A Binding Site for Thrombin in the Apple 1 Domain of Factor XI

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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 gelfiltered platelets promoted the activation of factor XI (60 nM) by thrombin (0.02-10 nM, EC 50 ϳ100 pM, threshold concentration ϳ10 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 M) or prothrombin (1.2 M) and CaCl 2 (2 mM). The maximum rates of factor XI activation achieved in the presence of activated gel-filtered platelets were 30 nM⅐min ؊1 with thrombin, 6 nM⅐min ؊1 with factor XIIa and 2 nM⅐min ؊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 M) 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 thrombinmediated initiation of intrinsic coagulation in vivo.Human coagulation factor XI is a disulfide-linked homodimer consisting of two identical polypeptide chains each containing 607 amino acids. Factor XI is present in human plasma as a zymogen that requires proteolytic activation to develop serine protease activity (1-5). It circulates in human plasma in a noncovalent complex with high molecular weight kininogen (HK) 1 (6) and can be activated by three biologically relevant proteases: factor XIIa, factor XIa, and thrombin (1,7,8). The primary structure of factor XI has been determined (9, 10), including the identification of four tandem repeat sequences, designated Apple (A1, A2, A3, and A4) domains in the heavy chain region of factor XI. Binding sites (11-15) for thrombin, the Kringle 2 domain of prothrombin, and HK are present in the A1 domain, whereas both heparin-and plateletbinding sites exist within the A3 domain (16 -18) and a binding site for factor XIIa is located in the A4 domain (19).Factor XI can participate in the contact phase of blood coagulation in a reaction that requires the presence of anionic surfaces for optimal activation in vitro by factor XIIa (6,[20][21][22]. However, deficiencies in factor XII, prekallikrein, and HK are not associated with hemostatic abnormalities, whereas a deficiency in factor XI produces abnormal bleeding complications (23-25). Therefore, it has been suggested that the physiologically relevant pathway for factor XI activation might constitute feedback activation either by thrombin or by factor XIa (7,8,15). All three proteases cleave each monomer of factor XI at the Arg 369 -Ile 370 bond generating the new amino-terminal sequence (IVGG) of the cataly...

mentioning

confidence: 99%

Thrombin-mediated Feedback Activation of Factor XI on the Activated Platelet Surface Is Preferred over Contact Activation by Factor XIIa or Factor XIa

Baglia¹,

Walsh²

2000

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“…The key residues identified were further characterized by dose-response studies, and their importance was confirmed by FXI activation in the presence of activated platelets. Although dextran sulfate is commonly employed in studies of FXI activation by thrombin (8,9,20), it should be noted that activated platelets and dextran sulfate may promote thrombincatalyzed FXI activation by similar but non-identical mechanisms because the binding site for the activated platelet surface has been localized to the A3 domain of FXI (14,23), whereas binding to dextran sulfate is mediated through the A1 domain (24).…”

Section: Discussionmentioning

confidence: 99%

“…At least one thrombin-binding site on GPIb␣ has been localized to residues 269 -287 (54) and is mediated by electrostatic interactions and perhaps direct contacts. FXI binding to GPIb␣ through its A3 domain is achieved when one of its homodimers is first complexed with HMWK or prothrombin (14,23,24). Once bound to the GPIb-IX-V complex by one of its monomers, FXI can interact with an adjacent thrombin molecule through the A1 domain of its other, free monomer (12).…”

Section: I-ppack-thrombin To Glycocalicin In the Presence Of Various mentioning

confidence: 99%

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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

Yun

Baglia²,

Myles

et al. 2003

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Activation of factor XI (FXI) by thrombin on stimulated platelets plays a physiological role in hemostasis, providing additional thrombin generation required in cases of severe hemostatic challenge. Using a collection of 53 thrombin mutants, we identified 16 mutants with <50% of the wild-type thrombin FXI-activating activity in the presence of dextran sulfate. These mutants mapped to anion-binding exosite (ABE) I, ABE-II, the Na ؉ -binding site, and the 50-insertion loop. Only the ABE-II mutants showed reduced binding to dextran sulfate-linked agarose. Selected thrombin mutants in ABE-I (R68A, R70A, and R73A), ABE-II (R98A, R245A, and K248A), the 50-insertion loop (W50A), and the Na ؉ -binding site (E229A and R233A) with <10% of the wildtype activity also showed a markedly reduced ability to activate FXI in the presence of stimulated platelets. The ABE-I, 50-insertion loop, and Na ؉ -binding site mutants had impaired binding to FXI, but normal binding to glycocalicin, the soluble form of glycoprotein Ib␣ (GPIb␣). In contrast, the ABE-II mutants were defective in binding to glycocalicin, but displayed normal binding to FXI. Our data support a quaternary complex model of thrombin activation of FXI on stimulated platelets. Thrombin bound to one GPIb␣ molecule, via ABE-II on its posterior surface, is properly oriented for its activation of FXI bound to a neighboring GPI␣ molecule, via ABE-I on its anterior surface. GPIb␣ plays a critical role in the co-localization of thrombin and FXI and the resultant efficient activation of FXI.

“…Their studies indicate that FXI apple domain A1 binds to HK via a sequence segment (41) that mirrors the corresponding HK attachment site of PPK A1 (15). In addition FXI apple A1 binds to thrombin (42), an important endogenous activator of FXI in blood coagulation (43,44). FXI domain A3 has been shown to bind to platelets (45) and heparin (46), and domain A4 to FXIIa (47).…”

Section: Fig 5 Transfer Of Ppk Apple Domain A2 Increases Hk Bindingmentioning

confidence: 99%

Mapping of the Discontinuous H-kininogen Binding Site of Plasma Prekallikrein

Renné¹,

Dedio²,

Meijers³

et al. 1999

Plasma prekallikrein, a zymogen of the contact phase system, circulates in plasma as heterodimeric complex with H-kininogen. The binding is mediated by the prekallikrein heavy chain consisting of four apple domains, A1 to A4, to which H-kininogen binds with high specificity and affinity (K D ‫؍‬ 1.2 ؋ 10 ؊8 M). Previous work had demonstrated that a discontinuous kininogen-binding site is formed by a proximal part located in A1, a distal part exposed by A4, and other yet unidentified portion(s) of the kallikrein heavy chain. To detect relevant binding segment(s) we recombinantly expressed single apple domains and found a rank order of binding affinity for kininogen of A2 > A4 Ϸ A1 > A3. Removal of single apple domains in prekallikrein deletion mutants reduced kininogen binding by 21 (A1), 64 (A2), and 24% (A4), respectively, whereas deletion of A3 was without effect. Transposition of homologous A2 domain from prekallikrein to factor XI conferred high-affinity kininogen binding from the former to the latter. The principal role of A2 for H-kininogen docking to the prekallikrein heavy chain was further substantiated by the finding that cleavage of a single peptide bond in A2 drastically diminished the H-kininogen binding affinity. Furthermore, the epitope of monoclonal antibody PKH6 which blocks kallikrein-kininogen complex formation with an IC 50 of 8 nM mapped to the center portion of domain A2. Our data indicate that domain A2 and two flanking sequence segments of A1 and A4 form a discontinuous binding platform for H-kininogen on the prekallikrein heavy chain. Domain-specific antibodies directed to these critical sites efficiently interfered with contact phase-induced bradykinin release from H-kininogen.