Suppressing protein Z-dependent inhibition of factor Xa improves coagulation in hemophilia A.

Essentials Protein Z (PZ) catalyzes PZ-dependent proteinase inhibitor (ZPI) inactivation of factor (F)Xa. Gene-deletion of PZ or ZPI improves coagulation in hemophilia (FVIII knockout) mice. A PZ blocking antibody enhances thrombin generation in human hemophilia plasma. Suppression of the PZ/ZPI pathway may ameliorate the phenotype of severe hemophilia. SUMMARY: Background Hemostasis requires a balance between procoagulant and anticoagulant factors. Hemophiliacs bleed because of a procoagulant deficiency. Targeted reduction in the activity of endogenous anticoagulant pathways is currently being investigated as a means of improving hemostasis in hemophilia. Protein Z (PZ) is a cofactor that serves as a catalyst for PZ-dependent protease inhibitor (ZPI) inactivation of activated factor X at phospholipid surfaces. Objectives To evaluate the effects of PZ or ZPI gene deletion in hemophilic mice, and of blocking PZ in human hemophilic plasma. Methods A tail vein rebleeding assay (TVRB) was developed on the basis of the serial disruption of clots forming over a period of 15 min following tail vein laceration in an anesthetized mouse. Wild-type (WT)/FVIII knockout FVIIIKO, PZ knockout PZKO/FVIIIKO and ZPI knockout ZPIKO/FVIIIKO mice were evaluated in this model, and their plasmas were tested in thrombin generation assays. A mAb against PZ was evaluated in human hemophilic plasma thrombin generation assays. Results The numbers of clot disruptions (mean ± standard error of the mean) in the TVRB were: 4.0 ± 0.9 for WT/FVIIIKO mice; 23.8 ± 1.1 for WT/FVIIIKO mice supplemented with 100% FVIII; 15.2 ± 1.1 for PZKO/FVIIIKO mice; and 14.7 ± 1.2 for ZPIKO/FVIIIKO mice. Thrombin generation in PZKO/FVIIIKO and ZPIKO/FVIIIKO mouse plasmas was similar to that in FVIIIKO plasma supplemented with ~ 15% recombinant FVIII. A mAb against PZ added to human hemophilic plasma enhanced thrombin generation to an extent similar to the addition of ~ 15% FVIII. Conclusions Blockade of the PZ/ZPI system may be sufficient to ameliorate the phenotype of severe hemophilia.

Re-evaluation of mouse tissue factor pathway inhibitor and comparison of mouse and human tissue factor pathway inhibitor physiology.

Essentials Mouse models are often used to define roles of tissue factor pathway inhibitor (TFPI) in man. TFPI isoform-specific KOs reveal unexpected differences between mouse and human TFPI physiology. Mouse plasma contains 20 times more TFPI than man, derived from TFPIγ, a form not found in man. TFPIγ null mice, expressing only TFPI isoforms α and ß, may better reflect the human situation. SUMMARY: Background Mouse models can provide insight into the pathophysiology of human thrombosis and hemostasis. Tissue factor pathway inhibitor (TFPI) regulates coagulation through protein S (PS)-enhanced factor (F) Xa inhibition and FXa-dependent inhibition of FVIIa/tissue factor (TF) activity. TFPI is expressed as isoforms α and ß in man, and α, ß and γ in the mouse. Objective Assess the reliability of extending TFPI-related studies in mice to humans. Method Compare mouse and human TFPI physiology using a variety of methods. Results Mouse TFPI and human TFPI are similar in regard to: (i) the mechanisms for FVIIa/TF and FXa inhibition; (ii) TFPIα is a soluble form and TFPIß is glycosyl phosphatidyl inositol (GPI) membrane anchored; (iii) the predominant circulating form of TFPI in plasma is lipoprotein-associated; (iv) low levels of TFPIα circulate in plasma and increase following heparin treatment; and (v) TFPIα is the isoform in platelets. They differ in that: (i) mouse TFPI circulates at a ~20-fold higher concentration; (ii) mouse lines with isolated isoform deletions show this circulating mouse TFPI is derived from TFPIγ; (iii) sequences homologous to the mouse TFPIγ exon are present in many species, including man, but in primates are unfavorable for splicing; and (iv) tandem mass spectrometry (MS/MS) detects sequences for TFPI isoforms α and ß in human plasma and α and γ in mouse plasma. Conclusion To dissect the pathophysiological roles of human TFPIα and TFPIß, studies in TFPIγ null mice, expressing only α and ß, only α or only ß should better reflect the human situation.

The role of tissue factor/factor VIIa in the pathophysiology of acute thrombotic formation.

Tissue factor (TF) is the essential cofactor for the coagulation protease factor VIIa (FVIIa), initiating the coagulation cascade. The role of TF in thrombotic diseases is becoming increasingly evident. Recent findings suggest that inhibition of TF/FVIIa activity could be important in the prevention of clinical sequelae associated with plaque rupture or vessel damage that exposes TF to blood. Furthermore, selective inhibitors of TF/FVIIa may be associated with less bleeding risk than other antithrombotic agents. Several TF/FVIIa inhibitors are in development, including the protein-based inhibitors (such as NAPc2, Corsevin M, FFR-FVIIa, and Tifacogin). Research into the development of small molecule inhibitors is on-going, but is at a less advanced stage.

Structural requirements for TFPI-mediated inhibition of neointimal thickening after balloon injury in the rat.

The intimal thickening that follows vascular injury is inhibited by periprocedural tissue factor pathway inhibitor (TFPI) treatment in animal models. TFPI is a multivalent Kunitz-type protease inhibitor that inhibits factor Xa via its second Kunitz domain and the factor VIIa/tissue factor (TF) complex via its first Kunitz domain. The basic C-terminus of TFPI is required for the binding of TFPI to cell surfaces and cell-bound TFPI mediates the internalization and degradation of factor X and the down regulation of surface factor VIIa/TF activity. The C-terminus of TFPI is also required for its reported direct inhibition of smooth muscle cell proliferation in vitro. To examine the structural requirements for the inhibition of neointimal formation by TFPI, several TFPI-related proteins were tested in the rat carotid angioplasty model: 1) XK(1), a hybrid protein containing the N-terminal portion of factor X and the first Kunitz domain of TFPI that directly inhibits factor VIIa/TF; 2) TFPI(WT), the full-length TFPI molecule that inhibits factor Xa and factor VIIa/TF and binds cell surfaces; 3) TFPI(K36I), an altered form of TFPI that inhibits factor Xa, but not factor VIIa/TF, and binds cell surfaces; 4) TFPI(13-161), a truncated form of TFPI that inhibits factor VIIa/TF but interacts with factor Xa poorly and does not bind to cell surfaces. Seven day infusions of XK(1), TFPI(WT), and high levels of TFPI(K36I) begun the day before balloon-induced vascular injury produced a significant reduction in the intimal hyperplasia measured 28 days after angioplasty. The infusion of high concentrations of TFPI(13-161) was ineffective in this model. These in vivo results directly mirror the ability of each TFPI-related protein to inhibit tissue thromboplastin-induced coagulation in rat plasma: XK(1) approximately TFPI(WT)>TFPI(K36I)>>TFPI(13-161). The studies confirm the important role of TF-mediated coagulation in the smooth muscle proliferation and neointimal thickening that follows vascular injury and suggest that the anticoagulant effect alone of TFPI and TFPI-related proteins is sufficient to explain their therapeutic action.

Inhibitory activity of unsaturated fatty acids and anacardic acids toward soluble tissue factor-factor VIIa complex.

Five compounds, which inhibited the amidolytic activity of soluble tissue factor/activated factor VII complex (sTF/VIIa), were isolated from two traditional Chinese medicinal plants commonly used in the treatment of cardiovascular and cerebrovascular diseases. The active compounds were found to be linolenic, linoleic, and oleic acids from roots of Salvia miltiorrhiza; and two anacardic acids, 6-(8'Z-pentadecenyl)- and 6-(10'Z-heptadecenyl)-salicylic acids, from leaves of Ginkgo biloba. The IC50 values were in the range 30-80 micromol/L. Palmitic acid, isolated from roots of Salvia miltiorrhiza, and 2-[(3',7',11',15'-tetramethyl)-2'E,6'E,10'E, 14'E-hexadecatetraenyl]-1,4-hydroquinone, isolated from the marine sponge Adocia viola, did not inhibit sTF/VIIa. Further expansion of the structure-activity relationship to include anacardic acids, 6-(8'Z,11'Z-heptadecadienyl)- and 6-(8'Z, 11'Z, 14'Z-heptadecatrienyl)-salicylic acids from leaves of Anacardium spondias, and other fatty acids demonstrated that at least one cis double bond was essential for inhibitory activity, and that fatty acids containing two or three cis double bonds were optimal. Evidence from preincubation studies implied that these fatty acids may exert their effect by binding to VIIa and consequently preventing binding of sTF to VIIa.

Long-term inhibition of murine experimental autoimmune encephalomyelitis using CTLA-4-Fc supports a key role for CD28 costimulation.

T cell activation involves not only recognition of antigen presented by the MHC, but also nonspecific interactions termed "costimulation." The costimulatory molecules B7-1 and B7-2 are ligands on antigen-presenting cells for the CD28 and CTLA-4 receptors on T cells. Previously, a fusion protein consisting of human CTLA-4 linked to human Fc was shown to bind B7-1 and B7-2 with high avidity and to prevent specific T cell activation. Here we investigated the effects of a recombinant fusion protein consisting of the extracellular domain of human CTLA-4 bound to mouse IgG2a Fc (CTLA-4-Fc) upon experimental autoimmune encephalomyelitis, a T cell-mediated disease that serves as a model for multiple sclerosis. CTLA-4-Fc prevented experimental autoimmune encephalomyelitis in 26 of 28 CTLA-4-Fc-treated mice (median maximum score 0), whereas 28 of 30 mice treated with control mouse IgG2a developed disease (median maximum score 2.75). Less inflammation and virtually no demyelination or axonal loss occurred in CTLA-4-Fc-treated compared with control-treated mice. Activated splenocytes from CTLA-4-Fc-treated mice were able to transfer disease adoptively to naive recipients. These results indicate a key role for the B7/CD28 system in the development of actively induced murine experimental autoimmune encephalomyelitis, suggesting an area of investigation with therapeutic potential for multiple sclerosis.

Complementary DNA sequencing of canine tissue factor pathway inhibitor reveals a unique nanomeric repetitive sequence between the second and third Kunitz domains.

Tissue factor pathway inhibitor (TFPI) is a factor Xa-dependent inhibitor of the factor VIIa-tissue factor complex of blood coagulation. The primary amino acid sequence of canine TFPI has been deduced from cDNA sequences obtained using the techniques of reverse transcription followed by amplification using PCR and conventional screening of a canine endothelial cell cDNA library. The open reading frame for canine TFPI encodes a signal peptide of 28 amino acids followed by a 40.7 kDa protein of 368 amino acids. Similar to human, rat and rabbit TFPI, canine TFPI contains a negatively-charged cluster of amino acids at its mature amino-terminus, followed by three Kunitz-type proteinase inhibitory domains and a cluster of positively-charged amino acids near its carboxy-terminus. In contrast to other TFPIs, following its second Kunitz-type proteinase inhibitory domain canine TFPI contains an additional amino acid insert which includes a nanomeric peptide-sequence repeated six times. Recombinant canine TFPI was expressed in both bacterial- and insect cell-expression systems for functional analysis and the generation of antibodies. The recombinant canine TFPI inhibits tissue factor-induced coagulation in an in vitro canine system. Immunoprecipitation of TFPI from canine plasma, followed by Western-blot analysis, tentatively identifies canine TFPI as an 80,000 kDa protein. Anti-peptide antibodies raised to the nanomeric peptide repeat immunoprecipitate an identical, cross-reactive, 80,000 kDa protein.

Structure of the human lipoprotein-associated coagulation inhibitor gene. Intro/exon gene organization and localization of the gene to chromosome 2.

Lipoprotein-associated coagulation inhibitor (LACI) is a multivalent, Kunitz-type proteinase inhibitor which appears to play an important role in the regulation of hemostasis. LACI directly inhibits factor Xa, and, in a Xa-dependent fashion, also inhibits the factor VIIa-tissue factor catalytic complex. Hybridization of a LACI cDNA probe to DNA isolated from a panel of human-mouse somatic cell hybrids containing different human chromosomes localized the human LACI gene to chromosome 2. In situ hybridization to metaphase chromosomes further mapped the gene to the region 2q31----2q32.1. Exons of the human LACI gene were cloned from genomic or chromosome 2-specific phage libraries and sequenced, including approximately 500 base pairs of 5' upstream DNA. The 5' DNA did not contain a prototypical TATAA box or CCAAT sequence, and attempts to identify a unique site for the initiation of transcription were unsuccessful in that primer extension and S1 nuclease protection analysis indicate multiple transcription initiation sites for LACI messages. Comparing the gene sequence with LACI cDNA sequences indicates that the gene contains nine exons and that alternative splicing can occur, resulting in the absence of exon 2 in the 5' untranslated region of some messages. The three Kunitz domains in LACI are encoded on separate exons. Introns which interrupt coding sequences all occur in the same codon phase interrupting the first and second bases of the codon triplets. The data are consistent with LACI evolving by a combination of gene segment duplications and exon shuffling.

The lipoprotein-associated coagulation inhibitor.

TF mediated initiation of coagulation appears to play a critical role in normal hemostasis and probably pathologic thrombosis as well. Although teleological considerations would seem to suggest that a specific regulator of this process should exist, and although the presence in plasma of such an inhibitor was documented many years ago, it was not until the past five years that the inhibitor was characterized and its mechanism of action defined. LACI produces factor Xa-dependent feedback initiation of the VIIa/TF catalytic complex. The mechanism of this feedback inhibition is novel. First, LACI, a multi-headed protease inhibitor, binds factor Xa, a product of VIIa/TF catalysis, at one of its inhibitory domains. The Xa-LACI complex, possibly acting as a pseudosubstrate, then is able to bind to VIIa/TF in an appropriate conformation such that a second inhibitory domain of LACI is positioned to interact with factor VIIa in the VIIa/TF complex. Whether such a unique means of eliciting feedback inhibition in a protease cascade is repeated in nature is unknown. The existence of LACI appears to help explain the clinical need for both "extrinsic" and "intrinsic" coagulation pathways. In addition, data to the present are consistent with the notion that, in normal hemostasis at least, TF is responsible for an initial burst of factor Xa generation which provides sufficient thrombin to induce the aggregation of platelets and the activation of the critical coagulation cofactors factor V and factor VIII. Ultimate and persistent hemostasis, however, appears to require the continued production of additional factor Xa through the action of factor IXa and factor VIII. The fact that patients with factor XI deficiency suffers a variable but usually mild bleeding diathesis suggests that under certain conditions the initial burst of factor IXa formed through the action of VIIa/TF is insufficient and supplemental factor IXa generated by factor XIa is needed for normal hemostasis. The mechanism by which this factor XIa is generated in vivo, however, has not been determined. We stress that the predicted in vivo role of LACI is simply that--a prediction based on its known in vitro properties. Documentation of its physiologic importance remains to be provided and is an area of active research. Further, although significant progress has been made over the past few years in the characterization of LACI, many questions remain unanswered. For example: What is the mechanism for LACI's association with lipoproteins in plasma? What function, if any, does the third Kunitz-type protease inhibitor domain in LACI serve? (ABSTRACT TRUNCATED AT 400 WORDS)

Endogenous phosphorylation of the lipoprotein-associated coagulation inhibitor at serine-2.

Lipoprotein-associated coagulation inhibitor (LACI) inhibits activated Factor X (Xa) directly and, in an Xa-dependent fashion, inhibits Factor VIIa-tissue factor (TF), presumably by forming a quaternary Xa-LACI-VIIa-TF complex. LACI isolated from the conditioned media of HepG2 cells grown in the presence of [32P]orthophosphate was observed to be covalently phosphorylated. Dephosphorylation of 32P-LACI with phosphatase resulted in an almost complete removal of the radiolabel. Phosphoamino acid analysis of the purified 32P-LACI established that the phosphorylation occurred on (a) serine residue(s). At its N-terminus, LACI contains a cluster of acidic residues C-terminal to the serine-2 residue. Such a site is characteristic of the sites phosphorylated by casein kinase II (CKII) in protein substrates. Edman degradation of endogenously labelled 32P-LACI revealed that the serine-2 residue was a major site of phosphorylation. Phosphorylation of purified LACI by bovine CKII was observed to occur in vitro; amino acid sequence analysis demonstrated that CKII phosphorylated LACI at the serine-2 residue. Recombinant LACI expressed from mouse C127 fibroblasts transfected using a bovine-papilloma-virus expression vector was found to be endogenously phosphorylated. By using site-directed mutagenesis, an altered form of LACI was produced in which the serine-2 residue had been changed to alanine. This altered LACI, although expressed in similar quantity to the wild-type LACI, was not detectably phosphorylated. Using the altered LACI in functional studies demonstrated that a serine residue at position 2, and thus the phosphorylation of this site, was not essential for LACI's inhibition of Xa and VIIa-TF activities.

Inhibition of factor VIIa-tissue factor coagulation activity by a hybrid protein.

Lipoprotein-associated coagulation inhibitor (LACI) appears to inhibit tissue factor (TF)-induced blood coagulation by forming a quaternary inhibitory complex containing factor Xa, LACI, factor VIIa, and TF. A genetically engineered hybrid protein consisting of the light chain of factor Xa and the first Kunitz-type inhibitor domain of LACI is shown to directly inhibit the activity of the factor VIIa-TF catalytic complex. Unlike inhibition of factor VIIa-TF activity by native LACI, inhibition by the hybrid protein is not dependent on factor Xa. In an assay of TF-induced coagulation, 50% TF inhibition occurs with hybrid protein at 35 nanograms per milliliter, whereas LACI at 2.5 micrograms per milliliter is required for an equivalent effect. gamma-Carboxylation of glutamic acid residues in the factor Xa light chain portion of the hybrid protein is required for inhibitory activity, indicating that the first Kunitz-type domain of LACI alone is not sufficient for inhibition of factor VIIa-TF.

Purification and characterization of the lipoprotein-associated coagulation inhibitor from human plasma.

The lipoprotein-associated coagulation inhibitor (LACI) has been isolated from human plasma using a combination of hydrophobic, ion-exchange, and affinity chromatography. The final purification required was greater than 500,000-fold with a yield of 13%. Plasma LACI, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, contains major bands at 40 and 46 kDa and minor bands at 55, 65, 75, 90, and approximately 130 kDa. All of the molecular weight forms are recognized by antibodies to LACI's amino and carboxyl termini and are able to inhibit the factor VII(a)-tissue factor complex and factor Xa. Plasma LACI, reduced with beta-mercaptoethanol, migrates on sodium dodecyl-sulfate-polyacrylamide gel electrophoresis as a doublet at 42 kDa and has an amino-terminal sequence essentially identical to that of HepG2 LACI. The difference in size between reduced plasma LACI (42 kDa) and HepG2 LACI (47 kDa) may be related to differing degrees of N-linked glycosylation. The 46-kDa and larger forms of unreduced plasma LACI are associated with apolipoprotein A-II (apoA-II) in mixed disulfide linkages. Studies using isolated lipoproteins show that low density lipoprotein (LDL) contains primarily the 40-kDa form of LACI, whereas high density lipoprotein (HDL) contains primarily the 46-kDa form of LACI (LACI/apoA-II complexes). Gel filtration of a fresh plasma sample showed approximately 50% of plasma LACI to be associated with LDL/very low density lipoprotein, 44% with HDL, and the remaining 6% to not be associated with lipoproteins.

Identification of the 1.4 kb and 4.0 kb messages for the lipoprotein associated coagulation inhibitor and expression of the encoded protein.

Lipoprotein-Associated Coagulation Inhibitor (LACI) is a factor Xa dependent inhibitor of the factor VII(a)/Tissue Factor catalytic complex. Deduced from partial cDNA sequence, LACI's amino acid sequence has recently been reported. Northern blot analysis showed LACI cDNA hybridizes to RNAs of 1.4 and 4.0 kb in size. To complete the characterization of the LACI message(s), overlapping LACI cDNAs were isolated from a human endothelial cell library. Sequence analysis revealed the clones' inserts span 4023 bases of sequence, consisting of 381 bases of 5' untranslated sequence, an open reading frame of 912 bases, 2682 bases of 3' untranslated sequence and 48 bases of poly(A) sequence. In addition, a short 1.4 kb insert which encodes for LACI was found to contain 49 bases of 3' untranslated sequence and a 3' poly(A) tail. The 1.4 kb of sequence is contained in the 4.0 kb sequence, except for 14 bases of 5' sequence, suggesting that the LACI messages arise by the use of alternative termination and polyadenylation signals during processing. Northern blot analysis of RNA isolated from cells treated with actinomycin D showed both RNA species appear to be relatively stable. Using a bovine papilloma virus vector, LACI cDNA was transfected into mouse C127 fibroblasts. The recombinant LACI is recognized by polyclonal anti-LACI IgG, binds to factor Xa and inhibits VII(a)/Tissue Factor activity in a similar fashion as LACI purified from HepG2 cell conditioned media.

Functional significance of the Kunitz-type inhibitory domains of lipoprotein-associated coagulation inhibitor.

Blood coagulation can be initiated when factor VII or VIIa, a plasma protease, binds to its essential cofactor, tissue factor (TF), and proteolytically activates factors IX and X, triggering a cascade of events which eventually leads to the formation of thrombin and a fibrin clot. Plasma contains a lipoprotein-associated coagulation inhibitor (LACI) which inhibits activated factor X (Xa) directly and, in a Xa-dependent way, inhibits VII(a)/TF activity, presumably by forming a quaternary Xa/LACI/VII(a)/TF complex. Sequence analysis of complementary DNA clones has shown that LACI contains three tandemly repeated Kunitz-type serine protease inhibitory domains. To investigate the relationship between these Kunitz structures and LACI function, we have used site-directed mutagenesis to produce altered forms of LACI in which the residue at the active-site cleft of each Kunitz domain has been individually changed. The second Kunitz domain is required for efficient binding and inhibition of Xa, and both Kunitz domains 1 and 2 are required for the inhibition of VIIa/TF activity; but alteration of the active-site residue of the third Kunitz domain has no significant effect on either function. We propose that in the putative inhibitory complex, Kunitz domain 1 is bound to the active site of VII(a)/TF and that Kunitz domain 2 is bound to Xa's active site.