The effect of leukocyte elastase on tissue factor pathway inhibitor.

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type inhibitor that directly inhibits factor Xa and, in a factor Xa-dependent fashion, also inhibits the factor VIIa/tissue factor (TF) catalytic complex. The Kunitz-2 domain in TFPI is needed for the binding and inhibition of factor Xa, while the Kunitz-1 domain appears to be responsible for binding factor VIIa in a quaternary factor Xa-TFPI-factor VIIa/TF inhibitory complex. Human leukocyte elastase (HLE) proteolytically cleaves TFPI between threonine-87 and threonine-88 within the polypeptide that links the Kunitz-1 and Kunitz-2 domains in the TFPI molecule. HLE treatment not only affects the ability of TFPI to inhibit factor VIIa/TF, but also dramatically reduces its inhibition of factor Xa. Both purified HLE and stimulated neutrophils regenerate TF activity from a preformed factor Xa-TFPI-factor VIIa/TF inhibitory complex. Kinetic analysis suggests that HLE cleavage does not effect the affinity of the initial encounter interaction between factor Xa and TFPI, whereas it markedly reduces the affinity of the final factor Xa:TFPI complex with Ki (final) values for untreated and HLE-treated TFPI of 58 pmol/L and 4.4 nmol/L, respectively. Thus, an epitope in the amino-terminal region of TFPI or a conformation of the TFPI molecule that requires the presence of this region is needed in concert with the Kunitz-2 domain to produce optimal inhibition of factor Xa by TFPI.     

Ixolaris, a novel recombinant tissue factor pathway inhibitor (TFPI) from the salivary gland of the tick, Ixodes scapularis: identification of factor X and factor Xa as scaffolds for the inhibition of factor VIIa/tissue factor complex

Saliva of the hard tick and Lyme disease vector, Ixodes scapularis, has a repertoire of compounds that counteract host defenses. Following sequencing of an I scapularis salivary gland complementary DNA (cDNA) library, a clone with sequence homology to tissue factor pathway inhibitor (TFPI) was identified. This cDNA codes for a mature protein, herein called Ixolaris, with 140 amino acids containing 10 cysteines and 2 Kunitz-like domains. Recombinant Ixolaris was expressed in insect cells and shown to inhibit factor VIIa (FVIIa)/tissue factor (TF)-induced factor X (FX) activation with an inhibitory concentration of 50% (IC(50)) in the picomolar range. In nondenaturing gel, Ixolaris interacted stoichiometrically with FX and FXa but not FVIIa. Ixolaris behaves as a fast-and-tight ligand of the exosites of FXa and gamma-carboxyglutamic acid domainless FXa (des-Gla-FXa), increasing its amidolytic activity. At high concentration, Ixolaris attenuates the amidolytic activity of FVIIa/TF; however, in the presence of DEGR-FX or DEGR-FXa (but not des-Gla-DEGR-FXa), Ixolaris becomes a tight inhibitor of FVIIa/TF as assessed by recombinant factor IX (BeneFIX) activation assays. This indicates that FX and FXa are scaffolds for Ixolaris in the inhibition of FVIIa/TF and implies that the Gla domain is necessary for FVIIa/TF/Ixolaris/FX(a) complex formation. Additionally, we show that Ixolaris blocks FXa generation by endothelial cells expressing TF. Ixolaris may be a useful tool to study the structural features of FVIIa, FX, and FXa, and an alternative anticoagulant in cardiovascular diseases.     

Physiological concentrations of tissue factor pathway inhibitor do not inhibit prothrombinase

Tissue factor pathway inhibitor (TFPI) is a Kunitz-type serine proteinase inhibitor that directly inhibits factor Xa and, in a factor Xa dependent manner, inhibits the factor VIIa/tissue factor catalytic complex. The inhibitory effect of TFPI in prothrombin activation assays using purified components of the prothrombinase complex was examined. When factor Xa is added to mixtures containing TFPI, prothrombin, calcium ions, and nonactivated platelets or factor V and phospholipids, TFPI significantly reduces subsequent thrombin generation, and the inhibitory effect is enhanced by heparin. If factor Xa is preincubated with calcium ions and thrombin-activated platelets or factor Va and phospholipids to permit formation of prothrombinase before the addition of prothrombin and physiologic concentrations of TFPI (< 8 nmol/L), minimal inhibition of thrombin generation occurs, even in the presence of heparin. Thus, contrary to results in amidolytic assays with chromogenic substrates, prothrombinase is resistant to inhibition by TFPI in the presence of its physiological substrate, prothrombin. Higher concentrations of TFPI (approximately 100 nmol/L), similar to those used in animal studies testing for therapeutic actions of TFPI, do effectively block prothrombinase activity.     

Tissue factor pathway inhibitor: the carboxy-terminus is required for optimal inhibition of factor Xa.

Tissue factor pathway inhibitor (TFPI) is a multivalent Kunitz-type protease inhibitor that binds to and inactivates factor Xa directly, and in a factor Xa-dependent fashion inhibits the factor VIIa/tissue factor catalytic complex. TFPI is a slow, tight-binding, competitive, and reversible inhibitor of factor Xa, in which the formation of an initial encounter complex between TFPI and factor Xa is followed by slow isomerization to a final, tightened complex. Wild-type recombinant TFPI (rTFPI), expressed in mouse C127 cells, separates into two forms on heparin-agarose chromatography that elute at 0.3 mol/L and 0.6 mol/L NaCl. Western blot analysis shows that both forms contain the N-terminus of full-length TFPI, but only rTFPI(0.6) is recognized by an antibody directed against the C-terminus. rTFPI(0.3) and rTFPI(0.6) inhibit factor Xa with 1:1 stoichiometry and inhibit factor VIIa/tissue factor equally in an endpoint-type assay. However, rTFPI(0.6) is a more potent inhibitor than rTFPI(0.3) of coagulation in normal plasma induced by either factor Xa or tissue factor. The initial inhibition of factor Xa (less than 5 seconds) produced by rTFPI(0.6) is several-fold greater than that produced by rTFPI(0.3), presumably reflecting a lower Ki of the immediate encounter complex between factor Xa and TFPI. The differential effect of these forms of TFPI on tissue factor-induced coagulation in normal plasma appears to be directly related to their ability to inhibit factor Xa. To confirm the role of the C-terminal region of TFPI in optimal factor Xa inhibition, a carboxy-terminal mutant of rTFPI, which is truncated after leucine 252 and thus lacks the basic sequence K T K R K R K K Q R V K (residues 254-265), was expressed in C127 cells. This form of rTFPI elutes from heparin-agarose at 0.28 mol/L NaCl and inhibits factor Xa at a rate that is slower than rTFPI(0.3). The Ki(final)s for factor Xa inhibition by rTFPI(0.6), rTFPI(0.3), and rTFPI1-252 are 3.1 +/- 0.6, 19.6 +/- 0.8, and 19.6 +/- 3.0 pmol/L, respectively.     

Tissue factor pathway inhibitor and the revised hypothesis of blood coagulation

The recent rediscovery, isolation, and characterization of an endogenous coagulation inhibitor termed tissue factor pathway inhibitor (TFPI) has provided new insight into the regulation of in vivo coagulation. TFPI is a multivalent, Kunitz-type, protease inhibitor that directly binds and inactivates factor Xa and, in a factor-Xa-dependent fashion, produces feedback inhibition of the factor VIIa-tissue factor catalytic complex. The demonstrated in vitro properties of TFPI have led to the formulation of a revised theory of blood coagulation. In the revised model, coagulation proceeds through a single pathway rather than the alternative and redundant "extrinsic" and "intrinsic" pathways that had previously been postulated.     

Localization of a factor VIII-inhibiting antibody epitope to a region between residues 338 and 362 of factor VIII heavy chain.

We have used a recombinant DNA epitope library to localize the binding region of a factor VIII (FVIII) monoclonal antibody that neutralizes coagulant activity. The antibody, C5, has previously been described and has been shown to have a FVIII neutralizing potency of 1488 Bethesda units per mg of purified immunoglobulin. A recombinant DNA epitope library was constructed from short, random FVIII cDNA fragments and immunologically screened with C5 to identify bacteriophage expressing the antigenic determinant. The isolation and characterization of immunoreactive bacteriophage restricted the C5 epitope to the overlapping or shared DNA sequence of nine different clones and corresponded to amino acid residues 338-362 of the mature FVIII peptide. The defined epitope is between the proposed activated protein C cleavage site (Arg-336) and thrombin cleavage site (Arg-372) on the amino-terminal 90-kDa FVIII heavy-chain subunit. The identification of the epitope of an inhibiting anti-FVIII antibody between two critical cleavage sites suggests that this amino acid sequence plays a role in regulating FVIII coagulant activity.     

Evidence that plasma lipoproteins inhibit the factor VIIa-tissue factor complex by a different mechanism that extrinsic pathway inhibitor

Factor VIIa participates in blood clotting by activating factor X and/or factor IX by limited proteolysis. The proteolytic activity of factor VIIa is absolutely dependent on a lipoprotein cofactor designated tissue factor. We have examined the ability of purified preparations of human plasma high density, low density and very low density lipoproteins, as well as apolipoproteins A-I and A-II, to inhibit the factor VIIa-tissue factor mediated activation of either factor X or factor IX before and after treatment of the lipoprotein preparation with polyclonal antibody directed against partially- purified human plasma extrinsic pathway inhibitor (EPI). In the absence of anti-EPI IgG, HDL, LDL, VLDL, and apolipoprotein A-II noncompetitively inhibited factor X activation by factor VIIa-tissue factor with apparent Ki values of 3.39 mumol/L, 124 nmol/L, 33 nmol/L, and 10.5 mumol/L, respectively. Apolipoprotein A-I had no effect on this reaction. The inhibitory activity of HDL, LDL, VLDL, and apolipoprotein A-II in this reaction was unaffected by the presence of high levels of anti-EPI IgG. In the absence of exogenous factor Xa, none of the lipoproteins studied inhibited the activation of factor IX using the tritiated peptide release assay. In the presence of added factor Xa (1 nmol/L), LDL and VLDL, but not HDL and apolipoprotein A- II, inhibited the activation of factor IX by factor VIIa-tissue factor. This inhibition was completely blocked by prior incubation of the lipoprotein with anti-EPI IgG indicating association of EPI with these particles. Taken collectively, our data indicate that HDL, LDL, and VLDL, at or below their plasma concentration, each selectively inhibits the factor VIIa-tissue factor mediated activation of factor X by a mechanism that appears to be distinct from extrinsic pathway inhibitor. These lipoproteins may not only play a role in the regulation of extrinsic blood coagulation, but may also selectively promote the activation of factor IX by factor VIIa-tissue factor in vivo at low tissue factor concentrations.     

Overexpression of glycosyl phosphatidylinositol-anchored tissue factor pathway inhibitor-1 inhibits tissue factor activity.

The cellular initiation of coagulation by the tissue factor (TF)-activated factor VII complex is transiently inhibited by endogenous tissue factor pathway inhibitor-1 (TFPI-1), whereas exogenously added TFPI-1 is targeted to a degradation pathway. This study investigates the relevance of glycosyl phosphatidylinositol (GPI) anchoring for the anticoagulant properties of TFPI-1. Experiments were performed with the human cell line ECV304 using liposomal gene transfer. For GPI anchoring of TFPI-1 we used a fusion protein of TFPI-1 and the GPI attachment sequence of decay-accelerating factor (GPI-TFPI-1), and compared it with wild-type TFPI-1. We measured TF and TFPI-1 surface expression by flow cytometry and TF proteolytic activity by a chromogenic assay for activated factor X generation. After transfection of GPI-TFPI-1, surface expression of TFPI-1 increased to 134 +/- 9% of mock transfected cells (mean +/- SEM, P = 0.004), and transfection with wild-type TFPI-1 did not significantly alter TFPI-1 surface expression. After transfection with GPI-TFPI-1, TF activity was reduced by 18 +/- 9% compared with mock transfections (P = 0.003), whereas after transfection with TFPI-1 wild type no significant inhibition was observed. This effect was not due to altered TF expression. GPI anchoring is an essential prerequisite for surface expression of TFPI-1 and inhibition of TF activity. Gene transfer of GPI-anchored TFPI, therefore, may be an efficient tool to inhibit local TF-induced coagulation.     

Molecular cloning of cDNA coding for the gamma subunit of Torpedo acetylcholine receptor.

From the electric organ of Torpedo californica, we purified mRNA that, when translated in vitro, produces polypeptides immunoprecipitable by antibodies against purified acetylcholine receptor. A novel cloning system [Okayama, H. & Berg, P. (1982) Mol. Cell. Biol. 2, 161-170] was used to produce a cDNA library from this mRNA. This library contained clones with receptor sequences identified by differential hybridization and hybridization-selection. We describe a clone of 2,030 base pairs with sequences appropriate for the amino-terminal amino acids of the gamma subunit of acetylcholine receptor. This clone contains 82 bases 5' of the codon for the amino-terminal amino acid of the mature protein. A portion of this sequence codes for a methionine followed by a 16-amino acid polypeptide that is contiguous to the amino-terminal amino acid of the mature protein and that has the characteristics of a leader peptide. The cDNA insert hybridizes to a 2,100-base RNA present in electric organ but not in the brain of T. californica.     

Amino acid sequence of trocarin, a prothrombin activator from Tropidechis carinatus venom: its structural similarity to coagulation factor Xa.

Among snake venom procoagulant proteins, group II prothrombin activators are functionally similar to blood coagulation factor Xa. We have purified and partially characterized the enzymatic properties of trocarin, the group II prothrombin activator from the venom of the Australian elapid, Tropidechis carinatus (rough-scaled snake). Prothrombin activation by trocarin is enhanced by Ca2+, phospholipids, and factor Va, similar to that by factor Xa. However, its amidolytic activity on peptide substrate S-2222 is significantly lower. We have determined the complete amino acid sequence of trocarin. It is a 46,515-Dalton glycoprotein highly homologous to factor Xa and shares the same domain architecture. The light chain possesses an N-terminal Gla domain containing 11 gamma-carboxyglutamic acid residues, followed by two epidermal growth factor (EGF)-like domains; the heavy chain is a serine proteinase. Both chains are likely glycosylated: the light chain at Ser 52 and the heavy chain at Asn 45. Unlike other types of venom procoagulants, trocarin is the first true structural homologue of a coagulation factor. It clots snake plasma and thus may be similar, if not identical, to snake blood coagulation factor Xa. Unlike blood factor Xa, it is expressed in high quantities and in a nonhepatic tissue, making snake venom the richest source of factor Xa-like proteins. It induces cyanosis and death in mice at 1 mg/kg body weight. Thus, trocarin acts as a toxin in venom and a similar, if not identical, protein plays a critical role in hemostasis.     

Cloning and characterization of two cDNAs coding for human von Willebrand factor.

A cDNA library was prepared in lambda gt11 bacteriophage from poly(A)+ RNA isolated from primary cultures of endothelial cells from human umbilical vein. Approximately 2.5 million independent recombinants were screened and 2 of those were found to synthesize a fusion protein with beta-galactosidase that reacted with rabbit antibody against human von Willebrand factor. Comparison of the amino acid sequence translated from the cDNA insert of the two clones with the amino acid sequence determined by Edman degradation of the protein established that both phage isolates code for von Willebrand factor. The first clone (lambda HvWF1) contained an insert of 404 nucleotides that corresponded to amino acid residues 1-110 in the mature protein circulating in blood, in addition to a portion (24 amino acids) of a prepro leader sequence. The second cDNA clone (lambda HvWF3) contained an insert of 4.9 kilobases that coded for the carboxyl-terminal 1525 amino acids of von Willebrand factor, a stop codon of TGA, 134 nucleotides of 3' noncoding sequence, and a poly(A) tail of 150 nucleotides. The two clones together code for greater than 80% of the molecule circulating in blood. The same carboxyl-terminal lysine residue was identified in the mature protein as well as in the cDNA, indicating that all of the proteolytic processing that occurs during the biosynthesis and assembly of von Willebrand factor is associated with the amino-terminal portion of the precursor protein. The amino acid sequence of von Willebrand factor indicates the presence of two different internal gene duplications and one triplication. These repetitive amino acid sequences account for about one-half of the amino acids present in the mature protein. The tetrapeptide sequence of Arg-Gly-Asp-Ser, which mediates the cell attachment and platelet binding activity of fibronectin, was also identified in the carboxyl-terminal portion of von Willebrand factor.     

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.     

Breaching the conformational integrity of the catalytic triad of the serine protease plasmin: localized disruption of a side chain of His-603 strongly inhibits the amidolytic activity of human plasmin.

Site-directed mutagenesis has been used to construct a cDNA that encodes a recombinant variant human plasminogen (hPg) containing a Pro-611-->Ile mutation (MrhPg). The mutein was expressed in recombinant baculovirus-infected Spodoptera frugiperda cells (IPLB-SF-21AE), and purified. After activation of this zymogen to its corresponding form of the serine protease plasmin (MrhPm), this latter enzyme was essentially inactive toward an amide plasmin substrate, most likely from alteration of the spatial relationships of the active-site His-603 to its partners of the catalytic triad, Asp-646 and Ser-741. Partial amidolytic activity of MrhPm was restored as a consequence of imidazole addition to the assay medium, due to an increase in the catalytic constant kcat of the enzyme. The serine protease inhibitor, diisopropylphosphofluoridate, when preincubated with MrhPm, did not inhibit restoration of its amidolytic activity with imidazole, whereas diisopropylphosphofluoridate did inhibit the amidolytic activity of MrhPm in the presence of imidazole. This result implies that His-603 directly influences the nucleophilic character of Ser-741. When imidazole as pretreated with alpha-N-tosyl-L-lysine chloromethyl ketone, the ability of this imidazole solution to restore amidolytic activity to MrhPm was eliminated, suggesting that N alpha-(p-tosyl)lysine chloromethyl ketone directs into the binding pocket a derivatized form of imidazole, which is ineffective as an His-603 substitute. These results indicate that the conformational reorientation of His-603 results in a malfunctional catalytic triad in the serine protease MrhPm, thus leading to an inactive enzyme despite the presence of all three essential amino acids of the catalytic triad. Addition of extramolecular imidazole restores a portion of the amidolytic activity of this mutant enzyme. These data also argue for an enzyme mechanism in which the active-center His-603 residue directly influences the nucleophilicity of the active-site Ser 741 residue.     

Reversible regulation of tissue factor-induced coagulation by glycosyl phosphatidylinositol-anchored tissue factor pathway inhibitor

Endothelial and tumor cells synthesize tissue factor pathway inhibitor (TFPI-1), which regulates tissue factor (TF) function by TF. VIIa. Xa. TFPI-1 quaternary complex formation (where VIIa and Xa are coagulation factors) and by translocation of these complexes into glycosphingolipid-rich microdomains of the cell membrane. Recombinant TFPI-1 added exogenously to cells is targeted to a degradation pathway. This study analyzes whether quaternary complex formation with endogenous TFPI-1 results also in internalization and degradation. We demonstrate that endogenous TFPI-1 and recombinant TFPI-1 differ in their distribution on the cell surface. Recombinant TFPI-1 is found in phospholipid- and glycosphingolipid-rich membrane domains, whereas endogenous TFPI-1 preferentially localizes to glycosphingolipid-rich microdomains. On quaternary complex formation, endogenous TFPI-1 remains protease sensitive and accessible for antibodies on intact cells, demonstrating that it is not appreciably internalized. Rather, regulation of TF by TFPI-1 is restored within 12 hours, consistent with dissociation of quaternary complexes on the cell surface. Endogenous TFPI-1 can be released from the cell surface by phospholipase treatment, indicating that TFPI-1 either is a glycosyl phosphatidylinositol (GPI)-anchored protein or binds to a GPI-linked receptor. We demonstrate that expression of a recombinant GPI-anchored form of TFPI-1 targets TF. VIIa complexes to glycosphingolipid-rich membrane fractions. Thus, GPI anchoring of TFPI-1 is sufficient for regulation of TF. VIIa complex function by a pathway of reversible inhibition rather than internalization and degradation.     

Anticoagulant versus amidolytic activity of tissue factor pathway inhibitor in coronary artery disease.

In view of raised levels of endothelial markers in coronary artery disease (CAD), the aim of the present study was to investigate the status of tissue factor pathway inhibitor (TFPI), another endothelium-associated glycoprotein and coagulation protease inhibitor, in CAD. The intravascular pool of TFPI is heterogeneous with respect to assay-dependent activity. While the standard amidolytic assay works well with both full-length and truncated (lipoprotein-associated) TFPI, the anticoagulant assay works better with the former. The anticoagulant activity of TFPI can be estimated using dilute tissue factor (TF) to trigger clotting of plasma. In the present study, recombinant TF diluted 2,000-fold was used to initiate coagulation. A dose-dependent shortening of clotting time of normal plasma pools with polyclonal antibody against the C-terminal but not the N-terminal peptide of TFPI demonstrated the importance of the C-terminal region, and hence that of full-length TFPI, in conferring its anticoagulant activity, corroborating current opinion. As a further confirmation, the C-terminal peptide itself prolonged dilute TF clotting time of normal pooled plasma in a concentration-dependent manner. The amidolytic and anticoagulant activities of TFPI were determined in 20 patients with clinically and angiographically assessed CAD and in 68 asymptomatic controls. The mean +/- SD ages of patients and controls were 54.9 +/- 10.3 and 48.8 +/- 11.6 years, respectively, the difference being statistically significant (P = 0.04). The mean TFPI activity measured by amidolytic assay was comparable for patients and controls (1.2 +/- 0.3 and 1.3 +/- 0.5 U/ml, respectively). However, the dilute TF clotting time was 115 +/- 26 s in patients, against 99 +/- 10 s in controls (P < 0.0001, irrespective of age adjustment). Since none of the patients had received heparin or had coagulation factor deficiency that may interfere with the assay, prolongation of clotting time may be attributed to the presence of TFPI, particularly the full-length form. To verify this inference, 33 extra aliquots left over from 88 samples (62.5%), 21 from controls and 12 from patients, were incubated with 1:10 diluted antibody against the C-terminal peptide of TFPI prior to dilute TF assay. The mean clotting time of both patients and controls decreased, and the between-group difference leveled (90 +/- 10 versus 88 +/- 20 s for controls and patients, respectively; P = 0.841). The mean drop in clotting time was 9% for the controls and 24% for the patients. This illustrates the specificity of dilute TF assay for full-length TFPI and supports the conclusion that relative to lipoprotein-associated TFPI, the proportion of the full-length form was possibly greater in patients with CAD. Contribution of lipoprotein-associated TFPI to the overall anticoagulant activity by its activated factor X-dependent inhibition of activated factor VII-TF complex seems less important considering the similar between-group mean amidolytic activities.     

Tissue factor pathway inhibitor and protease nexin-1 are major factor Xa binding proteins on the HepG2 cell surface

Previous studies indicated that human factor Xa bound to a human hepatocellular carcinoma cell line (HepG2) that constitutively synthesizes a factor V/Va molecule. Factor Xa binding to this cell line was not measurably affected by pretreatment of the cells with anti- factor V IgG and to a large extent (approximately 70%) was calcium- independent, suggesting the presence of cell-surface binding proteins specific for factor Xa other than factor V/Va. In the present study, we have further characterized the interaction of factor Xa with the HepG2 cell and performed chemical cross-linking and immunoprecipitation studies to determine the identity of the HepG2 surface protein(s) interacting with factor Xa. Initial studies demonstrated that HepG2- bound 125I-factor Xa was not significantly displaced by unlabeled factor Xa blocked at the active site with dansyl-L-glutamyl-glycyl-L- arginine (DEGR)-chloromethyl ketone (DEGR-Xa), whereas DEGR-Xa effectively inhibited prothrombinase activity of cell-bound factor Xa (Ki = 5 nmol/L). Essentially no 125I-DEGR-Xa binding to the HepG2 cells was observed, suggesting that an intact factor Xa active site was a prerequisite for binding. 125I-factor Xa binding to HepG2 cells was inhibited approximately 70% by pretreatment of the cells with anti- tissue factor pathway inhibitor (TFPI) IgG in the presence or absence of calcium ions, but was without effect on the expression of prothrombinase activity. Immunoprecipitation of 125I-factor Xa chemically cross-linked to its cell-surface binding protein with anti- factor X IgG followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed a complex with an apparent molecular weight of 96,000. An identical molecular weight complex was observed following immunoprecipitation of this radiolabeled complex with anti- TFPI IgG. In addition to TFPI, approximately 30% of cell-bound factor Xa appears to form a covalent complex with HepG2 cell-surface protease nexin-1 (PN-1) as shown by pretreatment of the HepG2 cell with murine anti-PN-1 IgG. These results suggest that approximately 1% to 2% of the factor Xa interacts with HepG2 cell-surface factor V/Va to form a productive prothrombinase complex, while the remaining factor Xa forms a non-productive complex with either TFPI or PN-1.     

Purification and partial amino acid sequence of asialo murine granulocyte-macrophage colony stimulating factor.

A procedure utilizing reversed-phase high-performance liquid chromatography is described for the purification of asialo granulocyte-macrophage colony stimulating factor (asialo-GM-CSF) from mouse lung-conditioned medium. In the purification, the partially purified factor was treated with neuraminidase to reduce charge heterogeneity due to variable degrees of sialation. Three active forms of the asialo factor were separated by the final reversed-phase liquid chromatography step. These each gave a single major band and several minor bands on polyacrylamide gel electrophoresis and had similar amino acid compositions. The specific activity of purified murine asialo-GM-CSF was approximately 8 X 10(9) colonies per mg of protein. Amino acid sequence determination of the major form gave a single amino-terminal sequence, which has been used to develop oligonucleotide probes for the isolation of two cDNA clones encoding GM-CSF. The nucleotide sequence of these two clones gave a deduced amino acid sequence almost identical with that determined for the amino terminus of asialo-GM-CSF and an amino acid composition very similar to that for asialo-GM-CSF.     

The beta subunit of the mitochondrial processing peptidase from rat liver: cloning and sequencing of a cDNA and comparison with a proposed family of metallopeptidases.

Most nuclearly encoded mitochondrial proteins are synthesized with amino-terminal leader peptides that are removed by the mitochondrial processing peptidase (MPP) after translocation. Earlier we reported cloning and sequencing of a cDNA for the larger subunit (MPP alpha subunit) of this enzyme from rat liver mitochondria. We have now completed the cloning and sequencing of a cDNA encoding the smaller subunit of the enzyme (MPP beta subunit) from the same source. The cDNA consists of 1570 bp: 17 bp of 5'-untranslated sequence, 1467 bp of coding sequence, and 86 bp of 3'-untranslated sequence. The predicted protein consists of 489 amino acid residues, including a 45-amino acid leader peptide at the amino terminus and a 444-amino acid mature protein. The amino acid sequences of four tryptic peptides derived from purified MPP beta subunit precisely match those predicted by the cDNA sequence, as does the predicted mature amino terminus. The amino-terminal sequence is typical of a mitochondrial leader peptide, with eight positively charged arginine residues and a single negatively charged aspartate residue. When the amino acid sequence of rat MPP beta subunit is compared with sequences in the protein data bases, significant homology is found with the protease-enhancing protein of Neurospora crassa, the smaller subunit of MPP from Saccharomyces cerevisiae, and the core I protein of bovine ubiquinol:cytochrome c reductase. Lower homology is found with other members of a recently proposed class of endoproteases, which includes human insulinase and protease III from Escherichia coli.     

The role of tissue factor pathway inhibitor-2 in cancer biology

Tissue factor pathway inhibitor-2 (TFPI-2), a member of the Kunitz-type serine proteinase inhibitor family, is a structural homologue of tissue factor pathway inhibitor (TFPI). The expression of TFPI-2 in tumors is inversely related to an increasing degree of malignancy, which may suggest a role for TFPI-2 in the maintenance of tumor stability and inhibition of the growth of neoplasms. TFPI-2 inhibits the tissue factor/factor VIIa (TF/VIIa) complex and a wide variety of serine proteinases including plasmin, plasma kallikrein, factor XIa, trypsin, and chymotrypsin. Aberrant methylation of TFPI-2 promoter cytosine-phosphorothioate-guanine (CpG) islands in human cancers and cancer cell lines was widely documented to be responsible for diminished expression of mRNA encoding TFPI-2 and decreased or inhibited synthesis of TFPI-2 protein during cancer progression. Furthermore, an aberrantly spliced variant of TFPI-2 mRNA (designated asTFPI-2) was detected, which represents an untranslated form of TFPI-2. The levels of asTFPI-2 were very low or undetectable in normal cells but markedly upregulated in neoplastic tissue. TFPI-2 functions in the maintenance of the stability of the tumor environment and inhibits invasiveness and growth of neoplasms, as well as metastases formation. TFPI-2 has also been shown to induce apoptosis and inhibit angiogenesis, which may contribute significantly to tumor growth inhibition. Restoration of TFPI-2 expression in tumor tissue inhibits invasion, tumor growth, and metastasis, which creates a novel possibility of cancer patient treatment. However, more information is still needed to define the precise role of TFPI-2 in human tumor biology.