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L V Rao

Publications and source records attributed to L V Rao.

At least 37 records · Page 2Linked to original sources
PubMed1998

Tissue factor pathway inhibitor in tetracycline-induced pleuritis in rabbits.

Pleural fibrin deposition that promotes loculation and fibrosis after pleural injury is initiated by tissue factor (TF). In this study, we sought to determine if tissue factor pathway inhibitor (TFPI), an inhibitor of the TF-factor VIIa complex, was likewise expressed in tetracycline (TCN)-induced pleural injury and, if so, whether TFPI was locally elaborated. Pleural fluid TFPI activity approximated that of plasma by 24 h and doubled by 3 days after intrapleural TCN. By contrast, pleural fluid coagulation factors VII and V remained below plasma concentrations at these intervals. Immunohistochemical studies demonstrated TF, TFPI and fibrin localized in pleural and subpleural tissues and within intrapleural adhesions. TFPI activity and mRNA were also elaborated by rabbit pleural mesothelial cells and lung fibroblasts. TFPI is locally expressed and pleural fluid TFPI exceeds plasma levels during TCN-induced pleural injury. Resident cells as well as extravasation likely contribute to intrapleural TFPI. TFPI expression temporally and anatomically approximates that of TF and may limit TF-induced fibrin deposition in evolving TCN-induced pleuritis.

Animals↗
PubMed1998

Tissue factor on cells.

Tissue factor (TF), a cell surface glycoprotein, serves as the cellular receptor for either activated or non-activated factor VII [FVII(a)] and it is the formation of TF-FVII(a) complexes on cell surfaces which triggers the coagulation cascade in vivo. TF procoagulant functional expression on cell surfaces can be regulated by at least three distinct major mechanisms: (1) transcriptional regulation of TF gene expression; (2) cell membrane alterations in cells expressing TF; and (3) neutralization of TF-activated factor VII (FVIIa) activity by plasma inhibitors. The TF gene, which is not normally expressed in vascular cell types, can be induced by several pathophysiological stimuli, particularly those elaborated upon in inflammation and cancer. However, some of the stimuli elaborated in these pathological processes, e.g. basic fibroblast growth factor, suppress the induced expression of TF in endothelium. Not all TF molecules expressed on cell surfaces are functional even though they have the ability to bind to FVII(a). The availability of anionic phospholipids on cell membranes in the vicinity of TF and the spatial localization of TF within the cell membrane influence the functional activity of TF. Once TF-FVII(a) complexes are assembled on cell surfaces, at least two plasma inhibitors, TF pathway inhibitor and antithrombin III play an important role in regulating the TF-FVII(a) functional activity by inhibiting the activation of factor VII bound to TF and by inhibiting the catalytic activity of TF-FVIIa complexes. The availability of heparan sulphate proteoglycans with anticoagulant activity on cell surfaces plays an important role in enhancing the activity of the inhibitors. This manuscript summarizes the mechanisms by which TF functional expression on cells is regulated with a particular emphasis on the recent findings of the authors and their collaborators.

Cell Membrane↗
PubMed1998

Interaction of activated factor VII and active site-inhibited activated factor VII with tissue factor.

The coagulation cascade is initiated by binding of plasma activated or non-activated factor VII [FVII(a)] to cell surface tissue factor (TF). TF-induced coagulation plays a primary role not only in haemostasis but also in the pathogenesis of various thrombotic disorders. Recent studies with animal model systems showed that the administration of active site-inhibited FVIIa (FVIIai) blocked TF-FVIIa-induced fibrin and thrombus formation. These data suggest that FVIIai competes with plasma FVII(a) for a limited number of TF sites expressed on cells either constitutively or induced after the perturbation. To obtain insights into the mechanism(s) by which FVIIai is effective in inhibiting TF-FVIIa induced coagulation in vivo, we compared the interaction of FVIIai and FVIIa with TF using a variety of competition assays and direct binding assays. The TF-FVIIa amidolytic activity competition assay showed that FVIIai bound with a threefold higher affinity than that of FVIIa to TF relipidated in phosphatidylcholine (PC) vesicles, whereas no significant differences were found between FVIIa and FVIIai binding to TF if it had been relipidated in mixed phospholipid vesicles containing PC and phosphatidylserine (PS). When FVIIa and FVIIai binding to TF was analysed in a FXa generation assay, we found that FVIIai bound to TF in PCPS vesicles with two- to fivefold higher affinity than that of FVIIa, whereas the affinity of FVIIai for TF in PC vesicles was seven- to 10-fold higher than that of FVIIa. Direct binding analysis to TF, immobilized on a sensor chip or on a cell surface, showed a faster association and a slower dissociation of FVIIai to TF compared with that of FVIIa. Equilibrium binding to cell surface TF showed that the affinity of FVIIai was fivefold higher than that of FVIIa to non-functional TF, whereas both FVIIa and FVIIai bound functional TF with the same high affinity. The enhanced affinity of FVIIai to TF, particularly to non-functional TF, would make FVIIai a valuable reagent to block TF-induced coagulation before it is triggered by cell injury or a pathological stimuli.

Binding Sites↗
PubMed1997

Binding of factor VIIa to tissue factor induces alterations in gene expression in human fibroblast cells: up-regulation of poly(A) polymerase.

Tissue factor (TF) is the cellular receptor for an activated form of clotting factor VII (VIIa) and the binding of factor VII(a) to TF initiates the coagulation cascade. Sequence and structural patterns extracted from a global alignment of TF confers homology with interferon receptors of the cytokine receptor super family. Several recent studies suggested that TF could function as a genuine signal transducing receptor. However, it is unknown which biological function(s) of cells are altered upon the ligand, VIIa, binding to TF. In the present study, we examined the effect of VIIa binding to cell surface TF on cellular gene expression in fibroblasts. Differential mRNA display PCR technique was used to identify transcriptional changes in fibroblasts upon VIIa binding to TF. The display showed that VIIa binding to TF either up or down-regulated several mRNA species. The differential expression of one such transcript, VIIa-induced up-regulation, was confirmed by Northern blot analysis. Isolation of a full-length cDNA corresponding to the differentially expressed transcript revealed that VIIa-up-regulated gene was poly(A) polymerase. Northern blot analysis of various carcinomas and normal human tissues revealed an over expression of PAP in cancer tissues. Enhanced expression of PAP upon VIIa binding to tumor cell TF may potentially play an important role in tumor metastasis.

Base Sequence↗
PubMed1997

Incorporation of an active site inhibitor in factor VIIa alters the affinity for tissue factor.

Recent studies showed that the administration of active site-inhibited factor VIIa blocked factor VIIa/tissue factor-induced fibrin and thrombus formation in ex vivo and in vivo model systems. These studies suggest that inactivated factor VIIa competes efficiently with plasma factor VII(a) for a limited number of tissue factor sites. In the present study, we compared the interactions of factor VIIa and active site-inhibited factor VIIa with tissue factor. Competition studies of factor VIIa and active site-inhibited factor VIIa in a factor X activation assay showed that the affinity of the latter for relipidated tissue factor was 5-fold higher than that of factor VIIa. Radioligand binding studies with a human bladder carcinoma cell line (J82) and surface plasmon resonance studies using soluble tissue factor demonstrated a faster association and a slower dissociation for the active site-inhibited factor VIIa. Studies of equilibrium binding to cell surface tissue factor showed that the affinity of active site-inhibited VIIa was 5-fold higher than that of factor VIIa to non-functional tissue factor sites, whereas both inactivated factor VIIa and factor VIIa bound to functional tissue factor sites with the same high affinity. Comparison of the CD spectra of factor VIIa and active site-inactivated factor VIIa revealed structural differences in the protease domain. The potential physiological implications of these findings are discussed.

Amino Acid Chloromethyl Ketones↗
PubMed1997

Mechanisms of activity of lupus anticoagulants.

Lupus anticoagulants comprise a heterogenous group of circulating immunoglobulins that are associated with an increased risk of thrombosis. It was believed earlier that these antibodies were directed against anionic phospholipids. Recent findings, however, establish that the autoantibodies in lupus anticoagulants are not directed against "native" anionic phospholipids but rather to modified phospholipids, lipid-protein adducts, and certain phospholipid binding proteins. Despite a marked increase in the number of citations in recent years on laboratory and clinical manifestations of lupus anticoagulants, the pathogenetic mechanisms involved remain unknown. This review focuses on antigen specificities of autoantibodies in lupus anticoagulants and the mechanisms by which these antibodies contribute to an increased thrombotic risk in patients with lupus anticoagulant.

Adsorption↗
PubMed1997

Inhibition of tissue factor gene activation in cultured endothelial cells by curcumin. Suppression of activation of transcription factors Egr-1, AP-1, and NF-kappa B.

Binding of plasma factor VII(a) to tissue factor (TF) initiates the coagulation cascade. In health, TF is not expressed in endothelial cells. However, endothelial cells express TF in response to lipopolysaccharide (LPS), tumor necrosis factor-alpha (TNF alpha), and other biological stimuli. TF expression by endothelial cells is implicated in thrombotic disorders in patients with a variety of clinical disorders. In the present study, we demonstrate that curcumin (diferulolylmethane), a known anticarcinogenic and anti-inflammatory agent, inhibited phorbol 12-myristate 13-acetate (PMA), LPS, TNF alpha, and thrombin-induced TF activity and TF gene transcription in human endothelial cells. The present data show that curcumin prevented the activation of c-Rel/p65, which is essential for TF gene activation in endothelial cells, by impairing the proteolytic degradation inhibitor protein, I kappa B alpha. The data also show that curcumin downregulated AP-1 binding activity. The present studies are the first to demonstrate that PMA, but not LPS, TNF alpha, and thrombin, induced Egr-1 binding to the second serum-responsive region (SRR-2) of TF promoter and that curcumin inhibited the PMA-induced Egr-1 binding to SRR-2. Overall, the data suggest that the anticarcinogenic and anti-inflammatory properties of curcumin may be related to its ability to inhibit cellular gene expression regulated by transcription factors NF-kappa B, AP-1, and Egr-1.

Cells, Cultured↗
PubMed1997

Acidic and basic fibroblast growth factors suppress transcriptional activation of tissue factor and other inflammatory genes in endothelial cells.

Tissue factor (TF) is a transmembrane receptor that serves as a cofactor for factor VIIa and initiates the extrinsic pathway of blood coagulation. Under normal physiological conditions, TF is expressed in extravascular and perivascular cells but not in vascular endothelial cells and monocytes. TF can be induced in these cells by inflammatory regulators and other stimulators, such as LPS, thrombin, oxidized lipoproteins, and certain growth factors. An earlier study showed that growing primary cultures of human umbilical vein endothelial cells (HUVECs) with endothelial cell growth supplement (ECGS) and heparin had impaired the ability of monolayers to express surface membrane TF activity after perturbation. The mechanism by which ECGS suppressed TF activity was not known. In the present study, we investigated the effect of recombinant acidic and basic fibroblast growth factors (aFGF and bFGF) on the induction of TF in a HUVEC cell line and a fibroblast cell line. Both aFGF and bFGF suppressed the phorbol myristate acetate-induced expression of TF in endothelial cells but not the serum-induced expression of TF in fibroblast cells. Diminished expression of the cell surface TF activity observed in endothelial cells grown with aFGF or bFGF was due to the accumulation of a lower number of TF mRNA transcripts. TF mRNA stability was not altered in HUVECs grown with aFGF or bFGF. Nuclear run-on experiments revealed that the transcription of TF and several other genes that play an important role in inflammation and angiogenesis was reduced in the endothelial cells that were cultured with aFGF or bFGF. The diminished expression of TF may be part of a generalized response of endothelial cells to FGF that facilitates migration of endothelial cells during angiogenesis.

Cells, Cultured↗
PubMed1996

Mechanism and effects of the binding of lupus anticoagulant IgG and prothrombin to surface phospholipid.

We report here experiments on how lupus anticoagulant antibodies (LA IgG) that react with prothrombin bind to surface phospholipid and affect prothrombin's affinity for surface phospholipid and activation to thrombin. LA IgG was purified by protein A chromatography from the plasma of 16 patients of whom four had associated hypoprothrombinemia and 10 had experienced thrombosis. Many LA IgG bound, in the absence of phospholipid and calcium, not only to immobilized prothrombin but to both prothrombin 1 and fragment 1, which established at least an oligoclonal origin of LA IgG. No LA IgG bound to thrombin. Although prothrombin and Ca2+ were required to support binding of LA IgG to immobilized phosphatidylserine (PS), prothrombin at higher concentrations inhibited binding, presumably by competing with prothrombin/LA IgG complexes for PS binding sites. Prothrombin 1, which cannot bind to PS, also inhibited binding of many LA IgG to PS, presumably by forming competing soluble prothrombin 1/LA IgG complexes. Despite their ability to react with prothrombin independent of phospholipid, LA IgG enhanced binding of prothrombin to immobilized phospholipid and to cultured human umbilical vein endothelial cells. Prothrombin bound with LA IgG to the surface of endothelial cell monolayers could be activated to thrombin after supernatant prothrombin and LA IgG were washed away. The relation is discussed of these observations to a hypothesis that LA IgG mediated concentration of prothrombin on cell surface phospholipid represents a mechanism by which LA IgG could increase thrombotic risk.

Animals↗
PubMed1996

Plasma PIVKA proteins in rabbits given warfarin.

The presence of partially carboxylated forms of the vitamin K dependent coagulation factors (PIVKA) was evaluated in the plasma of rabbits treated with warfarin. Excess antigen over activity as measured in rabbit specific assays was taken as evidence for PIVKA. Our data confirm a previous report of the absence of plasma PIVKA prothrombin. In contrast, plasma PIVKA factors VII, IX, and X were demonstrable. A striking excess of plasma factor IX antigen over activity was measured and a large fraction of the factor IX antigen persisted in the plasma after its adsorption with barium citrate.

Animals↗
PubMed1996

Studies of the activation of factor VII bound to tissue factor.

Experiments were performed to evaluate activation of factor VII bound to relipidated tissue factor (TF) in suspension and to TF constitutively expressed on the surface of an ovarian carcinoma cell line (OC-2008). Activation was assessed by measuring cleavage of 125I-factor VII and by the ability of unlabeled factor VII to catalyze activation of a variant factor IX molecule that, after activation, cannot back-activate factor VII. Factor Xa was found to effectively activate factor VII bound to TF relipidated in either acidic or neutral phospholipid vesicles. Autoactivation of factor VII bound to TF in suspension was dependent on the preparation of TF apoprotein used and the technique of its relipidation. This highlights the need for caution in extrapolating data from TF in suspension to the activation of factor VII bound to cell surfaces during hemostasis. A relatively slow activation of factor VII bound to OC-2008 monolayers in the absence of added protease was observed consistently. Antithrombin in the presence or absence of heparin prevented this basal activation, whereas TF pathway inhibitor (TFPI/factor Xa complexes had only a limited inhibitory effect. Adding a substrate concentration of factor X markedly enhanced basal activation of factor VII, but both TFPI/factor Xa and antithrombin/heparin abolished this enhancement. Overall, our data are compatible with the hypothesis that not all factor VII/TF complexes formed at a site of tissue injury are readily activated to factor VIIa (VIIa)/TF complexes during hemostasis. The clinical significance of this is discussed.

Cell Membrane↗
PubMed1996

Gonadotropin-releasing hormone agonist influences absolute levels of lymphocyte subsets in vivo in male mice.

Our earlier studies have demonstrated that gonadotropin-releasing hormone (GnRH) agonists suppress immune system function in female mice. No systematic studies regarding the effect of gender on immune system function following GnRH agonist treatment, however, have been reported. This study, therefore, investigated sequential changes in lymphocyte subsets in 3- and 10-week-old male mice following agonist or placebo administration. Changes in immunophenotypic expression of lymphocytes from thymus, bone marrow, spleen, and blood were analysed at periodic intervals. Upon agonist administration, plasma testosterone levels were significantly increased in pre-pubertal mice, but were significantly decreased in post-pubertal males. Absolute thymic weights, thymocytes and T subsets were significantly increased from the third week regardless of gonadal status. Blood lymphocyte subsets showed a decreasing trend after agonist administration in pre-pubertal males, whereas no differences were observed in post-pubertal males. No significant differences were observed in spleen cells after agonist administration. These studies, together with earlier observations in female mice indicate that GnRH agonist effects on the immune system, are independent of steroid hormone levels. In contrast to suppressive effects in females, GnRH agonist induce no change or ultimately enhanced lymphocyte counts in males, indicating differential effects on the immune system between males and females. This may have important implications for the treatment of various diseases.

Animals↗
PubMed1996

Ligand-induced protease receptor translocation into caveolae: a mechanism for regulating cell surface proteolysis of the tissue factor-dependent coagulation pathway.

The ability to regulate proteolytic functions is critical to cell biology. We describe events that regulate the initiation of the coagulation cascade on endothelial cell surfaces. The transmembrane protease receptor tissue factor (TF) triggers coagulation by forming an enzymatic complex with the serine protease factor VIIa (VIIa) that activates substrate factor X to the protease factor Xa (Xa). Feedback inhibition of the TF-VIIa enzymatic complex is achieved by the formation of a quaternary complex of TF-VIIa, Xa, and the Kunitz-type inhibitor tissue factor pathway inhibitor (TFPI). Concomitant with the downregulation of TF-VIIa function on endothelial cells, we demonstrate by immunogold EM that TF redistributes to caveolae. Consistently, TF translocates from the Triton X-100-soluble membrane fractions to low-density, detergent-insoluble microdomains that inefficiently support TF-VIIa proteolytic function. Downregulation of TF-VIIa function is dependent on quaternary complex formation with TFPI that is detected predominantly in detergent-insoluble microdomains. Partitioning of TFPI into low-density fractions results from the association of the inhibitor with glycosyl phosphatidylinositol anchored binding sites on external membranes. Free Xa is not efficiently bound by cell-associated TFPI; hence, we propose that the transient ternary complex of TF-VIIa with Xa supports translocation and assembly with TFPI in glycosphingolipid-rich microdomains. The redistribution of TF provides evidence for an assembly-dependent translocation of the inhibited TF initiation complex into caveolae, thus implicating caveolae in the regulation of cell surface proteolytic activity.

Blood Coagulation↗
PubMed1996

Cells and the activation of factor VII.

Binding of factor VII to tissue factor (TF) present on or released from cells initiates TF-dependent coagulation in vivo. Earlier data obtained with relipidated TF suspensions led to the hypothesis that all factor VII bound to limiting TF sites exposed after tissue injury could be rapidly activated to VIIa/TF complexes by low concentrations of factor Xa, IXa, and VIIa. However, newer data on the activation of factor VII bound to TF expressed on cultured cell monolayers support the hypothesis that not all VII/TF complexes formed at a site of tissue injury are readily activated to VIIa/TF complexes during hemostasis. This is possibly related to why recombinant factor VIIa can bypass impaired IXa/VIIIa/phospholipid complex function in hemostasis.

Blood Cells↗
PubMed1995

Tissue factor residues Lys165 and Lys166 are essential for rapid formation of the quaternary complex of tissue factor.VIIa with Xa.tissue factor pathway inhibitor.

The extrinsic coagulation pathway is initiated by the binding of plasma factor VII(a) (VIIa) to the cell surface receptor tissue factor (TF), which serves as the cofactor for the ligand protease VIIa in the activation of macromolecular substrate factors X and IX. The catalytic function of the TF.VIIa complex is regulated by a specific Kunitz-type inhibitor, tissue factor pathway inhibitor (TFPI), which forms a stoichiometric complex with the serine protease factor Xa (Xa), resulting in greatly accelerated inhibition of the extrinsic initiation complex as compared to free inhibitor. In the present study we identify specific residues in the TF-VIIa complex that are involved in the factor Xa-mediated acceleration of TFPI inhibitory function. VIIa residue Arg290, which contributes to extended recognition of macromolecular substrate factor X, is not involved in the interaction with the TFPI.Xa complex. In contrast, TF residues Lys165 and Lys166, which are important for the activation of factor X, are required for the accelerated inhibition of the TF.VIIa complex by TFPI mediated by factor Xa. These data indicate that similar interactions contribute to the assembly of substrate factor X as well as of product Xa after complex formation with TFPI, suggesting a central role for the carboxyl-terminal structural module of TF in regulating the proteolytic activity of TF.VIIa.

Blood Coagulation↗
PubMed1995

Mechanism of antithrombin III inhibition of factor VIIa/tissue factor activity on cell surfaces. Comparison with tissue factor pathway inhibitor/factor Xa-induced inhibition of factor VIIa/tissue factor activity.

Recent studies have shown that antithrombin III (AT III)/heparin is capable of inhibiting the catalytic activity of factor VIIa bound either to relipidated tissue factor (TF) in suspension or to TF expressed on cell surfaces. We report studies of the mechanism of which by AT III inhibits factor VIIa bound to cell surface TF and compare this inhibitory mechanism with that of tissue factor pathway inhibitor (TFPI)-induced inhibition of factor VIIa/TF. AT III alone and AT III/heparin to a greater extent reduced factor VIIa bound to cell surface TF. Our data show that the decrease in the amount of factor VIIa associated with cell surface TF in the presence of AT III was the result of (1) accelerated dissociation of factor VIIa from cell surface TF after the binding of AT III to factor VIIa/TF complexes and (2) the inability of the resultant free factor VIIa-AT III complexes to bind effectively to a new cell surface TF site. Binding of TFPI/factor Xa to cell surface factor VIIa/TF complexes markedly decreased the dissociation of factor VIIa from the resultant quaternary complex of factor VIIa/TF/TFPI/factor Xa. Addition of high concentrations of factor VIIa could reverse the AT III-induced inhibition of cell surface factor VIIa/TF activity but not TFPI/factor Xa-induced inhibition of factor VIIa/TF activity.

Antithrombin III↗
PubMed1995

Evidence for an essential role of tissue factor dependent blood coagulation in the pathogenesis of the local Shwartzman reaction.

Adherence and aggregation of leukocytes within the vessels of a prepared skin site has been shown to be essential for the pathogenesis of the local Shwartzman reaction (LSR) (Argenbright and Barton: J Clin Invest 89: 259, 1992). We have now performed experiments in rabbits to evaluate whether coagulation within the vessels of a prepared site is a second requirement for the reaction. Skin sites were prepared by an intradermal injection of endotoxin 24 hours before a provoking intravenous injection of endotoxin. Thirteen control rabbits all developed the LSR. Seven of 12 rabbits given warfarin to achieve anticoagulation approximating that used therapeutically in humans before the provoking injection were protected against the LSR (p = 0.003). Five of nine rabbits given anti-rabbit factor X IgG before the provoking injection to yield mean values in individual rabbits of between 7% and 18% plasma factor X activity were protected against the LSR (p = 0.009). Six of 11 rabbits given anti-rabbit factor VII IgG before the provoking injection to yield mean values in individual rabbits of between < 0.5% and 2.2% were protected against the LSR (p = 0.007). Four rabbits failed to develop the LSR at an endotoxin-prepared skin site when an infusion of tissue factor (TF) causing substantial intravascular coagulation was substituted for a provoking injection of endotoxin. It would appear that two events are required for the pathogenesis of the LSR provoked by endotoxin: formation of aggregated masses of WBC in the prepared skin vessels and deposition of fibrin due to TF-initiated coagulation.

Animals↗