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J. Biol. Chem., Vol. 275, Issue 27, 20514-20519, July 7, 2000

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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^§¶

From  The Sol Sherry Thrombosis Research Center, ^§ Departments of Medicine and Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140

Received for publication, January 19, 2000, and in revised form, April 3, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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, EC₅₀ ~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 high molecular weight kininogen (45 nM) and ZnCl₂ (25 µM) or prothrombin (1.2 µM) and CaCl₂ (2 mM). The maximum rates of factor XI activation achieved in the presence of activated gel-filtered platelets were 30 nM·min¹ with thrombin, 6 nM·min¹ with factor XIIa and 2 nM·min¹ with factor XIa. Values of turnover number calculated at various enzyme concentrations (0.05-1 nM) were 24-167 (mean = 86) min¹ for thrombin, 4.6-50 (mean = 21) min¹ for factor XIIa, and 1.3-14 (mean = 8) min¹ 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 gel-filtered 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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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)¹ (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 platelet-binding 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 Arg³⁶⁹-Ile³⁷⁰ bond generating the new amino-terminal sequence (IVGG) 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, because both HK and fibrinogen can inhibit thrombin-catalyzed activation of factor XI in the presence of dextran sulfate (26, 27). However, 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 Zn²⁺ ions or in the presence of prothrombin and Ca²⁺ ions; 2) both prothrombin (in the presence of Ca²⁺) and HK (in the presence of Zn²⁺) 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.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- Human prothrombin, human factor XIIa, and human factor XIa were purchased from Hematologic Technologies Inc. (Essex Junction, VT). Human -thrombin (28,000 NIH units/mg) and human fibrinogen were purchased from Enzyme Research Laboratories (South Bend, IN). The thrombin receptor agonist peptide SFLLRN-amide was synthesized on an Applied Biosystems (Foster City, CA) 430A synthesizer and purified via reverse-phase HPLC to >99% homogeneity. All reagents and materials used for SDS-polyacrylamide gel electrophoresis were purchased from Bio-Rad Laboratories, Inc. (Nelville, NY). IODO-GEN vials (20-µg coating) were obtained from Pierce (Rockford, IL). Hepes, Sepharose 2B-CL, Sephadex G-25 (fine), Tris-HCl, sodium phosphate, Trizma (Tris base), bovine serum albumin, and sodium metabisulfite were purchased from Sigma.

Purification of Proteins-- Factor XI (250 units/mg of 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 units/mg) was purified by the method of Kerbiriou and Griffin (31).

Radiolabeling of Protein-- Purified factor XI was radiolabeled with ¹²⁵I by a minor modification (15) of the IODO-GEN method to a specific activity of 5 × 10⁶ cpm/µg. The radiolabeled protein retained >98% of its biological activity.

Protein Analysis-- Protein concentrations were determined by the Bio-Rad (Richmond, CA) dye-binding assay according to the instructions provided by the manufacturer. Polyacrylamide gel electrophoresis in SDS was performed as described previously (15). Gels were stained and dried onto paper, and autoradiograms were prepared from the dried gels using intensifier screens (DuPont Cronex Lighting-Plus Screens, mounted on Spectrolene cassettes, Reliance X-Ray, Inc., Oreland, PA). X-Omat-AR film (Eastman Kodak Inc.) 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, average M_r = 500,000; Sigma) 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--dioleoyl-phosphatidylchlorine, 1:3 ratio), prepared as described previously (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 absorbance at 405 nM (A₄₀₅). In experiments with thrombin as a factor XI activator, hirudin (25 units/ml) (Sigma) 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 (nanomolar) by the time (minutes): 2 min 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 the substrate was converted to factor XIa. To confirm the formation of factor XIa, samples of identical incubation mixtures with added ¹²⁵I-labeled factor XI were examined by SDS-gel electrophoresis (15). Autoradiography of SDS-gels were performed to visualize the proteolytic cleavage of ¹²⁵I-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-Tyrode buffer (126 mM NaCl, 2.7 mM KCl, 1 mM MgCl₂, 0.38 mM NaH₂PO₄, 5.6 mM dextrose, 6.2 mM sodium Hepes, 8.9 mM Hepes free acid, 0.1% bovine serum albumin), 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).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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), ZnCl₂ (25 µM), and CaCl₂ (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). 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 × 10⁸/ml) and rates of factor XI activation by thrombin (data not shown). A platelet concentration of 1.0 × 10⁸/ml was arbitrarily chosen for all the present experiments, because it approximates the physiological concentration in circulating blood. Activated gel-filtered platelets were shown to promote the activation of factor XI by thrombin (Fig. 1A) at rates 2- to 3-fold greater than those obtained with dextran sulfate (Fig. 1B) in the presence of HK (45 nM) and ZnCl₂ (25 µM). As shown in Table I, similar results were obtained when HK and ZnCl₂ were substituted with prothrombin (1.2 µM) and CaCl₂ (2 mM) as cofactors for factor XI binding to activated platelets (15). When dextran sulfate was used as a surface, the cofactors (HK plus ZnCl₂ or prothrombin plus CaCl₂) were excluded from the incubation mixture, because 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 ZnCl₂ (25 µM) or prothrombin (1.2 µM) and CaCl₂ (2 mM), the EC₅₀ (enzyme concentration at which 50% of activity was achieved) was ~0.1 nM (~0.01 units/ml) thrombin in the presence of either activated platelets or dextran sulfate.

View larger version (25K): [in this window] [in a new window]   Fig. 1.   Effects of activated platelets or dextran sulfate on the rate of activation of factor XI (60 nM) by thrombin (0.016-1.25 nM). A, gel-filtered platelets (1 × 108 platelets/ml, activated with thrombin receptor peptide, SFLLRN-amide, 25 µM) were incubated with ZnCl2 (25 µM), CaCl2 (2 mM), and HK (45 µM) in the presence or absence of factor XI (60 nM) for 5 min after which thrombin (0.016-1.25 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 ± S.E. (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 (). B, data shown are mean values ± S.E. (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 ().

Activation of Factor XI by Factor XIIa or Factor XIa in the Presence of Activated Platelets or Dextran Sulfate-- Because 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. Fig. 2 shows the activation of factor XI by factor XIIa in the presence of activated platelets (Fig. 2A) or dextran sulfate (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 (Fig. 3) demonstrated that activated platelets (Fig. 3A) 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 (Fig. 3B).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Effects of activated platelets or dextran sulfate on the rate of activation of factor XI by factor XIIa (0.016-1.25 nM). A, gel-filtered platelets (1 × 108 platelets/ml, activated with thrombin receptor peptide, SFLLRN-amide, 25 µM) were incubated with ZnCl2 (25 µM), CaCl2 (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 ± S.E. (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 (). B, data shown are mean values ± S.E. (n = 3) of 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 ().

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Effects of activated platelets or dextran sulfate on the rate of activation of factor XI (60 nM) by factor XIa (0.016-1.25 nM). A, gel-filtered platelets (1 × 108 platelets/ml, activated with thrombin receptor peptide, SFLLRN-amide, 25 µM) were incubated with ZnCl2 (25 µM), CaCl2 (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 ± S.E. (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 (). B, data shown are mean values ± S.E. (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 ().

Fig. 4 shows the initial rates of factor XI activation at various concentrations of the three enzymes in the presence of activated platelets (Figs. 4, A and B) or dextran sulfate (Fig. 4C) as a surface. Results with activated platelets are shown with HK (45 nM), CaCl₂ (2 mM), and ZnCl₂ (25 µM) as cofactors for factor XI binding to platelets (Fig. 4A) or with prothrombin (1.2 µM) and CaCl₂ (2 mM) added to facilitate factor XI binding to platelets (Fig. 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.

View larger version (18K): [in this window] [in a new window]   Fig. 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 Figs. 1, 2, and 3. A, data shown are mean values ± S.E. (n = 3) of measures of initial rates (nanomolar/min) of factor XIa formation with activated platelets in the presence of HK (45 nM), CaCl2 (2 µM), and ZnCl2 (25 mM) at various enzyme concentrations for: thrombin (), factor XIIa (), or factor XIa (). B, data shown are mean values ± S.E. (n = 3) of measures of initial rates (nanomolar/min) of factor XIa formation with activated platelets in the presence of prothrombin (1.2 µM) and CaCl2 (2 mM) at various enzyme concentrations for: thrombin (), factor XIIa (), or factor XIa (). C, data shown are mean values ± S.E. (n = 3) of measures of initial rates (nanomolar/min) of factor XIa formation with dextran sulfate at various enzyme concentrations for: thrombin (), factor XIIa (), and factor XIa ().

Comparison of Proteolytic Cleavage of Factor XI on Polyacrylamide Gel Electrophoresis 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 Figs. 1A, 2A, and 3A, samples of identical incubation mixtures with added ¹²⁵I-labeled factor XI (at one 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 (Fig. 5A) and for the extent of proteolytic cleavage of factor XI by measuring the intensity of the 50- and 30-kDa bands (heavy and light chains) by densitometry (Kodak Digital Scientific Program, Kodak) and direct quantitation of factor XI cleavage products (Fig. 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.

View larger version (22K): [in this window] [in a new window]   Fig. 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 Figs. 1, 2, and 3. Amidolytic assays were performed as described under "Methods," and results are shown in A. Percentage cleavage of factor XI was determined by densitometry of autoradiographs of SDS gels, and results are shown in B.

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 I 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 I).

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Intensive investigations have focused on the mechanism and physiological significance of thrombin-catalyzed factor XI activation (7, 8, 15, 26, 27, 34). Because no bleeding disorders are associated with deficiencies of factor XII, prekallikrein, 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 (26, 27) proceed in plasma. Although it has been shown that factor XI is activated at accelerated rates by thrombin in a purified system containing 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- 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 CaCl₂ (2 mM) was able to replace HK (45 nM in the presence of ZnCl₂, 25 µM) as a cofactor for the specific, reversible, high affinity binding of factor XI to platelets (15). Finally, prothrombin and CaCl₂ (2 mM) could substitute for HK and ZnCl₂ 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 prekallikrein.

Because factor XI is also activated by factors XIa and 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 factors XIIa and 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 with factor XIIa, and ~2 nM/min with factor XIa. Values of turnover numbers (k_cat) calculated from data presented in Figs. 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) min¹ for thrombin, 4.6-50 (mean = 21) min¹ for factor XIIa, and 1.3-21 (mean = 8) min¹ 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, because 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 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 thrombin-mediated 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). Because it has been demonstrated that thrombin can bind to glycoprotein Ib with high affinity (36) and we have preliminary evidence that factor XI can also bind to glycoprotein Ib/IX with high affinity,² 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, EC₅₀ ~100 pM; threshold concentration, ~10 pM). These results can explain the absence of hemostatic 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.

	ACKNOWLEDGEMENTS

We are grateful to Virginia Sheaffer and Patricia Pileggi for their assistance in manuscript preparation.

	FOOTNOTES

^* This study was supported by Research Grants HL46213, HL56153, and HL56914 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed: Sol Sherry Thrombosis Research Ctr., Temple University School of Medicine, 3400 N. Broad St., Philadelphia, PA 19140. Tel.: 215-707-4375; Fax: 215-707-3005; E-mail: pnw@astro.ocis.temple.edu.

Published, JBC Papers in Press, April 25, 2000, DOI 10.1074/jbc.M000464200

² F. A. Baglia, D. Sinha, K. O. Badellino, and P. N. Walsh, unpublished results.

	ABBREVIATIONS

The abbreviation used is: HK, high molecular weight kininogen.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				J. Jesty and E. Beltrami Positive Feedbacks of Coagulation: Their Role in Threshold Regulation Arterioscler. Thromb. Vasc. Biol., December 1, 2005; 25(12): 2463 - 2469. [Abstract] [Full Text] [PDF]

				D. Navaneetham, L. Jin, P. Pandey, J. E. Strickler, R. E. Babine, S. S. Abdel-Meguid, and P. N. Walsh Structural and Mutational Analyses of the Molecular Interactions between the Catalytic Domain of Factor XIa and the Kunitz Protease Inhibitor Domain of Protease Nexin 2 J. Biol. Chem., October 28, 2005; 280(43): 36165 - 36175. [Abstract] [Full Text] [PDF]

				L. Jin, P. Pandey, R. E. Babine, J. C. Gorga, K. J. Seidl, E. Gelfand, D. T. Weaver, S. S. Abdel-Meguid, and J. E. Strickler Crystal Structures of the FXIa Catalytic Domain in Complex with Ecotin Mutants Reveal Substrate-like Interactions J. Biol. Chem., February 11, 2005; 280(6): 4704 - 4712. [Abstract] [Full Text] [PDF]

				T. Shi, B. Giannakopoulos, G. M. Iverson, K. A. Cockerill, M. D. Linnik, and S. A. Krilis Domain V of {beta}2-Glycoprotein I Binds Factor XI/XIa and Is Cleaved at Lys317-Thr318 J. Biol. Chem., January 14, 2005; 280(2): 907 - 912. [Abstract] [Full Text] [PDF]

				F. A. Baglia, C. N. Shrimpton, J. Emsley, K. Kitagawa, Z. M. Ruggeri, J. A. Lopez, and P. N. Walsh Factor XI Interacts with the Leucine-rich Repeats of Glycoprotein Ib{alpha} on the Activated Platelet J. Biol. Chem., November 19, 2004; 279(47): 49323 - 49329. [Abstract] [Full Text] [PDF]

				F. A. Baglia, D. Gailani, J. A. Lopez, and P. N. Walsh Identification of a Binding Site for Glycoprotein Ib{alpha} in the Apple 3 Domain of Factor XI J. Biol. Chem., October 29, 2004; 279(44): 45470 - 45476. [Abstract] [Full Text] [PDF]

				S. J.H. Wielders, S. Beguin, H. C. Hemker, and T. Lindhout Factor XI-Dependent Reciprocal Thrombin Generation Consolidates Blood Coagulation when Tissue Factor Is Not Available Arterioscler. Thromb. Vasc. Biol., June 1, 2004; 24(6): 1138 - 1142. [Abstract] [Full Text] [PDF]

				T. Shi, G. M. Iverson, J. C. Qi, K. A. Cockerill, M. D. Linnik, P. Konecny, and S. A. Krilis {beta}2-Glycoprotein I binds factor XI and inhibits its activation by thrombin and factor XIIa: Loss of inhibition by clipped {beta}2-glycoprotein I PNAS, March 16, 2004; 101(11): 3939 - 3944. [Abstract] [Full Text] [PDF]

				T. H. Yun, F. A. Baglia, T. Myles, D. Navaneetham, J. A. Lopez, P. N. Walsh, and L. L. K. Leung Thrombin Activation of Factor XI on Activated Platelets Requires the Interaction of Factor XI and Platelet Glycoprotein Ib{alpha} with Thrombin Anion-binding Exosites I and II, Respectively J. Biol. Chem., November 28, 2003; 278(48): 48112 - 48119. [Abstract] [Full Text] [PDF]

				F. A. Baglia, C. N. Shrimpton, J. A. Lopez, and P. N. Walsh The Glycoprotein Ib-IX-V Complex Mediates Localization of Factor XI to Lipid Rafts on the Platelet Membrane J. Biol. Chem., June 6, 2003; 278(24): 21744 - 21750. [Abstract] [Full Text] [PDF]

				T. R. Baird and P. N. Walsh Factor XI, but Not Prekallikrein, Blocks High Molecular Weight Kininogen Binding to Human Umbilical Vein Endothelial Cells J. Biol. Chem., May 30, 2003; 278(23): 20618 - 20623. [Abstract] [Full Text] [PDF]

				T. R. Baird and P. N. Walsh The Interaction of Factor XIa with Activated Platelets but Not Endothelial Cells Promotes the Activation of Factor IX in the Consolidation Phase of Blood Coagulation J. Biol. Chem., October 4, 2002; 277(41): 38462 - 38467. [Abstract] [Full Text] [PDF]

				D. M. Monroe, M. Hoffman, and H. R. Roberts Platelets and Thrombin Generation Arterioscler. Thromb. Vasc. Biol., September 1, 2002; 22(9): 1381 - 1389. [Abstract] [Full Text] [PDF]

				T. R. Baird and P. N. Walsh Activated Platelets but Not Endothelial Cells Participate in the Initiation of the Consolidation Phase of Blood Coagulation J. Biol. Chem., August 2, 2002; 277(32): 28498 - 28503. [Abstract] [Full Text] [PDF]

				A. Zivelin, F. Bauduer, L. Ducout, H. Peretz, N. Rosenberg, R. Yatuv, and U. Seligsohn Factor XI deficiency in French Basques is caused predominantly by an ancestral Cys38Arg mutation in the factor XI gene Blood, April 1, 2002; 99(7): 2448 - 2454. [Abstract] [Full Text] [PDF]