Proc. Nati. Acad. Sci. USA Vol. 89, pp. 11189-11193, December 1992 Biochemistry Fibrinogen blocks the autoactivation and thrombin-mediated activation of factor XI on dextran sulfate (coagulation/kininogen/contact system) CHERYL F. SCOTT AND ROBERT W. COLMAN* Sol Sherry Thrombosis Research Center, Temple University School of Medicine, 3400 North Broad Street, Philadelphia, PA 19140 Communicated by Irving M. Klotz, August 10, 1992

ABSTRACT The intrinsic pathway of blood coagulation is formation of fibrin-one component of the hemostatic plug. activated when factor XIa, one of the three contact-system Several persons have been described in the literature who enzymes, is generated and then activates factor IX. Factor XI lack either prekallikrein (2, 14-16) or factor XII (1, 7), and has been shown to be efficiently activated in vitro by surface- three cases of HK deficiency were reported in 1975 (17-19). bound factor XIIa after factor XI is transported to the surface These individuals did not reveal a history of abnormal by its cofactor, high molecular weight kininoen (HK). How- bleeding, and all had been discovered by routine preoperative ever, individuals lacking any of the three contact-system pro- coagulation tests. Patients with factor XI deficiency, how- teins-namely, factor XII, prekallikrein, and 11K-do not ever, may manifest hemostatic abnormalities, although they suffer from bleeding abnormalities. This mystery has led are variable and usually mild, as compared with individuals several investigators to search for an "alternate" activation lacking coagulation factors further down on the cascade. The pathway for factor XI. Recently, factor XI has been reported paradox of normal hemostasis in individuals with undetect- to be autoactivated on the soluble "surface" dextran sulfate, able amounts of one of the contact factors has led many and thrombin was shown to accelerate the autoactivation. investigators to search for an alternate pathway by which However, it was also reported that HK, the cofactor for factor factor XIa is generated in the absence of factor XIIa. Two XIIa-mediated activation of factor XI, actually diminishes the recent publications have implicated thrombin as the missing thrombin-catalyzed activation rate offactor XI. Nonetheless, it link (20, 21). Factor XI was reported to be autoactivated on was suggested that thrombin was a more efficient activator than the soluble negatively charged "surface" dextran sulfate factor XIIa. In this report we investigated the effect of fibrin- -(DS), and thrombin was found to enhance that activation (20, ogen, the major coagulation protein in plasma, on the activa- 21). However, both groups of investigators demonstrated tion rate of factor XI. Fibrinogen, the preferred substrate for that HK, the activation cofactor for factor XI, actually thrombin in plasma, virtually prevented autoactivation of attenuates the thrombin-mediated activation offactor XI (20, factor XI as well as the thrombin-mediated activation offactor 21). Despite this observation, thrombin was claimed to be XI, while having no effect on factor XIIa-catalyzed activation. superior to factor XIIa in its ability to activate factor XI, and HK dramatically curtailed the autoactivation of factor XI in a revision of the coagulation cascade was proposed (21). addition to the thrombin-mediated activation. These data However, the effect of competing substrates for thrombin in XI in plasma, especially its preferred substrate, fibrinogen, was not indicate that factor would not be autoactivated a plasma considered in the revised mechanism. environment, and thrombin would, therefore, be unlikely to In this report, we have investigated the effect offibrinogen potentiate the activation. We believe that the "myIsI path- and HK on the autoactivation rate offactor XI on DS as well way" for factor XI activation remains an enigma that warrants as on the thrombin and factor XIIa-mediated activation rate further investigation. of factor XI on DS. Our data indicate that fibrinogen abol- ishes, and HK dramatically curtails, both the autoactivation The initiation of the intrinsic pathway of blood coagulation and the thrombin-mediated activation offactor XI on DS. In involves four proteins that make up the contact-activation contrast, HK augments factor XIIa-mediated activation of system-namely, the zymogens factor XII (Hageman factor) factor XI, and fibrinogen does not influence this reaction. We (1), prekallikrein (Fletcher factor) (2), factor XI (plasma also show that thrombin is not a significant contributor to the thromboplastin antecedent) (3), and the cofactor-substrate, activation of factor XI in plasma, which contains both HK high molecular weight kininogen (HK) (4). This system is and fibrinogen. These results have been presented in abstract triggered in vitro by negatively charged surfaces or polymers form (22). to which factor XII can bind (5); factor XII then changes conformation (6) and expresses enzymatic activity (factor XIIa) (7). Both factor XI (8) and prekallikrein (9) circulate in MATERIALS AND METHODS plasma complexed with separate molecules ofHK that, upon Materials. Highly purified human factor XIIa (prepared activation to HKa by kallikrein (10), transport them to an from homogeneous factor XII, activated with DS, which was appropriate surface where factor XIIa awaits to activate each removed after activation), HK (>90% single chain in SDS/ zymogen. Once prekallikrein is activated by factor XIIa to 10%o polyacrylamide gel electrophoresis, 12.5 units/mg), kallikrein (10) (the active enzyme) (11), it in turn can rapidly fibrinogen (homogeneous in nonreducing SDS/5% polyacryl- activate additional molecules offactor XII (12), which serves amide gel electrophoresis and greater than 95% clottable), to amplify the contact system. Following activation offactor and corn trypsin inhibitor (single band on SDS/110 poly- XI by surface-bound factor XIIa (3), the resulting factor XIa acrylamide gel electrophoresis) were obtained from Enzyme can rapidly activate factor IX (13), which sets off a chain of Research Laboratories (South Bend, IN). S-2366 (

The publication costs of this article were defrayed in part by page charge Abbreviations: DS, dextran sulfate; HK, high molecular weight payment. This article must therefore be hereby marked "advertisement" kininogen. in accordance with 18 U.S.C. §1734 solely to indicate this fact. *To whom reprint requests should be addressed.

11189 Downloaded by guest on September 28, 2021 11190 Biochemistry: Scott and Colman Proc. Natl. Acad Sci. USA 89 (1992) (Franklin, OH). Human thrombin (2900 units/mg) was a generous gift of John W. Fenton 11 (23). DS (Mr = 500,000), Polybrene (hexadimethrine bromide), and recombinant hir- udin were purchased from Sigma. Immulon 2 microplates, used as reaction vessels because they were found not to promote contact activation, were purchased from Dynatech. 0.10 Preparation ofFactor XI. In initial experiments it was noted that traces of Polybrene had carried over to later steps in the E 0.08 purification offactor XI and interfered with the DS-mediated 0.06 activation of factor XI (data not shown). It was therefore essential to prepare factor XI without Polybrene at any step 0.04 of the purification for subsequent experiments. Polybrene- free factor XI was supplied by Enzyme Research Laborato- 0.02 ries (South Bend, IN) and was homogeneous in SDS/10%o 0.00 polyacrylamide gel electrophoresis with a specific activity of O 1 2 3 4 5 6 7 164 units/mg by coagulant assay. Thrombin, nM Activation of Factor XI. Factor XI (60 nM final concentra- tion) was incubated in 0.6-ml polypropylene microcentrifuge FIG. 1. Determination of k" for the activation of factor XI by tubes at 370C in buffer A (50 mM Tris HCl, pH 7.4/0.15 M thrombin. Factor XI (60 nM) was incubated with buffer A (o) or three different cocentrations of thrombin (-). The ti,2 was calculated for NaCl/1 mM EDTA/0.02% NaN3/0.1% bovine serum albu- each reaction and was divided by In 2 to determine k'. The slope for min). In experiments utilizing HK, factor XI was preincu- the k' of the thrombin/DS-mediated activation of factor XI was bated with HK for 5 min at 230C before other reactants were determined by regression analysis on a Macintosh computer with added. Fibrinogen or additional buffer A was added, followed Cricket Graph. This analysis yielded the line k' = 0.01433 + 0.02574 by either factor XIIa or thrombin. Immediately after the x [thrombin], with r2 = 0.999. Note that o indicates the k' value for addition of enzyme, DS (1 ,.g/ml final concentration) was the autoactivation of factor XI. added and the tubes were mixed and placed at 3rC. At each indicated time, 15 uld of reaction mixture was transferred to was fitted by linear regression analysis, the line intersected a microplate well containing 20 1.d ofbuffer A with Polybrene the y axis at 0.014 min-1 in the absence ofthrombin. This rate (to neutralize the DS) at 200 ,.ug/ml, corn trypsin inhibitor (to constant represents the contribution of the autoactivation to inhibit factor XIIa) at 100 nM, and hirudin (to inactivate the total activation offactor XI. The k' forthe autoactivation thrombin) at 100 units/ml. This mixture was allowed to offactor XI, determined in a separate experiment and found incubate at 230C for at least 15 min prior to assaying for factor to be 0.0144 min-1 (Fig. 1, 0), was in good agreement with the XIa (see below). extrapolated value. These data indicate that the contribution Assay of Factor XIa Activity. An additional 65 tp1 of buffer of the autoactivation is small in comparison with the throm- A was added to each sample in the microplate well, followed bin/DS-mediated activation, and it can be ignored when by 50 Al of 4 mM S-2366, a substrate that is readily cleaved calculating the second-order rate constant. The k" for the by factor XIa (24). After 30 min at 230C, 100 ul of 2% citric thrombin/DS-mediated activation of factor XI was found to acid was added to terminate the hydrolysis. The plates were be 22 AM-'-min-' (Table 1). read at 405 nm on a microplate reader (Bio-Tek, EL 311, Burlington, VT). Kinetics for the Activation of Factor XI. The conversion of RESULTS AND DISCUSSION factor XI to XIa by thrombin or factor XIIa can be regarded Effect of HK on the Thrombln/DS-Medlated Activation of as irreversible because -AFis large for proteolytic reactions. Factor XI. Previous publications revealed that HK, the At high thrombin or factor XIIa concentrations, the second- cofactor of factor XI, attenuates the ability of thrombin to order rate constant (k") can be determined from the pseudo- activate factor XI (20, 21) in a concentration-dependent first-order reaction manner. We investigated this effect, using a fixed concen- factor XI -- factor tration ofHK (650 nM, which is the concentration in plasma) XIa, and three concentrations of thrombin (Fig. 2). At each which is described by the pseudo-first-order rate equation thrombin concentration, we observed a dramatic decrease in 1.0- Xla,. In - = Ik't9 [11 XIa.ax Mat 0.8- where XIama.. is the A A4o5/30 min produced by complete activation of factor XI in the incubation mixture, XIa, is the E, 0.6- / // + amount of factor XIa generated at time t, and k' is the pseudo-first-order rate constant. Under conditions where the concentration of activating enzyme [E], thrombin or factor XIIa, is constant, a second-order rate constant can be deter- mined from the relation kit = 0.0 k'/[E], [21 0 5 1 0 1 5 20 where k' is determined for various [E]. A plot ofthe left side Time, min of Eq. 1 vs. t at each [E] will produce a slope, k', from which FIG. 2. Effect ofHK on the thrombin/DS-mediated activation of k" is calculated, knowing the concentration of the activating factor XI. Factor XI (60 nM) was incubated with 2.09 nM (o, *), 4.18 enzyme (thrombin or factor XIIa) (Eq. 2). nM (o, *), or 6.27 nM (A, *) thrombin in the presence (e, *, A) or When the k' for the activation offactor XI by thrombin was absence (o, o, A) of 650 nM HK. The data represent averages of plotted vs. the thrombin concentration (Fig. 1) and the curve duplicate determinations. Downloaded by guest on September 28, 2021 Biochemistry: Scott and Colman Proc. Natl. Acad. Sci. USA 89 (1992) 11191 the rate of factor XI activation, suggesting that substantial factor XI activation could not occur in the presence of HK unless the concentration ofthrombin were extremely high. In preliminary studies using factor XI that contained traces of Polybrene, a less pronounced effect of HK was observed (data not shown), in agreement with a previous report (20). We therefore utilized Polybrene-free factor XI for all exper- iments in this report. Effect of Fibrinogen or HK on the DS-Mediated Autoacti- vation of Factor XI. We previously reported that, while fibrinogen is a substrate for factor XIa in purified systems, factor XIa does not cleave fibrinogen in plasma (25). This finding suggests that fibrinogen is a comparatively poor substrate for factor XIa. With this in mind, we tested whether fibrinogen could influence the autoactivation rate of factor 0 20 40 60 80 XI. Fibrinogen, at its plasma concentration (8.8 AuM), was Time, min added to 60 nM factor XI, DS was added, and the mixture was incubated at 370C alongside tubes containing factor XI and FIG. 4. Effect of HK and/or fibrinogen on the thrombin/DS- DS without fibrinogen (Fig. 3). Autoactivation of factor XI mediated activation offactor XI. Thrombin (20.9 nM) was incubated with factor XI (60 nM) and DS at 1 1ig/ml alone (e) or with 650 nM occurred in the absence of fibrinogen, in agreement with HK (o), 8.8 A&M fibrinogen (n), or both HK and fibrinogen (A). The previous reports (20, 21), but was abolished in the presence data points represent averages of duplicate determinations. of fibrinogen. We also tested the effect of HK at its plasma concentration (650 nM) on the autoactivation rate of factor differences were observed in the presence or absence of XI, because not only is HK a cofactor for factor XIa fibrinogen at any concentration of DS in the incubation generation, it also is a substrate for factor XIa (26). HK mixture (Fig. 5), suggesting that this mode ofactivation could dramatically decreased the autoactivation rate of factor XI. occur in a plasma environment and that fibrinogen was not Effect of Fibrinogen and/or HK on the Rate of Throm- with bin/DS-Mediated Activation ofFactor XI. Because fibrinogen interfering the enzymatic action offactor XIa on S-2366. is the most abundant substrate for thrombin in plasma (27), Comparison of the Rate of Activation of Purified Factor XI and because fibrinogen was able to block the autoactivation and Factor XI in Plasma by Thrombin/DS. Factor XII- of factor XI (Fig. 3), we investigated the effect of fibrinogen deficient plasma was subjected to a mild acid treatment (29) on the rate of thrombin-catalyzed activation of factor XI. to inactivate plasma protease inhibitors as well as to render Gly-Pro-Arg-Pro (8.6 mM) was included in the reaction the fibrinogen nonclottable. DS (1 ,ug/ml) and thrombin (20.9 mixture to curtail the polymerization offibrin (28), and it was nM) were incubated with either acid-treated factor XII- also included in the reaction mixtures without fibrinogen. deficient plasma or purified factor XI at a final concentration Thrombin (20.9 nM) was incubated at 370C with 60 nM factor of 10 nM in polypropylene microcentrifuge tubes at 370C. At XI and DS in the presence or absence of HK and/or fibrin- various times, 15 Al was transferred into a microplate con- ogen (Fig. 4). HK dramatically curtailed the activation rate of taining 10 /l of Polybrene at 75 ,ug/ml and 10/41 of hirudin at factorXI, and fibrinogen completely prevented the thrombin- 50 units/ml. After incubation for 10 min, 65 1ul of buffer was mediated activation of factor XI on DS in both the presence added to each well, followed by 50 /L4 of 4 mM S-2366. After and absence of HK. These results were comparable to the 30 min, the hydrolysis was terminated by 100 ,ul of 2% citric effects offibrinogen and HK on the rate of autoactivation of acid. As shown in Fig. 6, thrombin/DS failed to activate factor XI (Fig. 3). factor XI in plasma, whereas it completely activated the Effect of Fibrinogen on the Factor XIIa/DS-Mediated Ac- purified factor XI. These data indicate that factor XI would tivation of Factor XI. Because fibrinogen affected both the autoactivation and thrombin-mediated activation offactor XI 100 on DS (Figs. 3 and 4), we tested the effect offibrinogen on the E DS/factor XIIa/HK-mediated activation of factor XI. No ._E E "6

C- + Buffer 0 x E 0.3 CU 00 0 co cU 'D 0.2- 0 IL + HK 0 2 4 6 8 10 +Fg DS, ug/ml FIG. 5. Effect of fibrinogen on the factor XIIa/DS-mediated 40 60 1 00 activation offactor XI in the presence of HK. Factor XI (60 nM) was Time, min incubated for 19 min with factor XIIa (0.3 nM), various concentra- tions ofDS, and 650 nM HK in the presence (e) or absence (o) of8.8 FIG. 3. Effect of HK or fibrinogen on the autoactivation rate of 1uM fibrinogen. The data, average of duplicate determinations, are factor XI on DS. Factor XI (60 nM) was incubated with DS at 1 ,ug/ml expressed as percent maximal factor XI activation observed, as (e), DS plus 650 nM HK (o), or DS plus 8.8 /AM fibrinogen (o). The determined by the DS concentration that produced the greatest data represent averages of duplicate determinations. amount of factor XIa activity. Downloaded by guest on September 28, 2021 11192 Biochemistry: Scott and Colman Proc. Nad. Acad. Sci. USA 89 (1992) fibrinogen 146-fold. These data, as well as the data presented 0.30 in Fig. 6, indicate that in plasma, where fibrinogen and HK are abundant, factor XI would not be autoactivated or become activated by thrombin. 0.25 Naito and Fujikawa reported that thrombin could activate C factor XI in plasma in the presence of sulfatides (20). This E0.20 conclusion was derived from an experment performed with factor XII-deficient plasma that had been artificially depleted g 0.15 of factor XI by affinity chromatography and compared with the same depleted plasma after factor XI was added at 1 0.10 unit/ml. A slight difference in clotting times was seen at thrombin concentrations below 400 ng/ml, but no differences 0.05 were seen above 400 ng/ml. This experiment was performed at pH 7.9 and the time that the thrombin-sulfatide mixture 0.00' was in contact with the deficient plasma (3 min) was insuf- 0 5 10 15 Time, min ficient to produce substantial activation of factor XI in a purified system (20). They reported in the same study that FIG. 6. Comparison ofthe rate ofactivation ofpurified factor XI thrombin at 100 ng/ml, when incubated with sulfatides, and factor XI in plasma by thrombin/DS. Purified factor XI (15 nM) activated less than 10% of total factor XI in 3 min at 370C (o) or acid-treated factor XII-deficient plasma (o; final concentration when purified factor XI was used (20). Furthermore, no = 25%) was incubated with DS (1 jug/ml) and throbin (20.9 nM) at 3TC for various times. The factor XIa that was generated was direct evidence was presented in the plasma experiment that determined by hydrolysis of S-2366 for 30 min. factor XI was actually activated (20). We conclude from our data that activation of factor XI in plasma is not a likely not be activated in plasma by thrombin in the presence of a consequence of thrombin in the presence of both HK and negatively charged surface. fibrinogen. Comparison ofthe k" for the Activation of Factor XI on DS. The fact that thrombin can activate factor XI in vitro is The k" for the activation offactor XI by factor XIIa plus DS interesting, but the importance of thrombin as a physiologic was found to be 110 iLM-1-min-1 in the absence of HK, and activator in plasma may have been overstated (21). Perhaps this decreased to 1.5 FM-l-min-1 upon addition offibrinogen thrombin can activate factor XI on the surfaces ofblood cells (Table 1). In contrast, the k" doubled upon addition of HK, or vessel walls. Further investigation is needed to determine to 220 AM-Lmin-', and was similar when fibrinogen was whether or not thrombin plays a role, in vivo, in the activation included in the mixture with HK. The high k" for the factor of factor XI. However, until further evidence is provided XIIa/DS-mediated activation of factor XI in the absence of indicating that thrombin can indeed activate factor XI in vivo, of the factor XIIa-catalyzed achange in our understanding ofthe coagulation cascade may HK represents a combination not, as yet, be justified. The solution to the paradox of activation and the autoactivation of factor XI. We reached contact factor-deficient individuals havingapparently normal this conclusion because the addition offibrinogen decreased hemostatic mechanisms remains to be elucidated. However, the k" to 1.5 ,uM-1-min-1, which is similar to the slow the fact that tissue factor plus factor VIIa can activate factor activation rate that was seen for the factor XIIa-mediated IX (31) provides a possible explanation why patients with activation offactor XI in the absence ofDS (data not shown). factor XI deficiency show a mild and inconsistent impairment Thus, in the absence of HK, factor XIIa potentiates the of hemostasis. The importance of the contact system in the autoactivation of factor XI much in the same way that inflammatory response is, most likely, far greater than its thrombin enhances this reaction. importance in coagulation. The k" for the thrombin-mediated release offibrinopeptide A from fibrinogen is 790 .uM-1'min1 (30), whereas the k" for We thank Drs. Robin A. Pixley, Edward P. Kirby, and Peter N. the thrombin/DS activation of factor XI is 22 ,.LM-1min-1 Walsh ofthis institution for their critique of this manuscript and Ms. (Table 1), indicating that the preference of thrombin for Rita Stewart for her skillful preparation of the manuscript. We are fibrinogen is at least 35-fold greater than for factor XI. grateful to Michael Morris ofEnzyme Research Laboratories for the However, the presence of 8.8 ,uM fibrinogen decreased the preparation ofPolybrene-free factor XI used in this study. This work thrombin/DS-mediated cleavage of factor XI to an immea- was supported by a Specialized Center of Research in Thrombosis surable rate. This effect may be due to a combined effect of HL 45486 from the National Institutes of Health. fibrinogen on the autoactivation of factor XI (Fig. 3) and the 1. Ratnoff, 0. D. & Colopy, J. (1955) J. Clin. Invest. 34, 602-613. preference of thrombin for the substrate fibrinogen, as com- 2. Wuepper, K. D. (1973) J. Exp. Med. 138, 1345-1355. pared with its preference for factor XI. HK decreased the 3. Ratnoff, E. D., Davie, E. W. & Mallet, D. L. (1961) J. Clin. thrombin/DS-mediated activation of factor XI by three or- Invest. 40, 803-819. ders ofmagnitude (Table 1), whereas it augmented the factor 4. Schiffman, S. & Lee, P. (1975) J. Clin. Invest. 56, 1082-1092. XMIa-mediated activation of factor XI in the presence of 5. Revak, S. D., Cochrane, C. G. & Griffin, J. H. (1977) J. Clin. Invest. 59, 1167-1175. Table 1. Comparison of the second-order rate constants (k") for 6. Cochrane, C. G. & Griffin, J. H. (1979) Am. J. Med. 67, the DS-mediated activation of factor XI by factor XIIa 657-664. 7. Ratnoff, 0. D. (1966) in Progress in Haematology, eds. Brown, and thrombin at 370C E. B. & Moore, C. B. (Grune & Stratton, New York), Vol. 5, pp. 204-245. 8. Thompson, R. E., Mandle, R., Jr., & Kaplan, A. P. (1977) J. Additions Factor XIIa Thrombin Clin. Invest. 60, 1376-1380. DS 110.00 22.00 9. Mandle, R., Jr., Colman, R. W. & Kaplan, A. P. (1976) Proc. DS + fibrinogen 1.50 0 Nati. Acad. Sci. USA 73, 4179-4183. DS + HK 220.00 0.03 10. Scott, C. F., Silver, L. D., Schapira, M. & Colman, R. W. (1984) J. Clin. Invest. 73, 954-92. DS + HK + fibrinogen 210.00 0 11. Kaplan, A. P. & Austen, K. F. (1970) J. Immunol. 105, 802- Fibrinogen was 8.8 IAM; HK was 650 nM. 811. Downloaded by guest on September 28, 2021 Biochemistry: Scott and Colman Proc. Nati. Acad. Sci. USA 89 (1992) 11193 12. Cochrane, C. G., Revak, S. D. & Wuepper, K. D. (1973) J. 22. Scott, C. F. & Colman, R. W. (1992) FASEB J. 6, A1320. Exp. Med. 138, 1564-1583. 23. Fenton, J. W., II, Fasco, M. J., Stackrow, A. B., Aronson, 13. Osterud, B., Bouma, B. N. & Griffin, J. H. (1978) J. Biol. D. L., Young, A. M. & Finlayson, J. S. (1977) J. Biol. Chem. Chem. 253, 5946-5951. 252, 3587-3598. 14. Hathaway, W. E., Belhanson, L. P. & Hathaway, H. S. (1965) 24. Scott, C. F., Sinha, D., Seaman, F. S., Walsh, P. N. & Col- Blood 26, 521-532. man, R. W. (1984) Blood 63, 42-50. 15. Weiss, A. S., Gallin, J. I. & Kaplan, A. P. (1974) J. Clin. 25. Scott, C. F., Mentzer, R. L., Budzynski, A. Z. & Colman, Invest. 53, 622-633. R. W. (1986) Arch. Biochem. Biophys. 249, 480-488. 16. Saito, H., Goodnough, L. T., Soria, J., Soria, C., Aznar, J. & 26. Scott, C. F., Silver, L. D., Purdon, A. D. & Colman, R. W. Espana, F. (1981) N. Engl. J. Med. 305, 910-914. (1985) J. Biol. Chem. 260, 10856-10863. 17. Lacombe, M. J., Varet, B. & Levy, J. P. (1975) Blood 46, 27. Lorand, L. (1951) Nature (London) 167, 992-994. 761-768. 28. Greenberg, C. S., Miraglia, C. C., Rickles, F. R. & Shuman, 18. Saito, H., Ratnoff, 0. D., Waldmann, R. & Abraham, J. P. M. A. (1985) J. Clin. Invest. 75, 1463-1470. (1975) J; Clin. Invest. 55, 1082-1089. 29. Colman, R. W., Mattler, L. & Sherry, S. (1967) J. Clin. Invest. 19. Colman, R. W., Bagdasarian, A., Talamo, R. C., Scott, C. F., 48, 23-32. Seavey, M., Guimaraes, J. A., Pierce, J. V. & Kaplan, A. P. 30. Hogg, P. J. & Jackson, C. M. (1990) J. Biol. Chem. 265, (1975) J. Clin. Invest. 56, 1650-1662. 248-255. 20. Naito, K. & Fujikawa, K. (1991)J. Biol. Chem. 266, 7353-7358. 31. Osterud, B. & Rapaport, S. I. (1977) Proc. Natl. Acad. Sci. 21. Gailani, D. & Broze, G. J., Jr. (1991) Science 253, 909-912. USA 74, 5260-5264. Downloaded by guest on September 28, 2021