©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Identification and Characterization of a Binding Site for Platelets in the Apple 3 Domain of Coagulation Factor XI (*)

(Received for publication, September 30, 1994; and in revised form, January 13, 1995)

Frank A. Baglia (1) Bradford A. Jameson (3) Peter N. Walsh (1) (2)(§)

From the  (1)The Sol Sherry Thrombosis Research Center and (2)Departments of Medicine and Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140 and the (3)Thomas Jefferson University, Philadelphia, Pennsylvania 19107

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Activated platelets expose a specific, reversible high affinity (K 10 nM) binding site (n 1500 sites/platelet) for factor XI that requires the presence of high molecular weight kininogen (HK) and ZnCl(2) (Greengard, J. S., Heeb, M. J., Ersdal, E., Walsh, P. N., and Griffin, J. H.(1986) Biochemistry 25, 3884-3890). Synthetic, conformationally constrained peptides from four tandem repeat (Apple) domains were tested for their capacity to inhibit I-factor XI binding to platelets. A peptide from the Apple 3 (A3) domain (Asn-Arg) inhibits factor XI binding to platelets in the presence of HK (42 nM), CaCl(2) (2 mM), and ZnCl(2) (25 µM), with a K 10 nM which is identical to the Kfor factor XI binding to platelets. A peptide from the A1 domain (Phe-Ser) partially inhibits factor XI binding to platelets (K 6 µM) by inhibiting factor XI binding to HK, whereas peptides from the A2 and A4 domains have no effect. Using computer modeling for rational design, conformationally constrained peptides were synthesized (Pro-Gln, Thr-Leu, and Ser-Ser) each of which acted alone and synergistically when added together to inhibit factor XI binding to platelets. Finally, the I-labeled A3 domain peptide (Asn-Arg) was found to bind to thrombin-activated platelets in a specific, reversible, and saturable manner. Thus, the sequence of amino acids Asn-Arg of the A3 domain of factor XI comprises a contact surface for interaction with a platelet receptor.


INTRODUCTION

Factor XI, an important component in the contact phase of intrinsic coagulation, is a homodimer (160,000 daltons) consisting of two identical disulfide-linked polypeptide chains each of which is cleaved at a single peptide bond by factor XIIa to give rise to factor XIa(1, 2) . This glycoprotein circulates in plasma as a complex with its cofactor high molecular weight kininogen (HK)(^1)(3, 4) . The activation of factor XI involves the proteins factor XII, prekallikrein, and HK. It has been shown that these proteins are assembled on negatively charged surfaces and form a contact factor complex capable of generating factor XIa activity on the surface. Previous studies, from which this model was derived, employed nonphysiological charged surfaces such as kaolin, glass, or dextran sulfate(5) . Useful as these studies have been for elucidating molecular events in contact activation, little is known of physiological sites involved in intrinsic coagulation. It has been demonstrated in experiments using highly purified contact factors and platelets that activated platelets in the presence of HK promote the proteolytic activation of factor XI by factor XIIa(6) . It was demonstrated that the proteolytic cleavage products of activated factor XI are associated with platelets(6) . In addition, it was determined that both HK (7) and factor XI (8) bind specifically and with high affinity to the surface of stimulated human platelets in the presence of Zn and Ca ions(8) . The present study was undertaken to examine the interactions of factor XI with the platelet surface and thereby determine which domain(s) may serve as a contact site of interaction. Four repeat sequences (designated A1, A2, A3, and A4 or Apple domains) are present in the heavy chain of factor XI. Their primary structure has been elucidated from the sequence of a cDNA insert coding for factor XI(2) . We have previously reported evidence for the presence of a HK-binding site in the A1 domain, a substrate binding site for factor IX in the A2 domain, and recently a binding site for factor XIIa in the A4 domain(9, 10, 11, 12) . Evidence for a specific high affinity binding site in the A3 domain of the heavy chain of factor XI that is an important contact site for the interaction with platelets is reported in the present study.


MATERIALS AND METHODS

Purification of Proteins

Factor XI, purified from human plasma by immunoaffinity chromatography using a monoclonal antibody to factor XI(9) , had a specific activity of 250 units/mg protein. HK (specific activity, 15 units/mg) was purified by the method of Kerbiriou and Griffin(13) . Both purified proteins appeared homogeneous by SDS-polyacrylamide gel electrophoresis.

Radiolabeling of Factor XI and A3 Peptide N235-R266

Purified factor XI and a tyrosinated peptide comprising a sequence of amino acids (YNLCLLKTSESGLPSTRIKKSKALSGFSLQSCRY) representing residues Asn-Arg from the A3 domain were radiolabeled by a minor modification of the IODO-GEN method (14) to a specific activity of 5-6.2 times 10^6 cpm/µg for factor XI and 0.3 times 10^6 cpm/µg for the A3 peptide. The radiolabeled factor XI retained >98% of its biological activity as measured by the capacity of radiolabeled factor XI to correct factor XI activity in a coagulation assay (see below). Also when coagulation assays (see below) were carried out in the presence of gel-filtered platelets, I-Asn-Arg was identical to unlabeled Asn-Arg in its inhibitory capacity.

Coagulation Assays

Factor XI activity was assayed by a minor modification (15, 16) of the kaolin-activated partial thromboplastin time using appropriate congenitally deficient substrate plasmas, results of which were quantitated on double logarithmic plots of clotting time versus concentrations of pooled normal plasma. Factor XI coagulation assays were carried out either in the presence of gel-filtered activated platelets (see below) or in the presence of phospholipids (0.04% inosithin, Associated Concentrate Inc., Woodside, NY).

Peptide Synthesis

Peptides were synthesized on an Applied Biosystems 430A Peptide Synthesizer (Foster City, CA) by a modification of the procedure described by Kent and Clark-Lewis (17) as described previously(16) . The sequences of the synthetic peptides utilized in this study are given in Table 1. Some of these have been previously published(9, 10, 11, 12) . The thrombin receptor peptide, SFLLRN-amide(18) , was also synthesized for use as a platelet agonist in binding studies.



Reduction and Alkylation and Refolding of Peptides

Peptides were dissolved in deionized water as a 100 µg/ml solution in a flask containing a stir bar in order to refold peptides containing cysteine residues. After the pH was adjusted to 8.5 with NH(4)OH, the solution was allowed to stir at 5 °C for at least 3 days. The solution was then lyophilized. Alternatively, peptides were reduced with dithiothreitol (7 mM) and alkylated with iodoacetamide (5 mM) as described previously(16) .

High Performance Liquid Chromatography (HPLC)

We utilized an HPLC system from Waters (Waters 600 Gradient Module, model 740 Data Module, model 46K Universal Injector, and Lambda-Max model 481 Detector, Milford, MA). Reverse-phase chromatography was performed using a Waters C8 µBondapak column, whereas gel filtration was carried out using a Waters Protein-Pak 60 column as described previously(9, 10, 11, 12, 16) .

Characterization of Synthetic Peptides

All peptides used in this study were examined by HPLC (both reverse-phase and gel filtration), and all demonstrated a single homogeneous peak (data not shown). This demonstrates the presence of a single homogeneous mixture of refolded peptides and not a mixed population of diverse polymers. The results were the same after reduction and alkylation of these same peptides. In addition, all peptides were examined for free-SH groups using the Ellman reagent, 5,5`-dithiobis(2-nitrobenzoic acid)(19) . It was determined that there was less than 0.02 mol of free-SH/mol of peptide, which further verifies that these refolded peptides were homogeneous preparations consisting of intramolecular disulfide-bonded peptide.

Mass Spectrometry

Fast atom bombardment mass spectrometry was used to assess homogeneity and molecular mass of each synthesized peptide with M(r) < 2,000. Fast atom bombardment was performed as described previously(12, 16) . Matrix-assisted laser desorption flash ionization (MALDI) was carried out as described previously (20) on peptides with M(r) > 2,000. The spectra of these peptides revealed masses almost identical to the calculated values (i.e. within 0.5%) with a single ion species observed in each case. The calculated (^2)and observed masses of the synthesized peptides were as follows: Phe-Ser, 3404 and 3408.9; Ala-Ala, 4777 and 4787.7; Asn-Arg, 3425 and 3413.5; Ala-Gly, 3457 and 3466.8; Pro-Gln, 568 and 568.5; Thr-Leu, 584 and 584.1; Ser-Ser, 1611 and 1609.1; Ser-Lys, 779 and 780.5; Gln-Asn, 1459 and 1460; and Ala-Ser, 826 and 827.6.

Computer Modeling

A structural model of the A3 domain was constructed utilizing the Sybyl Software Package (Triobos and Associates, St. Louis, MO) and a Silicon Graphics Onyx Parallel Processing Supercomputer. A description of the modeling package and methods has been previously published(10, 11, 12, 16) . Information concerning cysteine disulfide constraints (21) was used to initiate model building after which extended energy minimization calculations were carried out according to previously published methods(10, 11, 12, 16) . Subdomains were selected for synthesis directly from our molecular model. Conformational constraints were built into selected peptides using artificially introduced cysteine pairs.

Preparation of Washed Platelets

Platelets were prepared as described(8) . Platelet-rich plasma obtained from citrated human blood was centrifuged, and the platelets were resuspended in calcium-free HEPES-Tyrodes buffer, 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).

Binding Experiments

Platelets were prewarmed and incubated at a concentration of (2, 3, 4) times 10^8/ml in calcium-free HEPES-Tyrodes buffer, pH 7.3, in a 1.5-ml Eppendorf plastic centrifuge tube with a mixture of radiolabeled factor XI, unlabeled factor XI, or factor XI-derived peptides, divalent cations, a thrombin receptor peptide (SFLLRN-amide, as a platelet agonist) (18) and HK, or other proteins. All incubations were performed at 37 °C without stirring the reaction mixture. At various times after addition of all the components, aliquots were removed (100 µl) and centrifuged through a mixture of silicone oils as described(8) . Unless otherwise stated, total binding is shown, uncorrected for any nonsaturable component.

Competition Experiments

Platelets were incubated with ZnCl(2) (25 µM), CaCl(2) (2 mM), thrombin receptor peptide, SFLLRN-amide (25 µM), HK (5.0 µg/ml), and I-factor XI (0.2 µg/ml) or the A3 peptide, I-N235-R266 (at various concentrations) and mixed with samples of various peptides and buffers. After 30 min, samples were centrifuged. Binding of I-factor XI was compared to control binding in the absence of competing peptides or proteins.

Calculation of Binding Constants and Number of Sites

Binding data were analyzed according to the method of Scatchard(22) . Points were averages of triplicate determinations. ``Nonspecific'' binding, apparent dissociation constants, and numbers of binding sites were calculated. Different concentrations of total ligand were made by mixing increasing amounts of unlabeled protein with a constant amount of radiolabeled protein. The nonspecific binding was calculated by assuming that it represented an infinitely nonsaturable component of the total binding(23) .


RESULTS

The Effects of Factor XI Apple Domain Peptides on Coagulation Assays Containing Platelets

We have previously shown that the A1 domain of factor XI contains the binding site for HK, the A2 domain contains a substrate binding site for factor IX, and in the A4 domain resides a binding site for factor XIIa(9, 10, 11, 12) . We have previously shown that the synthetic peptides from these domains inhibit coagulant activity(9, 10, 11, 12) . However, when we examined a peptide in the A3 domain homologous to the A1, A2, and A4 domains in its amino acid sequence, the A3 peptide (Asn-Arg) was required at high concentrations (K(i) = 1 times 10M) to inhibit coagulant activity using negatively charged surface phospholipids (Fig. 1). When platelets were used as a surface instead of phospholipids (inosithin 0.04%) the A3 peptide (Asn-Arg) was a potent inhibitor of coagulant activity (K(i) = 2 times 10). This experiment suggests that the A3 peptide may interact with the platelet surface, since it appears to be a platelet-specific inhibitor of blood coagulation. By comparison, peptides derived from the A1 (Phe-Ser), the A2 (Ala-Ile), and A4 (Ala-Gly) domains are as effective in inhibiting coagulant activity in the presence of phospholipids as in the presence of platelets indicating that the effects of these inhibitors are, in contrast to the A3 domain peptide, not specific to platelet surface-mediated coagulation (Fig. 1).


Figure 1: The effect of factor XI heavy chain-derived synthetic peptides on clotting activity. The clotting activity was determined, as described under ``Materials and Methods,'' as a function of the concentration of the peptide given in the abscissa. The symbols denote assays in which platelets (4.2 times 10^8 platelets/ml) (circle) were substituted for phospholipids (Delta).



Effects of Heavy Chain-derived Peptides on the I-Factor XI Binding to Platelets

The platelet-specific inhibition of coagulation by the A3 domain peptide described above suggests the possibility that the A3 domain of factor XI may interact directly with the platelet surface. It has been demonstrated previously that activated platelets can promote the proteolytic activation of factor XI by factor XIIa (6) by exposing a binding site for factor XI in the presence of HK and Zn ions(8) . Therefore, to determine whether the A3 peptide can compete for I-factor XI binding to platelets, washed and stimulated platelets were incubated, unstirred at 37 °C with various concentrations of A1, A2, A3, or A4 peptides in the presence of HK (42 nM), CaCl(2) (2 mM), and ZnCl(2) (25 µM). Fig. 2demonstrates that the factor XI A3 peptide Asn-Arg is a potent inhibitor of factor XI binding to platelets with a K(i) (10 nM) that is almost identical to that observed with unlabeled factor XI. By comparison, peptides from the A2 domain and from the A4 domain have no effect upon the binding of factor XI to platelets (Fig. 2). A peptide from the A1 domain (Phe-Ser) also partially inhibited factor XI binding to platelets with a K(i) 6 times 10M (Fig. 2). Since this A1 domain peptide inhibits factor XI binding to HK (9) and since the presence of HK is required for factor XI binding to platelets(8) , this result confirms the conclusion that complex formation between factor XI and HK is a prerequisite for the interaction of factor XI with platelets.


Figure 2: Effects of factor XI heavy chain synthetic peptides on the binding of I-factor XI binding to platelets. The effects of factor XI and various synthetic peptides on the binding of I-factor XI to platelets were examined including: factor XI (circle), Phe-Ser (Delta), Ala-Ala (box), Asn-Arg (down triangle), Ala-Gly (bullet). I-factor XI (0.2 µg/ml), gel-filtered platelets (4.2 times 10^8 platelets/ml), ZnCl(2) (25 µM), CaCl(2) (2 mM), thrombin peptide (25 µM), and HK (42 nM) were incubated for 30 min at 37 °C either with the designated peptide at the concentration designated or with buffer solution. Aliquots were removed and centrifuged as described under ``Materials and Methods.'' Each point is an average of triplicate determinations and the maximum variation of counts/min bound for each observation was <2% of total counts/min bound. One-hundred % binding of factor XI represents an average of 100,190 cpm bound whereas 0% binding of factor XI represents 0% bound after subtracting an average of 140 cpm representing a control in which labeled factor XI was incubated with platelets at 0 time.



Computer Modeling of the A3 Domain and Design of Conformationally Restrained Peptides

Computer modeling studies with all four Apple domains suggested that a similar pattern of folding might be present in each domain, A1-A4, each characterized by three stem-loop structures that might interact with one another to form solvent-accessible surfaces possibly utilized for binding factor XI ligands. These models utilized information concerning cysteine disulfide constraints to initiate model building after which extended energy minimizations were carried out. The model for the A3 domain using this method demonstrates a surface comprising residues, Pro-Ser which is shown in Fig. 3. Based on this model, rationally designed synthetic peptides were prepared in which cysteine residues were introduced so that the resulting disulfide bond might stabilize the loop structure in a conformation likely to resemble the native binding surface (see Table 1). These peptides were examined for their capacity to inhibit the binding of I-factor XI to platelets. The results (Fig. 4) demonstrate that all three peptides (Pro-Gln(c); Ser-Ser(c); Thr-Leu(c)) were potent inhibitors of factor XI binding to platelets with IC values shown in Table 2. When all three of these peptides were added together in an equimolar mixture a modest synergistic effect was observed since the IC of the combined peptides was 2 times 10M compared with 3 times 10M for Ser-Ser(c), 10M for Pro-Gln(c), and 3 times 10M for Thr-Leu(c). This experiment demonstrates synergism since when the three peptides were added together at equimolar concentrations, their combined effect was greater than a simple additive effect, or the effect of each peptide alone. Three additional peptides from sequences of the A3 domain did not inhibit the binding of I-factor XI to platelets (Fig. 4). These three control peptides were chosen for synthesis on the basis of inspection of the model (Fig. 3) to represent amino acid sequences that are not predicted to comprise portions of the putative binding surface. For example, the peptide sequence Ala-Ser is predicted to be part of a loop structure exposed on the opposite face of the A3 domain as predicted by the model (Fig. 3) and was found to be inactive as an inhibitor of factor XI binding to platelets (Fig. 4). Although the peptide Ser-Lys contains part of the sequence of the active peptide loop Ser-Ser, it lacks the amino acids Ser-Ser predicted on the basis of the model (Fig. 3) to comprise the active binding surface and in fact was also found to be inactive (at 10M) in preventing factor XI binding to platelets whereas the peptide Ser-Ser inhibited this binding 50% at a concentration of 3 times 10M. This experiment suggests that the three peptide loop structures depicted in Fig. 3closely resemble the conformation of the A3 domain that forms a surface utilized for binding a platelet membrane receptor.


Figure 3: Molecular model of the A3 domain of the heavy chain of factor XI. The molecular model was constructed utilizing the Sybyl Software Package (Triobos and Ass., St. Louis, MO) and a Silicon Graphics Onyx Parallel Processing Supercomputer (see ``Materials and Methods''). Using the primary structure of the A3 domain (2) and its known disulfide linkages (21) and energy minimization calculations, a plausible three-dimensional structure was calculated. The figure depicts the protein backbone with the positions of amino acids numbered according to their sequence in the mature protein.




Figure 4: Effect of factor XI heavy chain synthetic peptides on the binding of I-factor XI to platelets. This experiment examines the effects of the following peptides on the binding of I-factor XI to platelets: Pro-Gln(c) (circle); Thr-Leu(c) (Delta); Ser-Ser(c) (box); Ala-Ser(c) (bullet); Gln-Asn(c) (); Ser-Lys(c) (); the following peptides were added in combination at equimolar concentrations: Pro-Gln(c) plus Thr-Leu(c) plus Ser-Ser(c) (circle). I-factor XI (0.2 µg/ml), gel filtered platelets (4.2 times 10^8 platelets/ml), ZnCl(2) (25 µM), CaCl(2) (2 mM), thrombin receptor peptide, SFLLRN-amide (25 µM), and HK (42 nM) were incubated for 30 min at 37 °C either with the designated peptide(s) at the concentration designated or with buffer solution. Aliquots were removed, and separation of bound from free ligand was accomplished as described under ``Materials and Methods.'' Each point is an average of triplicate determinations. When I-factor XI was incubated with platelets at 0 time, the amount of I-factor XI bound was <1% of the control value (incubated for 30 min), and the maximum variation of counts/min bound for each experimental observation was <2% of total counts/min bound. One-hundred % binding of factor XI represents an average of 100,580 cpm bound whereas 0% binding of factor XI represents 0 cpm bound after subtracting an average of 120 cpm representing the negative control in which I-factor XI was incubated with platelets at 0 time.





I-Asn-Arg Binding to Platelets

Since peptide Asn-Arg competes with I-factor XI for platelet-binding sites, the direct interaction of this peptide with the platelet surface was examined. In order to label this peptide with I we added a tyrosine residue to each end of the synthesized peptide. To determine whether this tyrosinated, iodinated peptide, I-Asn-Arg binds to platelets, and washed platelets were incubated unstirred at 37 °C with labeled peptide in the presence or absence of ZnCl(2) (25 µM), CaCl(2) (2 mM), human HK (5 µg/ml or 42 nM), and the thrombin receptor peptide, SFLLRN-amide (25 µM). The incubation mixture was sampled at varying time intervals, and aliquots were centrifuged through silicone oil barriers to separate platelets from unbound proteins. Fig. 5demonstrates that binding to stimulated platelets in the presence or absence of HK, ZnCl(2), and CaCl(2) reaches a plateau in 20 min. If ZnCl(2) or CaCl(2) or HK was omitted the same amount of binding was observed. Thus, unlike Ilabeled factor XI which requires HK, Zn, and Ca to bind platelets the I-labeled Asn-ArAsn-Argg peptide does not require these ions or cofactors.


Figure 5: Binding of I-Asn-Arg to platelets. Platelets were incubated without stirring at 37 °C with I-Asn-Arg (1.85 µM or 6.34 µg/ml), HK (5 µg/ml), ZnCl(2) (25 µM), CaCl(2) (2 mM), and thrombin receptor peptide, SFLLRN-amide (25 µM) (), all the above components in the absence of ZnCl(2) (circle), in the absence of CaCl(2) (box), or in the absence of HK (bullet). At the times indicated, aliquots were removed and centrifuged as described under ``Materials and Methods.'' Each point is an average of triplicate determinations and the maximum variation of counts/min bound for each experimental observation was <2% of total cpm bound.



Specificity and Reversibility of I-Asn-Arg Peptide Binding

In order to evaluate the specificity and reversibility of the I-Asn-Arg interaction with platelets, the ability of factor XI and prekallikrein to compete for binding sites was studied. Radiolabeled Asn-Arg was mixed with the buffer or 100 µg/ml aliquots of proteins in the presence of 5 µg/ml (42 nM) HK, 25 µM ZnCl(2), 2 mM CaCl(2), or 25 µM of thrombin peptide (SFLLRN-amide). Unlabeled factor XI competed with the A3 domain peptide for platelet-binding sites since only 34.6% (equivalent to nonspecific binding) of the I-Asn-Arg remained platelet-bound in the presence of a 100-fold molar excess of unlabeled factor XI (Table 3). Furthermore, the addition of either unlabeled factor XI or unlabeled A3 domain peptide (Asn-Arg), added in 100-fold molar excess after achieving equilibrium, resulted in rapid and complete dissociation of the bound I-Asn-Arg peptide (data not shown). Neither prekallikrein nor a prekallikrein-derived synthetic A3 peptide Asn-Arg (see Table 1) (which is 58% identical in amino acid sequence to the factor XI Asn-Arg A3 domain peptide) competed with I-Asn-Arg binding to platelets (Table 3). These data confirm the specificity data provided in Table 2.



Saturability of I-Asn-Arg Peptide Binding

Since the binding to platelets of the A3 domain peptide, I-Asn-Arg, is specific and reversible, saturation binding studies were carried out. Saturable binding of the A3 peptide, I-Asn-Arg, to thrombin-stimulated platelets was observed (Fig. 6A). Nonspecific binding, as calculated from competition of unlabeled factor XI was subtracted at each point. The results demonstrate that saturable binding was achieved at factor XI concentrations above the plasma concentration (25 nM) of factor XI. When the saturation data were analyzed by the method of Scatchard(22) , an apparent dissociation constant (K(d)) of 21.1 nM and a total of 1,035 binding sites was calculated (Fig. 6B).


Figure 6: Saturable, specific binding of I-Asn-Arg to platelets. Platelets were incubated at 37 °C with ZnCl(2) (25 µM), CaCl(2) (2 mM), thrombin receptor peptide, SFLLRN-amide (25 µM), HK (42 nM), and mixtures of I-factor XI Asn-Arg and unlabeled factor XI at various concentrations. Binding was determined at 30 min. A, amount of Asn-Arg bound at different input concentrations. Specific binding (Delta) is shown after subtracting the amount bound in the presence of a large molar excess of unlabeled factor XI. B, Scatchard analysis of the data shown in A. The line is best fit of data from triplicate samples. The apparent dissociation constant and number of binding sites were calculated as parameters.




DISCUSSION

Stimulated platelets have been demonstrated to promote the assembly of contact factors leading to factor XI activation (6) in part by exposing binding sites for factor XI(8) . Specific, high affinity factor XI binding occurs at physiological levels of the metal ions ZnCl(2) and CaCl(2) when HK is present(8) . It was demonstrated that binding required platelet stimulation and was specific, reversible, and saturable(8) . Scatchard analysis of the binding yielded approximately 1,500 binding sites/platelet with an apparent dissociation constant of approximately 10 nM(8) . The similarity between the concentrations of the metal ions optimal for factor XI binding and those optimal for HK binding (7) suggests the possibility that factor XI and HK may form a complex on the platelet surface as they do in solution and on negatively charged surfaces(5) . The purpose of the present study was to investigate the role of the Apple domains in the binding of factor XI to platelets and to delineate a sequence of amino acids which may indirectly or directly bind the platelet surface.

One important conclusion derived from our study is that platelets make a unique and an important contribution to the in vitro clotting process since the A3 peptide (Asn-Arg) was a potent inhibitor of clotting activity in the presence of platelets but not in the presence of phospholipids (Fig. 1). This key experiment also gave us a clue as to the role of the A3 domain in the interaction of factor XI with the platelet surface. The results of this and other experiments reported here support the hypothesis that a sequence of amino acids (Pro-Ser) in the A3 domain of the factor XI heavy chain region contains three antiparallel beta-strands connected by beta-turns, which comprise a continuous surface that interacts with a platelet receptor. The evidence supporting this possibility is listed as follows: 1) an A3-derived peptide (Asn-Arg) inhibits I-factor XI binding to platelets with a K(i) (10 nM) equivalent to that observed with unlabeled factor XI (Fig. 2), suggesting that the peptide as well as factor XI may bind directly to the platelet surface. 2) A model of the A3 domain derived from energy minimization computations (Fig. 3) predicts the presence of three beta-stranded loop structures (Pro-Gln, Thr-Leu, and Ser-Ser) that may fold together to form a solvent-accessible surface comprising a binding site for platelets. 3) Based on this model three conformationally constrained peptides were synthesized all of which were found to inhibit the binding of I-factor XI to platelets (Fig. 4). 4) Using equimolar mixtures of the three folded peptides, it was found that the inhibitory effects were mildly synergistic (Fig. 4). 5) A tyrosinated and radiolabeled A3 domain peptide (Asn-Arg) encompassing this putative solvent-accessible surface is able to bind directly to the activated platelet surface (Fig. 5), in a specific and saturable manner (Fig. 6) in the absence of HK and the metal ions CaCl(2) and ZnCl(2) which are required for factor XI binding to platelets (Fig. 5).

We have used computer modeling to predict the secondary and tertiary structures of the A3 domain of factor XI that appears to form a contact surface with platelets. In the absence of defined structural information from x-ray crystallography or nuclear magnetic resonance studies, this model was derived from molecular dynamic, energy minimization calculations which suggested that this domain may have a structural motif consisting of three antiparallel beta-strands connected by beta-turns. This construct was used to design synthetic peptides conformationally constrained with disulfide bonds. Thus, the model has been used as a tool to generate an hypothesis about the possible secondary and tertiary structure of the A3 domain that was then tested in functional studies of factor XI binding to platelets. Our results are consistent with the possibility that the predicted structural motif (Fig. 3) has some validity. However, the model is not presented as evidence of the three-dimensional structure of the A3 domain, which can only be obtained using physical techniques.

The optimal binding of factor XI to activated platelets requires the presence of Zn ions, calcium ions, and the cofactor HK (8) . The fact that HK binding to platelets also requires Zn and Ca ions, but occurs in the absence of factor XI(7) , suggests the possibility that the factor XI-HK complex that circulates in plasma (3, 4) may bind to activated platelets via the platelet binding site for HK(7) . However, the A3 domain-derived peptide Asn-Arg is able to bind directly to platelets in the absence of HK, CaCl(2), and ZnCl(2) (Fig. 5). Moreover, not only are the unlabeled peptide (Asn-Arg) and factor XI identical in their capacity to inhibit I-factor XI binding to platelets (Fig. 2), but the iodinated peptide binds directly to a similar number of sites (n = 1,035) as factor XI with similar affinity (K(d) 21 nM; Fig. 6). This suggests that the A3 domain contains a solvent-accessible surface (Asn-Arg) that contains all the structural information required for binding of factor XI to the platelet surface. We have also demonstrated that the A1 domain peptide (Phe-Ser) is a less potent, partial inhibitor of factor XI binding to platelets (Fig. 2). We have shown that this peptide comprises the binding site for HK in the A1 domain of factor XI(9, 11) . Thus, the binding of factor XI to platelets requires an interaction of the A1 domain with HK in the presence of ZnCl(2). In contrast, the interaction of platelets with the A3 domain peptide (Asn-Arg), and thus with the A3 domain of factor XI, appears to be direct and does not require ions or cofactors. It therefore seems reasonable to suggest that the binding of HK to factor XI may regulate the binding of factor XI directly to a platelet receptor, possibly by inducing a conformational alteration in factor XI that exposes the A3 domain-binding site. Thus, the initial event in the interaction of factor XI with platelets is the binding of factor XI to HK through the A1 domain followed by conformational changes in the heavy chain which permit the interaction of the A3 domain with platelets. The binding of HK to its own receptor on the activated platelet surface (7) then may serve to further stabilize the factor XIbulletHK complex on the platelet surface, or alternatively, the two proteins may even dissociate from one another once individually bound to platelets although there is no evidence to either confirm or refute this possibility.

The plasma protein prekallikrein contains four tandem repeat Apple domains which have 58% amino acid identity with the Apple domains of factor XI(2) . Our studies show that neither prekallikrein nor its A3 domain peptide, Asn-Arg, which is 53% identical to our factor XI A3 domain peptide (Fig. 7), is capable of competing with factor XI for platelet binding sites. This observation provides strong evidence of the specificity of factor XI binding to platelets. In addition, we reasoned that an examination of the A3 domain in prekallikrein might give us important clues about which amino acids are present on the surface of the factor XI A3 domain that are involved in binding to platelets. As shown in Fig. 7, although a 53% amino acid sequence identity exists between factor XI and prekallikrein in the region between amino acids 235 and 266, there is one stretch of nine amino acids(249-257) that are entirely different in factor XI compared with prekallikrein. Interestingly, this sequence corresponds closely with the sequence of the most active of the three conformationally constrained A3 peptides, Ser-Ser ( Table 1and Table 2and Fig. 4). Inspection of the molecular model of the A3 domain (Fig. 3) suggests that a surface loop structure consisting of residues KKSKAL(252-257) might comprise the primary platelet binding site in factor XI and that additional amino acid sequences contained within the region Pro-Arg (i.e. Pro-Gln, and Thr-Leu) might provide accessory platelet-binding sites.


Figure 7: Comparison of amino acid sequences of portions of the Apple 3 domains of factor XI and prekallikrein (PK). The positions that have identical residues are boxed. The primary structure of the A3 domain in factor XI and prekallikrein (2) was utilized.




FOOTNOTES

*
This study was supported by Research Grants HL46213, HL45486 and HL25661 from the National Institutes of Health and by the W. W. Smith Charitable Trust. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 Center, Temple University School of Medicine, 3400 N. Broad St., Philadelphia, PA 19140. Tel.: 215-707-4375; Fax: 215-707-3005.

(^1)
The abbreviations used are: HK, high molecular weight kininogen; HPLC, high performance liquid chromatography; A1, A2, A3, A4, Apple domains; cpm, counts/minute.

(^2)
Masses were obtained by addition of the average masses of amino acids which were determined using the atomic weights of the elements (C = 12.011, H = 1.0079, N = 14.0067, O = 15.9994, S = 32.06).


ACKNOWLEDGEMENTS

We are grateful to Patricia Pileggi for assistance in manuscript preparation.


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