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F A Baglia

Publications and source records attributed to F A Baglia.

At least 19 recordsLinked to original sources
PubMed2001

Defective binding of factor XI-N248 to activated human platelets.

Variants of factor XI containing Gln226 to Arg (Q226 to R) and Ser248 to Asn (S248 to N) substitutions were first identified in an African American family with a history of excessive bleeding. The substitutions have recently been identified in unrelated individuals, suggesting they are relatively common. Both amino acids are located in the third apple domain of factor XI, an area implicated in binding interactions with factor IX and activated platelets. Recombinant factor XI-R226 and factor XI-N248 were compared with wild-type factor XI in assays for factor IX activation or platelet binding. Factor XI-R226 activates factor IX with a Michaelis-Menten constant (K(m)) about 5-fold greater than wild-type protein. The catalytic efficiency of factor IX activation is similar to wild-type protein, however, due to an increase in the turnover number (k(cat)) for the reaction. Iodinated factor XI-N248 binds to activated platelets with a dissociation constant (K(d)) more than 5-fold higher than wild-type protein (55 nM and 10 nM, respectively). Activation of factor XI-N248 by thrombin in the presence of activated platelets is slower and does not progress to the same extent as activation of the wild-type protein under similar conditions. Factor XI-N248 activates factor IX normally in a purified protein system and has relatively normal activity in activated partial thromboplastin time (aPTT) assays. Factor XI-N248 is the first factor XI variant described with a clear functional difference compared with wild-type protein. Importantly, the defect in platelet binding would not be detected by routine clinical evaluation with an aPTT assay.

Amino Acid Substitution↗
PubMed2000

A binding site for the kringle II domain of prothrombin in the apple 1 domain of factor XI.

Previously we defined binding sites for high molecular weight kininogen (HK) and thrombin in the Apple 1 (A1) domain of factor XI (FXI). Since prothrombin (and Ca(2+)) can bind FXI and can substitute for HK (and Zn(2+)) as a cofactor for FXI binding to platelets, we have attempted to identify a prothrombin-binding site in FXI. The recombinant A1 domain (rA1, Glu(1)-Ser(90)) inhibited the saturable, specific and reversible binding of prothrombin to FXI, whereas neither the rA2 domain (Ser(90)-Ala(181)), rA3 domain (Ala(181)-Val(271)), nor rA4 domain (Phe(272)-Glu(361)) inhibited prothrombin binding to FXI. Kinetic binding studies using surface plasmon resonance showed binding of FXI (K(d) approximately 71 nm) and the rA1 domain (K(d) approximately 239 nm) but not rA2, rA3, or rA4 to immobilized prothrombin. Reciprocal binding studies revealed that synthetic peptides (encompassing residues Ala(45)-Ser(86)) containing both HK- and thrombin-binding sites, inhibit (125)I-rA1 (Glu(1)-Ser(90)) binding to prothrombin, (125)I-prothrombin binding to FXI, and (125)I-prothrombin fragment 2 (Ser(156)-Arg(271)) binding to FXI. However, homologous prekallikrein-derived peptides (encompassing Pro(45)-Gly(86)) did not inhibit FXI rA1 binding to prothrombin. The peptides Ala(45)-Arg(54), Phe(56)-Val(71), and Asp(72)-Ser(86), derived from sequences of the A1 domain of FXI, acted synergistically to inhibit (125)I-rA1 binding to prothrombin. Mutant rA1 peptides (V64A and I77A), which did not inhibit FXI binding to HK, retained full capacity to inhibit rA1 domain binding to prothrombin, and mutant rA1 peptides Ala(45)-Ala(54) (D51A) and Val(59)-Arg(70) (E66A), which did not inhibit FXI binding to thrombin, retained full capacity to inhibit rA1 domain binding to prothrombin. Thus, these experiments demonstrate that a prothrombin binding site exists in the A1 domain of FXI spanning residues Ala(45)-Ser(86) that is contiguous with but separate and distinct from the HK- and thrombin-binding sites and that this interaction occurs through the kringle II domain of prothrombin.

Binding Sites↗
PubMed2000

The role of high molecular weight kininogen and prothrombin as cofactors in the binding of factor XI A3 domain to the platelet surface.

We have reported that prothrombin (1 microm) is able to replace high molecular weight kininogen (45 nm) as a cofactor for the specific binding of factor XI to the platelet (Baglia, F. A., and Walsh, P. N. (1998) Biochemistry 37, 2271-2281). We have also determined that prothrombin fragment 2 binds to the Apple 1 domain of factor XI at or near the site where high molecular weight kininogen binds. A region of 31 amino acids derived from high molecular weight kininogen (HK31-mer) can also bind to factor XI (Tait, J. F., and Fujikawa, K. (1987) J. Biol. Chem. 262, 11651-11656). We therefore investigated the role of prothrombin fragment 2 and HK31-mer as cofactors in the binding of factor XI to activated platelets. Our experiments demonstrated that prothrombin fragment 2 (1 microm) or the HK31-mer (8 microm) are able to replace high molecular weight kininogen (45 nm) or prothrombin (1 microm) as cofactors for the binding of factor XI to the platelet. To localize the platelet binding site on factor XI, we used mutant full-length recombinant factor XI molecules in which the platelet binding site in the Apple 3 domain was altered by alanine scanning mutagenesis. The recombinant factor XI with alanine substitutions at positions Ser(248), Arg(250), Lys(255), Leu(257), Phe(260), or Gln(263) were defective in their ability to bind to activated platelets. Thus, the interaction of factor XI with platelets is mediated by the amino acid residues Ser(248), Arg(250), Lys(255), Leu(257), Phe(260), and Gln(263) within the Apple 3 domain.

Amino Acids↗
PubMed2000

Thrombin-mediated feedback activation of factor XI on the activated platelet surface is preferred over contact activation by factor XIIa or factor XIa.

To study the pathways for initiation of intrinsic blood coagulation, activated human platelets were compared with dextran sulfate as surfaces for factor XI activation by factor XIIa, factor XIa, or thrombin. Activated gel-filtered platelets promoted the activation of factor XI (60 nm) by thrombin (0.02-10 nm, EC(50) approximately 100 pm, threshold concentration approximately 10 pm) at initial rates 2- to 3-fold greater than those obtained with dextran sulfate in the presence of either high molecular weight kininogen (45 nm) and ZnCl(2) (25 micrometer) or prothrombin (1.2 micrometer) and CaCl(2) (2 mm). The maximum rates of factor XI activation achieved in the presence of activated gel-filtered platelets were 30 nm.min(-1) with thrombin, 6 nm.min(-1) with factor XIIa and 2 nm.min(-1) with factor XIa. Values of turnover number calculated at various enzyme concentrations (0.05-1 nm) were 24-167 (mean = 86) min(-1) for thrombin, 4.6-50 (mean = 21) min(-1) for factor XIIa, and 1.3-14 (mean = 8) min(-1) for factor XIa. A physiological concentration of fibrinogen (9.0 micrometer) inhibited factor XI activation by thrombin (but not by factor XIIa) in the presence of dextran sulfate but not in the presence of gel-filtered platelets. Compared with factors XIIa and XIa, thrombin is the preferred factor XI activator, and activated platelets are a relevant physiological surface for thrombin-mediated initiation of intrinsic coagulation in vivo.

Blood Platelets↗
PubMed2000

Factor XI binding to activated platelets is mediated by residues R(250), K(255), F(260), and Q(263) within the apple 3 domain.

To localize the platelet binding site on factor XI, rationally designed, conformationally constrained synthetic peptides were used to compete with [(125)I]factor XI binding to activated platelets. The major platelet binding energy resided within the sequence of amino acids T(249)-F(260). Homology scanning, using prekallikrein amino acid substitutions within the synthetic peptide T(249)-F(260), identified a major role for R(250) in platelet binding. Inhibition of [(125)I]factor XI binding to activated platelets by the recombinant Apple 3 domain of factor XI and inhibition by unlabeled factor XI were identical, whereas the recombinant Apple 3 domain of prekallikrein had little effect. A "gain-of-function" chimera in which the C-terminal amino acid sequence of the Apple 3 domain of prekallikrein was replaced with that of factor XI was as effective as the recombinant Apple 3 domain of factor XI and unlabeled factor XI in inhibiting [(125)I]factor XI binding to activated platelets. Alanine scanning mutagenic analysis of the recombinant Apple 3 domain of factor XI indicated that amino acids R(250), K(255), F(260), and Q(263) (but not K(252) or K(253)) are important for platelet binding. Thus, the binding energy mediating the interaction of factor XI with platelets is contained within the C-terminal amino acid sequence of the Apple 3 domain (T(249)-V(271)) and is mediated in part by amino acid residues R(250), K(255), F(260), and Q(263).

Amino Acid Sequence↗
PubMed1998

A binding site for heparin in the apple 3 domain of factor XI.

Since heparin potentiates activated factor XI (FXIa) inhibition by protease nexin-2 by providing a template to which both proteins bind (Zhang, Y., Scandura, J. M., Van Nostrand, W. E., and Walsh, P. N. (1997) J. Biol. Chem. 272, 26139-26144), we examined binding of factor XI (FXI) and FXIa to heparin. FXIa binds to heparin (Kd approximately 0.7 x 10(-9) M) >150-fold more tightly than FXI (Kd approximately 1.1 x 10(-7) M). To localize the heparin-binding site on FXI, rationally designed conformationally constrained synthetic peptides were used to compete with 125I-FXI binding to heparin. A peptide derived from the Apple 3 (A3) domain of FXI (Asn235-Arg266) inhibited FXI binding to heparin (Kd approximately 3.4 x 10(-6) M), whereas peptides from the A1 domain (Phe56-Ser86), A2 domain (Ala134-Ala176), and A4 domain (Ala317-Gly350) had no such effect. The recombinant A3 domain (rA3, Ala181-Val271) inhibited FXI binding to heparin (Ki approximately 1.4 x 10(-7) M) indicating that all the information necessary for FXI binding to heparin is contained entirely within the A3 domain. The A3 domain also contains a platelet-binding site (Asn235-Arg266), consisting of three surface-exposed loop structures, Pro229-Gln233, Thr741-Leu246, and Thr249-Phe260 (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1995) J. Biol. Chem. 270, 6734-6740). Only peptide Thr249-Phe260 (which contains a heparin binding consensus sequence, RIKKSKA) inhibits FXI binding to heparin (Ki = 2.1 x 10(-7) M), whereas peptides Pro229-Gln233 and Thr241- Leu246 had no effect. Fine mapping of the heparin-binding site using prekallikrein analogue amino acid substitutions of the synthetic peptide Thr249-Phe260 and alanine scanning of the recombinant A3 indicated that the amino acids Lys252 and Lys253 are important for heparin binding. Thus, the sequence Thr249-Phe260 which contains most of the binding energy for FXI interaction with platelets also mediates the binding of FXI to heparin.

Amino Acid Sequence↗
PubMed1998

Tissue-specific expression of functional platelet factor XI is independent of plasma factor XI expression.

Platelet factor XI is an alternatively spliced product of the factor XI gene expressed specifically within megakaryocytes and platelets as an approximately 1.9-kb mRNA transcript (compared with approximately 2.1 kb in liver cells) lacking exon V. Flow cytometry with an affinity-purified factor XI antibody, with PAC1 antibody (to the GPIIb/IIIa complex on activated platelets), and with S12 antibody (to P-selectin, an alpha-granule membrane protein expressed on the platelet surface during secretion) on platelets activated with ADP, thrombin, thrombin receptor peptide (SFLLRN amide), or collagen at various concentrations exposed platelet factor XI and PAC1 antibody binding in parallel. Unactivated platelets expressed approximately 40% of total platelet factor XI but no PAC1 binding sites. Enhanced membrane exposure of platelet factor XI is independent of alpha-granule secretion, because ADP and collagen exposed platelet factor XI but no S12 binding sites. Platelets from four patients with plasma factor XI deficiency (<0.04 U/mL) had normal constitutive and activation-dependent expression of platelet factor XI. Well-washed platelets from normal and from factor XI-deficient donors incubated with low concentrations of thrombin (0. 05 to 0.1 U/mL) corrected the clotting defect observed with factor XI-deficient plasma. Thus, functionally active platelet factor XI is differentially expressed on platelet membranes in a tissue-specific manner both constitutively and in a concentration-dependent fashion by various agonists in the absence of detectable plasma factor XI.

Adult↗
PubMed1998

Prothrombin is a cofactor for the binding of factor XI to the platelet surface and for platelet-mediated factor XI activation by thrombin.

To study the physiological significance of thrombin as an initiator of intrinsic blood coagulation, activated human platelets were compared with dextran sulfate as a surface for thrombin-catalyzed factor XI activation. Activated gel-filtered platelets promoted factor XI activation by thrombin at initial rates 2-5-fold greater than dextran sulfate in the presence of high molecular weight kininogen (HK, 45 nM), ZnCl2 (25 microM), and CaCl2 (2 mM), conditions optimal for factor XI binding to platelets. Physiological concentrations of HK (636 nM) inhibited factor XI activation by thrombin in a concentration-dependent manner, and this inhibition was reversed by prothrombin (1-3 microM) and by prothrombin fragment 1.2 (PF1.2), but not by prothrombin fragment 1 (PF1). Since prothrombin and PF1.2 (but not PF1) also displaced HK from its binding site on the Apple 1 domain of factor XI, we conclude that the Kringle II domain of prothrombin competes with HK for binding to the Apple 1 domain of factor XI. Prothrombin (1-3 microM) and PF1.2 (but not PF1) in the presence of CaCl2 (2 mM) were able to replace HK (45 nM) in the presence of ZnCl2 (25 microM) as a cofactor for the specific, reversible, high-affinity (Kd approximately 25 nM) binding of factor XI to 947 +/- 150 sites per platelet. This binding is mediated by residues Asn 235-Arg 266 in the Apple 3 domain since a conformationally constrained, synthetic peptide analogue of this sequence inhibits both factor XI binding to activated platelets and platelet-mediated, thrombin-catalyzed factor XI activation in the presence of prothrombin and CaCl2. Finally, prothrombin (1.2 microM) and CaCl2 (2 mM) could substitute for HK (45 nM) and ZnCl2 (25 microM) in promoting optimal rates of thrombin-catalyzed factor XI activation on the platelet surface, thereby initiating the intrinsic coagulation pathway by mechanisms completely independent of the contact phase proteins, factor XII, HK, and prekallikrein.

Binding Sites↗
PubMed1996

A binding site for thrombin in the apple 1 domain of factor XI.

Previously we defined a binding site for high molecular weight kininogen (HK) in the A1 domain of factor XI (FXI). Since thrombin can activate FXI and HK inhibits the activation of FXI by thrombin, we have identified a thrombin binding site in FXI. Both the recombinant A1 domain (Glu1-Ser90) and a synthetic peptide (Phe56-Ser86) containing the HK binding site inhibited FXI activation by thrombin. Both a monoclonal antibody, 5F7, recognizing the A1 domain, and the rA1 domain were shown to be competitive inhibitors of thrombin-catalyzed FXI activation. The peptides Ala45-Arg54 and Val59-Arg70 acted synergistically to inhibit FXI activation by thrombin. Mutant rA1 domain constructs (Val64 --> Ala and Ile77 --> Ala), which do not inhibit FXI binding to HK, retain full capacity to inhibit FXI activation by thrombin. The peptide Ala45-Arg54 inhibited thrombin-catalyzed FXI activation, whereas it had no effect on FXI binding to HK. In contrast, the peptide Asn72-Leu83 (which inhibited FXI binding to HK) did not inhibit FXI activation by thrombin. Thus, a thrombin binding site exists in the A1 domain of FXI spanning residues Ala45-Arg70 that is contiguous with but separate and distinct from the HK binding site. These sites may regulate which ligand is bound to FXI and through which pathway FXI is activated.

Amino Acid Sequence↗
PubMed1995

The Apple 1 and Apple 4 domains of factor XI act synergistically to promote the surface-mediated activation of factor XI by factor XIIa.

Binding sites for high molecular weight kininogen (HK) and for factor XIIa are present in the Apple 1 (A1) and the A4 domains of factor XI, respectively. To define the roles of these two sites in surface-mediated factor-XI activation we prepared conformationally constrained synthetic peptides and recombinant A1 domain (rA1) and determined their effects on the activation of factor XI by factor XIIa in the presence of HK and either kaolin or dextran sulfate. Surface-mediated factor-XI activation by factor XIIa was inhibited by a conformationally constrained A4 peptide (Ala317-Gly350), by an A1 peptide (Phe56-Ser86), and by rA1 (Glu1-Ser90). When used in combination at equimolar concentrations, rA1 and A4 peptide were 10-fold more effective than either one alone in inhibiting surface-mediated activation of factor XI by factor XIIa. The A4 peptide was a competitive inhibitor of factor XIIa amidolytic activity and a noncompetitive inhibitor of factor-XI activation by factor XIIa, whereas rA1 and the A1 peptide did not inhibit factor XIIa. The rA1 domain inhibited factor XI binding to HK, whereas the A4 peptide did not. We conclude that specific sequences exposed on the surfaces of the A1 (Val59-Lys83) and A4 (Ala317-Gly350) domains of factor XI act synergistically to promote surface-mediated factor-XI activation by factor XIIa in the presence of HK by binding factor XI to surface-bound HK (A1 domain) and by binding factor XIIa near the cleavage site (Arg369-Ile370) of factor XI (A4 domain).

Dextran Sulfate↗
PubMed1995

Identification and characterization of a binding site for platelets in the Apple 3 domain of coagulation factor XI.

Activated platelets expose a specific, reversible high affinity (Kdapp congruent to 10 nM) binding site (n congruent to 1500 sites/platelet) for factor XI that requires the presence of high molecular weight kininogen (HK) and ZnCl2 (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 125I-factor XI binding to platelets. A peptide from the Apple 3 (A3) domain (Asn235-Arg266) inhibits factor XI binding to platelets in the presence of HK (42 nM), CaCl2 (2 mM), and ZnCl2 (25 microM), with a Ki congruent to 10 nM which is identical to the Kd for factor XI binding to platelets. A peptide from the A1 domain (Phe56-Ser86) partially inhibits factor XI binding to platelets (Ki congruent to 6 microM) 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 (Pro229-Gln233, Thr241-Leu246, and Ser248-Ser261) each of which acted alone and synergistically when added together to inhibit factor XI binding to platelets. Finally, the 125I-labeled A3 domain peptide (Asn235-Arg266) was found to bind to thrombin-activated platelets in a specific, reversible, and saturable manner. Thus, the sequence of amino acids Asn235-Arg266 of the A3 domain of factor XI comprises a contact surface for interaction with a platelet receptor.

Amino Acid Sequence↗
PubMed1994

Binding of high-molecular-mass kininogen to the Apple 1 domain of factor XI is mediated in part by Val64 and Ile77.

We have previously demonstrated the presence of a binding site for high-molecular-mass kininogen (HK), spanning residues Val59-Lys83, in the first Apple (A1) domain in the heavy-chain region of factor XI. We have now prepared conformationally constrained synthetic peptides and recombinant A1 domain (rA1) constructs to identify the specific amino acid residues that constitute the HK-binding site. Expression of the A1 domain (Glu1-Ser90) was achieved in a bacterial expression system following PCR amplification of the A1 domain from factor XI cDNA and ligation into an expression plasmid. The rA1 inhibited factor XI binding to HK [Ki approximately (2-3) x 10(-7) M] in a manner indistinguishable from purified factor XI, indicating that all the information necessary for binding HK is contained within the A1 domain. To identify specific amino acid residues involved in binding HK, conformationally constrained peptides were synthesized containing conservative amino acid substitutions at residues suspected to contain side chains involved in binding, including Val64-->Ala, Glu66-->Ala, Arg73-->Ala and Ile77-->Ala. Because normal results were obtained with all peptides with the exception of Val64-->Ala and Ile77-->Ala, which failed to compete normally with factor XI for binding to HK, we prepared two mutant rA1 domains (Val64-->Ala and Ile77-->Ala) by PCR-based site-directed mutagenesis, both of which exhibited diminished capacity to inhibit factor XI binding to HK. Competition studies with prekallikrein (PK) and a PK-dependent synthetic peptide suggested that PK and factor XI have a common surface in the A1 domain for binding HK of which Val64 is a part. We conclude that the binding of factor XI to HK is mediated at least in part by Val64 and Ile77 in the A1 domain of factor XI.

Amino Acid Sequence↗
PubMed1993

Identification and characterization of a binding site for factor XIIa in the Apple 4 domain of coagulation factor XI.

Previously we have characterized a binding site for high M(r) kininogen in the first of four tandem-repeat (Apple) domains within the heavy chain region of factor XI (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1990) J. Biol. Chem. 265, 4149-4154; Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1991) J. Biol. Chem. 267, 4247-4252), whereas a substrate binding site for factor IX was localized to the second Apple (A2) domain (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1991) J. Biol. Chem. 266, 24190-24197). To define the factor XI domain that binds factor XIIa, we have screened a panel of synthetic peptides for their capacity to inhibit factor XI activation by factor XIIa. Peptide Gly326-Lys357 (located in the A4 domain) is a noncompetitive inhibitor of factor XI activation by factor XIIa (Ki = 3.75 microM), whereas structurally similar peptides from the A1, A2, and A3 domains were required at > 1000-fold higher concentrations for similar effects. The same peptide (Gly326-Lys357) is a competitive inhibitor of factor XIIa amidolytic activity (Ki = 3.8 microM) suggesting that it binds near the active site of factor XIIa. Computer modeling was used to predict the secondary and tertiary structure of the A4 domain of factor XI that interacts with factor XIIa. Rationally designed, conformationally constrained peptides were synthesized comprising residues Ala317-Gly326, Lys331-Lys340, and Gly344-Gly350, which act in concert to inhibit factor XI-activation by factor XIIa. Finally, a conformationally constrained peptide spanning residues Ala317-Gly350 inhibits factor XIIa-catalyzed factor XI activation 50% at a concentration of 5 x 10(-7) M. These results, interpreted in the context of the model, suggest that the sequence of amino acids from Ala317 through Gly350 of the heavy chain of the A4 domain of factor XI contains three peptide structures, possibly consisting of three antiparallel beta-strands that together comprise a contact surface for interacting with factor XIIa.

Amino Acid Sequence↗
PubMed1992

Fine mapping of the high molecular weight kininogen binding site on blood coagulation factor XI through the use of rationally designed synthetic analogs.

Using immunological and chemical cleavage techniques, we have previously identified a domain contained within residues Phe56-Ser86 in the first tandem repeat (A1) of the heavy chain of factor XI which binds high Mr kininogen (Baglia, F. A., Jameson, B. A., and Walsh, P. N. (1990) J. Biol. Chem. 265, 4149-4154). We have now chemically synthesized peptides from corresponding homologous regions in the second (A2), third (A3), and fourth (A4) tandem repeats of the heavy chain (A2: Asn145-Ala176; A3: Asn235-Arg266; and A4: Gly326-Lys357). These peptides had no effect on the binding of factor XI to high Mr kininogen. Because of a lack of detailed structural information for the A1 domain, a molecular model of this region was constructed. This hypothetical model made distinct and testable predictions regarding potential surfaces and concomitant secondary structure. Specifically, the resulting structure depicted two juxtaposed beta-stranded stem-loops that, in conjunction with biological information, constitute a candidate surface for contact with high Mr kininogen. The hypothetical A1 model was, consequently, used as a predictive template in the rational design of two synthetic peptides (Val59-Arg70 and Asn72-Lys83). When both these peptides were added together and the binding of factor XI to high Mr kininogen was examined, a synergistic inhibitory effect was observed compared with each peptide added individually. Our data are consistent with the notion that the sequence of amino acids from Val59-Lys83 of the heavy chain of factor XI contains two antiparallel beta-strands connected by beta-turns that together comprise a continuous surface utilized for the binding of high Mr kininogen.

Amino Acid Sequence↗
PubMed1991

Identification and chemical synthesis of a substrate-binding site for factor IX on coagulation factor XIa.

We have previously used monoclonal antibodies to identify an epitope on the heavy chain of factor XIa that is a substrate-binding site for factor IX (Sinha, D., Seaman, F.S., and Walsh, P.N. (1987) Biochemistry 26, 3768-3775; Baglia, F.A., Sinha, D., and Walsh, P.N. (1989) Blood 74, 244-251). To define the factor XIa domain that binds factor IX, we have now screened a panel of factor XI heavy chain-derived synthetic peptides for their capacity to inhibit the formation of an activation peptide reflecting factor IX activation by factor XIa. Peptide Asn145-Ala176 (which is located in the second tandem repeat or A2 domain of the factor XI heavy chain) is a competitive inhibitor of factor IX activation by factor XIa with a Ki of 30 nM, whereas structurally similar peptides in the A1, A3, and A4 domains were required at 10-1000-fold higher concentrations for similar effects, and a synthetic peptide identical with a highly homologous region of the heavy chain A2 domain of prekallikrein (Tyr143-Ala176) had no effect on factor IX activation by factor XIa. Because detailed structural information is lacking, a potential three-dimensional structure for the factor XI A2 domain was calculated based on its sequence information in conjunction with previously determined structural constraints. The resulting structure depicted three juxtaposed beta-stranded stem-loops that, based on biological information, constitute a candidate surface for contact with factor IX. The A2 model was therefore used as a template in the rational design of three synthetic peptides (Ala134-Ile146 (peptide a), Leu148-Arg159 (peptide b), and Ile160-Leu172 (peptide c]. When peptides a and b or a and c were added together and the activation of factor IX by factor XIa was examined, a synergistic inhibitory effect was observed, compared with each peptide added individually, whereas peptides b and c showed additive effects. Our data suggest that the sequence of amino acids from Ala134 through Leu172 of the heavy chain of factor XI contains three antiparallel beta-strands connected by beta-turns that together comprise a continuous surface utilized for the binding of factor IX.

Amino Acid Sequence↗
PubMed1990

Localization of the high molecular weight kininogen binding site in the heavy chain of human factor XI to amino acids phenylalanine 56 through serine 86.

We have previously demonstrated that a monoclonal antibody (5F7) directed against the heavy chain region of factor XI inhibits the binding of factor XI to high molecular weight kininogen (high Mr kininogen) and the surface-mediated proteolytic activation of factor XI by factor XIIa in the presence of high Mr kininogen. In order to identify the structural domain of factor XI that binds high Mr kininogen, CNBr-digested factor XI was passed over a 5F7 antibody affinity column. One of two CNBr peptides that bound to this 5F7 affinity column inhibited binding of 125I-factor XI to high Mr kininogen, as did intact factor XI. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of an inhibitory peptide purified by high performance liquid chromatography revealed an Mr of 10,000-15,000. Gas-phase sequencing of this peptide revealed the following amino-terminal sequence: X-X-Val-Thr-Gln-Leu-Leu-Lys-Asp-Thr. These data together with the amino acid composition of the isolated peptide indicate that both the epitope recognized by antibody 5F7 and at least a portion of the high Mr kininogen binding site are contained within the amino-terminal portion of factor XI comprising residues Glu-1 through Met-102. Further cleavage of this peptide with o-iodosobenzoic acid at a tryptophanyl peptide bond revealed that an Mr 5,000 peptide (with the amino-terminal sequence Trp-Phe-Thr-Cys-Val-Leu) bound to a high Mr kininogen affinity column and inhibited binding of 125I-factor XI to high Mr kininogen. Finally, a synthetic peptide comprising residues Phe-56 through Ser-86 inhibited 125I-factor XI binding to high Mr kininogen. These experiments strongly suggest that the high Mr kininogen binding site is contained within the domain in the heavy chain region of factor XI comprising residues Phe-56 through Ser-86.

Amino Acid Sequence↗