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W Kisiel

Publications and source records attributed to W Kisiel.

At least 73 records · Page 4Linked to original sources
PubMed1996

[Appendiceal abscess in the third gestational trimester of pregnancy, complications pre and postoperatively].

Delayed surgical intervention connected with misdiagnosis of preterm labour and urinary tract infection caused in gravida 3 in 34th gestational week appendiceal abscess, septic shock, stillbirth by cesarean section, necessity of hysterectomy, recidivism of multi peritoneal and pleural abscesses. Although the patient was rescued the retrospective pro memoria considerations of our procedure are regarded.

Abscess↗
PubMed1995

Molecular cloning, expression, and partial characterization of two novel members of the ovalbumin family of serine proteinase inhibitors.

A human placental lambda gt11 cDNA library was screened for sequences encoding proteins related to human proteinase inhibitor 6 (PI6), and two plaques were identified that displayed weak hybridization at high stringency. Isolation and characterization of the DNA inserts revealed two novel sequences encoding proteins composed of 376 and 374 amino acids with predicted molecular masses of approximately 42 kDa. The novel proteins displayed all of the structural features unique to the ovalbumin family of intracellular serpins including the apparent absence of a cleavable N-terminal signal sequence. The degree of amino acid sequence identity between the novel serpins and PI6 (63-68%) significantly exceeds that of any other combination of known intracellular serpins. The two novel serpins encoded by the two novel cDNA sequences have been designated as proteinase inhibitor 8 (PI8) and proteinase inhibitor 9 (PI9). The putative reactive center P1-P1' residues for PI8 and PI9 were identified as Arg339-Cys340 and Glu340-Cys341, respectively. PI9 appears to be unique in that it is the first human serpin identified with an acidic residue in the reactive center P1 position. In addition, the reactive center loop of PI9 exhibits 54% identity with residues found in the reactive center loop of the cowpox virus CrmA serpin. Two PI8 transcripts of 1.4 kilobases (kb) and 3.8 kb were detected by Northern analysis in equal and greatest abundance in liver and lung, while the 1.4-kb mRNA was in excess over the 3.8-kb mRNA in skeletal muscle and heart. Two PI9 transcripts of 3.4 and 4.4 kb were detected in equal and greatest abundance in lung and placenta and were weakly detected in all other tissues. PI8 and PI9 were expressed in baby hamster kidney and yeast cells, respectively. Immunoblot analyses using rabbit anti-PI6 IgG indicated the presence of PI8 in the cytosolic fraction of stably transfected cells that formed an SDS-stable 67-kDa complex with human thrombin. PI9 was purified to homogeneity from the yeast cell lysate by a combination of heparin-agarose chromatography and Mono Q fast protein liquid chromatography and migrated as a single band in SDS-polyacrylamide gel electrophoresis with an apparent molecular mass of 42 kDa. Purified recombinant PI9 failed to inhibit the amidolytic activities of trypsin, papain, thrombin, or Staphylococcus aureus endoproteinase Glu-C and did not form an SDS-stable complex when incubated with thrombin. The cognate intracellular proteinases that interact with PI8 and PI9 are unknown.

Amino Acid Sequence↗
PubMed1995

Full-length human tissue factor pathway inhibitor inhibits human activated protein C in the presence of heparin.

Previous studies have demonstrated that tissue factor pathway inhibitor (TFPI) purified from a hepatoma cell line failed to inhibit human activated protein C (APC) or human thrombin. In the present study, we have examined the ability of full-length TFPI and a truncated form of TFPI lacking the third Kunitz-type domain and C-terminal tail (TFPI1-161) to inhibit the amidolytic activity of human APC in the presence and absence of heparin. TFPI readily inhibited APC amidolytic activity only in the presence of heparin, whereas TFPI1-161 failed to inhibit APC amidolytic activity in the presence or absence of heparin. Optimal inhibition of APC by TFPI was observed at 1 U/ml heparin. The results of competition studies between factor Xa and APC for inhibition by TFPI in the presence of heparin suggested that the second Kunitz-type domain in TFPI was responsible for the inhibition of APC.

Anticoagulants↗
PubMed1995

Enzymatic removal of sialic acid from human factor IX and factor X has no effect on their coagulant activity.

Factor IX and factor X have sialic acid in O-linked and N-linked oligosaccharides on their activation peptides, and a terminal sialic acid is found on a recently described O-linked tetrasaccharide at Ser-61 in the light chain of human factor IXa. In studies presented here, the potential role of sialic acid residues in mediating activity of human coagulation factors IX and X was tested after enzymatic removal of sialic acid residues. In contrast to previous reports, treatment of factor IX or factor IXa with recombinant sialidase did not decrease the rate of factor IX activation or proteolytic properties of human factor IXa. The activation rates of factor IX and desialated factor IX were indistinguishable when treated with factor XIa, with factor VIIa/tissue factor complex, and with the factor X activating enzyme from Russell's viper venom. Desialated human factor IXa showed full activity in the non-activated partial thromboplastin time assay and retained full "tenase" activity in a coupled amidolytic assay. Similar experiments with human factor X showed no detectable loss of clotting activity in the prothrombin time assay after desialation. Additionally, desialated human factor X was cleaved by the factor X activating enzyme from Russell's viper venom and intrinsic tenase at the same rate as untreated factor X when analyzed by SDS-polyacrylamide gel electrophoresis. These studies have shown that factor IX and factor X clotting activity are not dependent on sialic acid content. Further studies are needed to determine whether desialated factor IX binds to endothelial cells, and whether factors IX and X are more rapidly cleared from circulation or have altered susceptibility to proteolysis after enzymatic removal of sialic acid.

Factor IX↗
PubMed1995

Hepsin, a putative membrane-associated serine protease, activates human factor VII and initiates a pathway of blood coagulation on the cell surface leading to thrombin formation.

Previous studies have shown that hepsin is a putative membrane-associated serine protease that is required for cell growth (Torres-Rosado, A., O'Shea, K. S., Tsuji, A., Chou, S.-H., and Kurachi, K. (1993) Proc. Natl. Acad. Sci. U.S. A. 90, 7181 7185). In the present study, we have transfected baby hamster kidney (BHK) cells with a plasmid containing the cDNA for human hepsin and examined these cells for their ability to activate several blood coagulation factors including factors X, IX, VII, prothrombin, and protein C. Little, if any, proteolytic activation of factors X, IX, prothrombin, or protein C was observed when these clotting factors were incubated with hepsin-transfected cells. On the other hand, hepsin-transfected cells proteolytically activated significant concentrations of human factor VII in a time- and calcium-dependent manner, whereas essentially no activation of factor VII was observed in BHK cells transfected with plasmid lacking the cDNA for hepsin. The factor VII activating activity in the hepsin-transfected BHK cell line was confined exclusively to the total membrane fraction and was inhibited > 95% by antibody raised against a fusion protein consisting of maltose-binding protein and the extracellular domain of human hepsin. An active site factor VII mutant, S344A factor VII, was cleaved as readily as plasma-derived factor VII by hepsin-transfected cells, indicating that factor VII was not converted to factor VIIa autocatalytically on the cell surface. In contrast, an activation cleavage site factor VII mutant, R152E factor VII, was not cleaved by hepsin-transfected cells, suggesting that factor VII and S344A factor VII were activated on these cells by cleavage of the Arg152-Ile153 peptide bond. In the copresence of factor VII and factor X, hepsin-transfected BHK cells supported the formation of factor Xa. In addition, in the copresence of factor VII, factor X, and prothrombin, hepsin-transfected BHK cells supported the formation of thrombin. These results strongly suggest that membrane-associated hepsin converts zymogen factor VII to factor VIIa, which in turn, is capable of initiating a coagulation pathway on the cell surface that ultimately leads to thrombin formation.

Animals↗
PubMed1995

A simple chromogenic substrate assay of tissue factor pathway inhibitor activity in plasma and serum.

A simple chromogenic substrate assay for the quantitation of tissue factor pathway inhibitor (TFPI) activity in plasma or serum samples was developed. After immobilization on microtiter plates for 20 hours at 4 degrees C, a commercial thromboplastin was incubated for 1 hour at room temperature with 1 U/mL of a prothrombin complex concentrate (Protromplex). After washing, solid-phase Factor Xa activity was measured by a chromogenic substrate (S-2222). Factor Xa generation was progressively inhibited when increasing amounts (1-12 microL) of heated serum or plasma, and recombinant TFPI (1-5 ng/mL), were coincubated with Protromplex. Inhibition by serum or plasma was abolished by anti-TFPI polyclonal antibodies. Plasma levels of TFPI in 25 healthy volunteers were found to be 0.98 +/- 0.19 U/mL (range 0.71-1.52), with an intra- and inter-assay coefficient of variation of 10.7 and 11.1%, respectively. The use of a recombinant human thromboplastin improved the sensitivity and reproducibility of the assay. Plasma levels of TFPI were found to be normal in 10 women at the end of their pregnancies, in 10 patients receiving oral anticoagulant therapy, and in 10 diabetic patients. Significantly higher levels were detected in 10 patients with chronic liver disease and in 10 patients with unexplained juvenile thrombosis. In patients with cardiovascular disease, a 7-day treatment with subcutaneous standard heparin increased TFPI activity. The availability of a simple and rapid assay to measure TFPI that does not require purified coagulation proteins may facilitate studies of the pathophysiologic relevance of this inhibitor.

Adolescent↗
PubMed1995

Internalization and degradation of recombinant human coagulation factor VIIa by the human hepatoma cell line HuH7.

Previous studies demonstrated that several normal and transformed cultured human cell lines specifically bind human coagulation factors VII and VIIa via tissue factor. In the present study, we show that 125I-radiolabeled recombinant human factor VIIa (125I-rFVIIa) binds to a human hepatoma cell line (HuH7). In the presence of rabbit polyclonal anti-human tissue factor apoprotein IgG, binding of 125I-rFVIIa to the HuH7 cells was decreased approximately 60%, suggesting of tissue factor-independent binding sites for 125I-rFVIIa on these cells. The binding isotherm of 125I-rFVIIa for the HuH7 cells in the presence of anti-tissue factor IgG exhibited a hyperbolic profile and was time-, temperature- and calcium-dependent. Furthermore, binding at 4 degrees C was specific, dose-dependent and saturable. Scatchard analysis of the binding data demonstrated a single class of binding sites with a dissociation constant (Kd) of 3.2 nM and 27,000 binding sites per cell. At 4 degrees C, 125I-rFVIIa bound to, and eluted from, the cell was indistinguishable from offered 125I-rFVIIa as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. The molecular properties of the tissue factor-independent binding protein were studied by using the cleavable cross-linking agent 3,3'-dithiobis(sulfosuccinimidylpropionate). A cross-linking product of 125I-rFVIIa and a cell surface protein with an apparent M(r) approximately 100,000 was observed. The cross-linking reaction was strongly inhibited by a 100-fold molar excess of unlabeled rFVIIa, but not by rabbit polyclonal anti-human tissue factor apoprotein IgG, indicating that cross-linking does not involve the extracellular domain of tissue factor.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma, Hepatocellular↗
PubMed1994

Molecular cloning, expression, and partial characterization of a second human tissue-factor-pathway inhibitor.

Previous studies have shown that tissue-factor-pathway inhibitor (TFPI) is an important regulator of the extrinsic pathway of blood coagulation through its ability to inhibit factor Xa and factor VIIa-tissue factor activity. We describe the molecular cloning and expression of a full-length cDNA that encodes a molecule, designated TFPI-2, that has a similar overall domain organization and considerable primary amino acid sequence homology to TFPI. After a 22-residue signal peptide, the mature protein contains 213 amino acids with 18 cysteines and two canonical N-linked glycosylation sites. The deduced sequence of mature TFPI-2 revealed a short acidic amino-terminal region, three tandem Kunitz-type domains, and a carboxyl-terminal tail highly enriched in basic amino acids. Northern analysis indicates that TFPI-2 is transcribed in umbilical vein endothelial cells, liver, and placenta. TFPI-2 was expressed in baby hamster kidney cells and purified from the serum-free conditioned medium by a combination of heparin-agarose chromatography, Mono Q FPLC, Mono S FPLC, and Superose 12 FPLC. Purified TFPI-2 migrated as a single band in SDS/PAGE and exhibited a molecular mass of 32 kDa in the presence and absence of reducing agent. The amino-terminal sequence of recombinant TFPI-2 was identical to that predicted from the cDNA. Despite its structural similarity to TFPI, the purified recombinant TFPI-2 failed to react with polyclonal anti-TFPI IgG. Preliminary studies indicated that purified recombinant TFPI-2 strongly inhibited the amidolytic activities of trypsin and the factor VIIa-tissue factor complex. In addition, the inhibition of factor VIIa-tissue factor amidolytic activity by recombinant TFPI-2 was markedly enhanced in the presence of heparin. TFPI-2 at high concentrations weakly inhibited the amidolytic activity of human factor Xa, but had no measurable effect on the amidolytic activity of human thrombin.

Amino Acid Sequence↗
PubMed1994

A peptide derived from the amino terminus of endothelial-monocyte-activating polypeptide II modulates mononuclear and polymorphonuclear leukocyte functions, defines an apparently novel cellular interaction site, and induces an acute inflammatory response.

Endothelial-monocyte-activating polypeptide II (EMAP II) is a novel mediator isolated from conditioned medium of methylcholanthrene A-induced tumor cells which modulates properties of endothelial cells, mononuclear phagocytes (MPs), and polymorphonuclear leukocytes (PMNs) in vitro and induces an acute inflammatory response in vivo. A synthetic peptide comprising 15 residues from the N-terminal region (residues 6-20) was shown to induce directional migration of MPs and PMNs, with half-maximal effect at approximately 200-250 pM, whereas a peptide from the C terminus of EMAP II, as well as other irrelevant peptides, were without effect. Modulation of cellular phenotype by EMAP II-derived peptide was suggested by peptide-induced elevation of cytosolic free calcium concentration in fura-2-loaded MPs and PMNs and by stimulation of peroxidase release in PMNs. Consistent with these in vitro data, EMAP II-derived N-terminal peptide-albumin conjugates injected into the mouse footpad elicited inflammatory cell tissue infiltration, whereas albumin alone or EMAP II-derived C-terminal peptide conjugated to albumin incited little response. Binding of 125I-labeled EMAP II-derived peptide (residues 12-20) to MPs was saturable (Kd approximately 200 pM) and was blocked in a dose-dependent manner by the addition of intact EMAP II and unlabeled EMAP II-derived peptides (residues 6-20 and 12-20), whereas interleukin 1, tumor necrosis factor, formyl-methionyl-leucinyl-phenylalanine, or irrelevant peptides were without effect. Cross-linking of 125I-EMAP II-derived peptide (residues 12-20) by disuccinimidyl suberate to human MPs demonstrated a band, approximately 73 kDa, on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 125I-EMAP II-derived peptide also demonstrated specific binding to human PMNs and murine RAW cells. These data indicate that the N-terminal region of EMAP II defines a biologically active locus of the molecule which interacts with target cells via a potentially novel cellular receptor.

Amino Acid Sequence↗
PubMed1994

Complementary DNA cloning and kinetic characterization of a novel intracellular serine proteinase inhibitor: mechanism of action with trypsin and factor Xa as model proteinases.

The full-length cDNA encoding a novel human intracellular serine proteinase inhibitor has been sequenced and found to encode a 376 amino acid protein (M(r) approximately 42.5K) that we designate as cytoplasmic antiproteinase. Analysis of the primary structure revealed that the cytoplasmic antiproteinase has the majority of structural motifs conserved among the greater superfamily of serine proteinase inhibitors, or serpins. On the basis of several criteria such as amino acid identity and the absence of a classical N-terminal signal peptide, the cytoplasmic antiproteinase represents a new member of the intracellular serpin family. Further inspection of the cytoplasmic antiproteinase amino acid sequence identified three potential N-glycosylation sites and Arg341-Cys342 as the reactive site P1-P1' residues, respectively. We have also employed the slow binding kinetic approach to detail the mechanism of bovine trypsin and human factor Xa inhibition by the novel cytoplasmic antiproteinase. Inhibition of trypsin by the cytoplasmic antiproteinase was preceded by a two-step mechanism corresponding to the formation of an initial loose complex, followed by an isomerization step to a more stable, tight complex. The binding of the cytoplasmic antiproteinase to trypsin occurred with a second-order association rate constant of 2.8 x 10(6) M-1 s-1 and an overall equilibrium constant of 22.5 pM, demonstrating that the factor is a potent inhibitor of this proteinase. Under the appropriate conditions, the tight complex between trypsin and the cytoplasmic inhibitor was reversible, indicated by an exponential regeneration of proteinase amidolytic activity from the preformed complex. Therefore, the tight complex appears to be stabilized predominantly by reversible bonds that form between trypsin and the cytoplasmic inhibitor. In contrast to the inhibition of trypsin, the inhibition of factor Xa amidolytic activity by the cytoplasmic antiproteinase followed a single-step binding mechanism. The apparent first-order rate constant for factor Xa inhibition was found to increase as a linear function of the inhibitor concentration range studied. Formation of the inhibitory complex between factor Xa and the cytoplasmic antiproteinase occurred with a second-order association rate constant of approximately 1.3 x 10(5) M-1 s-1 and a equilibrium constant of 3.7 nM. These findings suggests that the cytoplasmic inhibitor may initially encounter significant energy barriers for proper alignment with the substrate binding cleft of factor Xa. However, once aligned, the reaction proceeds rapidly to a tight factor Xa.inhibitor complex that dissociates at a slow rate.

Amino Acid Sequence↗
PubMed1993

Isolation and characterization of an intracellular serine proteinase inhibitor from a monkey kidney epithelial cell line.

A recent report described a thrombin inhibitory activity in the soluble fraction of human placenta and the cytosolic fraction of K562 cells. Isolation and characterization of the functionally inactive 35-38-kDa placental form of this protein revealed that it was a novel serine proteinase inhibitor (Coughlin, P. B., Tetaz, T., and Salem, H. H. (1993) J. Biol. Chem. 268, 9541-9547). In the present study, we observed a 67-kDa sodium dodecyl sulfate (SDS)-stable complex when 125I-thrombin was incubated with the cytosolic fraction of a monkey kidney epithelial cell line, BSC-1. This complex was not observed in either the particulate cell fraction extracted with 0.2% Triton X-100 or medium conditioned by cells, suggesting that the thrombin-complexing factor is confined to the cytoplasm. The cytoplasmic antithrombin activity was purified to apparent homogeneity from the cytosol of BSC-1 cells previously pulsed with [35S]methionine by a combination of heparin-agarose chromatography, Mono Q fast protein liquid chromatography, and anhydrotrypsin-Affi-Gel 10 affinity chromatography. Analysis of the affinity-purified preparation by SDS-polyacrylamide gel electrophoresis and fluorography revealed a single protein with an apparent molecular mass of 38 kDa. The purified 38-kDa protein inhibited the amidolytic activities of thrombin, trypsin, urokinase, and factor Xa but not that of elastase. Incubation of the 38-kDa protein with excess thrombin identified approximately 60% of the labeled 38-kDa protein in an SDS-stable 67-kDa complex. The purified 38-kDa inhibitor was cleaved with cyanogen bromide and the isolated peptides subjected to microsequencing. Amino acid sequence obtained for a region within this protein exhibited significant homology with human antithrombin III and plasminogen activator inhibitors 1 and 2. This homologous peptide contained the full complement of residues designated as highly conserved in helix F of the greater serine proteinase inhibitor superfamily. In addition, an internal sequence of GGGGDIHQGF was found in the monkey cytoplasmic inhibitor, which is identical to that reported for an internal sequence of the human placental inhibitor. These findings confirm the existence of a novel cytoplasmic serine proteinase inhibitor in mammalian cells and provide additional details of its molecular properties. The physiological function of this novel serine proteinase inhibitor in cytoplasm is unknown.

Amino Acid Sequence↗
PubMed1993

Structure of human des(1-45) factor Xa at 2.2 A resolution.

The structure of a large molecular fragment of factor Xa that lacks only a Gla (gamma-carboxyglutamic acid) domain (N-terminal 45 residues) has been solved by X-ray crystallography and refined at 2.2 A resolution to a crystallographic R-value of 0.168. The fragment identity was clearly established by automated Edman degradation. X-ray structure analysis confirmed the biochemical characterization and also revealed that the N-terminal epidermal growth factor (EGF)-like domain is flexibly disordered in crystals. The second EGF module, however, is positionally ordered making contacts with the catalytic domain. The overall folding of the catalytic domain is similar to that of alpha-thrombin, excluding the insertion loops of the latter with respect to simpler serine proteinases. The C-terminal arginine of the A-chain interacts in a substrate-like manner with the S1 specificity site of the active site of a crystallographically neighboring molecule. Based on this interaction and the structure of D-PheProArg methylene-thrombin, a model of the commonly used dansylGluGlyArg methylene inhibitor-factor Xa interaction is proposed. The region of factor Xa corresponding to the fibrinogen recognition site of thrombin has a reversed electrical polarity to the anion binding fibrinogen recognition site of thrombin but possesses a site similar to the Ca2+ binding site of trypsin and other serine proteinases. The structure of the C-terminal EGF domain of factor Xa is the first to be determined crystallographically. Its folding has been comprehensively compared with similar domains determined by NMR. Although the A-chain makes 44 contacts at less than 3.5 A with the catalytic domain, only 16 involve the EGF module. In addition, the A-chain makes 30 intermolecular contacts with a neighboring catalytic domain.

Amino Acid Sequence↗
PubMed1993

Isolation and characterization of proteolytic fragments of human factor VIIa which inhibit the tissue factor-enhanced amidolytic activity of factor VIIa.

The interaction of circulating factor VII/VIIa with tissue factor presented by cells in extravascular tissues represents the initial event in the extrinsic pathway of blood coagulation. To determine the tissue factor binding domains in human factor VIIa, we have subjected recombinant human factor VIIa to tryptic digestion and isolated two proteolytic fragments (molecular mass = 32 and 20 kDa) by a combination of immunoaffinity chromatography and reversed phase high performance liquid chromatography (HPLC) which strongly inhibits the tissue factor-enhanced amidolytic activity of factor VIIa and inhibits the activation of factor X by factor VIIa in the presence of tissue factor. The 32-kDa factor VIIa fragment consisted of residues 1-137/143 from the light chain of factor VIIa connected by a disulfide bond to residues 153-277 from the heavy chain. The 20-kDa factor VIIa fragment consisted of residues 1-137 of the light chain of factor VIIa in disulfide linkage with residues 248-266 of the heavy chain. The 32- and 20-kDa factor VIIa fragments inhibited the tissue factor apoprotein-enhanced factor VIIa amidolytic activity with Ki values of 35 and 65 nM, respectively. The Ki values for the inhibition of relipidated tissue factor apoprotein-enhanced factor VIIa amidolytic activity by the 32- and 20-kDa factor VIIa fragments were 70 and 610 nM, respectively. Factor X activation by factor VIIa-relipidated tissue factor was inhibited half-maximally by the 32- and 20-kDa factor VIIa fragments at 65 and 680 nM concentrations, respectively. Equilibrium binding studies indicated that the 32- and 20-kDa factor VIIa fragments interacted with cell surface tissue factor expressed on J82 cells in a specific and saturable manner with Kd values of 30 and 64 nM, respectively. In addition, a peptide consisting of residues 1-109 from the light chain of factor VIIa obtained by reduction and HPLC of the 20-kDa factor VIIa fragment retained inhibitory activity, but the selective removal of the gamma-carboxyglutamic acid domain from the 20-kDa factor VIIa fragment by cathepsin G cleavage resulted in the complete loss of inhibitory activity in this fragment. Our data strongly suggest that the epidermal growth factor-like domains covalently linked to the gamma-carboxyglutamic acid domain in factor VIIa constitute the high affinity tissue factor binding domain in this molecule.

Amino Acid Sequence↗
PubMed1993

Tumor cell procoagulant and urokinase expression in carcinoma of the ovary.

BACKGROUND: An association between cancer and increased blood coagulation has been observed for many years. Generally, there is an equilibrium between the coagulation system (fibrin deposition) and the fibrinolytic system (degradation of fibrin by enzymes). However, in malignant disease such as ovarian carcinoma, this equilibrium is disrupted, resulting in the abnormal activation of coagulation or hypercoagulability. Also, evidence indicates that various components of these pathways may contribute to the disorderly characteristics of malignancy, such as proliferation, invasion, and metastasis. PURPOSE: Our purpose was to define the mode of interaction of tumor cells in ovarian carcinoma with both the coagulation (procoagulant-initiated) and fibrinolysis (urokinase-type plasminogen activator-initiated) (u-PA) pathways. METHODS: Studies were performed on acetone-methylbenzoate-xylene-fixed tissue prepared from fresh resected primary tumor specimens from 15 patients with cystic epithelial ovarian carcinoma. None of the patients had received prior treatment. Antibodies were tested on control and tumor tissues in concentrations that provided maximum staining intensity with minimum background staining. Laboratory immunohistochemical techniques used purified, monospecific antibodies to detect coagulant antigens. Tests were performed utilizing antibodies to recombinant human tissue factor; factor VII; factor X; factor XIIIA; high-molecular-weight and low-molecular-weight forms of u-PA; tissue-type plasminogen activator; plasminogen; and the plasminogen activator inhibitors 1, 2, and 3. Monoclonal antibodies used for specific antigen detection included 1-8C6 (fibrinogen), T2G1 (fibrin), and EBM-11 (macrophage-specific). RESULTS: The ovarian tumor cells expressed urokinase-type plasminogen activator in a pattern that was variable in intensity and distribution. Tumor cell plasminogen was not detected. Tumor cells also expressed tissue factor and coagulation pathway intermediates that resulted in local thrombin generation as evidenced by the conversion of fibrinogen (present in tumor connective tissue) to fibrin that was found to hug the surfaces of tumor nodules and individual tumor cells. Detected fibrin could not be accounted for on the basis of necrosis or a local inflammatory cell infiltrate. CONCLUSIONS: These results are consistent with the existence of a dominant tumor cell-associated procoagulant pathway that leads to thrombin generation and hypercoagulability in carcinoma of the ovary. IMPLICATIONS: In ovarian carcinoma the procoagulant pathway may contribute to tumor progression. Clinical trials of therapeutic drugs capable of limiting local coagulability (anticoagulants, protease inhibitors) are indicated in this tumor type.

Blood Coagulation Factors↗
PubMed1993

Inhibitory properties of full-length and truncated recombinant tissue factor pathway inhibitor (TFPI). Evidence that the third Kunitz-type domain of TFPI is not essential for the inhibition of factor VIIa-tissue factor complexes on cell surfaces.

Human tissue factor pathway inhibitor (TFPI) is a plasma protease inhibitor that consists of three tandem Kunitz-type inhibitor domains flanked by a negatively charged NH2 terminus and a positively charged COOH-terminal tail. Previous studies have shown that the first and second Kunitz-type domains in TFPI are involved in the inhibition of factor VIIa and factor Xa activity, respectively. In the present study, we have compared the inhibitory properties of full-length recombinant TFPI and a truncated form of TFPI lacking the third Kunitz-type domain and COOH-terminal tail (TFPI1-161) with respect to inhibition of factor VIIa-tissue factor complexes on the surface of a human bladder carcinoma cell line J82. Full-length TFPI and TFPI1-161 were kinetically indistinguishable with respect to neutralization of the proteolytic activity of preformed complexes of factor VIIa-tissue factor on the J82 cell surface in the absence of factor Xa. Equimolar amounts of factor Xa augmented the anticoagulant activity of both preparations of TFPI to the same extent, and both preparations of TFPI were equally effective in inhibiting factor VIIa-tissue factor amidolytic activity in solution phase. In addition, plasma concentrations of both forms of TFPI, in stoichiometric complex with factor Xa, inhibited cell surface factor VIIa-tissue factor proteolytic activity markedly faster than plasma levels of antithrombin III, even in the presence of 1 unit/ml heparin. The results of displacement studies suggested slight differences in the affinity of the two TFPI molecules for the cell surface in that approximately 5% of a VIIa.TF.Xa.TFPI1-161 quaternary complex on J82 cells was displaceable from the cell surface by high concentrations of factor VIIa (10-100 nM), whereas only 1-2% of a VIIa.TF.Xa.TFPI complex was displaceable under comparable conditions. Pretreatment of the cells with TFPI/Xa alone or together with R152E factor VII, followed by factor VIIa treatment, revealed significant differences in the two TFPI forms with respect to the degree with which offered factor VIIa could restore factor X activation on the cell surface. These differences notwithstanding, our collective findings indicate that the third Kunitz-type domain and/or COOH-terminal tail of TFPI is not essential for the inhibition of cell surface factor VIIa-tissue factor complexes and suggests that TFPI1-161 may be a useful therapeutic agent in the treatment of thromboembolic episodes.

Antithrombin III↗
PubMed1993

Distribution of tissue factor pathway inhibitor in normal and malignant human tissues.

Specific antibodies to tissue factor pathway inhibitor (TFPI) were used in immunohistochemical procedures to determine the distribution of TFPI in normal and neoplastic human tissues. TFPI was restricted to megakaryocytes and the endothelium of the microvasculature in normal and abnormal tissues, but was not found in the endothelium of larger vessels or in hepatocytes. TFPI was also detected in macrophages in the villi of term placenta. Tumor-associated macrophages in several types of malignancy that we have shown previously to express a complete tissue factor-initiated pathway of coagulation and thrombin generation also manifested TFPI. By contrast, malignant cells in small cell carcinoma of the lung, renal cell carcinoma, and malignant melanoma that we have shown previously to express coagulation factors together with tumor cell-associated fibrin formation failed to stain for TFPI. We postulate that TFPI may be lacking from the latter malignancies because of the absence of the appropriately configured tissue factor-factor VIIa-factor Xa complex required for TFPI binding.

Capillaries↗
PubMed1993

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.

Amyloid beta-Protein Precursor↗