Since heparin potentiates activated factor XI (FXIa) inhibition by protease nexin-2 by providing a template to which both proteins bind (Zhang, Y., Scandura, J. M., Van Factor XI (FXI) 1 is a zymogen that circulates in plasma in a non-covalent complex with high molecular weight kininogen (HMWK) (1, 2) and participates in the contact phase of blood coagulation (3). The active enzyme, activated factor XI (FXIa), is a trypsin-like serine protease that is generated when FXI is cleaved by FXIIa (3), thrombin, or FXIa (4, 5) at an internal Arg 369 -Ile 370 bond (2). Upon activation, FXIa is capable of converting FIX into its active form, FIXa (2, 6). FIXa, in the presence of FVIII and platelets, can activate FX (7,8), resulting in the generation of thrombin and, subsequently, a fibrin clot.The structure of FXI is unique among the plasma coagulation enzymes (9, 10), since it exists as a homodimer consisting of two subunits each of which contains 607 amino acids. During proteolytic activation, each of these subunits is cleaved to generate a heavy chain of 369 amino acids and a light chain or catalytic domain of 238 amino acids. The heavy chain of FXI provides binding surfaces for several blood coagulation proteins and is organized into four tandem repeat Apple domains designated A1, A2, A3, and A4 (9, 10). Each of these four domains contains 90 to 91 amino acids, the sequences of which are 23-34% identical. The A1 domain provides a binding site for HMWK (11), a protein that promotes the activation of FXI by FXIIa (12). Both the A2 domain (13) and the A3 domain (14) have been implicated in the binding of FXIa to its substrate, FIX. The A4 domain contains Cys 321 that is responsible for the dimerization of FXI (15,16). In addition, the A4 domain binds to FXIIa (10).Examination of the cellular site of FXI activation by FXIIa has resulted in evidence that platelets promote the proteolytic activation of FXI by FXIIa (17) and that FXI, in the presence of zinc ions, calcium ions, and HMWK, binds to activated platelets in a specific, reversible, and saturable manner with a dissociation constant (K d ) of ϳ10 nM (18). Previous work from this laboratory showed that a synthetic peptide (Asn 235 -Arg 266 ) from the A3 domain inhibited 125 I-FXI binding to platelets (inhibition constant (K i ) ϭ 10 nM) in the presence of HMWK, ZnCl 2 , and CaCl 2 (19,20). Hence, these studies indicate that the A3 domain mediates FXI binding to platelets.A second possible site of FXI activation is the endothelial cell surface. Berrettini et al. (21) demonstrated that FXI binds to endothelial cells in the presence of HMWK, CaCl 2 , and ZnCl 2 with a K d(app) ϳ4.5 nM, whereas FXIa binds with higher affinity (K d(app) ϳ1.5 nM). The binding of FXI to the endothelial cell surface may be of functional significance in localizing coagulation to the site of vascular injury since activation of FXI can occur on the endothelial cell surface (21), where proteoglycans such as heparan sulfate are known to be present. Both Naito and Fujikawa (4) and Gailani an...

Tumor necrosis factor-like weak inducer of apoptosis (TWEAK) is a member of the tumor necrosis factor superfamily of structurally related cytokines. TWEAK acts on responsive cells via binding to a cell surface receptor named Fn14. Recent studies have demonstrated that TWEAK can stimulate numerous cellular responses including cell proliferation, migration, and proinflammatory molecule production. It has also been reported that TWEAK can stimulate blood vessel formation in the rat cornea angiogenesis assay, but it is presently unknown whether this cytokine could play a role in the pathological angiogenesis associated with human diseases such as cancer, rheumatoid arthritis, and diabetic retinopathy. In the present study we investigated whether TWEAK was expressed in human tumors and whether it could promote tumor growth and angiogenesis in vivo. TWEAK mRNA expression was detected in many tumor types by cDNA array hybridization analysis, and TWEAK protein expression was confirmed in human colon cancer tissue by immunohistochemistry. As an initial approach to address whether TWEAK might act as a tumor angiogenesis factor, we established several human embryonic kidney cell lines that constitutively secrete a soluble TWEAK protein and examined their growth properties in vitro and in vivo. We found that although TWEAK-overexpressing cells do not have a growth advantage in vitro, they form larger and more highly vascularized tumors in athymic mice when compared with control, vector-transfected cells. This result suggests that the TWEAK-Fn14 signaling system may be a potential regulator of human tumorigenesis.

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 ...

To localize the platelet binding site on factor XI, rationally designed, conformationally constrained synthetic peptides were used to compete with [(125)I]factor XI binding to activated platelets. The major platelet binding energy resided within the sequence of amino acids T(249)-F(260). Homology scanning, using prekallikrein amino acid substitutions within the synthetic peptide T(249)-F(260), identified a major role for R(250) in platelet binding. Inhibition of [(125)I]factor XI binding to activated platelets by the recombinant Apple 3 domain of factor XI and inhibition by unlabeled factor XI were identical, whereas the recombinant Apple 3 domain of prekallikrein had little effect. A "gain-of-function" chimera in which the C-terminal amino acid sequence of the Apple 3 domain of prekallikrein was replaced with that of factor XI was as effective as the recombinant Apple 3 domain of factor XI and unlabeled factor XI in inhibiting [(125)I]factor XI binding to activated platelets. Alanine scanning mutagenic analysis of the recombinant Apple 3 domain of factor XI indicated that amino acids R(250), K(255), F(260), and Q(263) (but not K(252) or K(253)) are important for platelet binding. Thus, the binding energy mediating the interaction of factor XI with platelets is contained within the C-terminal amino acid sequence of the Apple 3 domain (T(249)-V(271)) and is mediated in part by amino acid residues R(250), K(255), F(260), and Q(263).