Fibrinogen blocks the autoactivation and thrombin-mediated

Transcription

1 Proc. Nati. Acad. Sci. USA Vol. 89, pp , 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, 34 North Broad Street, Philadelphia, PA 1914 Communicated by Irving M. Klotz, August 1, 1992 ABSTRACT The intrinsic pathway of blood coagulation is activated when factor XIa, one of the three contact-system enzymes, is generated and then activates factor IX. Factor XI has been shown to be efficiently activated in vitro by surfacebound factor XIIa after factor XI is transported to the surface by its cofactor, high molecular weight kininoen (HK). However, individuals lacking any of the three contact-system proteins-namely, factor XII, prekallikrein, and 11K-do not suffer from bleeding abnormalities. This mystery has led several investigators to search for an "alternate" activation pathway for factor XI. Recently, factor XI has been reported to be autoactivated on the soluble "surface" dextran sulfate, and thrombin was shown to accelerate the autoactivation. However, it was also reported that HK, the cofactor for factor XIIa-mediated activation of factor XI, actually diminishes the thrombin-catalyzed activation rate of factor XI. Nonetheless, it was suggested that thrombin was a more efficient activator than factor XIIa. In this report we investigated the effect of fibrinogen, the major coagulation protein in plasma, on the activation rate of factor XI. Fibrinogen, the preferred substrate for thrombin in plasma, virtually prevented autoactivation of factor XI as well as the thrombin-mediated activation of factor XI, while having no effect on factor XIIa-catalyzed activation. HK dramatically curtailed the autoactivation of factor XI in addition to the thrombin-mediated activation. These data indicate that factor XI would not be autoactivated in a plasma environment, and thrombin would, therefore, be unlikely to potentiate the activation. We believe that the "myisi pathway" for factor XI activation remains an enigma that warrants further investigation. The initiation of the intrinsic pathway of blood coagulation involves four proteins that make up the contact-activation system-namely, the zymogens factor XII (Hageman factor) (1), prekallikrein (Fletcher factor) (2), factor XI (plasma thromboplastin antecedent) (3), and the cofactor-substrate, high molecular weight kininogen (HK) (4). This system is triggered in vitro by negatively charged surfaces or polymers 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 plasma complexed with separate molecules of HK that, upon activation to HKa by kallikrein (1), transport them to an appropriate surface where factor XIIa awaits to activate each zymogen. Once prekallikrein is activated by factor XIIa to kallikrein (1) (the active enzyme) (11), it in turn can rapidly activate additional molecules of factor XII (12), which serves to amplify the contact system. Following activation of factor XI by surface-bound factor XIIa (3), the resulting factor XIa can rapidly activate factor IX (13), which sets off a chain of reactions known as the "coagulation cascade" and leads to 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 solely to indicate this fact. formation of fibrin-one component of the hemostatic plug. Several persons have been described in the literature who lack either prekallikrein (2, 14-16) or factor XII (1, 7), and three cases of HK deficiency were reported in 1975 (17-19). These individuals did not reveal a history of abnormal bleeding, and all had been discovered by routine preoperative coagulation tests. Patients with factor XI deficiency, however, may manifest hemostatic abnormalities, although they are variable and usually mild, as compared with individuals lacking coagulation factors further down on the cascade. The paradox of normal hemostasis in individuals with undetectable amounts of one of the contact factors has led many investigators to search for an alternate pathway by which factor XIa is generated in the absence of factor XIIa. Two recent publications have implicated thrombin as the missing link (2, 21). Factor XI was reported to be autoactivated on the soluble negatively charged "surface" dextran sulfate -(DS), and thrombin was found to enhance that activation (2, 21). However, both groups of investigators demonstrated that HK, the activation cofactor for factor XI, actually attenuates the thrombin-mediated activation of factor XI (2, 21). Despite this observation, thrombin was claimed to be superior to factor XIIa in its ability to activate factor XI, and a revision of the coagulation cascade was proposed (21). However, the effect of competing substrates for thrombin in plasma, especially its preferred substrate, fibrinogen, was not considered in the revised mechanism. In this report, we have investigated the effect of fibrinogen and HK on the autoactivation rate of factor XI on DS as well as on the thrombin and factor XIIa-mediated activation rate of factor XI on DS. Our data indicate that fibrinogen abolishes, and HK dramatically curtails, both the autoactivation and the thrombin-mediated activation of factor XI on DS. In contrast, HK augments factor XIIa-mediated activation of factor XI, and fibrinogen does not influence this reaction. We also show that thrombin is not a significant contributor to the activation of factor XI in plasma, which contains both HK and fibrinogen. These results have been presented in abstract form (22). MATERIALS AND METHODS Materials. Highly purified human factor XIIa (prepared from homogeneous factor XII, activated with DS, which was removed after activation), HK (>9% single chain in SDS/ 1%o polyacrylamide gel electrophoresis, 12.5 units/mg), fibrinogen (homogeneous in nonreducing SDS/5% polyacrylamide gel electrophoresis and greater than 95% clottable), and corn trypsin inhibitor (single band on SDS/11 polyacrylamide gel electrophoresis) were obtained from Enzyme Research Laboratories (South Bend, IN). S-2366 (<Glu-Pro- Arg p-nitroanilide) was purchased from Kabi/Pharmacia Abbreviations: DS, dextran sulfate; HK, high molecular weight kininogen. *To whom reprint requests should be addressed

2 1119 Biochemistry: Scott and Colman (Franklin, OH). Human thrombin (29 units/mg) was a generous gift of John W. Fenton 11 (23). DS (Mr = 5,), Polybrene (hexadimethrine bromide), and recombinant hirudin were purchased from Sigma. Immulon 2 microplates, used as reaction vessels because they were found not to promote contact activation, were purchased from Dynatech. Preparation of Factor XI. In initial experiments it was noted that traces of Polybrene had carried over to later steps in the purification of factor XI and interfered with the DS-mediated activation of factor XI (data not shown). It was therefore essential to prepare factor XI without Polybrene at any step of the purification for subsequent experiments. Polybrenefree factor XI was supplied by Enzyme Research Laboratories (South Bend, IN) and was homogeneous in SDS/1%o polyacrylamide gel electrophoresis with a specific activity of 164 units/mg by coagulant assay. Activation of Factor XI. Factor XI (6 nm final concentration) was incubated in.6-ml polypropylene microcentrifuge tubes at 37C in buffer A (5 mm Tris HCl, ph 7.4/.15 M NaCl/1 mm EDTA/.2% NaN3/.1% bovine serum albumin). In experiments utilizing HK, factor XI was preincubated with HK for 5 min at 23C before other reactants were added. Fibrinogen or additional buffer A was added, followed by either factor XIIa or thrombin. Immediately after the addition of enzyme, DS (1,.g/ml final concentration) was added and the tubes were mixed and placed at 3rC. At each indicated time, 15 uld of reaction mixture was transferred to a microplate well containing 2 1.d of buffer A with Polybrene (to neutralize the DS) at 2,.ug/ml, corn trypsin inhibitor (to inhibit factor XIIa) at 1 nm, and hirudin (to inactivate thrombin) at 1 units/ml. This mixture was allowed to incubate at 23C for at least 15 min prior to assaying for factor XIa (see below). Assay of Factor XIa Activity. An additional 65 tp1 of buffer A was added to each sample in the microplate well, followed by 5 Al of 4 mm S-2366, a substrate that is readily cleaved by factor XIa (24). After 3 min at 23C, 1 ul of 2% citric acid was added to terminate the hydrolysis. The plates were read at 45 nm on a microplate reader (Bio-Tek, EL 311, Burlington, VT). Kinetics for the Activation of Factor XI. The conversion of factor XI to XIa by thrombin or factor XIIa can be regarded as irreversible because -AFis large for proteolytic reactions. At high thrombin or factor XIIa concentrations, the secondorder rate constant (k") can be determined from the pseudofirst-order reaction factor XI -- factor XIa, which is described by the pseudo-first-order rate equation In XIa.ax Xla,. - Ik't9 Mat = where XIama.. is the A A4o5/3 min produced by complete activation of factor XI in the incubation mixture, XIa, is the 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 determined from the relation kit = k'/[e], where k' is determined for various [E]. A plot of the left side of Eq. 1 vs. t at each [E] will produce a slope, k', from which k" is calculated, knowing the concentration of the activating enzyme (thrombin or factor XIIa) (Eq. 2). When the k' for the activation offactor XI by thrombin was plotted vs. the thrombin concentration (Fig. 1) and the curve [11 [21 Proc. Natl. Acad Sci. USA 89 (1992).1 E O Thrombin, nm FIG. 1. Determination of k" for the activation of factor XI by thrombin. Factor XI (6 nm) was incubated with buffer A (o) or three different cocentrations of thrombin (-). The ti,2 was calculated for each reaction and was divided by In 2 to determine k'. The slope for the k' of the thrombin/ds-mediated activation of factor XI was determined by regression analysis on a Macintosh computer with Cricket Graph. This analysis yielded the line k' = x [thrombin], with r2 =.999. Note that o indicates the k' value for the autoactivation of factor XI. was fitted by linear regression analysis, the line intersected the y axis at.14 min-1 in the absence ofthrombin. This rate constant represents the contribution of the autoactivation to the total activation offactor XI. The k' for the autoactivation of factor XI, determined in a separate experiment and found to be.144 min-1 (Fig. 1, ), was in good agreement with the extrapolated value. These data indicate that the contribution of the autoactivation is small in comparison with the thrombin/ds-mediated activation, and it can be ignored when calculating the second-order rate constant. The k" for the thrombin/ds-mediated activation of factor XI was found to be 22 AM-'-min-' (Table 1). RESULTS AND DISCUSSION Effect of HK on the Thrombln/DS-Medlated Activation of Factor XI. Previous publications revealed that HK, the cofactor of factor XI, attenuates the ability of thrombin to activate factor XI (2, 21) in a concentration-dependent manner. We investigated this effect, using a fixed concentration ofhk (65 nm, which is the concentration in plasma) and three concentrations of thrombin (Fig. 2). At each thrombin concentration, we observed a dramatic decrease in E,.6- / / / FIG. 2. Effect of HK on the thrombin/ds-mediated activation of factor XI. Factor XI (6 nm) was incubated with 2.9 nm (o, *), 4.18 nm (o, *), or 6.27 nm (A, *) thrombin in the presence (e, *, A) or absence (o, o, A) of 65 nm HK. The data represent averages of duplicate determinations.

3 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 (2). We therefore utilized Polybrene-free factor XI for all experiments in this report. Effect of Fibrinogen or HK on the DS-Mediated Autoactivation 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 XI. Fibrinogen, at its plasma concentration (8.8 AuM), was added to 6 nm factor XI, DS was added, and the mixture was incubated at 37C alongside tubes containing factor XI and DS without fibrinogen (Fig. 3). Autoactivation of factor XI occurred in the absence of fibrinogen, in agreement with previous reports (2, 21), but was abolished in the presence of fibrinogen. We also tested the effect of HK at its plasma concentration (65 nm) on the autoactivation rate of factor XI, because not only is HK a cofactor for factor XIa generation, it also is a substrate for factor XIa (26). HK dramatically decreased the autoactivation rate of factor XI. Effect of Fibrinogen and/or HK on the Rate of Thrombin/DS-Mediated Activation offactor XI. Because fibrinogen is the most abundant substrate for thrombin in plasma (27), and because fibrinogen was able to block the autoactivation of factor XI (Fig. 3), we investigated the effect of fibrinogen on the rate of thrombin-catalyzed activation of factor XI. Gly-Pro-Arg-Pro (8.6 mm) was included in the reaction mixture to curtail the polymerization of fibrin (28), and it was also included in the reaction mixtures without fibrinogen. Thrombin (2.9 nm) was incubated at 37C with 6 nm factor XI and DS in the presence or absence of HK and/or fibrinogen (Fig. 4). HK dramatically curtailed the activation rate of factor XI, and fibrinogen completely prevented the thrombinmediated activation of factor XI on DS in both the presence and absence of HK. These results were comparable to the effects of fibrinogen and HK on the rate of autoactivation of factor XI (Fig. 3). Effect of Fibrinogen on the Factor XIIa/DS-Mediated Activation of Factor XI. Because fibrinogen affected both the autoactivation and thrombin-mediated activation of factor XI on DS (Figs. 3 and 4), we tested the effect of fibrinogen on the DS/factor XIIa/HK-mediated activation of factor XI. No FIG. 3. E.3 co 'D.2- Biochemistry: Scott and Colman Buffer + HK +Fg 1 Effect of HK or fibrinogen on the autoactivation rate of factor XI on DS. Factor XI (6 nm) was incubated with DS at 1,ug/ml (e), DS plus 65 nm HK (o), or DS plus 8.8 /AM fibrinogen (o). The data represent averages of duplicate determinations. FIG. 4. Proc. Natl. Acad. Sci. USA 89 (1992) Effect of HK and/or fibrinogen on the thrombin/dsmediated activation of factor XI. Thrombin (2.9 nm) was incubated with factor XI (6 nm) and DS at 1 1ig/ml alone (e) or with 65 nm HK (o), 8.8 A&M fibrinogen (n), or both HK and fibrinogen (A). The data points represent averages of duplicate determinations. differences were observed in the presence or absence of fibrinogen at any concentration of DS in the incubation mixture (Fig. 5), suggesting that this mode of activation could occur in a plasma environment and that fibrinogen was not interfering with the enzymatic action of factor XIa on S Comparison of the Rate of Activation of Purified Factor XI and Factor XI in Plasma by Thrombin/DS. Factor XIIdeficient plasma was subjected to a mild acid treatment (29) to inactivate plasma protease inhibitors as well as to render the fibrinogen nonclottable. DS (1,ug/ml) and thrombin (2.9 nm) were incubated with either acid-treated factor XIIdeficient plasma or purified factor XI at a final concentration of 1 nm in polypropylene microcentrifuge tubes at 37C. At various times, 15 Al was transferred into a microplate containing 1 /l of Polybrene at 75,ug/ml and 1 /41 of hirudin at 5 units/ml. After incubation for 1 min, 65 1ul of buffer was added to each well, followed by 5 /L4 of 4 mm S After 3 min, the hydrolysis was terminated by 1,ul of 2% citric acid. As shown in Fig. 6, thrombin/ds failed to activate factor XI in plasma, whereas it completely activated the purified factor XI. These data indicate that factor XI would E._E E "6 C- x CU cu IL DS, ug/ml 8 1 FIG. 5. Effect of fibrinogen on the factor XIIa/DS-mediated activation of factor XI in the presence of HK. Factor XI (6 nm) was incubated for 19 min with factor XIIa (.3 nm), various concentrations of DS, and 65 nm HK in the presence (e) or absence (o) of 8.8 1uM fibrinogen. The data, average of duplicate determinations, are expressed as percent maximal factor XI activation observed, as determined by the DS concentration that produced the greatest amount of factor XIa activity.

4 11192 Biochemistry: Scott and Colman FIG C E.2 g ' Comparison of the rate of activation of purified factor XI and factor XI in plasma by thrombin/ds. Purified factor XI (15 nm) (o) or acid-treated factor XII-deficient plasma (o; final concentration = 25%) was incubated with DS (1 jug/ml) and throbin (2.9 nm) at 3TC for various times. The factor XIa that was generated was determined by hydrolysis of S-2366 for 3 min. not be activated in plasma by thrombin in the presence of a negatively charged surface. Comparison of the k" for the Activation of Factor XI on DS. The k" for the activation of factor XI by factor XIIa plus DS was found to be 11 ilm-1-min-1 in the absence of HK, and this decreased to 1.5 FM-l-min-1 upon addition of fibrinogen (Table 1). In contrast, the k" doubled upon addition of HK, to 22 AM-Lmin-', and was similar when fibrinogen was included in the mixture with HK. The high k" for the factor XIIa/DS-mediated activation of factor XI in the absence of HK represents a combination of the factor XIIa-catalyzed activation and the autoactivation of factor XI. We reached this conclusion because the addition of fibrinogen decreased the k" to 1.5,uM-1-min-1, which is similar to the slow activation rate that was seen for the factor XIIa-mediated activation offactor XI in the absence of DS (data not shown). Thus, in the absence of HK, factor XIIa potentiates the autoactivation of factor XI much in the same way that thrombin enhances this reaction. The k" for the thrombin-mediated release of fibrinopeptide A from fibrinogen is 79.uM-1'min1 (3), whereas the k" for the thrombin/ds activation of factor XI is 22,.LM-1min-1 (Table 1), indicating that the preference of thrombin for fibrinogen is at least 35-fold greater than for factor XI. However, the presence of 8.8,uM fibrinogen decreased the thrombin/ds-mediated cleavage of factor XI to an immeasurable rate. This effect may be due to a combined effect of fibrinogen on the autoactivation of factor XI (Fig. 3) and the preference of thrombin for the substrate fibrinogen, as compared with its preference for factor XI. HK decreased the thrombin/ds-mediated activation of factor XI by three orders of magnitude (Table 1), whereas it augmented the factor XMIa-mediated activation of factor XI in the presence of Table 1. Comparison of the second-order rate constants (k") for the DS-mediated activation of factor XI by factor XIIa and thrombin at 37C Additions Factor XIIa Thrombin DS DS + fibrinogen 1.5 DS + HK DS + HK + fibrinogen 21. Fibrinogen was 8.8 IAM; HK was 65 nm. Proc. Nad. Acad. Sci. USA 89 (1992) fibrinogen 146-fold. These data, as well as the data presented in Fig. 6, indicate that in plasma, where fibrinogen and HK are abundant, factor XI would not be autoactivated or become activated by thrombin. Naito and Fujikawa reported that thrombin could activate factor XI in plasma in the presence of sulfatides (2). This conclusion was derived from an experment performed with factor XII-deficient plasma that had been artificially depleted of factor XI by affinity chromatography and compared with the same depleted plasma after factor XI was added at 1 unit/ml. A slight difference in clotting times was seen at thrombin concentrations below 4 ng/ml, but no differences were seen above 4 ng/ml. This experiment was performed at ph 7.9 and the time that the thrombin-sulfatide mixture was in contact with the deficient plasma (3 min) was insufficient to produce substantial activation of factor XI in a purified system (2). They reported in the same study that thrombin at 1 ng/ml, when incubated with sulfatides, activated less than 1% of total factor XI in 3 min at 37C when purified factor XI was used (2). Furthermore, no direct evidence was presented in the plasma experiment that factor XI was actually activated (2). We conclude from our data that activation of factor XI in plasma is not a likely consequence of thrombin in the presence of both HK and fibrinogen. The fact that thrombin can activate factor XI in vitro is interesting, but the importance of thrombin as a physiologic activator in plasma may have been overstated (21). Perhaps thrombin can activate factor XI on the surfaces of blood cells or vessel walls. Further investigation is needed to determine whether or not thrombin plays a role, in vivo, in the activation of factor XI. However, until further evidence is provided indicating that thrombin can indeed activate factor XI in vivo, a change in our understanding ofthe coagulation cascade may not, as yet, be justified. The solution to the paradox of contact factor-deficient individuals having apparently normal hemostatic mechanisms remains to be elucidated. However, the fact that tissue factor plus factor VIIa can activate factor IX (31) provides a possible explanation why patients with factor XI deficiency show a mild and inconsistent impairment of hemostasis. The importance of the contact system in the inflammatory response is, most likely, far greater than its importance in coagulation. We thank Drs. Robin A. Pixley, Edward P. Kirby, and Peter N. Walsh of this institution for their critique of this manuscript and Ms. Rita Stewart for her skillful preparation of the manuscript. We are grateful to Michael Morris of Enzyme Research Laboratories for the preparation of Polybrene-free factor XI used in this study. This work was supported by a Specialized Center of Research in Thrombosis HL from the National Institutes of Health. 1. Ratnoff,. D. & Colopy, J. (1955) J. Clin. Invest. 34, Wuepper, K. D. (1973) J. Exp. Med. 138, Ratnoff, E. D., Davie, E. W. & Mallet, D. L. (1961) J. Clin. Invest. 4, Schiffman, S. & Lee, P. (1975) J. Clin. 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