Kinetics of the Factor XIa catalyzed activation of human blood coagulation Factor IX.

Sun

et al. 2001

Blood

Human coagulation factor XI (FXI) is a plasma serine protease composed of 2 identical 80-kd polypeptides connected by a disulfide bond. This dimeric structure is unique among blood coagulation enzymes. The hypothesis was tested that dimeric conformation is required for normal FXI function by generating a monomeric version of FXI (FXI/PKA4) and comparing it to wild-type FXI in assays requiring factor IX activation by activated FXI (FXIa). FXI/PKA4 was made by replacing the FXI A4 domain with the A4 domain from prekallikrein (PK). A dimeric version of FXI/PKA4 (FXI/PKA4-Gly326) was prepared as a control. Activated FXI/PKA4 and FXI/PKA4-Gly326 activate factor IX with kinetic parameters similar to those of FXIa. In kaolin-triggered plasma clotting assays containing purified phospholipid, FXI/PKA4 and FXI/PKA4-Gly326 have coagulant activity similar to FXI. IntroductionA paradigm in the field of blood coagulation is that the zymogen of a plasma protease is activated by limited proteolysis on a phospholipid surface, in the presence of a protein cofactor and divalent cations. [1][2][3][4][5] In vivo, appropriate phospholipid surfaces are provided by activated platelets and cell membranes of damaged tissues. Formation of surface-bound protease-substrate complexes increases the rate of zymogen activation, concentrates procoagulant reactions to sites of vessel injury, and minimizes spread of thrombogenic proteases beyond wound sites. An apparent exception to this model is the activation of factor IX by activated factor XI (FXIa). In in vitro coagulation systems, such as the activated partial thromboplastin time (aPTT) assay, activation of factor IX by FXIa requires calcium ions. [6][7][8] However, phospholipids known to promote activation of factor X and prothrombin, such as brain cephalin, have little effect on the reaction. 9,10 Furthermore, a candidate protein cofactor to promote surface assembly of a FXIa-based factor IX-activating complex has not been identified. These observations suggest, counterintuitively, that factor IX activation by FXIa proceeds to a significant extent in the fluid phase of blood.Zymogen factor XI (FXI) and FXIa do bind to activated platelets in a process that is saturable and reversible and that requires the protein cofactor high molecular weight kininogen (HK) and zinc ions. 11,12 Evidence strongly suggests that the platelet surface is a physiologic environment for reactions involving FXI. When bound to activated platelets, FXI activation by the proteases thrombin, factor XIIa, and FXIa is greatly accelerated. 13,14 Furthermore, prothrombin may substitute for HK as a cofactor for FXI/FXIa binding to platelets, 13,15 providing an explanation for the lack of excessive bleeding in patients congenitally deficient in HK. 16 Given these data and the observation that factor IX binds to activated platelets, 17 it is likely that the surface of activated platelets is a physiologic environment for activation of factor IX by FXIa.The FXI polypeptide is composed of an N-terminal noncatalytic heavy chain ...

Section: Factor IX Activation By Fxia In the Presence Of Plateletsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Model for a factor IX activation complex on blood platelets: dimeric conformation of factor XIa is essential

Sun

et al. 2001

Blood

“…The importance of the FIX Gla domain to FIX activation by factor XIa is less certain because the reaction appears to involve a mechanism distinctly different from typical vitamin K-dependent protease-substrate interactions. Although calcium is required (1,21,22), phospholipid has little influence on the process (1,11,23). Indeed, factor XIa lacks a Gla domain, suggesting that it may interact poorly with phospholipid (3,24,25).…”

mentioning

confidence: 99%

The Factor IX γ-Carboxyglutamic Acid (Gla) Domain Is Involved in Interactions between Factor IX and Factor XIa

Aktimur

Gabriel

Journal of Biological Chemistry

et al. 2003

During hemostasis, factor IX is activated to factor IXa␤ by factor VIIa and factor XIa. The glutamic acidrich ␥-carboxyglutamic acid (Gla) domain of factor IX is involved in phospholipid binding and is required for activation by factor VIIa. In contrast, activation by factor XIa is not phospholipid-dependent, raising questions about the importance of the Gla for this reaction. We examined binding of factors IX and IXa␤ to factor XIa by surface plasmon resonance. Plasma factors IX and IXa␤ bind to factor XIa with K d values of 120 ؎ 11 nM and 110 ؎ 8 nM, respectively. Recombinant factor IX bound to factor XIa with a K d of 107 nM, whereas factor IX with a factor VII Gla domain (rFIX/VII-Gla) and factor IX expressed in the presence of warfarin (rFIX-des␥) did not bind. An anti-factor IX Gla monoclonal antibody was a potent inhibitor of factor IX binding to factor XIa (K i 34 nM) and activation by factor XIa (K i 33 nM). In activated partial thromboplastin time clotting assays, the specific activities of plasma and recombinant factor IX were comparable (200 and 150 units/mg), whereas rFIX/VIIGla activity was low (<2 units/mg). In contrast, recombinant factor IXa␤ and activated rFIX/VIIa-Gla had similar activities (80 and 60% of plasma factor IXa␤), indicating that both proteases activate factor X and that the poor activity of zymogen rFIX/VII-Gla was caused by a specific defect in activation by factor XIa. The data demonstrate that factor XIa binds with comparable affinity to factors IX and IXa␤ and that the interactions are dependent on the factor IX Gla domain.

“…Activated factor XII (factor XIIa), thrombin, and factor XIa have all been shown to activate factor XI in vitro; however, the physiologic mechanisms responsible for conversion of factor XI to the active protease are not certain (2,4,5). Regardless of the mechanism of activation, it is generally agreed that factor XIa contributes to coagulation by activating factor IX by limited proteolysis in the presence of calcium ions (6,7). The factor XIa molecule is comprised of a pair of 35-kDa trypsin-like catalytic light chains and two 45-kDa non-catalytic heavy chains (8).…”

mentioning

confidence: 99%

Identification of a Factor IX Binding Site on the Third Apple Domain of Activated Factor XI

Sun¹,

Journal of Biological Chemistry

1996

105

144

Activated factor XI (factor XIa) participates in blood coagulation by activating factor IX. Previous work has demonstrated that a binding site for factor IX is present on the noncatalytic heavy chain of factor XIa (Sinha, D., Seaman, F. S., and Walsh, P. N. (1987) Biochemistry 26, 3768 -3775). Recombinant factor XI proteins were expressed in which each of the four apple domains of the heavy chain (designated A1 through A4) were individually replaced with the corresponding domain from the homologous but functionally distinct protease prekallikrein (PK). To identify the site of factor IX binding, the chimeric proteins were activated with factor XIIa and tested for their capacity to activate factor IX in plasma coagulation and purified protein assays. The chimera with the substitution in the third apple domain (factor XI/PKA3) had <1% of the coagulant activity of wild type factor XIa in a plasma coagulation assay, whereas the chimeras with substitutions in A1, A2, and A4 demonstrated significant activity (68 -140% of wild type activity). The K m for activation of factor IX by factor XIa/ PKA3 (12.7 M) is more than 30-fold higher than the K m for activation by wild type factor XIa or the other factor XI/PK chimeras (0.11-0.37 M). Two monoclonal antibodies (2A12 and 11AE) that recognize epitopes on the factor XI A3 domain were potent inhibitors of factor IX activation by factor XIa, whereas antibodies against the A2 (1A6) and A4 (3G4) domains were poor inhibitors. The data indicate that a binding site for factor IX is present on the third apple domain of factor XIa.Factor XI is the zymogen of a plasma serine protease which is a critical component of the intrinsic pathway of blood coagulation (1). Human factor XI is comprised of two identical 80-kDa polypeptides connected by a disulfide bond (2, 3). A single proteolytic cleavage in each polypeptide converts the zymogen to the active form of the enzyme, factor XIa. Activated factor XII (factor XIIa), thrombin, and factor XIa have all been shown to activate factor XI in vitro; however, the physiologic mechanisms responsible for conversion of factor XI to the active protease are not certain (2, 4, 5). Regardless of the mechanism of activation, it is generally agreed that factor XIa contributes to coagulation by activating factor IX by limited proteolysis in the presence of calcium ions (6, 7). The factor XIa molecule is comprised of a pair of 35-kDa trypsin-like catalytic light chains and two 45-kDa non-catalytic heavy chains (8). The heavy chain appears to be required for factor XI specific activities including the activation of factor IX (9, 10). Indeed, isolated factor XIa light chain has less than 5% of the factor IX activating activity of intact factor XIa, 1 even though both molecules cleave a chromogenic substrate similarly (9, 11).The human factor XI heavy chain is comprised of four apple domains repeated in tandem (designated A1, A2, A3, and A4), a feature shared with the structurally homologous but functionally distinct plasma protease prekallikrein (PK) (12). ...