The Role of High Molecular Weight Kininogen and Prothrombin as Cofactors in the Binding of Factor XI A3 Domain to the Platelet Surface

Abstract

We have reported that prothrombin (1 M) 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. Coagulation factor XI (FXI)1 is a disulfide-linked homodimeric protein (160,000 Da) (1) that is cleaved by thrombin, FXIIa, or FXIa at a single peptide bond (Arg 369 -Ile 370 ) to give rise to FXIa (2-5). The primary structures of four repeat sequences (designated A1, A2, A3, and A4 or Apple domains) present in the heavy chain region of FXI have been elucidated from the sequence of cDNA inserts coding for FXI (1). Evidence exists for the presence of a FIX binding site on the A2 domain (6) and at the N terminus of the A3 domain (7,8), and a binding site for FXIIa in the A4 domain (9). In plasma, FXI circulates in a complex with high molecular weight kininogen (HK) (10, 11), an interaction that requires the A1 domain of FXI (12, 13) and the light chain of HK (14). FXI can also bind to prothrombin, which can displace HK bound to FXI and which binds to a site (Ala 45 -Ser 86 ) in the A1 domain that is within the HK-binding site (15). In the presence of HK or prothrombin, FXI can bind reversibly and specifically to high affinity sites on the surface of stimulated human platelets in the presence of zinc and calcium ions (15, 16). More specifically, this laboratory has demonstrated that the A3 domain of FXI is essential in the binding of FXI to platelets, since a recombinant A3 (rA3) domain of FXI demonstrated specific, saturable binding and effectively competed with radiolabeled FXI in binding to the platelet surface (17). The consequence of FXI binding to activated platelets is an acceleration of the rate of FXI activation by thrombin, FXIIa, or FXIa (15).Since both prothrombin (15, 18) and HK (14, 16, 19) have been shown to bind both to platelets (15,16,18,19) and to FXI (14, 15), one objective of the present study was to examine the mechanism by which HK and prothrombin promote the binding of FXI to the activated platelet, i.e. to determine whether either the FXI⅐HK complex or the FXI⅐prothrombin complex initially binds as an encounter complex to platelets via HK or prothrombin, or whether, alternatively either HK or prothrombin binds only to the FXI A1 domain resulting in exposure of the A3 domain platelet binding site. Another goal of the present study was to define the amino acids within the A3 domain of FXI that mediate its binding to platelets. The results of these studies, utilizing site-directed mutational analysis of recombinant FXI (rFXI) confirm and extend previous studies utilizing conformationally constrained synthetic peptides and the rA3 domain of FXI, which identified Arg 250 , Lys 255 , Phe 260...

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

“…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).…”

“…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).…”

“…This possibility was substantiated by the seminal observations of Naito and Fujikawa (10) and Gailani and Broze (11) who demonstrated that FXI could be activated by thrombin in the presence of dextran sulfate. 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).…”

“…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.…”