Departments of Biochemistry and Medicine, University of Vermont, Burlington, .... FIG. 1. Hypothetical cleavage pathways during the activation offVIII by thrombin.
Proc. Nati. Acad. Sci. USA Vol. 86, pp. 6508-6512, September 1989 Biochemistry
Differential proteolytic activation of factor VIII-von Willebrand factor complex by thrombin (coagulation/hemophilia/proteolytic regulation)
DAVID C. HILL-EUBANKS, CARLO G. PARKER, AND PETE LOLLAR* Departments of Biochemistry and Medicine, University of Vermont, Burlington, VT 05405
Communicated by K. M. Brinkhous, May 26, 1989
Recently, we observed that both a purified 240-kDa fVIII heterodimer and isolated fVIIILC bind vWf with a stoichiometry of one molecule per vWf subunit (13). Cleavage of fVIIILC is associated with loss of the vWf binding function. Thus, the function of the cleavage of fVIIILc by thrombin may be to dissociate fVIII from vWf, whereas heavy-chain cleavage independently activates the cofactor function of fVIII. This predicts that intermediate I in Fig. 1 would be active in the absence of vWf. Also, if fVIII bound to vWf is unable to function as a cofactor, then vWf should inhibit the activity ofthis intermediate since it would bind vWf by means of its intact light chain. To test this hypothesis, we screened several snake venoms and subsequently isolated an enzyme from Bothrops jararacussu that produces this intermediate. In this report we examine the functional properties of the venom enzyme-modiflied fVIII in the presence and absence of vWf.
ABSTRACT Blood coagulation factor VIII (fVI) is a plasma protein that is decreased or absent in hemophilia A. It is isolated as a mixture of heterodimers that contain a variably sized heavy chain and a common light chain. Thrombin catalyzes the activation of fVIII in a reaction that is associated with cleavages in both types of chain. We isolated a serine protease from Bothropsjararacussu snake venom that catalyzes thrombin-like heavy-chain cleavage but not light-chain cleavage in porcine fVIII as judged by NaDodSO4/PAGE and N-terminal sequence analysis. Using a plasma-free assay of the ability of activated fVIII to function as a cofactor in the activation of factor X by factor IXa, we found that fVIII is activated by the venom enzyme. The venom enzyme-activated fVIII was isolated in stable form by cation-exchange HPLC. von Willebrand factor inhibited venom enzyme-activated fVIII but not thrombin-activated fVIII. These results suggest that the binding of fVIII to von Willebrand factor depends on the presence of an intact light chain and that activated fVIII must dissociate from von Willebrand factor to exert its cofactor effect. Thus, proteolytic activation of fVIII-von Willebrand factor complex appears to be differentially regulated by lightchain cleavage to dissociate the complex and heavy-chain cleavage to activate the cofactor function.
MATERIALS AND METHODS All procedures were carried out at room temperature unless indicated otherwise. Materials. Crude snake venoms, Mes, and L-histidine were purchased from Sigma. Thrombocytin was purchased from Calbiochem. Sephadex G-150 and Mono S (HR 5/5) were purchased from Pharmacia. Heparin-Sepharose was a gift from Alpha Therapeutics (Los Angeles). Porcine intestinal heparin was purchased from United States Biochemical. All other materials were reagent grade or better. Small unilamellar phosphatidylcholine/phosphatidylserine (75:25, wt/wt) vesicles were a gift from Sriram Krishnaswamy (University of Vermont). Freshly isolated, washed human platelets were a gift from Paula Tracy (University of Vermont). Coagulation Proteins. Porcine coagulation proteins (7, 14), 125I-labeled fVIII (1251-fVIII) (14), human fibrinogen (15), and vWf (16) were isolated according to published procedures. Porcine fVIII was assayed as described previously (8). The specific activity of I251-fVIII was 7 x 10 dpm/unit. Extinction Coefficients and Molecular Masses. The concentration of protein in crude venoms was estimated by measuring the absorbance at 280 nm and by assuming an extinction coefficient (e0.) of 1.0. Extinction coefficients for thrombin (14), fVIII and activated fVIII (7, 13, 17), and vWf (16) have been previously reported. The extinction coefficient of the purified BJV-VIIIcp was determined to be 1.6 by the method of van lersel et al. (18). Molecular masses used
Factor VIII (fVIII) and von Willebrand factor (vWf) are plasma proteins that circulate as a tightly bound noncovalent complex. Following proteolytic activation, fVIII functions as a cofactor in the activation of factor X by factor IXa along the intrinsic pathway of blood coagulation (for review, see ref. 1). vWf is necessary for normal adhesion of platelets to the vessel wall (for review, see ref. 2) and additionally prolongs the life of fVIII in plasma (3). fVIII is synthesized as a single-chain molecule, predominantly in hepatocytes (reviewed in ref. 4). The derived amino acid sequence of human fVIII reveals internal homology that defines three A regions or domains, two C domains, and a large B domain (Fig. 1) (5, 6). The domains are arranged in the sequence A1-A2-B-A3-C1-C2. Purified human and porcine fVIII consist of a mixture of heterodimers, each of which contains a variably sized N-terminal heavy chain and a common C-terminal light chain (fVIIILC) (Fig. 1) (6, 8, 9). Heterodimers result from variable proteolysis in the B domain, perhaps prior to secretion from the cell (10). In a system containing purified factors IXa and X and phospholipid, the isolated fVIII heterodimers possess no measurable cofactor activity (11). Thrombin activates fVIII in either the presence or absence of vWf. The development of cofactor activity is associated with heavy-chain cleavages at positions 372 and 740 and a single light-chain cleavage at position 1689 (12). The mechanism by which activated fVIII exerts its cofactor function is not known.
Abbreviations: fVIII, factor VIII; BJV-VIIIcp, Bothrops jararacussu venom fVIII coagulant protein; fVIIIA1 A2, heavy-chain species of fVIII that lacks the entire B domain; fVIIIAlA2/Lc, fVIII heterodimeric species that lacks the entire B domain; fVl11a1la, thrombin (factor IIa)-activated factor VIII; fVIIIaBJ, BJVVIIIcp-activated fVIII; fVIIIA1, fVIIIA2, and fVIIIA3 Cl c2 are subunits of fVIIIaIia that contain the indicated domains; fVIIILC, light chain of fVIII; vWf, von Willebrand factor. *To whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 6508
Biochemistry: Hill-Eubanks et al.
Proc. Natl. Acad. Sci. USA 86 (1989) r--------------------
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FIG. 1. Hypothetical cleavage pathways during the activation offVIII by thrombin. The sequence of domains for single-chain fVIII is shown at the top along with relevant amino acid numbers (5, 6). The stippled and filled blocks refer to the acidic regions between the Al and A2 domains and the B and A3 domains, respectively. Only two heterodimer forms of fVIII are shown for simplicity. Assuming that cleavage at position 740 is fast relative to other cleavages, two kinetic pathways are possible. Intermediate I is formed by cleavage between the Al and A2 domains.
Intermediate II is formed by cleavage of the filled block from the N terminus of the light chain. The final product is an active heterotrimer (7). We tested the hypothesis that intermediate I is active in the absence of vWf.
28 kDa (by NaDodSO4/PAGE); fVIII, 200 kDa (average of the heterodimeric mixture by NaDodSO4/ PAGE); fVIIIaIia and fVIIIaBJ, 160 kDa (7); thrombin, 37 kDa (14); vWf subunit, 270 kDa (19). Venom Screening Procedure. Crude, lyophilized snake venoms were dissolved in 0.15 M NaCl/0.02 M Tris Cl (pH 7.4) and stored at a final concentration of 2-15 mg/ml at -200C. Snake venoms (0.01 mg/ml) were incubated for 90 min with 125I-labeled fVIII (6 x 105 dpm/ml) in 0.15 M NaCl/0.02 M Tris Cl/0.01% Tween-80/5 mM CaC12 (pH 7.4). Proteolytic activity was analyzed with NaDodSO4/PAGE followed by autoradiography. Activated fVIII Assay. A chromogenic substrate assay of factor Xa production by an enzymatic complex consisting of factor IXa, phosphatidylcholine/phosphatidylserine vesicles, calcium, and limiting amounts of fVIII cleaved by BJV-VIIIcp or thrombin was used to measure activated fVIII as described previously (11). The measured value is the initial velocity of activation of factor X, which is linearly proportional to the activated fVIII in the sample. Isolation of fVITI Activator from B. janracussu Venom. Crude, lyophilized B. jararacussu venom (450 mg) was dissolved in 15 ml of buffer A (0.02 M Mes at pH 6.0) containing 0.1 M NaCl and 1 mM benzamidine and was applied to a Sephadex G-150 column (2.5 x 90 cm) at 0.4 ml/min. Fractions from this and successive steps were assayed for the desired proteolytic activity toward 125"-labeled fVIII. Fractions from the Sephadex G-150 column that contained activity were pooled and applied at 0.5 ml/min to a heparin-Sepharose column (2.5 x 20 cm) equilibrated in buffer A containing 0.1 M NaCl and 1 mM benzamidine. After the column was washed with buffer A containing 0.5 M NaCl and 1 mM benzamidine, the activator was eluted by using a 500-ml gradient of 0.5-1.5 M NaCl in buffer A at a flow rate of 1 ml/min. The activator emerged in 1.0 M NaCI. The preparation was dialyzed against buffer A containing 1 mM benzamidine and applied to a Mono S HR 5/5 column at 1.0 were BJV-VIIIcp,
a
ml/min. The activator was eluted by using a 20-ml linear gradient of 0.2-1.0 M NaCl. The fVIII activator emerged in 0.8 M NaCl and migrated as a single band when analyzed by NaDodSO4/PAGE. The purified enzyme is called B. jararacussu venom fVIII coagulant protein or BJV-VIIIcp. The major purification occurred during the heparinSepharose step since BJV-VIIIcp bound tightly to the resin. Approximately 5 mg of activator was recovered from 450 mg of crude venom. The apparent molecular mass of BJV-VIIIcp by NaDodSO4/PAGE was 28 kDa and 33 kDa under nonreducing and reducing conditions, respectively. After verifying freeze-thaw stability, BJV-VIIIcp was stored at -70°C. Isolation of BJV-VlllcrActivated fVIII and ThrombinActivated fVIII. We have recently found that thrombinactivated fVIII can be isolated by using Mono S chromatography in a form that is stable for at least several weeks at 4°C (7). The venom enzyme-activated fVIII, called fVIIIaBJ, was prepared by using the same procedure with slight modifications. Briefly, BJV-VIIIcp (20 nM) was reacted with fVIII (130 nM) in 0.16 M NaCI/0.01 M Tris Cl/5 mM CaCl2/0.01% Tween-80 (pH 7.4) until peak activity was reached. Heparin (5 units/ml) was added to stop the reaction. A sufficient volume of 0.03 M histidine hydrochloride/S mM CaCl2/ 0.01% Tween-80 (pH 6.0) was added to bring the final pH of the reaction mixture to 6.2. The reaction mixture was applied to a Mono S HR 5/5 column equilibrated in 0.1 M NaCl/0.01 M histidine hydrochloride/S mM CaCl2 (pH 6.0) at 2 ml/min. fVIIIaBJ was eluted with a 20-ml linear gradient of 0. 1-0.8 M NaCI at 1 ml/min. A single peak eluted at 0.64 M NaCl as judged by the absorbance at 280 nM. The activity of fVIIIaBJ was stable for weeks when stored in the elution buffer at 4°C. Electrophoresis. NaDodSO4/7% PAGE was done using the Laemmli buffer system (20) and silver staining of proteins (21) as described (22). Each lane was loaded with 400 ng of protein. Apparent molecular masses were determined by using molecular size standards from Bethesda Research Laboratories.
6510
Biochemistry: Hill-Eubanks et al.
N-Terminal Sequence Analysis of fVIla5j. fVIIIaBJ was exchanged into 0.05% NaDodSO4/1 mM dithiothreitol and concentrated to 0.25 mg/ml by repeated ultrafiltration and dilution with a Centricon 10 membrane. Following NaDodS04/PAGE and staining with Coomassie blue, the three major bands labeled LC, Al, and A2 in Fig. 4 were electroblotted onto a polyvinylidene difluoride membrane and sequenced as described (23). Platelet Aggregation and Fibrinogen Clotting Assay. Fibrinogen clotting activity was measured as previously described (22). Aggregation of washed human platelets by BJV-VIIIcp was studied in a BioData model PAP-3 aggregometer by using
Proc. Natl. Acad. Sci. USA 86 (1989)
40-
30
-
0
conditions essentially as described previously for the study of
platelet aggregation by thrombocytin (24, 25).
RESULTS We screened 65 commercially available crude snake venoms for an enzyme that catalyzes thrombin-like cleavages in fVIII heavy chains but not fVIIILC. All 8 venoms that were screened from snakes belonging to the Bothrops genus (B. jararacussu, B. jararaca, B. cotiara, B. atrox, B. alternata, B. medusa, B. neuwieldi, and B. nummifer) appeared to possess the desired activity. The least expensive venom, from B.jararacussu, was chosen for further characterization. Preliminary studies showed that cleavage of fVIII after incubation with crude venom was associated with the activation of fVIII. The fVIII activator in the venom, designated B. jararacussu venom fVIII coagulant protein or BJV-VIIIcp, was purified as described in Materials and Methods, yielding a product with an apparent molecular mass of 28 kDa that was greater than 95% pure as judged by NaDodSO4/PAGE. In addition to its activity toward fVIII, BJV-VIIIcp catalyzed the hydrolysis of the chromogenic substrate S2238 (HD-phenylalanyl-L-piperyl-L-arginyl-p-nitroanilide). BJVVIIIcp did not appear to be homologous to another Bothropsderived protein, batroxobin (26), since it had no fibrinogenolytic activity. However, BJV-VIIIcp caused aggregation of washed human platelets in a manner similar to that previously described for thrombocytin from B. atrox (24, 25), suggesting that these enzymes may be homologous. Benzamidine and diisopropyl fluorophosphate inhibited the activity of BJV-VIIIcp toward S2238 and fVIII, indicating that the enzyme is a trypsin-like serine protease. Additionally, incubation of BJV-VIIIcp with [3H]diisopropyl fluorophosphate, followed by NaDodSO4/PAGE, and autoradiography resulted in the appearance of a band that comigrated with the 28-kDa band. This suggests that the observed activity is not due to a trace contaminant in the preparation. The kinetics of activation of fVIII by BJV-VIIcp and thrombin were compared by using the assay described in Materials and Methods. Like thrombin, BJV-VIIIcp activates fVIII in a process that is characterized by a peak and decline of activity (Fig. 2). The mechanism underlying the decay of activity is not known but does not appear to be due to proteolysis (11, 14). The peak activity generated by BJVVIIQcp was 60%o of that generated by thrombin. In contrast, when BJV-VIIIcp was omitted, there was no detectable activity (less than 1% of the peak level). These results indicate that BJV-VIIIcp is a potent activator of fVIII but suggest that the activated species, designated fVIIIaBJ, may have less activity than thrombin-activated fVIII (fVlllaiia). Fig. 3 shows the proteolytic cleavage pattern of fVIII corresponding to the experiment described by Fig. 2. Cleavage of fVIII heavy chains by BJV-VIIIcp appears identical to that catalyzed by thrombin. The disappearance of heavychain species larger than fVIIIA1_A? is fast relative to the appearance of the final heavy-chain products, fVIIIA1 and fVIIIA2 (subunits of fVIIIaiia that contain the Al or A2 domain, respectively), indicating that fVIIIA1 A2 is present in
0
10
20
30
40
50
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FIG. 2. Activation of fNIMl by BJV-VIIIcp and thrombin. fVIII (130 nM) was reacted with BJV-VIIIcp (6 nM, o) or thrombin (0.1 nM, *) in 0.15 M NaCl/0.02 M Tris Cl/0.01% Tween-80 (pH 7.4). At various times, samples were removed, and activated fVIII was measured using a chromogenic substrate assay.
intermediates generated by both activators. fVIIIA1 A2 lacks the entire B domain and is present in the starting material. Considerable evidence indicates that cleavage between the Al and A2 domains is required for the activation of fVIII (7, 9, 12). Fragments containing the B domain are not visualized with this staining method (6). In contrast to thrombin, addition of BJV-VIIIcp to fVIII results in only the faint appearance of a band corresponding to the thrombin-cleaved light chain, fVIIIA3 Clc2. This indicates that a species of activated fVIII can form without cleavage of the light chain. This was demonstrated further by isolating fVIIIaBJ. We have recently shown that fVIIIaIIa can be isolated in stable form by cation-exchange HPLC (7). fVIIIaBj was isolated by using the same procedure (see Materials and Methods) and -^,M2
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FIG. 3. Cleavage of fVIII by BJV-VIIIcp and thrombin. Samples from the experiment described in Fig. 2 containing thrombin (Upper) or BJV-VIIIcp (Lower) were removed and analyzed by NaDodSO4/ 7% PAGE. HC, fVIII heavy chains; LC, fVIIILC; Al, fVIIIAl; A2,
fVIIIA2; A3-Cl-C2, fVIIIA3-c1-c2.
Biochemistry: HHI-Eubanks et al.
Al-A
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FIG. 4. Subunit structures of fVIIIaBJ and fVIIIaIIa. fVlIaBj (lane A) and fVIIIaIIa (lane B) were isolated by chromatography on Mono S and analyzed by NaDodSO4/7% PAGE. LC, fVIIILC; Al, fMIIIAl; A2, fVIIIA2; A3-C1-C2, fVIIIA2.c1C2.
was stable for at least several weeks at 40C. Fig. 4 shows a comparison of NaDodSO4/PAGE analysis of the fVIIIaBJ and fVIIIaIIa preparations. fVIIIaiia is a heterotrimer containing subunits fVIIIA1, fVIIIA2, and fVIIIA3 Cl c2 (7). The fVIIIaBJ preparation appears to consist of a heterotrimer containing fVIIILC, fVIIIA1, and fVIIIA2 as subunits. Additionally, there is a minor band corresponding to fVIIIAl-A2, indicating the presence of the fVIIIA1-A2/Lc (a heterodimeric species that lacks the entire B domain) in the preparation. Attempts to prepare fVIIIaBJ free of fVIIIA1-A2/Lc were unsuccessful since prolonged incubation of fVIII with BJVVIIIcp was associated with proteolytic degradation of fVIIIA1. The specific activity of the fVIIIaBe preparation was 40% of the fVIIIaIia preparation. Since fVIIIA1_A2/Lc has no detectable activity unless activated by thrombin (7, 9), its presence in the fVIIIaBJ preparation results in a slight underestimation of the functional activity of fVIIIaBJ. The fragments labeled LC, Al, and A2 in Fig. 4 were isolated and subjected to N-terminal sequence analysis (Table 1). The N-terminus of fVIIILc has not been modified by BJV-VIIIcp. This excludes an activation of fVIII by an electrophoretically invisible cleavage of a small peptide from the N terminus of fVIIILC. Additionally, BJV-VIIIcp, like thrombin, cleaves fVIII at Arg-372. vWf binds fVIIILc but does not appear to bind thrombincleaved fVIIILC (16). Thus, the cleavage of fVIIILc by thrombin may function to dissociate fVIII from vWf. If
dissociation of activated fVIII from vWf is required for intrinsic pathway factor X activation, then vWf should inhibit fVIIIaBJ but not fVIIIaiia. Using isolated fVIIIaIIa and fVIIIaBe, we found this to be the case. Fig. 5 shows that increasing concentrations of vWf cause progressive inhibition of fVIIIaBJ but have no effect on fVIIIaIIa. The inhibition curve is consistent with our previous finding that each subunit in multimeric vWf can bind a single fVIII heterodimer. The fact Table 1. N-terminal sequence analysis of subunits of fVIIIaBJ Human fVIIILc* 1649-EITRTTLQ Porcine fVIIILct DISLPTFQ
fVIIIaBJ LCO Human fVIIIaAl* Porcine fVIIIaAlt
6511
-
A3-C1-C2i-
A2
Proc. Natl. Acad. Sci. USA 86 (1989)
ISLP?FQ
1-ATRRYY ?IRRYY AI??YY fVIIIaBj A1t Human fVIIIaA2* 373-SVAKK Porcine fVIIIaA2t ?VAKK fVIIIaBj A2t SVAKK ?, Unidentified residues. Numbers preceding the sequences correspond to residue numbers in human fVIII. The single-letter code for amino acid residues is used (27). *Derived from human cDNA sequence (5). tFrom ref. 6. tBands defined in Fig. 4.
1 2 3 Mol of vWf subunit/mol fVIII
FIG. 5. Inhibition of fVIIIaBj by vWf. Purified, stable fVIIIajia (1 nM, *) or fVIIIaBJ (10 nM, *) was mixed with vWf for 10 sec in 0.15 M NaCI/0.02 M Tris Cl/0.01% Tween-80 (pH 7.4) at the indicated molar ratios. Longer incubations with vWf gave similar results. Activated fVII was measured by using a chromogenic substrate assay.
that the plot does not show straight line inhibition with an
x-intercept of 1 may indicate competition of phosphatidylcholine/phosphatidylserine vesicles for vWf (28). fVIIIaBJ also was inhibited by adding vWf to the activation mixture in the experiments described in Fig. 2. At the indicated times, aliquots of the activation mixtures were added to a solution containing a 4-fold molar excess of vWf subunits prior to diluting the activation mixture into the factor IXa/phosphatidylcholine/phosphatidylserine solution. The addition of vWf resulted in greater than 90% inhibition of fVIIIaBJ activity but had no effect on fVIIIaIIa activity (data not shown).
DISCUSSION Our results delineate distinct functional properties associated with cleavage of fVIII heavy chains and fVIIILC. In the presence of vWf, fVIII cleaved in both the light chain and heavy chains is required for the development of activity. However, in the absence of vWf, fVIIILC cleavage is not required to produce a species with substantial activity. fVIIIaBJ is inhibited by vWf, presumably since vWf binds to the intact fVIIILc and prevents fVIIIaBJ from binding phospholipid. These findings are in agreement with previous studies that demonstrated that the interaction of fVIII with vWf is light chain-mediated and that the acidic 41-residue Nterminal fragment that is cleaved from fVIIILc by thrombin plays a critical role in the binding of fVIII to vWf (16, 29). Both thrombin and BJV-VIIIcp cleave fVIII at Arg-372 between the Al and A2 domains of fVIII. Like thrombin, BJV-VIIIcp also appears to cleave fVIII at or near Arg-740. The exact position ofthis cleavage site has not been identified because the B-domain fragment has not been isolated and sequenced. That the activation of fVIII by BJV-VIIIcp is associated with cleavage at Arg-372 supports previous assertions that cleavage of fVIII between the Al and A2 domains is necessary for activation (7, 12, 30). It is interesting to note that activated factor V, which is homologous to fVIII, is a heterodimer (31), whereas fVIIIaIIa is a heterotrimer (7). The extra subunit in fVIII is generated by cleavage at Arg-372. There is an acidic region N-terminal to Arg-372 that is not homologous to factor V (32). Although activation ofthis region appears crucial, its function is not known.
6512
Biochemistry: Hill-Eubanks et al.
Our results do not support all of the conclusions by Pittman and Kaufman (30) from their analysis of site-directed mutants of human fVIII. They found that mutation of arginine to isoleucine at residue 372 resulted in a product that could not be activated by thrombin. Our results are consistent with this conclusion. However, they also found that mutation of arginine to isoleucine at residue 1689 resulted in a nonactivatable product. This is not consistent with our results. Since our product has been isolated in a stable form free of vWf, we think that the inactivity characterized by the mutation at 1689 results from the presence of vWf associated with fVIII or from a secondary structural change induced by the amino acid alteration. We thank Dr. Sriram Krishnaswamy for reviewing the manuscript. This work was supported by an American Heart Association Established Investigator Award to P.L., by a grant-in-aid from the National Institutes of Health (HL-40921), and by the Vermont Specialized Center of Research in Thrombosis (HL-35058). 1. Mann, K. G., Jenny, R. J. & Krishnaswamy S. (1988) Annu. Rev. Biochem. 57, 915-956. 2. Ruggeri, Z. M. & Zimmerman, T. S. (1985) Semin. Hematol. 22, 203-218. 3. Brinkhous, K. M., Sandberg, H., Garris, J. B., Mattson, C., Palm, M., Griggs, T. & Read, M. S. (1985) Proc. Nati. Acad. Sci. USA 82, 8752-8755. 4. Kane, W. H. & Davie, E. W. (1988) Blood 71, 539-555. 5. Vehar, G. A., Keyt, B., Eaton, D., Rodriguez, H., O'Brien, D. P., Rotblat, F., Oppermann, H., Keck, R., Wood, W. I., Harkins, R. N., Tuddenham, E. G. D., Lawn, R. M. & Capon, D. J. (1984) Nature (London) 312, 337-342. 6. Toole, J. J., Knopf, J. L., Wozney, J. M., Sultzman, L. A., Buecker, J. L., Pittman, D. D., Kaufman, R. J., Brown, E., Shoemaker, C., Orr, E. C., Amphlett, G. W., Foster, W. B., Coe, M. L., Knutson, G. J., Fass, D. N. & Hewick, R. M. (1984) Nature (London) 312, 342-347. 7. Lollar, P. & Parker, C. G. (1989) Biochemistry 28, 666-674. 8. Fass, D. N., Knutson, G. J. & Katzmann, J. A. (1982) Blood 59, 594-600. 9. Fay, P. J., Anderson, M. T., Chavin, S. I. & Marder, V. J. (1986) Biochim. Biophys. Acta 871, 268-278.
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