Abstract

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.

Highlights

  • Factor XI (FXI),[1] a protein that participates in the intrinsic pathway of blood coagulation, consists of two identical polypep

  • One objective of the present study is to identify the amino acid sequences in FXI that interact with prothrombin and Prothrombin Fragment 2 (PF2) in order to ascertain the relevant physiological mechanisms that promote activation of FXI by thrombin on the platelet surface

  • Binding of 125I-Prothrombin to Factor XI—Since contiguous but distinct binding sites for high molecular weight kininogen (HK) (Val59–Lys83) and thrombin (Ala45–Arg70) reside within the Apple 1 (A1) domain of FXI (18, 20), we previously examined the effects of prothrombin and its fragments on the binding of FXI to HK (14)

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Summary

Introduction

Factor XI (FXI),[1] a protein that participates in the intrinsic pathway of blood coagulation, consists of two identical polypep-. The amino-terminal portion of the FXI monomer consists of four tandem repeat sequences of 90 –91 residues, each with 6 –7 cysteine residues in a characteristic disulfide linkage termed Apple domains (4, 5). Our experiments support the notion that prothrombin as well as thrombin bind FXI at a site encompassing residues Phe56–Ser[86] that mediates HK binding to the A1 domain (14). One objective of the present study is to identify the amino acid sequences in FXI that interact with prothrombin and PF2 (containing kringle II) in order to ascertain the relevant physiological mechanisms that promote activation of FXI by thrombin on the platelet surface. The conclusion drawn from these studies is that prothrombin kringle II can bind to the FXI A1 domain at a site contiguous with but separate and distinct from the A1 domain site utilized by thrombin to bind to and activate FXI

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