JBC Papers in Press. Published on April 25, 2000 as Manuscript M000464200 REVISIONS ARE INDICATED IN BOLD TEXT Thrombin-Mediated Feedback Activation of Factor XI on the Activated Platelet Surface is Preferred over Contact Activation by Factor XIIa or Factor XIa* Frank A. Baglia and Peter N. Walsh The Sol Sherry Thrombosis Research Center Departments of Medicine and Biochemistry Temple University School of Medicine Philadelphia, Pennsylvania 19140 *This study was supported by research grants from the National Institute of Health (HL46213, HL56153 and HL56914) Author to whom correspondence should be addressed: Peter N. Walsh, M.D., Ph.D. Sol Sherry Thrombosis Research Center Temple University School of Medicine 3400 North Broad Street Philadelphia, PA 19140 Telephone Number: (215) 707-4375; Fax Number: (215) 707-3005; E-mail: pnw@astro.ocis.temple.edu 1
RUNNING TITLE: Thrombin is Preferred Activator of Factor XI 2
SUMMARY To study the pathways for initiation of intrinsic blood coagulation, activated human platelets were compared with dextran sulfate as surfaces for factor XI activation by factor XIIa, factor XIa or thrombin. Activated gel-filtered platelets promoted the activation of factor XI (60 nm) by thrombin (0.02-10 nm, EC 50 ~ 100 pm, threshold concentration ~ 10 pm) at initial rates 2- to 3-fold greater than those obtained with dextran sulfate in the presence of either HK (45 nm) and ZnCl 2 (25 µm) or prothrombin (1.2 µm) and CaCl 2 (2 mm). The maximum rates of factor- XI activation achieved in the presence of activated gel-filtered platelets were 30 nm min -1 with thrombin, 6 nm min -1 with factor XIIa and 2 nm min -1 with factor XIa. Values of turnover number calculated at various enzyme concentrations (0.05-1 nm) were 24 167 (mean = 86) min 1 for thrombin, 4.6-50 (mean = 21) min -1 for factor XIIa, and 1.3-14 (mean = 8) min 1 for factor XIa. A physiological concentration of fibrinogen (9.0 µm) inhibited factor-xi activation by thrombin (but not by factor XIIa) in the presence of dextran sulfate but not in the presence of gelfiltered platelets. Compared with factors XIIa and XIa, thrombin is the preferred factor XI activator and activated platelets are a relevant physiological surface for thrombin-mediated initiation of intrinsic coagulation in vivo. 3
INTRODUCTION Human coagulation factor XI is a disulfide-linked homodimer, consisting of two identical polypeptide chains each containing 607 amino acids. Factor XI is present in human plasma as a zymogen that requires proteolytic activation to develop serine protease activity (1-5). It circulates in human plasma in a noncovalent complex with high molecular weight kininogen (HK) (6), and can be activated by three biologically relevant proteases: factor XIIa, factor XIa, and thrombin (1,7,8). The primary structure of factor XI has been determined (9,10) including the identification of four tandem repeat sequences, designated Apple (A1, A2, A3 and A4) domains in the heavy chain region of factor XI. Binding sites (11-15) for thrombin, the Kringle 2 domain of prothrombin and HK are present in the A1 domain whereas both heparin- and plateletbinding sites exist within the A3 domain (16-18), and a binding site for factor XIIa is located in the A4 domain (19). Factor XI can participate in the contact phase of blood coagulation in a reaction that requires the presence of anionic surfaces for optimal activation in vitro by factor XIIa (6,20-22). However, deficiencies in factor XII, prekallikrein and HK are not associated with hemostatic abnormalities, whereas a deficiency in factor XI produces abnormal bleeding complications (23-25). Therefore, it has been suggested that the physiologically relevant pathway for factor-xi activation might constitute feedback activation either by thrombin or by factor XIa (7,8,15). All three proteases cleave each monomer of factor XI at the R 369 -I 370 bond generating the new amino terminal sequence (I-V-G-G) of the catalytic domain that then activates the catalytic triad of the serine protease. Although factor XI can be activated by thrombin, it has been suggested that this mechanism may not proceed in plasma since both HK and fibrinogen can inhibit thrombincatalyzed activation of factor XI in the presence of dextran sulfate (26,27). However, 4
experiments previously reported from our laboratory (15) demonstrate that: 1) activated platelets provide a surface that promotes thrombin-mediated factor-xi activation in the presence of either HK and Zn 2+ ions or in the presence of prothrombin and Ca 2+ ions; 2) both prothrombin (in the presence of Ca 2+ ) and HK (in the presence of Zn 2+ ) bind to the A1 domain of factor XI and promote factor XI binding to activated platelets; and, 3) platelet-mediated factor-xi activation by thrombin (in contrast with factor-xi activation by thrombin in the presence of dextran sulfate) is not inhibited by physiological concentrations of HK when prothrombin is present at physiological concentrations. These observations support the conclusion that the activated platelet, in contrast to dextran sulfate, comprises a physiologically relevant surface for initiation of intrinsic coagulation in vivo. The present study was undertaken to determine the preferred pathway and conditions required for factor-xi activation on the activated platelet surface. 5
EXPERIMENTAL PROCEDURES Materials. Human prothrombin, human factor XIIa and human factor XIa were purchased from Haematologic Technologies Inc. (Essex Junction, Vermont). Human α-thrombin (28,000 NIH U/mg) and human fibrinogen were purchased from Enzyme Research Laboratories (South Bend, Indiana). The thrombin receptor agonist peptide SFLLRN-amide was synthesized on an Applied Biosystems (Foster City, California) 430A synthesizer and purified via reversephase high performance liquid chromatography (HPLC) to >99% homogeneity. All reagents and materials used for SDS polyacrylamide gel electrophoresis were purchased from Bio-Rad Laboratories, Inc. (Nelville, New York). Iodogen vials (20 µg coating) were obtained from Pierce (Rockford, Illinois). N-(2-hydroxyethyl) piperazine-n'-2-ethane sulfonic acid (HEPES), Sepharose 2B-CL, Sephadex G25 (fine), tris-hcl, sodium phosphate, Trismas base, bovine serum albumin and sodium metabisulfite were purchased from Sigma Chemical Co. (St. Louis, Missouri). Purification of Proteins. Factor XI (250 U/mg protein) was purified from human plasma by immunoaffinity chromatography (28). Factor XI was assayed by minor modifications (29) of the kaolin-activated partial thromboplastin time (30). HK (specific activity 15 U/mg) was purified by the method of Kerbiriou and Griffin (31). Radiolabeling of Protein. Purified factor XI were radiolabeled with 125 I by a minor modification (15) of the iodogen method to a specific activity of 5 x 10 6 cpm/µg. The radiolabeled proteins retained >98% of their biological activity. Protein Analysis. Protein concentrations were determined by the Bio-Rad dye-binding assay according to the instructions provided by the manufacturer (Bio-Rad Laboratories, Richmond, CA). Polyacrylamide gel electrophoresis in SDS was performed as previously described (15). Gels were stained, dried onto paper, and autoradiograms were prepared from the 6
dried gels using intensifier screens (Dupont Cronex Lighting-Plus Screens, mounted on Spectrolene cassettes, Reliance X-Ray, Inc., Oreland, PA). X-Omat-AR film (Kodak Inc., Rochester, New York) was used and developed according to instructions provided with the film. Assays of Factor-XI Activation. Activation of factor XI (60 nm) by thrombin, factor XIIa and factor XIa (at various concentrations) was measured by chromogenic assay. Incubations were carried out at 37 C in 200 µl of Tris (50 mm), NaCl (150 mm), ph 7.3 with 1% bovine serum albumin and dextran sulfate (1 µg/ml, Av Mr = 500,000, Sigma Chemical Co., St. Louis, MO) or gel-filtered platelets (activated by incubation at 37 C for 1 min with the thrombin receptor activation peptide (SFLLRN-amide, 25 µm), or 2 µm phospholipid vesicles (consisting of phosphatidylserine and L-α-dioleoyl-phosphatidylchlorine, 1:3 ratio), prepared as previously described (32). After diluting to a final volume of 1 ml with Tris (50 mm), NaCl (150 mm), ph 7.3 with 1% bovine serum albumin containing 600 µm S-2366 (EPR para-nitroanilide, Chromogenix, Mölndal, Sweden), the amount of free para-nitroaniline was determined by measuring the change in absorbence at 405 nm (A405). In experiments with thrombin as a factor-xi activator, hirudin (25 U/ml) (Sigma Chemical Co., St. Louis, MO) was added at the end of the reaction to quench background amidolytic activity from thrombin. Low levels of enzymatic activity after 5-fold dilution of incubation mixtures containing no added factor XI were subtracted from all results to assure detection and measurement only of factor XIa generated during experimental incubations. The amount of factor XIa generated was assayed by reference to a standard curve constructed using purified factor XIa. Initial rates were obtained by visual estimation during the linear portion of the progress curves by dividing factor XIa formed (nm) by the time (min): two minutes for the enzymes thrombin and factor XIIa and 15-20 min for factor XIa at various concentrations. The linear portions of the progress curves were chosen to reflect conditions at which less than 50% of 7
the substrate was converted to factor XIa. To confirm the formation of factor XIa, samples of identical incubation mixtures with added 125 I-labeled factor XI were examined by SDS-gel electrophoresis (15). Autoradiography of SDS-gels were performed to visualize the proteolytic cleavage of 125 I-factor XI. Preparation of Washed Platelets. Platelets were prepared as described (16). Platelet-rich plasma obtained from citrated human blood was centrifuged and the platelets were resuspended in calcium-free Hepes-Tyrodes buffer (126 mm NaCl, 2.7 mm KCl, 1 mm MgCl 2, 0.38 mm NaH 2 PO 4, 5.6 mm dextrose, 6.2 mm sodium Hepes, 8.9 mm Hepes free acid, 0.1% BSA), ph 6.5, and gel-filtered on a column of Sepharose 2B equilibrated in calcium-free Hepes-Tyrodes buffer, ph 7.2. Platelets were counted electronically (Coulter Electronics, Hialeah, FL). 8
RESULTS The Activation of Factor XI by Thrombin in the Presence of Activated Platelets or Dextran Sulfate. We previously determined that activated platelets in the presence of added HK (45 nm), ZnCl 2 (25 µm) and CaCl 2 (2 mm) could promote the activation of factor XI by thrombin at initial rates 2- to 5-fold greater than those obtained in the presence of dextran sulfate (15). In order to extend these studies, we investigated the effects of various concentrations of thrombin (0.016-10 nm) on the activation of factor XI in the presence of activated platelets. The concentration of dextran sulfate (1 µg/ml) used in our experiments was found to be optimal in previous studies (7,8,15). In experiments with platelets there was a direct linear relationship between platelet concentration (0.2-1.0 x 10 8 /ml) and rates of factor-xi activation by thrombin (data not shown). A platelet concentration of 1.0 x 10 8 /ml was arbitrarily chosen for all the present experiments since it approximates the physiological concentration in circulating blood. Activated gel-filtered platelets were shown to promote the activation of factor XI by thrombin (Figure 1, Panel A) at rates 2- to 3-fold greater than those obtained with dextran sulfate (Figure 1, Panel B) in the presence of HK (45 nm) and ZnCl 2 (25 µm). As shown in Table 1, similar results were obtained when HK and ZnCl 2 were substituted with prothrombin (1.2 µm) and CaCl 2 (2 mm) as cofactors for factor XI binding to activated platelets (15). When dextran sulfate was used as a surface, the cofactors (HK plus ZnCl 2 or prothrombin plus CaCl 2 ) were excluded from the incubation mixture since they either did not affect rates of thrombin-mediated factor XI activation (i.e. with added prothrombin) or inhibited these rates (i.e. with added HK). These experiments reveal that in the presence of either cofactor HK (45 nm) and ZnCl 2 (25 µm) or prothrombin (1.2 µm) and CaCl 2 (2 mm) the EC 50 (enzyme concentration at which 50% of activity was achieved) was ~0.1 nm (~0.01 U/ml) thrombin in the presence of either activated platelets or dextran sulfate. 9
Activation of Factor XI by Factor XIIa or Factor XIa in the Presence of Activated Platelets or Dextran Sulfate. Since factor XI is activated by factor XIIa or factor XIa (autoactivation) in the presence of activated platelets or dextran sulfate (7,8,15,33), we compared these activators with thrombin to determine the preferred activator of factor XI. Figure 2 shows the activation of factor XI by factor XIIa in the presence of activated platelets (Panel A) or dextran sulfate (Panel B) as a surface. Dextran sulfate promotes the activation of factor XI by factor XIIa (0.016-10 nm) at initial rates 2- to 3-fold greater than those obtained with activated platelets. However, similar experiments performed with factor XIa (Figure 3) demonstrated that activated platelets (Panel A) promote the activation of factor XI by factor XIa (0.016-10 nm) at initial rates about the same as those obtained with dextran sulfate (Panel B). Figure 4 shows the initial rates of factor-xi activation at various concentrations of the three enzymes in the presence of activated platelets (Figures 4A and 4B) or dextran sulfate (Figure 4C) as a surface. Results with activated platelets are shown with HK (45 nm), CaCl 2 (2 mm) and ZnCl 2 (25 µm) as cofactors for factor XI binding to platelets (Figure 4A) or with prothrombin (1.2 µm) and CaCl 2 (2 mm) added to facilitate factor XI binding to platelets (Figure 4B). The maximum initial rates of factor-xi activation were achieved at enzyme concentrations of ~1 nm in the presence of activated platelets. These maximum rates were ~30 nm/min with thrombin, ~6 nm/min with factor XIIa and ~2 nm/min with factor XIa. The maximum rates of factor-xi activation achieved in the presence of dextran sulfate were ~10 nm/min with thrombin, ~15 nm/min for factor XIIa and ~1.75 nm/min with factor XIa. Comparison of Proteolytic Cleavage of Factor XI on PAGE versus Amidolytic Activity to Determine Factor XIa Formation by the Three Enzymes in the Presence of Activated Platelets. To confirm the formation of factor XIa in experiments similar to those shown in Figures 1A, 2A and 3A, samples of identical incubation mixtures with added 125 I-labeled factor XI (at one 10
enzyme concentration, 1.25 nm for either thrombin, factor XIIa or factor XIa) were examined by SDS gel electrophoresis. To confirm and quantitate the effects of the three enzymes at 1.25 nm, the incubation mixtures were examined by both factor XIa amidolytic activity (Figure 5A) and for the extent of proteolytic cleavage of factor XI by measuring the intensity of the 50 kda band and 30 kda band (heavy and light chains) by densitometry (Kodak Digital Scientific Program, Kodak Co., Rochester, NY) and direct quantitation of factor XI cleavage products (Figure 5B). A comparison of the rates of factor XI activation by the three enzymes shows a close correspondence between rates of generation of amidolytic activity versus proteolytic cleavage of factor XI, strongly supporting the conclusion that the amidolytic activity detected in our experiments represents factor XIa generation. Effects of Fibrinogen on Factor XI Activation. We have demonstrated that HK (45 nm) is a cofactor that facilitates factor XI binding to platelets and optimal rates of factor XIa formation with platelets (15). In the presence of high concentrations of HK up to the physiological concentration in plasma (~640 nm) we observed a concentration-dependent decrease in rates of factor-xi activation in the presence of dextran sulfate or activated platelets (15). However, the presence of prothrombin at physiological concentrations reversed this inhibition by HK of factor XIa generation (15). Similarly, it has been shown that fibrinogen at plasma concentrations (~9 µm) completely abolishes dextran sulfate-mediated activation of factor XI by thrombin (26). We, therefore, examined fibrinogen (9.0 µm) to determine its effect on thrombin-mediated activation of factor XI on the platelet surface. The data in Table 1 reveal that fibrinogen at plasma concentrations (9.0 µm) does not prevent thrombin-mediated activation of factor XI on the platelet surface, whereas it does prevent dextran sulfate mediated thrombin activation of factor XI as previously reported (26). Fibrinogen does not affect rates of factor XI activation by factor XIIa in the presence of dextran sulfate or activated platelets (Table 1). 11
DISCUSSION Intensive investigations have focused on the mechanism and physiological significance of thrombin-catalyzed factor-xi activation (7,8,15,26,27,34). Since no bleeding disorders are associated with deficiencies of factor XII, PK or HK, it has been suggested that thrombin rather than factor XIIa is the physiological activator of factor XI in vivo. However, conflicting lines of evidence have been published to support conclusions that the activation of factor XI by thrombin may (7,8,15,34) or may not proceed (26,27) in plasma. Although it has been shown that factor XI is activated at accelerated rates by thrombin in a purified system including dextran sulfate, HK and fibrinogen at their plasma concentrations inhibit thrombin-catalyzed factor-xi activation in the presence of dextran sulfate and other artificial surfaces (7,8,15,26,27). We have reported that activated platelets promote factor-xi activation by thrombin at initial rates 2-fold to 5-fold greater than dextran sulfate under conditions optimal for factor XI binding to platelets (15). Physiological concentrations of HK (640 nm) inhibited factor-xi activation by thrombin in a concentration-dependent manner, and this inhibition was reversed by 1-3 µm prothrombin (15). Furthermore, prothrombin (1-3 µm) in the presence of CaCl 2 (2 mm) was able to replace HK (45 nm in the presence of ZnCl 2, 25 µm) as a cofactor for the specific, reversible, high-affinity binding of factor XI to platelets (15). Finally, prothrombin and CaCl 2 (2 mm) could substitute for HK and ZnCl 2 in promoting optimal rates of thrombin-catalyzed factor-xi activation on the platelet surface. These studies suggest that the intrinsic coagulation pathway can be initiated by mechanisms independent of the contact phase proteins, factor XII, HK and PK. Since factor XI is also activated by factor XIa and factor XIIa (1,7,8), we were interested in comparing the three activators on the two surfaces that have been reported to be the most potent activating surfaces (dextran sulfate and activated platelets). Our present study supports the following conclusions: 1) thrombin is the preferred factor-xi activator as compared with 13
factor XIIa and factor XIa on the platelet surface; and 2) fibrinogen, a substrate for thrombin in plasma, does not prevent thrombin-mediated activation of factor XI on the platelet surface. Thus, at equimolar concentrations of the enzymes (i.e., thrombin, factor XIIa and factor XIa) required for optimal rates of factor-xi activation (i.e. ~1 nm), the maximum rates of factor-xi activation achieved in the presence of activated platelets were ~30 nm/min with thrombin, ~6 nm/min factor XIIa and ~2 nm/min with factor XIa. Values of turnover numbers (kcat) calculated from data presented in Figure 1-4 for factor-xi activation on activated platelets under these conditions (i.e., initial rates divided by added enzyme concentration) were 24-167 (mean = 86) min -1 for thrombin, 4.6-50 (mean = 21) min -1 for factor XIIa and 1.3-21 (mean = 8) min -1 for factor XIa. Previously, we reported that a specific binding site for platelets exists in the A3 domain of factor XI (16). We then determined that the A3 domain interacts with the platelet surface and mediates factor XI binding to the platelet surface which facilitates the enhancement by platelets of thrombin-catalyzed activation (15). The biochemical nature of the receptor on the platelet surface that binds factor XI is unknown. The mechanism by which interaction of factor XI with its platelet receptor might accelerate its activation by thrombin is unknown but might result from colocalization of enzyme and substrate on the platelet surface or might involve a change in the conformation of factor XI to render it a more favorable substrate. However, we have previously concluded that the mechanism by which activated platelets and dextran sulfate promote thrombin-catalyzed factor XI activation are different since the binding of factor XI to platelets occurs through the A3 domain, whereas binding to dextran sulfate is mediated through the A1 domain (15). Fibrinogen, the most abundant substrate for thrombin in plasma has been found to prevent thrombin-mediated activation of factor XI by dextran sulfate while having no effect on factor XIIa-catalyzed activation of factor XI (26). These data have been interpreted to suggest that 14
factor XI is unlikely to be activated in plasma by thrombin. We examined physiological concentrations of fibrinogen to determine its effect on thrombin-mediated activation of factor XI bound to the platelet surface. It was found that fibrinogen (9.0 µm) does not prevent thrombinmediated potentiation of factor-xi activation on the platelet surface, but does prevent dextran sulfate-mediated thrombin activation of factor XI by thrombin in confirmation of a previous report (26). Fibrinogen might be expected to compete with factor XI as a substrate for thrombin, so the fact that competitive inhibition of thrombin-mediated factor XI activation does not occur on the platelet surface is particularly important and interesting. The explanation for this result may be that both factor XI (K d ~ 10 nm) and thrombin (K d ~ 0.3 nm) bind to platelets with high affinity, whereas fibrinogen binds to thrombin (35) with much lower affinity (K d ~ 13 µm). Since it has been demonstrated that thrombin can bind to GPIbα with high affinity (36) and we have preliminary evidence that factor XI can also bind to GPIb/IX with high affinity (preliminary results, F.A. Baglia, D. Sinha, K.O. Badellino and P.N. Walsh), thrombin and factor XI may be colocalized on a high-affinity binding site on the activated platelet surface that is separate and distinct from the fibrinogen binding site on glycoprotein IIb/IIIa, thereby accounting for the absence of competitive inhibition by fibrinogen of thrombin-mediated factor XI activation on the activated platelet surface. In summary, the data presented in this paper support the conclusion that thrombin is an important activator of factor XI (4-fold more effective than factor XIIa; 11-fold more effective than factor XIa based on calculated values of turnover number) on the platelet surface. These findings further corroborate the conclusion that activated platelets can provide a physiologically relevant surface for the activation of factor XI by thrombin (at low concentrations EC 50 ~100 pm; threshold concentration ~10 pm). These results can explain the absence of hemostatic 15
abnormalities in patients with deficiencies of factor XII, HK and prekallikrein. The suggestion that this reaction could occur in a plasma and activated platelet environment is supported by the observation that the presence of activated platelets could also abrogate the inhibitory effect of fibrinogen (at physiological concentrations) in the activation of factor XI by thrombin on the platelet surface. We have previously reported that prothrombin (at physiological concentrations) can abolish the inhibitory effect of HK (at physiological concentrations) on the activation of factor XI by thrombin (15). Thereby, HK and fibrinogen would not block thrombin activation of factor XI on the platelet surface. Thus, these data support the hypothesis (7,8) that the activation of the intrinsic pathway in a revised model of blood coagulation occurs under physiological conditions when thrombin (at low concentrations) is initially generated via the tissue factor pathway and can activate factor XI on the activated platelet surface independent of other contact phase proteins. Acknowledgments -- The authors are grateful to Virginia Sheaffer and Patricia Pileggi for their assistance in manuscript preparation. 16
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Table 1 The Effects of Fibrinogen and Activated Platelets, Dextran Sulfate or Phospholipids on the Activation of Factor XI by Thrombin, Factor XIIa or Factor XIa a Enzyme Enzyme Concentration Fibrinogen IIa XIIa XIa Materials (nm) (µm) Initial Rate (nm/min) b No Surface 1.25 0.33 ± 0.029 0.5 ± 0.031 0.25 ± 0.019 Activated Platelets 1.25 -- 30.0 ± 3.2 5.8 ± 0.60 1.65 ± 0.13 HK (45 nm) 0 -- 1.25 ± 0.18 1.0 ± 0.09 0.25 ± 0.21 CaCl 2 (2 mm) 1.25 9.0 30.2 ± 2.6 6.1 ± 0.51 -- and ZnCl 2 (25 µm) Phosphatidylserine: Phosphatidylcholine (1:3) 2 µm 1.25 -- 0.61 ± 0.041 0.65 ± 0.04 0.22 ± 0.11 Activated Platelets 1.25 -- 28.9 ± 2.1 5.95 ± 0.62 1.89 ± 0.19 Prothrombin (1.2 µm) 0 -- 1.05 ± 0.11 0.98 ± 0.09 0.51 ± 0.02 CaCl 2 (2 mm) 1.25 9.0 29.1 ± 2.2 6.1 ± 0.45 -- Dextran Sulfate (1 µg/ml) 1.25 -- 10.1 ± 1.1 14 ± 2.1 1.65 ± 0.17 0 -- 1.15 ± 0.10 0.57 ± 0.035 0.59 ± 0.03 1.25 9.0 2.0 ± 0.19 13.5 ± 1.2 -- a b The experiment was performed and rate of Factor XIa formation was described under experimental procedures. Data shown represent the means ± standard deviation of three observations. 20
FIGURE LEGENDS FIGURE 1. Effects of activated platelets or dextran sulfate on the rate of activation of factor XI (60 nm) by thrombin (.016 nm - 1.25 nm). Panel A: Gel-filtered platelets (1 x 10 8 platelets/ml, activated with thrombin receptor peptide, SFLLRN-amide, 25 µm) were incubated with ZnCl 2 (25 µm), CaCl 2 (2 mm) and HK (45 µm) in the presence or absence of factor XI (60 nm) for 5 min after which thrombin (0.016 nm - 1.25 nm) was added and the mixture was further incubated at 37 C. The rate of factor XIa formation was determined as described under Experimental Procedures. Data shown are mean values ± SEM (n = 3) of measurements of factor XIa at various concentrations of thrombin: 0.016 nm ( ), 0.033 nm ( ), 0.066 nm ( ), 0.15 nm ( ), 0.3 nm (+), 0.4 nm ( ), and 1.25 nm ( ). Panel B: Data shown are mean values ± SEM (n = 3) of measurements of factor XIa with dextran sulfate (1 µg/ml), factor XI (60 nm) and various concentrations of thrombin: 0.016 nm ( ), 0.033 nm ( ), 0.066 nm ( ), 0.15 nm ( ), 0.3 nm (+), 0.4 nm ( ), and 1.25 nm ( ). FIGURE 2. Effects of activated platelets or dextran sulfate on the rate of activation of factor XI by factor XIIa (0.016 nm - 1.25 nm). Panel A: Gel-filtered platelets (1 x 10 8 platelets/ml, activated with thrombin receptor peptide, SFLLRN-amide, 25 µm) were incubated with ZnCl 2 (25 µm), CaCl 2 (2 mm) and HK (45 µm) in the presence or absence of factor XI (60 nm) for 5 min after which factor XIIa was added and the mixture was further incubated at 37 C. The rate of factor XIa formation was determined as described under Experimental Procedures. Data shown are mean values ±SEM (n = 3) of measurements of factor XIa at various concentrations of factor XIIa: 0.016 nm ( ), 0.033 nm ( ), 0.066 nm ( ), 0.15 nm ( ), 0.3 nm ( ), 0.425 nm ( ), and 1.25 nm ( ). Panel B: Data shown are mean values ±SEM (n = 3) of 21
measurements of factor XIa with dextran sulfate (1 µg/ml), factor XI (60 nm) and various concentrations of factor XIIa: 0.016 nm ( ), 0.033 nm ( ), 0.066 nm ( ), 0.15 nm ( ), 0.3 nm ( ), 0.425 nm ( ), and 1.25 nm ( ). FIGURE 3. Effects of activated platelets or dextran sulfate on the rate of activation of factor XI (60 nm) by factor XIa (0.016 nm - 1.25 nm). Panel A: Gel-filtered platelets (1 x 10 8 platelets/ml, activated with thrombin receptor peptide, SFLLRN-amide, 25 µm) were incubated with ZnCl 2 (25 µm), CaCl 2 (2 mm) and HK (45 µm) in the presence or absence of factor XI (60 nm) for 5 min after which factor XIa was added and the mixture was further incubated at 37 C. The rate of factor XIa formation was determined as described under Experimental Procedures. Data shown are mean values ±SEM (n = 3) of measurements of factor XIa at various concentrations of factor XIa: 0.016 nm ( ), 0.033 nm ( ), 0.066 nm ( ), 0.15 nm ( ), and 1.25 nm ( ). Panel B: Data shown are mean values ±SEM (n = 3) of measurements of factor XIa with dextran sulfate (1 µg/ml), factor XI (60 nm) and various concentrations of factor XIa: 0.016 nm ( ), 0.033 nm ( ), 0.066 nm ( ), 0.15 nm ( ), and 1.25 nm ( ). FIGURE 4. Initial rates of factor XI activation in the presence of activated platelets or dextran sulfate in the presence of three enzymes. Initial rates were calculated from experiments similar to those presented in Figures 1, 2 and 3. Panel A: Data shown are mean values ±SEM (n = 3) of measures of initial rates (nm/min) of factor XIa formation with activated platelets in the presence of HK (45 nm), CaCl 2 (2 µm) and ZnCl 2 (25 mm)at various enzyme concentrations for: thrombin ( ), factor XIIa ( ), or factor XIa ( ). Panel B: Data shown are mean values ±SEM (n = 3) of measures of initial rates (nm/min) of factor XIa formation with activated platelets in the presence of prothrombin (1.2 µm) and CaCl 2 (2 mm) at various enzyme concentrations for: 22
thrombin ( ), factor XIIa ( ) or factor XIa ( ). Panel C: Data shown are mean values ±SEM (n=3) of measures of initial rates (nm/min) of factor XIa formation with dextran sulfate at various enzyme concentrations for: thrombin ( ), factor XIIa ( ) and factor XIa ( ). FIGURE 5. Effects of thrombin ( ), factor XIIa ( ) and factor XIa ( ) (each enzyme at 1.25 nm) on the activation of factor XI in the presence of activated platelets. The details of the experiments are as presented in the legends of Figures 1, 2 and 3. Amidolytic assays were performed as described in Methods and results are shown in Panel A. Percent cleavage of factor XI was determined by densitometry of autoradiographs of SDS gels, and results are shown in Panel B. 23
60 50 A 40 FXIa (nm) 30 20 10 0 0 0.5 1 1.5 2 2.5 TIME(Min)
60 50 B 40 FXIa (nm) 30 20 10 0 0 1 2 3 4 5 6 Time(Min)
60 50 A 40 FXIa (nm) 30 20 10 0 0 2 4 6 8 10 12 TIME (Min)
60 50 B 40 FXIa (nm) 30 20 10 0 0 5 10 15 20 TIME (Min)
40 35 A 30 25 FXIa (nm) 20 15 10 5 0 0 5 10 15 20 25 TIME(Min)
35 30 B 25 FXIa (nm) 20 15 10 5 0 0 5 10 15 20 25 TIME(Min)
35 30 A Initial Rate (nm/min) 25 20 15 10 5 0 0.01 0.1 1 10 Enzyme Concentration (nm)
35 30 B Initial Rate (nm/min) 25 20 15 10 5 0 0.01 0.1 1 10 Enzyme Concentration (nm)
Initial Rate(nM/Min) 16 14 C 12 10 8 6 4 2 0 0.01 0.1 1 Enzyme Concentration (nm) 10
PERCENT AMIDOLYTIC ACTIVITY (%) 100 80 60 40 20 0 A 0 5 10 15 20 25 TIME(MIN)
100 B PERCENT CLEAVAGE (%) 80 60 40 20 0 Thrombin (1.25nM) FXIIa(1.25nM) FXIa(1.25nM) 0 5 10 15 20 25 TIME (MIN)
Thrombin-mediated feedback activation of factor XI on the activated platelet surface is preferred over contact activation by factor XIIa or factor XIa Frank A. Baglia and Peter N. Walsh J. Biol. Chem. published online April 25, 2000 Access the most updated version of this article at doi: 10.1074/jbc.M000464200 Alerts: When this article is cited When a correction for this article is posted Click here to choose from all of JBC's e-mail alerts