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).
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...
Fresh platelet preparations are utilized to treat a wide variety of wounds, although storage limitations and mixed results have hampered their clinical use. We hypothesized that concentrated lyophilized and reconstituted platelet preparations, preserved with trehalose, maintain and possibly enhance fresh platelets' ability to improve wound healing. We studied the ability of a single dose of trehalose lyophilized and reconstituted platelets to enhance wound healing when topically applied on full-thickness wounds in the genetically diabetic mouse. We compared these results with the application of multiple doses of fresh platelet preparations and trehalose lyophilized and reconstituted platelets as well as multiple doses of vascular endothelial growth factor (VEGF) and wounds left untreated. Trehalose lyophilized and reconstituted platelets, in single and multiple applications, multiple applications of fresh platelets and multiple applications of VEGF increased granulation tissue deposition, vascularity, and proliferation when compared with untreated wounds, as assessed by histology and immunohistochemistry. Wounds treated with multiple doses of VEGF and a single dose of freeze-dried platelets reached 90% closure faster than wounds left untreated. A single administration of trehalose lyophilized and reconstituted platelet preparations enhanced diabetic wound healing, therefore representing a promising strategy for the treatment of nonhealing wounds.
Previously we defined binding sites for high molecular weight kininogen (HK) and thrombin in the Apple 1 (A1) domain of factor XI (FXI). Since prothrombin (and Ca(2+)) can bind FXI and can substitute for HK (and Zn(2+)) as a cofactor for FXI binding to platelets, we have attempted to identify a prothrombin-binding site in FXI. The recombinant A1 domain (rA1, Glu(1)-Ser(90)) inhibited the saturable, specific and reversible binding of prothrombin to FXI, whereas neither the rA2 domain (Ser(90)-Ala(181)), rA3 domain (Ala(181)-Val(271)), nor rA4 domain (Phe(272)-Glu(361)) inhibited prothrombin binding to FXI. Kinetic binding studies using surface plasmon resonance showed binding of FXI (K(d) approximately 71 nm) and the rA1 domain (K(d) approximately 239 nm) but not rA2, rA3, or rA4 to immobilized prothrombin. Reciprocal binding studies revealed that synthetic peptides (encompassing residues Ala(45)-Ser(86)) containing both HK- and thrombin-binding sites, inhibit (125)I-rA1 (Glu(1)-Ser(90)) binding to prothrombin, (125)I-prothrombin binding to FXI, and (125)I-prothrombin fragment 2 (Ser(156)-Arg(271)) binding to FXI. However, homologous prekallikrein-derived peptides (encompassing Pro(45)-Gly(86)) did not inhibit FXI rA1 binding to prothrombin. The peptides Ala(45)-Arg(54), Phe(56)-Val(71), and Asp(72)-Ser(86), derived from sequences of the A1 domain of FXI, acted synergistically to inhibit (125)I-rA1 binding to prothrombin. Mutant rA1 peptides (V64A and I77A), which did not inhibit FXI binding to HK, retained full capacity to inhibit rA1 domain binding to prothrombin, and mutant rA1 peptides Ala(45)-Ala(54) (D51A) and Val(59)-Arg(70) (E66A), which did not inhibit FXI binding to thrombin, retained full capacity to inhibit rA1 domain binding to prothrombin. Thus, these experiments demonstrate that a prothrombin binding site exists in the A1 domain of FXI spanning residues Ala(45)-Ser(86) that is contiguous with but separate and distinct from the HK- and thrombin-binding sites and that this interaction occurs through the kringle II domain of prothrombin.
Variants of factor XI containing Gln226 to Arg (Q226 to R) and Ser248 to Asn (S248 to N) substitutions were first identified in an African American family with a history of excessive bleeding. The substitutions have recently been identified in unrelated individuals, suggesting they are relatively common. Both amino acids are located in the third apple domain of factor XI, an area implicated in binding interactions with factor IX and activated platelets. Recombinant factor XI-R226 and factor XI-N248 were compared with wildtype factor XI in assays for factor IX activation or platelet binding. Factor XI-R226 activates factor IX with a MichaelisMenten constant (K m ) about 5-fold greater than wild-type protein. The catalytic efficiency of factor IX activation is similar to wild-type protein, however, due to an increase in the turnover number (k cat ) for the reaction. Iodinated factor XI-N248 binds to activated platelets with a dissociation constant (K d ) more than 5-fold higher than wild-type protein (55 nM and 10 nM, respectively). Activation of factor XI-N248 by thrombin in the presence of activated platelets is slower and does not progress to the same extent as activation of the wild-type protein under similar conditions. Factor XI-N248 activates factor IX normally in a purified protein system and has relatively normal activity in activated partial thromboplastin time (aPTT) assays. Factor XI-N248 is the first factor XI variant described with a clear functional difference compared with wild-type protein. Importantly, the defect in platelet binding IntroductionCoagulation factor XI is the zymogen of a plasma protease that contributes to hemostasis by activating factor IX. 1 In congenital factor XI deficiency in humans, low plasma activity is almost always associated with a comparable loss of antigen (cross-reactive material-negative [CRM Ϫ ] deficiency). 2,3 Few factor XI-deficient patients with low activity and disproportionately high circulating levels of factor XI antigen (CRM ϩ deficiency) have been reported, 4-6 and the genetic abnormalities in these individuals have not been determined. This distinguishes factor XI deficiency from hemophilias A and B (deficiency of factor VIII or IX, respectively) and von Willebrand disease, where many circulating dysfunctional variants have been described. 7 Another peculiarity of factor XI deficiency that distinguishes it from hemophilia is the poor correlation between plasma factor XI activity and bleeding symptoms. 6,8,9 It is likely that several factors are responsible for this phenomenon. One possibility is that commonly used in vitro tests for factor XI activity do not accurately measure properties of the protein most important for in vivo hemostasis.Previously, we reported 2 amino acid substitutions in factor XI (Q226R and S248N), identified in an African American with a history of excessive bleeding and a modest deficiency in plasma factor XI activity. 10 This patient is a compound heterozygote, with the amino acid substitutions residing on separate alleles. The ...
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