Identification and Characterization of a Binding Site for Platelets in the Apple 3 Domain of Coagulation Factor XI

Abstract

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.

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

“…We have previously shown that FXI binds to the activated platelet surface through the A3 domain (12). In the present study we show that FXIa binds to platelets utilizing residues that reside outside the A3 domain.…”

“…However recent observations cast considerable doubt on the conclusion that activated platelets can promote the feedback activation of FXI by thrombin (35)(36)(37). Therefore, in a revised model of the interactions of FXI and FXIa with the activated platelet surface (Figure 7), dimeric FXI in complex with either HK or prothrombin is shown to expose a site within the A3 domain that binds to glycoprotein Ibα on the activated platelet surface (12)(13)(14)(15)19). Since this interaction has been demonstrated to be reversible (13), and since it has not been rigorously demonstrated whether it is the plateletbound or soluble form of FXI that is converted to FXIa, the activation of FXI by FXIIa, FXIa or thrombin is shown in Figure 7 to occur in solution.…”

“…FXI when complexed with HK in the presence of ZnCl 2 or prothrombin in the presence of CaCl 2 (13,14) binds to the activated platelet surface through the A3domain (12,15). 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).…”

“…Although the physiological surface that might potentiate FXI-activation by thrombin has not been definitively identified, it has been demonstrated that FXI binds to high-affinity (K D ~10 nM), saturable, specific receptors (B max ~1,500 sites/ platelet) on activated platelets, in the presence of HK and ZnCl 2 or prothrombin and CaCl 2 (12)(13)(14)(15). It is hypothesized that the formation of the FXI/HK or FXI/prothrombin complex leads to the exposure of residues within the Apple 3 (A3) domain that mediate FXI-binding (12,15) to activated platelets where FXI can be cleaved to FXIa by FXIIa (16) and possibly also by thrombin (17). In contrast, neither resting nor thrombin-activated human umbilical vein endothelial cells have any capacity to bind FXI or promote its activation by thrombin (18).…”

“…Two chimeras were generated, rFXI/PKA3 and rFXIa/PKA3, in which the A3 domain of FXI, the domain responsible for mediating the binding of FXI to the activated platelet surface (12,14), was replaced with the A3 domain of PK in order to study the binding of FXIa to platelets. We have examined the ability of FXI and FXIa as well as rFXI/ PKA3 and rFXIa/PKA3 chimeras to compete with 125 I-FXIa for binding to the activated platelet surface.…”