Factor XI Interacts with the Leucine-rich Repeats of Glycoprotein Ibα on the Activated Platelet
2004; Elsevier BV; Volume: 279; Issue: 47 Linguagem: Inglês
10.1074/jbc.m407889200
ISSN1083-351X
AutoresFrank A. Baglia, Corie N. Shrimpton, Jonas Emsley, Kouki Kitagawa, Zaverio M. Ruggeri, José A. López, Peter N. Walsh,
Tópico(s)Blood Coagulation and Thrombosis Mechanisms
ResumoFactor XI (FXI) binds specifically and reversibly to high affinity sites on the surface of stimulated platelets (Kd app of ∼10 nm; Bmax of ∼1,500 sites/platelet) utilizing residues exposed on the Apple 3 domain in the presence of high molecular weight kininogen and Zn2+ or prothrombin and Ca2+. Because the FXI receptor in the platelet membrane is contained within the glycoprotein Ibα subunit of the glycoprotein Ib-IX-V complex (Baglia, F. A., Badellino, K. O., Li, C. Q., Lopez, J. A., and Walsh, P. N. (2002) J. Biol. Chem. 277, 1662–1668), we utilized mocarhagin, a cobra venom metalloproteinase, to generate a fragment (His1–Glu282) of glycoprotein Ibα that contains the leucine-rich repeats of the NH2-terminal globular domain and excludes the macroglycopeptide portion of glycocalicin, the soluble extracytoplasmic portion of glycoprotein Ibα. This fragment was able to compete with FXI for binding to activated platelets (Ki of 3.125 ± 0.25 nm) with a potency similar to that of intact glycocalicin (Ki of 3.72 ± 0.30 nm). However, a synthetic glycoprotein Ibα peptide, Asp269–Asp287, containing a thrombin binding site had no effect on the binding of FXI to activated platelets. Moreover, the binding of 125I-labeled thrombin to glycocalicin was unaffected by the presence of FXI at concentrations up to 10-5m. The von Willebrand factor A1 domain, which binds the leucine-rich repeats, inhibited the binding of FXI to activated platelets. Thus, we examined the effect of synthetic peptides of each of the seven leucine-rich repeats on the binding of 125I-FXI to activated platelets. All leucine-rich repeat (LRR) peptides derived from glycoprotein Ibα were able to inhibit FXI binding to activated platelets in the following order of decreasing potency: LRR7, LRR1, LRR4, LRR5, LRR6, LRR3, and LRR2. However, the leucine-rich repeat synthetic peptides derived from glycoprotein Ibβ and Toll protein had no effect. We conclude that FXI binds to glycoprotein Ibα at sites comprising the leucine-rich repeat sequences within the NH2-terminal globular domain that are separate and distinct from the thrombin-binding site. Factor XI (FXI) binds specifically and reversibly to high affinity sites on the surface of stimulated platelets (Kd app of ∼10 nm; Bmax of ∼1,500 sites/platelet) utilizing residues exposed on the Apple 3 domain in the presence of high molecular weight kininogen and Zn2+ or prothrombin and Ca2+. Because the FXI receptor in the platelet membrane is contained within the glycoprotein Ibα subunit of the glycoprotein Ib-IX-V complex (Baglia, F. A., Badellino, K. O., Li, C. Q., Lopez, J. A., and Walsh, P. N. (2002) J. Biol. Chem. 277, 1662–1668), we utilized mocarhagin, a cobra venom metalloproteinase, to generate a fragment (His1–Glu282) of glycoprotein Ibα that contains the leucine-rich repeats of the NH2-terminal globular domain and excludes the macroglycopeptide portion of glycocalicin, the soluble extracytoplasmic portion of glycoprotein Ibα. This fragment was able to compete with FXI for binding to activated platelets (Ki of 3.125 ± 0.25 nm) with a potency similar to that of intact glycocalicin (Ki of 3.72 ± 0.30 nm). However, a synthetic glycoprotein Ibα peptide, Asp269–Asp287, containing a thrombin binding site had no effect on the binding of FXI to activated platelets. Moreover, the binding of 125I-labeled thrombin to glycocalicin was unaffected by the presence of FXI at concentrations up to 10-5m. The von Willebrand factor A1 domain, which binds the leucine-rich repeats, inhibited the binding of FXI to activated platelets. Thus, we examined the effect of synthetic peptides of each of the seven leucine-rich repeats on the binding of 125I-FXI to activated platelets. All leucine-rich repeat (LRR) peptides derived from glycoprotein Ibα were able to inhibit FXI binding to activated platelets in the following order of decreasing potency: LRR7, LRR1, LRR4, LRR5, LRR6, LRR3, and LRR2. However, the leucine-rich repeat synthetic peptides derived from glycoprotein Ibβ and Toll protein had no effect. We conclude that FXI binds to glycoprotein Ibα at sites comprising the leucine-rich repeat sequences within the NH2-terminal globular domain that are separate and distinct from the thrombin-binding site. Human factor XI (FXI), 1The abbreviations used are: FXI, human factor XI; HK, high molecular weight kininogen; GPIb, glycoprotein Ib; LRR, leucine-rich repeat; vWF, von Willebrand factor; PPACK, prolyl-phenyl-alanyl-arginyl-chloromethyl-ketone; BSA, bovine serum albumin; ABE, anion binding exosite.1The abbreviations used are: FXI, human factor XI; HK, high molecular weight kininogen; GPIb, glycoprotein Ib; LRR, leucine-rich repeat; vWF, von Willebrand factor; PPACK, prolyl-phenyl-alanyl-arginyl-chloromethyl-ketone; BSA, bovine serum albumin; ABE, anion binding exosite. a homodimeric coagulation protein, circulates in plasma as a complex with a non-enzymatic cofactor high molecular weight kininogen (HK) (1Baglia F.A. Badellino K.O. Ho D.H. Dasari V.R. Walsh P.N. J. Biol. Chem. 2000; 275: 31954-31962Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 2Binder B. Krug G. Vukovich T. Thromb. Diath. Haemorrh. 1975; 34: 354PubMed Google Scholar, 3Brunnee T. La Porta C. Reddigari S.R. Salerno V.M. Kaplan A.P. Silverberg M. Blood. 1993; 81: 580-586Crossref PubMed Google Scholar, 4Gailani D. Broze Jr., G.J. Science. 1991; 253: 909-912Crossref PubMed Scopus (631) Google Scholar, 5Gailani D. Broze Jr., G.J. Semin. Thromb. Hemostasis. 1993; 19: 396-404Crossref PubMed Scopus (33) Google Scholar, 6Kaplan A.P. Silverberg M. Dunn J.T. Miller G. Ann. N. Y. Acad. Sci. 1981; 370: 253-260Crossref PubMed Scopus (17) Google Scholar, 7Thompson R.E. Mandle Jr., R. Kaplan A.P. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4862-4866Crossref PubMed Scopus (72) Google Scholar). FXI can bind specifically and reversibly to high affinity sites on the surface of stimulated platelets in the presence of HK (and Zn2+ ions) or prothrombin (and Ca2+ ions) (8Baglia F.A. Walsh P.N. Biochemistry. 1998; 37: 2271-2281Crossref PubMed Scopus (91) Google Scholar, 9Greengard J.S. Heeb M.J. Ersdal E. Walsh P.N. Griffin J.H. Biochemistry. 1986; 25: 3884-3890Crossref PubMed Scopus (86) Google Scholar). We have demonstrated previously that the Apple 3 domain of FXI mediates the binding of FXI to platelets (10Baglia F.A. Walsh P.N. J. Biol. Chem. 2000; 275: 20514-20519Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 11Baglia F.A. Jameson B.A. Walsh P.N. J. Biol. Chem. 1995; 270: 6734-6740Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar), activated platelets promote optimal rates of FXI activation by thrombin in the presence of HK or prothrombin (8Baglia F.A. Walsh P.N. Biochemistry. 1998; 37: 2271-2281Crossref PubMed Scopus (91) Google Scholar), and FXI binds to the platelet glycoprotein (GP) Ib-IX-V complex (12Baglia F.A. Badellino K.O. Li C.Q. Lopez J.A. Walsh P.N. J. Biol. Chem. 2002; 277: 1662-1668Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar) in platelet membrane lipid rafts (13Baglia F.A. Shrimpton C.N. Lopez J.A. Walsh P.N. J. Biol. Chem. 2003; 278: 21744-21750Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), promoting its activation by thrombin (12Baglia F.A. Badellino K.O. Li C.Q. Lopez J.A. Walsh P.N. J. Biol. Chem. 2002; 277: 1662-1668Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 13Baglia F.A. Shrimpton C.N. Lopez J.A. Walsh P.N. J. Biol. Chem. 2003; 278: 21744-21750Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The platelet GPIb-IX-V complex is a large plasma membrane complex (∼25,000 copies/platelet) comprising four polypeptide chains, GPIbα, GPIbβ, GPIX, and GPV, arranged in a stoichiometry of 2:2:2:1, respectively (14Lopez J.A. Blood Coagul. Fibrinolysis. 1994; 5: 97-119Crossref PubMed Scopus (291) Google Scholar). This receptor is responsible for platelet adhesion to the site of injury, a function that it carries out by binding the von Willebrand factor (vWF). The GPIb-IX-V complex also binds thrombin with high affinity. Glycocalicin is the soluble form of the extracellular portion of GPIbα, which contains the NH2-terminal globular domain as well as the macroglycopeptide region. GPIbα, GPIbβ, GPIbIX, and GPIbV each contain an extracellular domain, a single transmembrane helix, and a short cytoplasmic trail. The binding sites within the complex for both vWF and thrombin reside within the first 300 amino acids of GPIbα (15De Marco L. Mazzucato M. Masotti A. Fenton J.W.D. Ruggeri Z.M. J. Biol. Chem. 1991; 266: 23776-23783Abstract Full Text PDF PubMed Google Scholar). GPIbα is also a member of a family of proteins containing leucine-rich repeat (LRR) sequences including a large number of proteins that are principally involved in mediating protein-protein interactions (16Hess D. Schaller J. Rickli E.E. Clemetson K.J. Eur. J. Biochem. 1991; 199: 389-393Crossref PubMed Scopus (34) Google Scholar). These LRR sequences are typically 22–28 amino acids long and occur in tandem repeats that are commonly flanked by disulfide loop structures (16Hess D. Schaller J. Rickli E.E. Clemetson K.J. Eur. J. Biochem. 1991; 199: 389-393Crossref PubMed Scopus (34) Google Scholar). The interaction between vWF and GPIbα is mediated by the A1 domain of vWF and the NH2-terminal domain of the GPIbα chain (17Cruz M.A. Handin R.I. Wise R.J. J. Biol. Chem. 1993; 268: 21238-21245Abstract Full Text PDF PubMed Google Scholar, 18Emsley J. Cruz M. Handin R. Liddington R. J. Biol. Chem. 1998; 273: 10396-10401Abstract Full Text Full Text PDF PubMed Scopus (235) Google Scholar, 19Uff S. Clemetson J.M. Harrison T. Clemetson K.J. Emsley J. J. Biol. Chem. 2002; 277: 35657-35663Abstract Full Text Full Text PDF PubMed Scopus (158) Google Scholar, 20Huizinga E.G. Tsuji S. Romijn R.A. Schiphorst M.E. de Groot P.G. Sixma J.J. Gros P. Science. 2002; 297: 1176-1179Crossref PubMed Scopus (492) Google Scholar, 21Dumas J.J. Kumar R. McDonagh T. Sullivan F. Stahl M.L. Somers W.S. Mosyak L. J. Biol. Chem. 2004; 279: 23327-23334Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). The x-ray crystal structures of the vWF A1 domain and GPIbα have been reported (20Huizinga E.G. Tsuji S. Romijn R.A. Schiphorst M.E. de Groot P.G. Sixma J.J. Gros P. Science. 2002; 297: 1176-1179Crossref PubMed Scopus (492) Google Scholar, 21Dumas J.J. Kumar R. McDonagh T. Sullivan F. Stahl M.L. Somers W.S. Mosyak L. J. Biol. Chem. 2004; 279: 23327-23334Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), as have those of the GPIbα-thrombin complex (22Celikel R. McClintock R.A. Roberts J.R. Mendolicchio G.L. Ware J. Varughese K.I. Ruggeri Z.M. Science. 2003; 301: 218-221Crossref PubMed Scopus (166) Google Scholar, 23Dumas J.J. Kumar R. Seehra J. Somers W.S. Mosyak L. Science. 2003; 301: 222-226Crossref PubMed Scopus (141) Google Scholar). The GPIbα NH2-terminal and COOH-terminal regions have been implicated in contributing to vWF binding (24Cauwenberghs. N. Vanhoorelbeke K. Vauterin S. Westra D.F. Romo G. Huizinga E.G. Lopez J.A. Berndt M.C. Harsfalvi J. Deckmyn H. Blood. 2001; 98: 652-660Crossref PubMed Scopus (78) Google Scholar, 25Shen Y. Romo G.M. Dong J.F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. Lopez J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar), whereas thrombin binding is centered around an anionic region containing three tyrosine residues (Tyr276, Tyr278, and Tyr279) that are post-translationally sulfated in the native receptor. This modification is essential for GPIbα binding to thrombin and may contribute to the interaction with vWF (26Dong J. Ye P. Schade A.J. Gao S. Romo G.M. Turner N.T. McIntire L.V. Lopez J.A. J. Biol. Chem. 2001; 276: 16690-16694Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 27Marchese P. Murata M. Mazzucato M. Pradella P. De Marco L. Ware J. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 9571-9578Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). Thus, the biological relevance and structural determinants of GPIbα interactions with thrombin and the A1 domain of vWF are relatively well understood. In contrast, although GPIbα appears to comprise the platelet receptor and physiological locus for the FXI colocalization with thrombin leading to initiation of the consolidation pathway of blood coagulation (8Baglia F.A. Walsh P.N. Biochemistry. 1998; 37: 2271-2281Crossref PubMed Scopus (91) Google Scholar, 9Greengard J.S. Heeb M.J. Ersdal E. Walsh P.N. Griffin J.H. Biochemistry. 1986; 25: 3884-3890Crossref PubMed Scopus (86) Google Scholar, 10Baglia F.A. Walsh P.N. J. Biol. Chem. 2000; 275: 20514-20519Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 11Baglia F.A. Jameson B.A. Walsh P.N. J. Biol. Chem. 1995; 270: 6734-6740Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 12Baglia F.A. Badellino K.O. Li C.Q. Lopez J.A. Walsh P.N. J. Biol. Chem. 2002; 277: 1662-1668Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 13Baglia F.A. Shrimpton C.N. Lopez J.A. Walsh P.N. J. Biol. Chem. 2003; 278: 21744-21750Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), relatively little is known about the structural determinants of FXI interactions with the GPIb-IX-V complex and its colocalization with thrombin. The present studies demonstrate that FXI and thrombin bind GPIbα at distinct sites, which suggest that GPIbα may allow the colocalization of FXI and thrombin for efficient activation of FXI on the platelet surface. Materials—Human FXI, human prothrombin, and human HK were purchased from Hematologic Technologies Inc. (Essex Junction, VT). Ristocetin was purchased from Sigma. Human α-thrombin (2,800 NIH units/mg) was purchased from Enzyme Research Laboratories (South Bend, IN). Methyl silicon oil (1 DC-200) and Hi phenyl silicon oil (125 DC-550) were purchased from William F. Nye Inc. (Fairhaven, MA). Carrier-free Na125I was from Amersham Biosciences. The thrombin receptor agonist peptide SFLLRN-amide and the LRR peptides (20–24 amino acids) (Table I) were synthesized at the Protein Chemistry Facility of the University of Pennsylvania on the Applied Biosystems 430A Synthesizer, and reverse-phase high performance liquid chromatography was used to purify it to >99% homogeneity (Foster City, CA). The potent thrombin inhibitor prolyl-phenylalanyl-arginyl-chloromethyl-ketone (PPACK) was purchased from Calbiochem (Indianapolis, IN). Active site-inhibited thrombin was prepared by incubation of a 10-fold excess of PPACK with α-thrombin for 1 h at 37 °C,and this mixture was dialyzed with Spectrophor tubing (3,500 Mr cut off; Spectrum Medical Industries, Los Angeles, CA) overnight in phosphate-buffered saline at 5 °C. All purified proteins appeared homogeneous after SDS-PAGE.Table ISynthetic peptides based on the known sequences of GPIbα, GPIbβ, and the Toll proteinProteinDerived sequencePeptide sequenceGPIbαLRR1, 36–59L H L S E N L L Y T F S L A T L M P Y T R L T QGPIbαLRR2, 60–81L N L D R A E L T K L Q V D G T L P V L G TGPIbαLRR3, 82–104L D L S H N Q L Q S L P L L G Q T L P A L T VGPIbαLRR4, 105–128L D V S F N R L T S L P L G A L R G L G E L Q EGPIbαLRR5, 129–152L Y L K G N E L K T L P P G L L T P T P K L E KGPIbαLRR6, 153–176L S L A N N N L T E L P A G L L N G L E N L D TGPIbαLRR7, 177–200L L L Q E N S L Y T I P K G F F G S H L L P F AGPIbα266–287T L G D E G D T D L YaO-sulfated D YaO-sulfated YaO-sulfated P E E D T E G DGPIbα269–280D E G D T D L YaO-sulfated D YaO-sulfated YaO-sulfated PGPIbαScrambled LRR7, 177–200A Q F L P L H I L E P L S N S G K L T Y F G L FGPIbβLRR, 35–58L V L T G N N L T A L P P G L L D A L P A L R TToll proteinLRR, 361–384L L E H Q V N L L S L D L S N N R L T H L S G Da O-sulfated Open table in a new tab Radiolabeling of FXI—Purified FXI and PPACK-thrombin were radiolabeled with 125I by a minor modification (8Baglia F.A. Walsh P.N. Biochemistry. 1998; 37: 2271-2281Crossref PubMed Scopus (91) Google Scholar) of the Iodogen method to a specific activity of ∼5 × 106 cpm/μg and ∼1 × 106 cpm/μg, respectively. The radiolabeled FXI retained >98% of its biological activity. Protein Analysis—Protein concentrations were determined by the Bio-Rad dye-binding assay according to the instructions provided by the manufacturer (Bio-Rad Laboratories). Preparation of Washed Platelets—Platelets were prepared from normal donors as described (9Greengard J.S. Heeb M.J. Ersdal E. Walsh P.N. Griffin J.H. Biochemistry. 1986; 25: 3884-3890Crossref PubMed Scopus (86) Google Scholar, 11Baglia F.A. Jameson B.A. Walsh P.N. J. Biol. Chem. 1995; 270: 6734-6740Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar, 28Ho D.H. Baglia F.A. Walsh P.N. Biochemistry. 2000; 39: 316-323Crossref PubMed Scopus (33) Google Scholar). 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, 8.8 mm Hepes-free acid, and 0.1% bovine serum albumin (BSA)), 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 (Coulter Electronics, Hialeah, FL). Preparation of Glycocalicin and Mocarhagin Fragment His1–Glu282 from Human Platelets—Glycocalicin was extracted from human platelets and purified as described previously (29Romo G.M. Dong J.F. Schade A.J. Gardiner E.E. Kansas G.S. Li C.Q. McIntire L.V. Berndt M.C. Lopez J.A. J. Exp. Med. 1999; 190: 803-814Crossref PubMed Scopus (287) Google Scholar). The mocarhagin fragment His1–Glu282 was prepared from intact glycocalicin and purified as described previously (30Ward C.M. Andrews R.K. Smith A.I. Berndt M.C. Biochemistry. 1996; 35: 4929-4938Crossref PubMed Scopus (180) Google Scholar). Platelet Binding Experiments—Platelets were pre-warmed to 37 °C and incubated at a concentration of 1 × 108/ml in calcium-free Hepes-Tyrode's buffer, pH 7.3, in a 1.5-ml Eppendorf plastic centrifuge tube with a mixture of radiolabeled FXI, divalent cations, a thrombin receptor (PAR-1) activation peptide (SFLLRN-amide) as a platelet agonist (9Greengard J.S. Heeb M.J. Ersdal E. Walsh P.N. Griffin J.H. Biochemistry. 1986; 25: 3884-3890Crossref PubMed Scopus (86) Google Scholar, 10Baglia F.A. Walsh P.N. J. Biol. Chem. 2000; 275: 20514-20519Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 28Ho D.H. Baglia F.A. Walsh P.N. Biochemistry. 2000; 39: 316-323Crossref PubMed Scopus (33) Google Scholar), and HK or other proteins as designated in the legends to Figs. 1, 5, and 6. All incubations were performed at 37 °C without stirring after an initial mixing of the reaction mixture. At various added FXI concentrations, aliquots were removed (100 μl) and centrifuged through a mixture of silicone oils as described (8Baglia F.A. Walsh P.N. Biochemistry. 1998; 37: 2271-2281Crossref PubMed Scopus (91) Google Scholar, 9Greengard J.S. Heeb M.J. Ersdal E. Walsh P.N. Griffin J.H. Biochemistry. 1986; 25: 3884-3890Crossref PubMed Scopus (86) Google Scholar, 10Baglia F.A. Walsh P.N. J. Biol. Chem. 2000; 275: 20514-20519Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 28Ho D.H. Baglia F.A. Walsh P.N. Biochemistry. 2000; 39: 316-323Crossref PubMed Scopus (33) Google Scholar). In competition binding experiments, the concentration of the competitor that displaced 50% of the bound 125I-FXI (IC50) was determined by plotting the amount of 125I-FXI bound to platelets versus the amount of competitor ligand added. The Ki was calculated using the equation IC50 = (1 ± [s]/Kd)Ki, where [s] is the concentration of 125I-FXI used in these experiments (held constant at 22 nm) and the Kd was the value (∼10 nm) determined from direct binding experiments.Fig. 5The effect of A1 domain vWF (wild-type) and vWF-A1 2B mutant I546V on the binding of FXI to activated platelets. The effects of vWF-A1 domains were examined, including wild-type A1 domain vWF (⋄), wild-type A1 domain vWF and ristocetin (1.5 μg/ml) (X), the vWF-A1 2B mutant I546V (♦), and the vWF-A1 2B mutant I546V and ristocetin (1.5 μg/ml) (). 125I-FXI (22 nm), gel-filtered platelets (1 × 108 platelets/ml), ZnCl2 (25 μm), CaCl2 (2 mm), the thrombin receptor activation peptide (25 μm), and HK (42 nm) were incubated for 30 min at 37 °C either with the designated peptide at the indicated concentration or with buffer solution. Aliquots were removed and centrifuged as described under "Experimental Procedures." Each point is the average of triplicate determinations. When 125I-FXI was incubated with platelets at time 0 (at the start of the incubation), the amount of 125I-FXI bound was <1% of the control value, and the maximum variation of counts per minute bound for each experimental observation was <2% of the total counts per minute bound. One hundred percent binding of FXI represents an average of 98,910 cpm bound, whereas 0% binding to FXI represents 0% bound after subtracting 150 cpm, representing the control in which 125I-FXI was incubated with platelets at time 0.View Large Image Figure ViewerDownload (PPT)Fig. 6The effect of LRR synthetic peptides on the binding of 125I-FXI to platelets. The effects of LRR synthetic peptides derived from sequences in GPIbα, GPIbβ, and the Toll protein were examined, including GPIbα LRR 36–59 (⋄), GPIbα LRR 60–81 (X), GPIbα LRR 82–104 (♦), GPIbα LRR 105–128 (), GPIbα LRR 129–152 (□), GPIbα LRR 153–176 (▴), GPIbα LRR 177–200 (•), GPIbα LRR 177–200 scrambled (▴), GPIbβ LRR 35–58 (▿), and Toll protein LRR 361–384 (▪). 125I-FXI (22 nm), gel-filtered platelets (1 × 10-8 platelets/ml), ZnCl2 (25 μm), CaCl2 (2 mm), the thrombin peptide (25 μm), and HK (42 nm) were incubated for 30 min at 37 °C either with the designated peptide at the indicated concentration or with buffer solution. Aliquots were removed and centrifuged as described under "Experimental Procedures." Each point is an average of triplicate determinations. 125I-FXI was incubated with platelets at time 0 (at the start of the incubation), the amount of 125I-FXI bound was <1% of the control value, and the maximum variation of counts per minute bound for each observation was <2% of total counts per minute bound. One hundred percent binding of FXI represents an average of 99,565 cpm bound, whereas 0% binding of FXI represents 0% bound after subtracting 198 cpm, representing the control in which labeled FXI was incubated with platelets at time 0.View Large Image Figure ViewerDownload (PPT) Solid Phase Binding of 125I-FXI or 125I-PPACK Thrombin to Glycocalicin—We utilized a modification of the method of DeCristofaro et al. (31De Cristofaro R. De C ia E. Landolfi R. Rutella S. Hall S.W. Biochemistry. 2001; 40: 13268-13273Crossref PubMed Scopus (44) Google Scholar) to examine the binding of 125I-FXI to platelet-bound glycocalicin. Wheat germ lectin (10 μg/ml) was coated on the wells of a solid phase plate (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 capacity of the sample wells was blocked by incubation for 2 h with 1% BSA in Hepes-buffered saline (150 mm NaCl and 3.5 mm Hepes, pH 7.2). 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, the 125I-FXI or 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 1470 Wallac Wizard gamma counter. Sulfated Tyrosine-containing Peptides—Solid phase synthesis of sulfated-tyrosines in peptides Thr266–Asp287 and Asp269–Pro280 (Table I) was accomplished by the method of Kitagawa et al. (32Kitagawa K. Aida C. Fujiwara H. Yagami T. Futaki S. Kogire M. Ida J. Inoue K. J. Org. Chem. 2001; 66: 1-10Crossref PubMed Scopus (54) Google Scholar). Expression of Wild-type and the Type 2B Mutant A1 Domain of vWF—The 508–709 recombinant fragments of vWF with either the wild-type sequence or the Ile546-Val type 2B mutation were expressed and purified as described previously (33Mohri H. Yoshioka A. Zimmerman T.S. Ruggeri Z.M. J. Biol. Chem. 1989; 264: 17361-17367Abstract Full Text PDF PubMed Google Scholar, 34Celikel R. Ruggeri Z.M. Varughese K.I. Nat. Struct. Biol. 2000; 7: 881-884Crossref PubMed Scopus (66) Google Scholar). The Effect of Glycocalicin and Mocarhagin Fragments on the Binding of FXI to Activated Platelets—Previous studies have determined that the FXI binding site on platelets is in the GPIbα subunit of the GPIb-IX-V complex for the following reasons: 1) because two GPIbα ligands, SZ2 (a monoclonal antibody agonist the-NH2 terminal of GPIbα) and bovine vWF, inhibit FXI binding to platelets; 2) because FXI was shown by surface plasmon resonance to bind specifically to glycocalicin in a Zn2+-dependent fashion; and 3) because glycocalicin could promote FXI activation by thrombin, another GPIbα ligand (12Baglia F.A. Badellino K.O. Li C.Q. Lopez J.A. Walsh P.N. J. Biol. Chem. 2002; 277: 1662-1668Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). It has been determined that a sulfated tyrosine/anionic sequence, Tyr276–Glu282, of GPIbα comprises a binding site for vWF and thrombin (21Dumas J.J. Kumar R. McDonagh T. Sullivan F. Stahl M.L. Somers W.S. Mosyak L. J. Biol. Chem. 2004; 279: 23327-23334Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar, 22Celikel R. McClintock R.A. Roberts J.R. Mendolicchio G.L. Ware J. Varughese K.I. Ruggeri Z.M. Science. 2003; 301: 218-221Crossref PubMed Scopus (166) Google Scholar, 24Cauwenberghs. N. Vanhoorelbeke K. Vauterin S. Westra D.F. Romo G. Huizinga E.G. Lopez J.A. Berndt M.C. Harsfalvi J. Deckmyn H. Blood. 2001; 98: 652-660Crossref PubMed Scopus (78) Google Scholar, 25Shen Y. Romo G.M. Dong J.F. Schade A. McIntire L.V. Kenny D. Whisstock J.C. Berndt M.C. Lopez J.A. Andrews R.K. Blood. 2000; 95: 903-910Crossref PubMed Google Scholar, 26Dong J. Ye P. Schade A.J. Gao S. Romo G.M. Turner N.T. McIntire L.V. Lopez J.A. J. Biol. Chem. 2001; 276: 16690-16694Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 27Marchese P. Murata M. Mazzucato M. Pradella P. De Marco L. Ware J. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 9571-9578Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar) and that the LRRs of GPIbα are important for binding vWF (26Dong J. Ye P. Schade A.J. Gao S. Romo G.M. Turner N.T. McIntire L.V. Lopez J.A. J. Biol. Chem. 2001; 276: 16690-16694Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 27Marchese P. Murata M. Mazzucato M. Pradella P. De Marco L. Ware J. Ruggeri Z.M. J. Biol. Chem. 1995; 270: 9571-9578Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). To determine the binding site for FXI in GPIbα, we utilized mocarhagin, a cobra venom metalloproteinase, that cleaves GPIbα at Glu282–Asp283 to generate a fragment, His1–Glu282, which contains the LRRs and the sulfated tyrosine/anionic sequences Tyr276–Glu282 and excludes the carbohydrate-rich macroglycopeptide. We examined the effect of glycocalicin and the His1–Glu282 fragment on the binding of FXI to activated platelets. Fig. 1 shows that the His1–Glu282 fragment competed for binding sites on FXI-activated platelets with an IC50 similar to that for glycocalicin (10 nm ± 0.65), suggesting that FXI binds to the NH2-terminal portion of glycocalicin. Also, neither of the two synthetic peptides with sulfated tyrosines (Thr266–Asp287 and Asp269–Pro280) had any effect on the binding of FXI to activated platelets, suggesting that FXI does not bind to the anionic domain containing sulfated tyrosines that is utilized for binding thrombin and vWF. FXI Binding to Glycocalicin or the Mocarhagin Fragment in the Presence of ZnCl2—To confirm the notion that FXI is binding to the NH2-terminal region of GPIbα, we performed direct binding experiments in a solid phase assay. Fig. 2 shows that FXI binds to the fragment His 1-Glu 282 with the same Kd app (∼10 nm ± 0.9) as intact glycocalicin in the presence of ZnCl2, whereas in the absence of ZnCl2 no FXI-binding to either glycocalicin or the His1–Glu282 fragment was observed. This confirms the conclusion that the NH2-terminal globular domain of GPIb mediates the binding of FXI to GPIb-IX-Vα. The Effect of the His1–Glu282 Fragment and Sulfated Peptides on the Binding of FXI to Glycocalicin—To further confirm our findings on the effect of the His1–Glu282 fragment and sulfated peptides on FXI binding to activated platelets, we examined their effect on the binding of FXI to glycocalicin in a solid phase assay. Fig. 3 shows that the His1–Glu282 fragment inhibited the binding of FXI to glycocalicin with an IC50 of 20 nm ± 1.8, whereas the sulfated peptides Thr266–Asp287 and Asp269–Pro280 had no effect. The Effect of FXI on the Binding of Thrombin to Glycocalicin—Because both FXI and thrombin bind to the NH2-terminal globular domain of GPIbα and because FXI interacts with thrombin through anion-binding exosite I (35Yun T.H. Baglia F.A. Myles T. Navaneetham D. Lopez J.A. Walsh P.N. Leung L.L. J. Biol. Chem. 2003; 278: 48112-48119Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar), we determined whether thrombin and FXI share a common binding site on GPIbα. Fig. 4 shows that FXI at concentrations up to 4 × 10-5m does not inhibit the binding of thrombin to glycocalicin, which suggests that FXI binds gly
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