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Originally published In Press as doi:10.1074/jbc.M306925200 on October 2, 2003 Originally published In Press as doi:10.1074/jbc.M306925200 on September 25, 2003 Originally published In Press as doi:10.1074/jbc.M306925200 on September 10, 2003

J. Biol. Chem., Vol. 278, Issue 48, 48112-48119, November 28, 2003
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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*

Thomas H. Yun{ddagger}§, Frank A. Baglia§, Timothy Myles{ddagger}, Duraiswamy Navaneetham¶, José A. López||, Peter N. Walsh¶**, and Lawrence L. K. Leung{ddagger}{ddagger}{ddagger}

From the {ddagger}Division of Hematology, Department of Medicine, Stanford University School of Medicine, Stanford, California 94305, the Sol Sherry Thrombosis Research Center and the **Departments of Medicine and Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, and the ||Thrombosis Research Section, Department of Medicine, Baylor College of Medicine, and the Houston Veterans Affairs Medical Center, Houston, Texas 77030

Received for publication, June 29, 2003 , and in revised form, September 10, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Activation of factor XI (FXI) by thrombin on stimulated platelets plays a physiological role in hemostasis, providing additional thrombin generation required in cases of severe hemostatic challenge. Using a collection of 53 thrombin mutants, we identified 16 mutants with <50% of the wild-type thrombin FXI-activating activity in the presence of dextran sulfate. These mutants mapped to anion-binding exosite (ABE) I, ABE-II, the Na+-binding site, and the 50-insertion loop. Only the ABE-II mutants showed reduced binding to dextran sulfate-linked agarose. Selected thrombin mutants in ABE-I (R68A, R70A, and R73A), ABE-II (R98A, R245A, and K248A), the 50-insertion loop (W50A), and the Na+-binding site (E229A and R233A) with <10% of the wild-type activity also showed a markedly reduced ability to activate FXI in the presence of stimulated platelets. The ABE-I, 50-insertion loop, and Na+-binding site mutants had impaired binding to FXI, but normal binding to glycocalicin, the soluble form of glycoprotein Ib{alpha} (GPIb{alpha}). In contrast, the ABE-II mutants were defective in binding to glycocalicin, but displayed normal binding to FXI. Our data support a quaternary complex model of thrombin activation of FXI on stimulated platelets. Thrombin bound to one GPIb{alpha} molecule, via ABE-II on its posterior surface, is properly oriented for its activation of FXI bound to a neighboring GPI{alpha} molecule, via ABE-I on its anterior surface. GPIb{alpha} plays a critical role in the co-localization of thrombin and FXI and the resultant efficient activation of FXI.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Human factor XI (FXI),1 unique among the coagulation proteases in the clotting cascade, circulates in blood in zymogen form as a disulfide-linked homodimer (1–3) in a noncovalent complex with high molecular weight kininogen (HMWK) (4). Each FXI monomer is composed of an N-terminal heavy chain and a C-terminal light chain containing the serine protease catalytic triad (1, 2). The heavy chain has four homologous subunits called Apple domains (designated A1, A2, A3, and A4, from the N terminus), which are not found in other coagulation proteases, with the exception of prekallikrein (3). Additionally, FXI is the only coagulation protease (with the exception of FXII and prekallikrein) that does not contain "Gla domains," sequences that are rich in {gamma}-carboxylated glutamic acids required for binding to phospholipids (5). However, despite the absence of Gla domains and their inability to interact with phospholipids, FXI and FXIa do bind to activated platelets in a specific and saturable manner (6, 7). Because of these distinctive structural features, it is not surprising that the mode of activation of FXI is quite different from that of the other coagulation enzymes.

There are three biologically relevant proteases that can activate FXI, i.e. FXIIa, FXIa, and thrombin (8, 9), all of which cleave FXI at the Arg369–Ile370 bond and expose the active-site catalytic triad. FXIIa, a component of the contact phase of blood coagulation, is unlikely to be a physiologically relevant activator because FXII deficiency is not associated with clinical bleeding (10, 11). Recent data suggest that thrombin is the physiological activator of FXI on the activated platelet (12–14). Normal hemostasis is initiated by the exposure of tissue factor at sites of vascular injury, followed by the formation of the FVII-tissue factor complex and the subsequent activation of FX. The resultant initial burst of thrombin generation is limited by the rapid and potent inhibition of the FVIIa-tissue factor-FXa complex by the tissue factor pathway inhibitor, but is sufficient to activate platelets, FVIII, FV, and FXI. The subsequent initiation of the consolidation (or intrinsic) pathway of coagulation by thrombin-mediated FXI activation on the activated platelet membrane leads to the sequential activation of FIX, FX, and prothrombin, generating a large secondary burst of thrombin. Thus, FXI activation is required for normal hemostasis only in cases of significant hemostatic challenge, which is consistent with clinical observations that patients with congenital FXI deficiency generally have much milder bleeding than those with FVIII deficiency (hemophilia A) (15, 16). Consistent with these observations is the demonstration that thrombin is more efficient than FXIIa or FXIa in activating FXI in vitro (13).

In a current model of FXI activation, the circulating FXI dimer is complexed with either HMWK (17–19) or prothrombin (18, 20), both of which bind to the A1 domain of FXI (21). Binding of HMWK or prothrombin appears to be required for the exposure of amino acids on the A3 domain of FXI, which binds to the glycoprotein Ib (GPIb)-IX-V complex on the activated platelet surface (12, 22). The FXI homodimer binds to GPIb{alpha} in the GPIb-IX-V complex through one of its monomers (12, 14, 23). Thrombin then binds to the A1 domain and activates FXI (20, 24). The binding site on thrombin for FXI has yet to be defined.

The active site of thrombin lies within a deep cleft (25, 26). Access to the active site is restricted by two surface loops, the 50-insertion loop (Leu45–Asn57, thrombin B chain numbering system) and the autolysis loop (Leu144–Gly155), that are situated on the northern and southern rims of the active-site cleft, respectively. Enzyme specificity is further defined by two ligand-binding sites (exosites) that are characterized by a high density of solvent-exposed basic residues. Many thrombin substrates, receptors, and inhibitors gain access to the active site by binding to either of the two exosites. For example, anion-binding exosite (ABE) I is important for binding to fibrinogen (27), fibrin (28), heparin cofactor II (29), PAR1 (30), thrombomodulin (31), and hirudin (32, 33), whereas ABE-II is involved in binding to platelet GPIb{alpha} (34), protease nexin I (35), and glycosaminoglycans such as heparin (14).

Utilizing a collection of thrombin mutants generated by alanine scanning site-directed mutagenesis, we previously mapped the interactions of thrombin with some of its key substrates, including protein C, thrombin-activable fibrinolysis inhibitor, and thrombomodulin (36), fibrinogen (37), FV (38), and FVIII (39). In this study, we used this collection of thrombin mutants to identify the key residues on thrombin required for FXI activation. Our data suggest a model of thrombin activation of FXI that requires the engagement of thrombin ABE-I and ABE-II by FXI and the platelet GPIb-IX-V complex, respectively.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Purified human FXI and HMWK were purchased from Hematologic Technologies (Essex Junction, VT). The expression and purification of wild-type (WT) and alanine-substituted mutant thrombins from stably transfected Chinese hamster ovary AA8 cells have been described in detail previously (36, 37). The concentration of active thrombin molecules was determined by titration with D-phenylalanylprolylarginyl chloromethyl ketone (PPACK; Calbiochem) using the chromogenic substrate H-D-Val-Leu-Arg-p-nitroanilide (S-2266, Chromogenix). A comparison of the catalytic activities of the purified WT and mutant thrombins toward the chromogenic substrate H-D-Phe-Pip-Arg-p-nitroanilide (S-2388, Chromogenix) has been described in detail (36, 38, 39). Dextran sulfate (average Mr 500,000) and cyanogen bromide-activated Sepharose 4B-agarose were purchased from Sigma. Dimethylmethylene blue was obtained from Serva Fine Chemicals (Long Island, NY). Active site-inhibited thrombin was prepared by incubation of a 10-fold excess of PPACK with thrombin for 1 h at 37 °C. This mixture was then dialyzed in Spectrapor tubing (Mr cutoff of 3500; Spectrum Medical Industries, Inc., Los Angeles, CA) overnight in phosphate-buffered saline at 4 °C. The thrombin receptor activation peptide (SFLLRN-amide) was synthesized on an Applied Biosystems 430A synthesizer and purified to >99% homogeneity by reverse-phase high pressure liquid chromatography.

Radiolabeling of Factor XI and PPACK-Thrombin—Purified FXI and PPACK-thrombin were radiolabeled with 125I by a minor modification (20) of the IODO-GEN method to specific activities of 5 x 106 and 2 x 106 cpm/µg, respectively. Radiolabeled FXI retained >98% of its biological activity.

Preparation of Glycocalicin—Glycocalicin was extracted from human platelets and purified as described previously (40).

Factor XI Activation in the Presence of Dextran Sulfate—Mutant thrombins were screened for their ability to activate FXI using a twostage chromogenic assay. Initially, 1 nM thrombin was incubated with 60 nM FXI for 10 min at 37 °C in 100 µl of assay buffer (50 mM Tris (pH 7.3), 150 mM NaCl, 0.1% bovine serum albumin (BSA), and 1 µg/ml dextran sulfate) to activate FXI. Thrombin activity was inhibited with 25 units/ml hirudin. FXIa activity was measured by combining 20 µl of the reaction mixture with 80 µl of 600 µM S-2366 and observing the change in absorbance at 405 nm on a Spectramax Plus microplate reader (Molecular Devices, Sunnyvale, CA). For 10 mutants demonstrating diminished (<50% compared with WT thrombin) FXI activation, dose-response curves were constructed over a range of thrombin concentrations using the above protocol. Linear initial rates of FXIa generation with respect to time and enzyme (thrombin) concentration were observed in the presence of dextran sulfate as previously reported (8, 9, 13). EC50 values were calculated using SigmaPlot software.

Preparation of Washed Platelets—Platelets were prepared as described (6, 14, 23). Platelet-rich plasma obtained from citrated human blood was centrifuged, and the platelets were resuspended in calcium-free Hepes/Tyrode's 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), and 0.1% BSA) at pH 6.5 and gel-filtered on a column of Sepharose 2B equilibrated in calcium-free Hepes/Tyrode's buffer (pH 7.2). Platelets were counted electronically with a Coulter counter (Coulter Electronics, Hialeah, FL).

Assay of Factor XI Activation in the Presence of Activated Platelets—Activation of FXI (60 nM) by thrombin (and thrombin mutants) was measured by chromogenic assay. Incubations were carried out at 37 °C in 200 µl of 50 mM Tris and 150 mM NaCl (pH 7.3) with 1% BSA and gel-filtered platelets (activated by incubation at 37 °C for 1 min with the thrombin receptor activation peptide SFLLRN-amide at 25 µM) in the presence of 42 nM HMWK, 2 mM CaCl2, and 25 µM ZnCl2. After dilution to a final volume of 1 ml with 50 mM Tris and 150 mM NaCl (pH 7.3) with 1% BSA containing 600 µM S-2366, the amount of free p-nitroanilide was determined by measuring the changes in absorbance at 405 nm. Hirudin (25 units/ml) was added at the end of the reaction to inhibit thrombin activity. Low levels of enzymatic activity after 5-fold dilution of the incubation mixture containing no added FXI were subtracted from all results to assure detection and measurement of only FXIa generated during experimental incubations. The amount of FXIa generated was assayed by reference to a standard curve constructed using purified FXIa. Linear initial rates of FXIa generation with respect to time and enzyme (thrombin) concentration were observed in the presence of activated platelets as previously reported (13). Dose-response curves were constructed over a range of thrombin concentrations to quantify rates of FXI activation by WT thrombin and by the 10 thrombin mutants identified as demonstrating diminished FXI activation during screening.

Thrombin-Dextran Sulfate Binding Assay—Dextran sulfate (average Mr 500,000) was coupled to cyanogen bromide-activated Sepharose 4B-agarose according to the manufacturer's instructions. 3 g of Washed resin was combined with 30 ml of 10 mg/ml dextran sulfate in coupling buffer at room temperature for 2.5 h. Bound dextran sulfate was quantified by dimethylmethylene blue assay (35) and determined to be 3.2 mg of dextran/mg of resin. WT and mutant thrombins were screened for their ability to bind to dextran sulfate cross-linked to Sepharose 4B-agarose. 500 µl of 10 nM WT or mutant thrombin solution was incubated at room temperature with 50 µl of a 1:2 slurry of dextran sulfate-cross-linked agarose. After 10 min, the mixtures were filtered through spin columns (2000 x g), and the resultant flow-through fractions were assayed for thrombin activity using the chromogenic substrate S-2238. 100 µl of flow-through fractions was incubated for 10 min with 100 µlof 0.4 mM S-2238, and the absorbance at 405 nm was measured at that point. Absorbance readings were determined to be linear within the prescribed thrombin concentration ranges. To quantify the amount of bound thrombin, these measurements were subtracted from the A450 nm of the flow-through fractions from an incubation using non-cross-linked Sepharose 4B. This value was then divided by the A405 nm of the flow-through fractions from the non-cross-linked Sepharose 4B incubation to obtain a percentage of thrombin bound specifically to dextran sulfate.

Solid-phase Binding of 125I-PPACK-Thrombin to Glycocalicin—We utilized a modified method of De Cristofaro et al. (41) to examine the binding of 125I-PPACK-thrombin to plate-bound glycocalicin. Wheat germ lectin (10 µg/ml) was coated on the wells of 96-well polystyrene trays (Immulon high protein capacity binding) and incubated overnight at 4 °C in 50 mM carbonate buffer (pH 9.50). The remaining binding sites in the sample wells were blocked by incubation with 1% BSA in Hepes-buffered saline for 2 h. After aspiration of the BSA solution, purified glycocalicin was added to the wells at a concentration of 20 µg/ml and incubated at 4 °C for 1 h. After aspiration, 125I-PPACK-thrombin was applied to the wells and incubated for 1 h at 37 °C. Each sample and blank well were washed with Hepes-buffered saline seven times for 1 min each, dried, and counted in a Wallac Wizard 1470 {gamma}-counter.

Solid-phase Binding of 125I-PPACK-Thrombin to Factor XI—We utilized a modified method of De Cristofaro et al. (41) to examine the binding of 125I-PPACK-thrombin to FXI. Wheat germ lectin was coated on the wells of 96-well polystyrene trays (Immulon high protein capacity binding) and incubated overnight at 4 °C in 50 mM carbonate buffer (pH 9.50). The remaining binding sites of the sample wells were blocked for 2 h with 1% BSA in Hepes-buffered saline. After aspiration of the BSA solution, purified FXI was added to the wells at a concentration of 20 µg/ml and incubated at 4 °C for 1 h. After aspiration, 125I-PPACK-thrombin and the thrombin mutants were applied to the wells and incubated for 1 h at 37 °C. Each sample and blank well were washed with Hepes-buffered saline seven times for 1 min each, dried, and counted in a Wallac Wizard 1470 {gamma}-counter. We utilized the Kaleidagraph program (Synergy Software, Reading, PA) to determine the Kd of the thrombin-FXI interaction using the equation y = (M0 x M1)/(M2 + M0), where M0 is the total ligand concentrations, M1 is Bmax, and M2 is Kd. In competition experiments, the concentration of competitor that displaced 50% of the bound 125I-thrombin (IC50) was determined by plotting the amount of 125I-thrombin bound to FXI or glycocalicin versus the amount of competitor ligand added.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Screening for Thrombin Mutants Defective in Dextran Sulfate-mediated Factor XI Activation—A site-directed mutagenesis strategy was used to determine residues on thrombin important for activating FXI in the presence of dextran sulfate. A collection of 53 thrombin mutants in which solvent-exposed polar and charged residues were mutated to alanine was screened for FXI-activating ability in the presence of dextran sulfate (Fig. 1). A total of 16 mutants mapped to ABE-I, ABE-II, the Na+-binding site, and the 50-insertion loop demonstrated <50% of the WT thrombin FXI-activating ability. Among these are seven ABE-I mutants, H66A, R68A, R70A, Y71A, R73A, K107A, and the double mutant K106A/K107A, with relative activating abilities ranging from 28 to 48% of that of WT thrombin. Five ABE-II mutants, R89A/R93A/E94A, R98A, R245A, K248A, and R245A/K248A/Q251A, showed greater deficiency in FXI activation, as their relative abilities were all <27%. Interestingly, the effects of the R245A and K248A mutations seem to be additive because the triple mutant R245A/K248A/Q251A displayed an activating ability of 3%, well less than that of either the R245A or K248A mutant (27 and 11%, respectively). Mutation of Gln251 does not contribute to the deficiency of the triple mutant because the single mutation did not cause significant loss of FXI-activating ability. The ABE-I and ABE-II mutants all have normal kcat/Km values for the substrate S-2238 (36, 38, 39), suggesting that their deficiency in activating FXI results from reduced binding to FXI and/or dextran sulfate rather than a deficiency in catalysis. The 50-insertion loop (Leu45–Asn57 loop) mutant W50A and the Na+-binding site mutants E229A and R233A also showed greatly diminished FXI-activating ability.



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  		FIG. 1.
Screen of mutant thrombin FXI-activating ability. FXI activation by thrombin mutants was carried out in the presence of dextran sulfate using a two-stage chromogenic assay as described under "Experimental Procedures." The ability of mutant thrombins to activate FXI in the presence of dextran sulfate is shown as a percentage relative to that of WT thrombin. Results represent the means ± S.D. of at least three separate experiments, each done in duplicate. Mutants averaging <50% relative activating ability are indicated by black bars. Thrombin numbering is based on the thrombin B chain.

 
Binding of Thrombin to Dextran Sulfate—Dextran sulfate has been postulated to mediate FXI activation by acting as a template for the formation of a ternary complex along with thrombin (8). To identify region(s) of thrombin responsible for binding to dextran sulfate, thrombin mutants were screened for their ability to bind to dextran sulfate-cross-linked agarose (Fig. 2). Only four mutants bound to dextran sulfate-agarose with 50% or less efficiency. These four mutants all mapped to ABE-II: R89A/R93A/E94A (45.6 ± 3.6%), R98A (34 ± 1.3%), K248A (50.6 ± 1.1%), and R245A/K248A/Q251A (23.7 ± 2.0%). All of the other mutants bound to dextran sulfate with percentages ranging from 55 to 80%, with WT thrombin binding at 76.7 ± 1.0%. As in the FXI activation screen, the ABE-II triple mutant R245A/K248A/Q251 demonstrated the largest deficiency compared with WT thrombin, and this deficiency was a result of the cumulative effects of the mutations at Arg245 and Lys248, but not at Gln251. These data clearly implicate ABE-II as the region responsible for binding to dextran sulfate and suggest that the impairment in FXI-activating ability found among the ABE-II mutants is due solely to inefficient binding to dextran sulfate.



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  		FIG. 2.
Comparison of WT and mutant thrombin abilities to bind to dextran sulfate. WT and thrombin mutants were incubated with either noncross-linked or dextran sulfate-cross-linked agarose, and the residual free thrombin activities were determined by S-2238 hydrolysis as described under "Experimental Procedures." Results are expressed as percent thrombin-bound (means ± S.D. of at least three separate experiments, each done in duplicate). Mutants that bound with <=50% efficiency are indicated by black bars, and WT thrombin is indicated by the hatched bar.

 
Thrombin Activation of Factor XI in the Presence of Dextran Sulfate—Dose-response curves were generated for 10 of the 16 thrombin mutants found deficient in activating FXI in the initial screen. The selection of these 10 single mutants is representative of the regions identified to be important in FXI activation from the initial activation screen. The EC50 values of the mutants corroborate the findings of the initial screen, with all of the mutants demonstrating <10% activating ability over WT thrombin (0.47 ± 0.05 nM). Among these, the ABE-I mutants

R68A (15.9 ± 1.9 nM) and R73A (15.5 ± 1.1 nM), the ABE-II mutant R98A (22.5 ± 3.0 nM), and the 50-insertion loop mutant W50A (23.0 ± 4.4 nM) demonstrated at least a 30-fold increase compared with WT thrombin.

Factor XI Activation in the Presence of Activated Platelets—To identify thrombin residues important for FXI activation under physiological conditions, dose-response curves and EC50 values for the same 10 deficient thrombin mutants were generated using activated platelets (in the presence of 42 nM HMWK, 2 mM CaCl2, and 25 µM ZnCl2) instead of dextran sulfate in the assay (Table I). EC50 values obtained in the presence of activated platelets were remarkably similar to those obtained from studies done in the presence of dextran sulfate. A notable exception was the ~5-fold discrepancy between the EC50 values for WT thrombin in the presence of activated platelets (0.1 ± 0.2 nM) compared with dextran sulfate (0.47 ± 0.05 nM). This difference most likely reflects the higher affinity of WT thrombin for activated platelets than for dextran sulfate.


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  		TABLE I
Thrombin Activation of FXI in the presence of dextran sulfate and activated platelets 10 mutant thrombins identified with impaired FXI-activating ability from the activation screen were selected for dose-response studies. Various concentrations (0.2–50 nM) of WT and mutant thrombins were used in the dextran sulfate or activated platelet (42 nM HMWK, 2 mM CaCl2, and 25 µM, ZnCl2)-supplemented FXI activation assays described under "Experimental Procedures" to generate dose-response curves. EC50 values were calculated using SigmaPlot 2000 software. The inverse of mutant EC values standardized to that of WT thrombin was used to generate a comparative measure of FXI-activating ability (% relative to WT). Results represent the means ± S.D. of at least two separate experiments, each done in duplicate.

 
Binding of Thrombin Mutants to Glycocalicin—Glycocalicin, the extracellular domain of GPIb{alpha}, was recently discovered to accelerate thrombin activation of FXI to a degree similar to that observed with activated platelets (12). Because GPIb{alpha} appears to be the platelet receptor for FXI co-localization with thrombin, the same 10 single thrombin mutants used in the dose-response studies were tested for their ability to competitively inhibit the binding of 125I-PPACK-thrombin to glycocalicin in a solid-phase assay as a measure of their ability to bind to GPIb{alpha}. Of these thrombin mutants tested, only the three ABE-II mutants R98A, R245A, and K248A demonstrated a significant reduction in their ability to bind to glycocalicin as demonstrated by their IC50 values (Fig. 3 and Table II). All three ABE-II mutants showed at least a 100-fold decrease in binding affinity, whereas the other mutants displayed comparable or enhanced binding to glycocalicin compared with WT thrombin. These data clearly corroborate the findings of the dextran sulfate binding screen and indicate the functional similarity of dextran sulfate and glycocalicin in the activation of FXI.



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  		FIG. 3.
Binding of 125I-PPACK-thrombin to glycocalicin in the presence of various concentrations of WT and mutant thrombins. 125I-PPACK-thrombin (22 nM) was incubated with WT or mutant thrombins. When glycocalicin was not bound to the wells of microtiter plates, the amount of 125I-PPACK-thrombin was <0.1% of the control value, and the maximum variation of counts/min bound for each experimental observation was <2% of the total counts/min bound. 100% binding of 125I-PPACK-thrombin represents binding in the absence of any added WT or mutant thrombin (mean = 20,182 cpm). Nonspecific binding as represented by 125I-PPACK-thrombin binding to wells coated with BSA (mean = 404 cpm) was subtracted from the total binding at each data point. Results shown represents the means ± S.E. of three experiments, each done in duplicate to determine the effects of WT thrombin ({circ}) and thrombin mutants R68A (ABE-I) ({blacksquare}), R70A (ABE-I) ({triangledown}), Y71A (ABE-I) ({triangleup}), R73A (ABE-I) (•), R98A (ABE-II) ({blacktriangleup}), R245A (ABE-II) ({square}), K248A (ABE-II) (), W50A (Leu45–Asn57 loop) ({diamondsuit}), E229A (Na+-binding site) (x), and R233A (Na+-binding site) ({diamond})on the binding of 125I-PPACK-thrombin to glycocalicin.

 


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  		TABLE II
Effect of thrombin mutants on the binding of 125I-PPACK-thrombin to glycocalicin and to FXI in a solid-phase competition assay 10 mutant thrombins defective in activating FXI were tested for their ability to competitively inhibit the binding of 125I-PPACK-thrombin to either glycocalicin or FXI bound to a solid support, and IC50 values were calculated as described under "Experimental Procedures." Mutant IC50 values were divided by the WT thrombin IC50 values to obtain binding percentages relative to WT thrombin Results represent the means ± S.D. of at least two separate experiments, each done in duplicate.

 
Binding of Thrombin to Factor XI—To establish an experimental basis for determination of thrombin exosites that mediate binding to FXI, thrombin was inactivated with PPACK and radiolabeled to determine direct binding to FXI in a solidphase assay. The results indicate that 125I-PPACK-thrombin bound FXI in a saturable manner, with a Kd(app) of 41.9 ± 4.9 nM (Fig. 4).



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  		FIG. 4.
Saturable binding of 125I-PPACK-thrombin to FXI. FXI bound to microtiter plates was incubated with 125I-PPACK-thrombin at various concentrations, and the amounts of 125I-PPACK-thrombin bound ({circ}) at different input concentrations were determined. Binding of 125I-PPACK-thrombin to uncoated wells was <0.1% of the total binding, and the maximum variation of counts/min bound for each experimental observation was <2% of the total counts/min bound. The amount of 125I-PPACK-thrombin bound to wells coated with BSA (negative control) was subtracted from the total binding. Results shown represent the means ± S.E. of three experiments, each done in duplicate.

 
Binding of Thrombin Mutants to Factor XI—The same 10 single thrombin mutants identified in the screening assay were tested for their ability to bind to FXI in a solid-phase competition assay. All of the ABE-I mutants displayed reduced binding affinity for FXI (increase in IC50) compared with WT thrombin, with R73A showing a 20-fold reduction (Fig. 5 and Table II). The three ABE-II mutants tested showed enhancement of or negligible reduction in binding ability compared with WT thrombin. The Na+-binding site mutants E229A and R233A and the 50-insertion loop mutant W50A were also found to be deficient in FXI binding. Taken together, these results indicate the involvement of ABE-I, the Leu45–Asn57 loop, and the Na+-binding site in direct interactions with FXI.



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  		FIG. 5.
Binding of 125I-PPACK-thrombin to FXI in the presence of various concentrations of WT and mutant thrombins. 125I-PPACK-thrombin (50 nM) was incubated with WT or mutant thrombins. 100% binding of 125I-PPACK-thrombin to FXI represents binding in the absence of any added WT or mutant thrombin (mean = 15,035 cpm). Nonspecific binding as represented by 125I-PPACK-thrombin binding to wells coated with BSA (mean = 301 cpm) was subtracted from the total binding at each data point. 125I-PPACK-thrombin binding to uncoated wells was <0.1% of the total value, and the maximum variation of counts/min bound for each experimental observation was <2% of the total counts/min bound. Results shown in A represent the effect of WT thrombin ({circ}) and thrombin mutants R68A (ABE-I) ({blacksquare}), R70A (ABE-I) ({triangledown}), Y71A (ABE-I) ({triangleup}), and R73A (ABE-I) (•). Results shown in B represent the effect of R98A (ABE-II) ({blacktriangleup}), R245A (ABE-II) ({square}), K248A (ABE-II) (), W50A (Leu45–Asn57 loop) ({diamondsuit}), E229A (Na+-binding site) (x), and R233A (Na+-binding site) ({diamond}) on the binding of 125I-PPACK-thrombin to glycocalicin. Results shown represent the means ± S.E. of three experiments, each done in duplicate.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Thrombin activation of FXI plays an important role in amplifying thrombin generation at sites of vascular injury. In this study, utilizing a collection of thrombin mutants, we have identified the key thrombin residues important in this process (Fig. 6). We used two assay methods for FXI activation by thrombin. The initial screening was performed in the presence of dextran sulfate because it is technically more straightforward (8). The key residues identified were further characterized by dose-response studies, and their importance was confirmed by FXI activation in the presence of activated platelets. Although dextran sulfate is commonly employed in studies of FXI activation by thrombin (8, 9, 20), it should be noted that activated platelets and dextran sulfate may promote thrombin-catalyzed FXI activation by similar but non-identical mechanisms because the binding site for the activated platelet surface has been localized to the A3 domain of FXI (14, 23), whereas binding to dextran sulfate is mediated through the A1 domain (24).



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  		FIG. 6.
Space-filling model of thrombin residues critical in activating FXI. The Rasmol Version 2.6 software package was used to depict thrombin (EC 3.4.21.5 [EC] ; Protein Data Bank code 1PPB [PDB] ) as a space-filling model with solvent removed. The 10 residues identified to be essential for efficient FXI activation are labeled using the single amino acid code. A, the classic frontal view of thrombin with the active-site cleft running horizontally, left to right, with the 50-insertion loop on the upper rim of the cleft occluding the active-site Ser205 (orange). The active site is shown with the bound active-site inhibitor PPACK as a brown stick model. Critical Na+-binding site residues are colored separately in shades of green, whereas the 50-insertion loop residue Trp50 is colored yellow. ABE-I residues are colored in shades of blue and are located to the right of the active-site cleft, whereas ABE-II is located to the left of the cleft. B, the same molecule rotated 90° to the right with critical ABE-II residues now visible and colored in shades of red.

 
Despite these differences, the key thrombin residues identified to be important in FXI activation in the presence of dextran sulfate were confirmed by the subsequent activated platelet assay system (Table I). All four major functional domains of thrombin were found to be involved in the process, including ABE-I, ABE-II, the 50-insertion loop, and the Na+-binding site (Fig. 6). The specific roles of the exosites in the FXI interaction were delineated by solid-phase binding experiments using mutants that were found to be defective in activating FXI in the initial activation screen. Four ABE-I single mutants with impaired FXI-activating ability (R68A, R70A, Y71A, and R73A) were chosen for further characterization and found to be deficient in the activated platelet assay system (Table I) and in binding to FXI (Fig. 5 and Table II), but demonstrated normal binding to glycocalicin (Fig. 3 and Table II). These single mutations within ABE-I could directly disrupt specific contacts or indirectly alter the local topology of ABE-I for the interaction of thrombin and FXI. Future x-ray crystallographic studies on the thrombin-FXI complex will help to resolve this issue.

In contrast to the ABE-I mutants, three ABE-II single mutants with impaired FXI-activating ability (R98A, R245A, and K248A) displayed no significant impairment in binding to FXI (Fig. 5 and Table II), whereas they showed major impairment in binding to glycocalicin (Fig. 3 and Table II). These results implicate ABE-I in binding to FXI and ABE-II in binding to glycocalicin. Recently, De Cristofaro et al. (34) found the following ABE-II residues to be involved in binding to GPIb{alpha}: Arg89, Arg94, Arg98, Arg245, and Arg248. These residues were also implicated in our dextran sulfate binding screen, suggesting similarity to the role of GPIb{alpha} in FXI activation. Furthermore, unlike the other ABE-II residues tested, substitution of the ABE-II residue Gln251 with alanine does not result in reduced binding to dextran sulfate or to GPIb{alpha} (34). This confirms a role for electrostatic effects in ABE-II binding to GPIb{alpha}, first suggested by its salt dependence (42). Whether binding to GPIb{alpha} allosterically modifies the structure of thrombin is still in question because conflicting results have been published indicating no effect on PAR1 peptide hydrolysis (42), but inhibitory effects on fibrinopeptide A release and D-Phe-Pro-Arg-p-nitroanilide hydrolysis (43) and, more recently, FVIII activation (44).

The present data indicate that Trp50 in the 50-insertion loop is important in mediating FXI activation in the presence of dextran sulfate or activated platelets (Table I). Substitution of alanine for Trp50 had no effect on thrombin binding to glycocalicin (Fig. 3 and Table II), but resulted in severe impairment of thrombin binding to FXI (Fig. 5 and Table II). Trp50 plays an important role in defining the apolar S2 subsite and has been shown to make substantial contacts in the thrombin complex with direct thrombin inhibitors such as hirudin (32), hemadin (45), and PPACK (26) as well as with fibrinopeptide A (46). Alanine substitution of Trp50 significantly perturbs thrombin activity with all of the substrates tested thus far, disrupting fibrinogen clotting and the activation of protein C, thrombin-activatable fibrinolysis inhibitor, FV, and FVIII (36–39, 47). However, it does not affect the ability of thrombin to bind the DNA thrombin aptamer or fibrin, both of which bind to a defined site in ABE-I (37, 47), suggesting that the insertion loop and the ABE-I site are functionally independent. The significant impairment of FXI activation by the W50A mutant is consistent with either loss of direct contact with FXI or disruption of the S2 subsite.

Consistent with all of our previous thrombin-substrate mapping studies (36–39) is the demonstration in this study of the importance of Glu229 and Arg233 at the Na+-binding site in FXI activation (Table I) and in thrombin binding to FXI (Fig. 5 and Table II), whereas in contrast, mutations of the Na+ site residues (E229A and R233A) led to normal or enhanced binding to glycocalicin (Fig. 3 and Table II). The Na+-binding site is adjacent to the autolysis loop that forms the southern rim of the active-site cleft (Fig. 6). Na+-bound thrombin ("fast" form) has a predominantly procoagulant activity, whereas Na+-free thrombin ("slow" form) has a predominantly anticoagulant property (48–50). Based on empiric screening using this collection of thrombin mutants, we previously identified the thrombin mutants E229A and E229K as essentially anticoagulant forms of the protease with markedly diminished procoagulant properties, but with substantial activity in activating protein C and thrombin-activatable fibrinolysis inhibitor (35, 36, 51, 52). Recently, a new crystal structure of thrombin in which the active site was not occupied was obtained (53). It reveals a previously unobserved closed conformation of thrombin, which satisfies the conditions of the slow form of thrombin, supporting the thesis that thrombin can switch between these two conformational states. The substantial loss of FXI activation by the E229A and R233A mutants is consistent with these previous findings. Alanine substitution at the Glu229 or Arg233 site may disrupt Na+ binding and significantly alter the active-site conformation, leading to the closed form of thrombin.

Thus, our current data suggest a quaternary complex model of thrombin activation of FXI on the activated platelet surface. Thrombin binds to the GPIb-IX-V complex via ABE-II on the posterior surface of thrombin and to FXI via ABE-I on its anterior face (Fig. 6). At least one thrombin-binding site on GPIb{alpha} has been localized to residues 269–287 (54) and is mediated by electrostatic interactions and perhaps direct contacts. FXI binding to GPIb{alpha} through its A3 domain is achieved when one of its homodimers is first complexed with HMWK or prothrombin (14, 23, 24). Once bound to the GPIb-IX-V complex by one of its monomers, FXI can interact with an adjacent thrombin molecule through the A1 domain of its other, free monomer (12). Thus, thrombin bound to one GPIb{alpha} molecule, via ABE-II on its posterior surface, is properly oriented for its activation of FXI bound to a neighboring GPIb{alpha} molecule, via ABE-I on its anterior surface. In this model, GPIb{alpha} plays a major role in co-localizing thrombin and FXI to form the quaternary complex. It would account for the inability of either fibrinogen or HMWK to inhibit this activation in the presence of activated platelets (13).

From our recent thrombin-substrate mapping studies, we found that the binding of thrombin to the GPIb-IX-V complex on the platelet surface is also important in thrombin cleavage of platelet GPV and PAR1.2 Thus, engagement of thrombin on GPIb-IX-V via ABE-II may facilitate the interaction of thrombin with its physiological substrates on the platelet surface, which may represent the first step in the thrombin-platelet interaction process. The GPIb{alpha}-bound thrombin is then in a position to cleave PAR1, GPV, and FXI or to bind another GPI{alpha} molecule, all mediated by ABE-I. The specific outcome of this thrombin-substrate interaction will depend on the relative accessibility of these substrates or ligands and other factors that are not yet defined. One notable difference in the activation of FXI by thrombin is that it preferentially occurs only on the activated platelet surface (12), whereas the cleavage of GPV or PAR1 takes place before platelet activation. Because platelet activation also results in a substantial reduction in the number of GPIb molecules from the platelet surface (55) and redistribution to the surface canalicular system (56), this would imply that the binding site for FXI is mediated by a new conformational state of GPIb{alpha} that is formed only after platelet activation. One possibility is that thrombin activation leads to redistribution and co-localization of a subpopulation of thrombin, GPIb-IX-V, and FXI in lipid rafts on activated platelets, which is responsible for the bulk of FXI activation (22). A prediction from this model is that part of the platelet-bound thrombin should be found in lipid rafts, along with the other components of this enzyme-cofactor-substrate complex. Recently, cleavage of GPV by thrombin has been shown to play a signaling role in platelet activation (57). Whether the cleavage of GPV also facilitates the exposure of the FXI-binding site in the GPIb-IX-V complex remains to be tested.


   FOOTNOTES
 
* This work was supported by Research Grants HL57530 (to L. L. K. L.), Research Grants HL46213, HL64943, and HL70683 (to P. N. W.), and Research Grant P5065967 (to J. A. L.) from the National Institutes of Health; an American Heart Association scientist development award (to T. M.); the Cheong Har Family Foundation; the Janet Hughes Fund; and the Francis and Arjay Miller Fund (to L. L. K. L.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

{ddagger}{ddagger} To whom correspondence should be addressed: Div. of Hematology, Dept. of Medicine, Stanford University School of Medicine, CCSR Bldg., Rm. 1155, 269 Campus Dr., Stanford, CA 94305. Tel.: 650-725-4036; Fax: 650-736-0974; E-mail: lawrence.leung{at}stanford.edu.

1 The abbreviations used are: FXI, Factor XI; HMWK, high molecular weight kininogen; GPIb, glycoprotein Ib; ABE, anion-binding exosite; PAR1, protease-activated receptor-1; WT, wild-type; PPACK, D-phenylalanylprolylarginyl chloromethyl ketone; BSA, bovine serum albumin. Back

2 T. Myles, unpublished data. Back



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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