xa (0.1 pg/ml, 2 nm) or (B) thrombin (0.1 Ujml) in the absence (0) or presence of von Willebrand factor at (0) 1 pg/ml or (m) 10 pg/ml. At the indicated time points, factor VIII (FVIII) coagulant activity was determined and expressed as percentage of the initial activity RESULTS Identijkation of factor VIIIproteolytic fragments Partial proteolysis of 1251-factor VIII by thrombin and factor Xa is shown in Fig. 1. The light chain is seen as a doublet of 78/80 kda and the heavy chain is a heterogeneous mixture of 90 kda and kDa bands (Fig. 1, lane 1). After incubation with factor Xa the following proteolytic degradation fragments were identified (Fig. 1, lane 3) : a doublet of 66/68 kda and a band of 60 kda, which are products of the factor-viii light chain and further bands of 27, 30, 40 and 42 kda, together with small peptides of 12, 20/21 (a doublet) and 23 kda. Cleavage of 1251-factor VIII with thrombin resulted in fewer proteolytic products: a doublet of kda was seen together with bands of 50, 42, and 23 kda (Fig. 1, lane 2). Activation of factor VIII by factor Xa Incubation of factor VIII with factor Xa resulted in a 1.6- fold increase of coagulant activity (Fig. 2A). Peak activity was reached after 2 min, which was followed by inactivation. When the incubation was repeated in the presence of increasing amounts of von Willebrand factor, the rate and extent of factor VIII activation was concomitantly decreased. In the presence of 10 pg/ml von Willebrand factor, factor VIII was completely protected against activation and inactivation by B *y---- 0,,,, 0 " 6 - I - * time (mid Fig. 3. Effect of von Willebrand factor on factor- VIII proteolysis by factor Xa. (A) '251-factor VIII was incubated at 37 C with factor Xa as described for Fig. 1. At the indicated times, samples were removed and added to electrophoresis sample buffer containing 0.4% SDS to stop the reaction. von Willebrand factor (25 pg/ml) was either absent (-vwf) or present (f vwf) during the incubation. The molecular mass of marker proteins (in kda) is indicated. (B) Densitometric scan of factor-viii heavy and light chains. The 90-kDa and kDa (heavy chain) and 78/80-kDa (light chain) bands were scanned and the intensity of each band relative to the total absorbance in each lane was expressed as percentage of the original intensity. (0) Heavy chains without von Willebrand factor; (0) light chain without von Willebrand factor; (A) heavy chains with von Willebrand factor; (A) light chain with von Willebrand factor factor Xa (Fig. 2A). von Willebrand factor did not inhibit the enzyme itself, since it did not influence the amidolytic activity of factor Xa towards the chromogenic substrate. Furthermore, inclusion of hirudin (2 Ujml) in the incubation mixture did not influence the results (data not shown). To demonstrate that the effect of von Willebrand factor is specific for the activation of factor VIII by factor Xa, we activated factor VIII with thrombin (Fig. 2B). Addition of von Willebrand factor did not influence the activation and subsequent inactivation of factor VIII. The inhibitory effect of von Willebrand factor towards the activation of factor VIII by factor Xa could be caused by either a modification of the cleaved factor VIIIa molecule that alters its coagulant activity, or by direct inhibition of the proteolytic cleavage of factor VIII by factor Xa. To test these possibilities, we incubated '251-factor VIII with factor Xa in
4 time (mid Fig. 4. Elution offuctor VIII from immobilized von Willebrund factor. 1Z51-factor VIII was bound to immobilized von Willebrand factor and treated with factor Xa. At the indicated times, the radioactivity of the supernatant was determined and the wells were washed and counted. (0) Released radioactivity; (0) radioactivity remaining bound; ( W) released during control incubation in the absence of factor Xa; (LI) radioactivity remaining bound during control incubation ;(+) radioactivity remaining bound after washing the well with 0.25 M CaC1,; (0) remaining radioactivity after incubation with factor Xa for 120 min and additional washing with 0.25 M CaCI, the absence or presence of von Willebrand factor and followed its degradation on SDS/polyacrylamide gels. Fig. 3 shows that the breakdown of factor-viii heavy and light chains is markedly delayed in the presence of von Willebrand factor. To illustrate this observation more clearly, we have scanned the intensity of the factor VIII bands on the autoradiogram (Fig. 3 B). While in the absence of von Willebrand factor 91 9'0 of the 80-kDa light chain and the kDa heavy chains had disappeared within 1 min, 81% of the heavy chain and 74% of the light chain were still intact after 1 min when von Willebrand factor was present. After 10 min of incubation, these values were 33% and 24% for the heavy and the light chains, respectively. This result indicates that the formation of the factor-viii - von-willebrand-factor complex protects factor VIII from proteolytic cleavage by factor Xa. Release of factor VIII from von Willebrand factor '251-factor VIII was bound to von Willebrand factor immobilized on polystyrene wells. The action of factor Xa on the factor-viii - von-willebrand-factor complex was studied by quantification of the radioactivity and electrophoretic analysis of the material released from and remaining bound to the wells. Fig. 4 shows the release of radioactivity into the supernatant. Approximately 75% of the initially bound radioactivity was released from the well after 2 h at 37 C. The remaining activity could not be removed by additional incubation of the well with 0.25 M CaCl,. This result indicates that the material that remained associated after treatment with factor Xa represents non-specific binding of the labeled protein. Also a control incubation of the von-willebrandfactor-bound 1251-factor VIII with the high calcium concentration resulted in dissociation of 72% of the radioactivity (Fig. 4). Fig. 5 shows that all of the factor VIII polypeptides associated with von Willebrand factor were proteolyzed and released from the well. The fragments that appeared in the supernatant were identical to the fragments that were generated by treatment of factor VIII in solution with factor Xa (Fig. 1). A parallel incubation in which factor Xa was replaced by thrombin (Fig. 5B) showed that both chains of factor VIII were cleaved to a lesser extent, resulting in a 68/70-kDa doublet light-chain product (instead of the 66/68-kDa doublet after factor Xa treatment), while the 60-kDa fragment was absent. Heavy-chain fragments consisted of 50-kDa band (not present after factor Xa cleavage) and 42-kDa and 23-kDa bands. Further proteolysis into 40-kDa and 30/27/21/12-kDa fragments, as seen with factor Xa, was absent. The 42-kDa fragment produced by thrombin remained partially bound to von Willebrand factor (Fig. 5A). This phenomenon was not observed after cleavage of '251-fa~tor VIII by factor Xa. DISCUSSION This study shows that the activation and subsequent inactivation of factor VIII by factor Xa is inhibited by von Willebrand factor. Gel electrophoretic analysis indicates that von Willebrand factor prevents the proteolytic cleavages of factor VIII that are necessary for activation by factor Xa. Cleavage and concomitant activation of factor VIII by thrombin is not influenced by von Willebrand factor. The latter thus hinders one of the positive feed-back loops which are responsible for amplification of the coagulation cascade. This raises the question of whether factor Xa that has been generated through the extrinsic pathway by factor VIIa and tissue factor can be a physiological activator of factor VIII. If not, traces of thrombin have to be formed before factor VIII can be activated and the explosive formation of factor Xa and thrombin can start. Other investigators have found varying effects of factor Xa on the factor-viii - von-willebrandfactor complex. Using bovine proteins, van Dieijen et al. [I] were unable to show activation of factor VIII by factor Xa. On the other hand, Hultin [I31 and also Mertens and Bertina [I41 studied the activation of factor X in a system of human proteins. They found that preincubation with trace amounts of exogenous factor Xa abolishes the lag phase which normally precedes the generation of factor Xa. These authors ascribe a critical role to factor-xa feedback in the activation of factor VIII. Mertens and Bertina demonstrated that factor VIIl can be activated by factor Xa in plasma and that the necessary factor Xa can be generated by the extrinsic pathway [14]. Similarly, Neuenschwander and Jesty [18] recently described that the factor-viii - von-willebrand-factor complex could be activated by factor Xa in the presence of either phospholipid vesicles or activated platelets. These authors, however, found shorter lag times when more purified factor VIII was used, which could support our own finding of the inhibitory effect of von Willebrand factor. The observed discrepancies may merely result from differences in concentration of factor Xa and/or factor VIII used in the various systems. The inhibitory effect can be overcome by using higher factor Xa concentrations (not shown); Fig. 3 shows that at the high factor Xa concentration used to cleave '251-factor VIII, proteolysis is not completely blocked by von Willebrand factor. All of the factor VIII fragments that are formed upon cleavage by factor Xa are released from von Willebrand Factor (Fig. 5). This is in contrast to our earlier observation [23] that the 42-kDa fragment generated after cleavage of factor VIII with thrombin remained bound to von Willebrand factor. In the present study, we find that the thrombin-generated fragment is only partially bound to von Willebrand factor, while the majority is released into the supernatant (Fig. 5). The reason for the different behaviour is not clear, since no major changes in experimental conditions were introduced.
5 233 I / C THR C THR Fig. 5. Proteolysis ofjuctor VIII bound to immobilized von Willebrand factor factor VIII, bound to immobilized von Willebrand factor, was treated with factor Xa. The fragments that were released from or remained bound to the well were analyzed by SDS/PAGE. The times (min) are indicated at the bottom. (A) Fragments remaining bound. C, control incubation of 120 min in the absence of factor Xa; THR, incubation of 120 min where thrombin replaced factor Xa. (B) Fragments released from the well. C, released during control incubation of 120 min in the absence of factor Xa; THR, incubation with thrombin. Molecular masses (kda) are indicated on the sides There remains an apparent difference, however, between the association of the von Willebrand factor with the 42-kDa fragment produced after thrombin or factor Xa cleavage. Factor Xa cleaves factor VIII at the same sites as thrombin [4], although some additional sites exist that are unique for factor Xa. The common site in the light chain is after amino acid residue Arg-1689 and the specific factor Xa site is after Arg We found that the 78/80-kDa light chain of factor VIII is degraded to a new doublet of 66/68-kDa. This doublet has a slightly higher mobility than the doublet observed after cleavage by thrombin (68/70-kDa) (Figs 1 and 5). This difference has not been noted before and the reason for this variation is unknown at present. We also observe the 60-kDa light-chain fragment that is generated through the unique factor Xa cleavage site at Arg Considering its molecular mass, the 12-kDa product may well represent the factor-xagenerated light-chain fragment. Preliminary immunoprecipitation studies employing monoclonal antibodies to the factor VIII heavy and light chains confirm this interpretation (not shown). Apart from the 42-kDa fragment described above, various other fragments are derived from the factor-v1i1 heavy chain. 23-kDa fragment is shared with the thrombincleavage products, but products of 20/21,27,30, and 40-kDa are unique for factor-xa cleavage. The appearance of the 40- kda peptide seems to correlate with disappearance of the 42-kDa fragment, but direct evidence for such a precursor/ product relationship is lacking. The 42-kDa fragment is derived from the carboxy- terminus of the heavy chain by cleavages at positions 740 and 372 [4]. No further proteolysis of this fragment has been reported, however. We do not observe the amino-terminal 50-kDa fragment of the heavy chain which is also produced by cleavage at position 740. Presumably, factor Xa rapidly degrades this fragment further, possibly producing the peptides of lower molecular mass that are seen in Figs 1 and 5. Thrombin activation of factor VIII is not influenced by von Willebrand factor. Furthermore, activation by thrombin results in a factor VIIIa species with a higher activity than after activation by factor Xa (Fig. 2). This difference was also observed by other investigators [4, 15, 17, 181. Eaton et al. [4] have ascribed the moderate activation by factor Xa to inactivation of factor VIIIa by cleavage of the 50-kDa polypeptide at position 336. Another difference between activation by thrombin and factor Xa is the dependency of factor-xa activation on the presence of phospholipid. Although cleavage of 1251-factor VIII with a high concentration of factor Xa was possible in the absence of phospholipid (not shown), the enhancement of coagulant activity required phospholipid. This observation is in accordance with previously described results [ We now speculate that the protective effect of von Willebrand factor on activation and inactivation of factor VIIl by factor Xa is caused by its ability to block the phospholipid-dependent binding of factor Xa to factor VIII. von Willebrand factor, which binds to the light chain of factor VIII [29, 311, has been shown to compete with phospholipids in binding to factor VIII [32]; we have recently described a similar effect of von Willebrand factor on the inactivation of factor VIII by activated protein C [27], a reaction which is also dependent on phospholipid. Further studies are required to characterize further the mechanism and physiological relevance of the phenomenon described in this study. This work was supported by the Netherlands Organization of Pure Research (ZWO). REFERENCES 1. Van Dieijen, G., Tans, G., Rosing, J. & Hemker, H. C. (1981) J. Biol. Chem. 256, Mammen, E. F. (1983) Sem. Thromb. Hemostas. 9, Fulcher, C. A., Roberts, J. R. & Zimmerman, T. S. (1983) Blood 61, Eaton, D., Rodriguez, H. & Vehar, C. A. (1986) Biochemistry 25,
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