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

Abstract

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 ...

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

“…In a current model of FXI activation, the circulating FXI dimer is complexed with either HMWK (17)(18)(19) or prothrombin (18,20), both of which bind to the A1 domain of FXI (21).…”

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

“…However, recent work suggests that the physiologic environment for this reaction may be the surface of activated platelets (46,49,50). In this environment, the dimeric structure of factor XI is necessary for normal FIX activation, possibly because one polypeptide of the dimer is required for binding to the platelet, whereas the other interacts with FIX (46,49,50).…”

Characterization of the H-kininogen-binding Site on Factor XI

“…Several recent studies have shed light on this conundrum. It has been shown that HK facilitates FXI binding to the surface of activated platelets, where it is rapidly activated to FXIa (18,20,43). Prothrombin may serve as a substitute for HK in this role, providing a plausible explanation as to why HK-deficient humans do not bleed (19).…”

“…These findings, in conjunction with the observation that P3 does not contribute to HK binding to PPK (25), support the premise that F3 is not involved in HK binding to FXI. Because F3 appears to be important for binding to other components of the coagulation mechanism, such as factor IX (35,45), platelet membranes (51), and heparin (52), it makes sense that it would not be involved in the interaction with HK (43). Taken together, our data are consistent with a model of a discontinuous HK-binding site jointly formed by F2, F1, and F4 of FXI, where domain F2 harbors the core binding sequence and F1 and F4 contribute directly and/or indirectly to this high affinity binding site.…”