Factor XI Binding to Activated Platelets Is Mediated by Residues R²⁵⁰, K²⁵⁵, F²⁶⁰, and Q²⁶³ within the Apple 3 Domain

Abstract

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).

Thrombin Activation of Factor XI on Activated Platelets Requires the Interaction of Factor XI and Platelet Glycoprotein Ibα with Thrombin Anion-binding Exosites I and II, Respectively

“…Preparation of Washed Platelets-Platelets were prepared as described (6,14,23). Platelet-rich plasma obtained from citrated human blood was centrifuged, and the platelets were resuspended in calciumfree Hepes/Tyrode's buffer (126 mM NaCl, 2.7 mM KCl, 1 mM MgCl 2 , 0.38 mM NaH 2 PO 4 , 5.6 mM dextrose, 6.2 mM sodium Hepes, 8.9 mM Hepes (free acid), and 0.1% BSA) at pH 6.5 and gel-filtered on a column of Sepharose 2B equilibrated in calcium-free Hepes/Tyrode's buffer (pH 7.2).…”

“…FXIIa, a component of the contact phase of blood coagulation, is unlikely to be a physiologically relevant activator because FXII deficiency is not associated with clinical bleeding (10,11). Recent data suggest that thrombin is the physiological activator of FXI on the activated platelet (12)(13)(14). Normal hemostasis is initiated by the exposure of tissue factor at sites of vascular injury, followed by the formation of the FVII-tissue factor complex and the subsequent activation of FX.…”

“…The FXI homodimer binds to GPIb␣ in the GPIb-IX-V complex through one of its monomers (12,14,23). Thrombin then binds to the A1 domain and activates FXI (20,24).…”

“…Many thrombin substrates, receptors, and inhibitors gain access to the active site by binding to either of the two exosites. For example, anionbinding exosite (ABE) I is important for binding to fibrinogen (27), fibrin (28), heparin cofactor II (29), PAR1 (30), thrombomodulin (31), and hirudin (32,33), whereas ABE-II is involved in binding to platelet GPIb␣ (34), protease nexin I (35), and glycosaminoglycans such as heparin (14).…”

A Catalytic Domain Exosite (Cys527−Cys542) in Factor XIa Mediates Binding to a Site on Activated Platelets

“…It is hypothesized that the formation of the FXI/HK or FXI/FII complex leads to the exposure of residues within the A3 domain that mediate FXI-binding to platelets (14,15). FXIa binds to high-affinity receptors on the activated platelet surface that are distinct from the receptors for FXI (24,25).…”