Hema Kothari
Research interests
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InterestsHuman Physiology
Publications
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2.41Impact points
Tissue Factor encryption and decryption: Facts and controversies.
Thrombosis research. 03/2012;
Tissue factor (TF)-initiated coagulation plays a critical role in both hemostatsis and thrombosis. It is generally believed that most of the tissue factor expressed on cell surfaces is maintained in a cryptic, i.e., coagulantly inactive state and an activation step (decryption) is required for the e... [more] Tissue factor (TF)-initiated coagulation plays a critical role in both hemostatsis and thrombosis. It is generally believed that most of the tissue factor expressed on cell surfaces is maintained in a cryptic, i.e., coagulantly inactive state and an activation step (decryption) is required for the expression of maximum TF procoagulant activity. However, what exactly constitutes cryptic or procoagulant TF, molecular differences between these two forms and mechanisms that are responsible for transformation from one to the other form are not entirely clear and remain highly controversial, thus are a matter of ongoing debate. This brief review discusses pertinent literature on TF encryption/decryption with specific emphasis on the role of membrane phospholipids and reduction/oxidation of the TF Cys186-Cys209 disulfide bond in regulating TF activity at cell surfaces.
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4.32Impact points
Lipoprotein receptor-related protein 1 regulates collagen 1 expression, proteolysis, and migration in human pleural mesothelial cells.
American journal of respiratory cell and molecular biology. 02/2012; 46(2):196-206.
The low-density lipoprotein receptor-related protein 1 (LRP-1) binds and can internalize a diverse group of ligands, including members of the fibrinolytic pathway, urokinase plasminogen activator (uPA), and its receptor, uPAR. In this study, we characterized the role of LRP-1 in uPAR processing, col... [more] The low-density lipoprotein receptor-related protein 1 (LRP-1) binds and can internalize a diverse group of ligands, including members of the fibrinolytic pathway, urokinase plasminogen activator (uPA), and its receptor, uPAR. In this study, we characterized the role of LRP-1 in uPAR processing, collagen synthesis, proteolysis, and migration in pleural mesothelial cells (PMCs). When PMCs were treated with the proinflammatory cytokines TNF-α and IL-1β, LRP-1 significantly decreased at the mRNA and protein levels (70 and 90%, respectively; P < 0.05). Consequently, uPA-mediated uPAR internalization was reduced by 80% in the presence of TNF-α or IL-1β (P < 0.05). In parallel studies, LRP-1 neutralization with receptor-associated protein (RAP) significantly reduced uPA-dependent uPAR internalization and increased uPAR stability in PMCs. LRP-1-deficient cells demonstrated increased uPAR t(1/2) versus LRP-1-expressing PMCs. uPA enzymatic activity was also increased in LRP-1-deficient and neutralized cells, and RAP potentiated uPA-dependent migration in PMCs. Collagen expression in PMCs was also induced by uPA, and the effect was potentiated in RAP-treated cells. These studies indicate that TNF-α and IL-1β regulate LRP-1 in PMCs and that LRP-1 thereby contributes to a range of pathophysiologically relevant responses of these cells.
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Tissue factor: mechanisms of decryption.
Frontiers in bioscience (Elite edition). 01/2012; 4:1513-27.
It is generally believed that only a small fraction of the tissue factor (TF) found on cell surfaces is active whereas the vast majority is cryptic in coagulation. It is unclear how cryptic TF differs from the coagulant active TF or potential mechanisms involved in transformation of cryptic TF to th... [more] It is generally believed that only a small fraction of the tissue factor (TF) found on cell surfaces is active whereas the vast majority is cryptic in coagulation. It is unclear how cryptic TF differs from the coagulant active TF or potential mechanisms involved in transformation of cryptic TF to the coagulant active form. Exposure of phosphatidylserine (PS) in response to various chemical or pathophysiological stimuli has been considered as the most potent inducer of TF decryption. In addition to PS, TF self-association and association with specialized membrane domains may also play a role in TF decryption. It has been suggested recently that protein disulfide isomerase regulates TF decryption through its oxidoreductase activity by targeting Cys186-Cys209 disulfide bond in TF extracellular domain or regulating the PS equilibrium at the plasma membrane. However, this hypothesis requires further validation to become an accepted mechanism. In this article, we critically review literature on TF encryption/decryption with specific emphasis on recently published data and provide our perspective on this subject.
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10.56Impact points
Factor VIIa bound to endothelial cell protein C receptor activates protease activated receptor-1 and mediates cell signaling and barrier protection.
Blood. 01/2011; 117(11):3199-208.
Recent studies have shown that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR), a cellular receptor for protein C and activated protein C, but the physiologic significance of this interaction is unclear. In the present study, we show that FVIIa, upon binding to EPCR on en... [more] Recent studies have shown that factor VIIa (FVIIa) binds to the endothelial cell protein C receptor (EPCR), a cellular receptor for protein C and activated protein C, but the physiologic significance of this interaction is unclear. In the present study, we show that FVIIa, upon binding to EPCR on endothelial cells, activates endogenous protease activated receptor-1 (PAR1) and induces PAR1-mediated p44/42 mitogen-activated protein kinase (MAPK) activation. Pretreatment of endothelial cells with FVIIa protected against thrombin-induced barrier disruption. This FVIIa-induced, barrier-protective effect was EPCR dependent and did not involve PAR2. Pretreatment of confluent endothelial monolayers with FVIIa before thrombin reduced the development of thrombin-induced transcellular actin stress fibers, cellular contractions, and paracellular gap formation. FVIIa-induced p44/42 MAPK activation and the barrier-protective effect are mediated via Rac1 activation. Consistent with in vitro findings, in vivo studies using mice showed that administration of FVIIa before lipopolysaccharide (LPS) treatment attenuated LPS-induced vascular leakage in the lung and kidney. Overall, our present data provide evidence that FVIIa bound to EPCR on endothelial cells activates PAR1-mediated cell signaling and provides a barrier-protective effect. These findings are novel and of great clinical significance, because FVIIa is used clinically for the prevention of bleeding in hemophilia and other bleeding disorders.
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10.56Impact points
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10.56Impact points
Cystine 186-cystine 209 disulfide bond is not essential for the procoagulant activity of tissue factor or for its de-encryption.
Blood. 03/2010; 115(21):4273-83.
Tissue factor (TF) on cell surfaces resides mostly in a cryptic state. It is not entirely clear how cryptic TF differs from procoagulantly active TF and how deencryption occurs. Here, we critically evaluated the importance of cystine 186-cystine 209 (Cys186-Cys209) bond formation for TF procoagulant... [more] Tissue factor (TF) on cell surfaces resides mostly in a cryptic state. It is not entirely clear how cryptic TF differs from procoagulantly active TF and how deencryption occurs. Here, we critically evaluated the importance of cystine 186-cystine 209 (Cys186-Cys209) bond formation for TF procoagulant activity and its de-encryption. Chinese hamster ovary cells transfected with TF(C186S), TF(C209S), or TF(C186S/C209S) expressed little procoagulant activity at the cell surface. TF monoclonal antibody and activated factor VII (FVIIa) binding studies showed that little TF protein was present at the cell surface in cells expressing mutant TF. Similar data were obtained in human umbilical vein endothelial cells (HUVECs) transduced to express TF(C186S), TF(C209S), or TF(C186S/C209S). Analysis of TF activity in HUVECs expressing similar levels of wild-type TF and TF(C186S/C209S) showed that TF mutant in the presence of saturating concentrations of FVIIa exhibited similar coagulant activity as that of wild-type TF. More importantly, treatment of HUVECs expressing TF(C186S/C209S) with HgCl(2) or ionomycin increased the cell-surface TF activity to the same extent as that of the wild-type TF. Our data provide clear evidence that TF lacking the Cys186-Cys209 bond is coagulantly active once it is complexed with FVIIa, and TF de-encryption does not require Cys186-Cys209 disulfide bond formation.
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10.56Impact points
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1.78Impact points
Possibility of membrane modification as a mechanism of antimony resistance in Leishmania donovani.
Parasitology international. 04/2007; 56(1):77-80.
Resistance to antimonials has become a clinical threat in the treatment of visceral leishmaniasis (VL). Unravelling the resistance mechanism needs attention to circumvent the problem of drug resistance. In one of the resistant isolates, we earlier identified a gene (PG1) implicated in antimony resis... [more] Resistance to antimonials has become a clinical threat in the treatment of visceral leishmaniasis (VL). Unravelling the resistance mechanism needs attention to circumvent the problem of drug resistance. In one of the resistant isolates, we earlier identified a gene (PG1) implicated in antimony resistance whose localization in the present study was confirmed on the pellicular plasma membrane of the parasite thereby indicating towards membrane modification as a mechanism of resistance in this resistant isolate.
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1.51Impact points
Translation of open reading frame in kinetoplast DNA minicircles of clinical isolates of L. donovani.
Parasitology research. 04/2007; 100(4):893-7.
Till today, it remains an enigma whether the open reading frames said to be transcribed in minicircle sequences are indeed translated into protein products or not. We establish a protein-coding gene in minicircle variable region of kinetoplast DNA from clinical isolates of Leishmania donovani. The p... [more] Till today, it remains an enigma whether the open reading frames said to be transcribed in minicircle sequences are indeed translated into protein products or not. We establish a protein-coding gene in minicircle variable region of kinetoplast DNA from clinical isolates of Leishmania donovani. The protein was expressed as an N-tagged green fluorescent protein (GFP) fusion protein in leishmanial expression system. Fluorescence microscopy of the transfectants carrying recombinant GFP construct showed the protein to be localized on the plasmalemma of the parasite. This shows that the minicircle transcript is indeed translated into a protein product in the parasite cell and further points toward probable biological function of minicircles in kinetoplastids.
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1.56Impact points
Prokaryotic expression, purification, and polyclonal antibody production against a novel drug resistance gene of Leishmania donovani clinical isolate.
Protein expression and purification. 02/2006; 45(1):15-21.
Diseases produced by protozoan parasites are one of the main causes of morbidity and mortality around the world, affecting millions of people. Among these, leishmaniasis has become the second most common cause of death and the problem is further complicated by the expansion of parasite resistance to... [more] Diseases produced by protozoan parasites are one of the main causes of morbidity and mortality around the world, affecting millions of people. Among these, leishmaniasis has become the second most common cause of death and the problem is further complicated by the expansion of parasite resistance to the conventional drugs. The high rate of therapeutic failure thus calls for new rational approaches to develop alternative drugs. Understanding resistance mechanisms may help identify new targets for drug development. So we present here the cloning, expression, purification, and antibody production of a gene implicated in imparting resistance to pentavalent antimony (SbV) in clinical isolates of kala azar with a view to gain insight into the novel mechanism of its drug resistance.
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1.56Impact points
Overexpression in Escherichia coli and purification of pteridine reductase (PTR1) from a clinical isolate of Leishmania donovani.
Protein expression and purification. 01/2005; 38(2):228-36.
Pteridine reductase 1 (PTR1) is part of a novel metabolic pathway in Leishmania associated with folate metabolism. Its main function is to salvage pterins but a second one is to reduce folates. The novelty and possible uniqueness of the pathway in which PTR1 is involved opens the possibility of deve... [more] Pteridine reductase 1 (PTR1) is part of a novel metabolic pathway in Leishmania associated with folate metabolism. Its main function is to salvage pterins but a second one is to reduce folates. The novelty and possible uniqueness of the pathway in which PTR1 is involved opens the possibility of developing specific inhibitors, which in combination with dihydrofolate reductase inhibitors could be highly effective against Leishmania. In order to increase our understanding of this putative important chemotherapeutic target, we present here the cloning, overexpression and purification of this enzyme from a clinical isolate of Leishmania donovani causing kala azar in India. This recombinant enzyme will set the basis for inhibition studies as well as for structure-function relationships.
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Possibility of membrane modification as a mechanism of antimony resistance in Leishmania donovani
Parasitology International.
Resistance to antimonials has become a clinical threat in the treatment of visceral leishmaniasis (VL). Unravelling the resistance mechanism needs attention to circumvent the problem of drug resistance. In one of the resistant isolates, we earlier identified a gene (PG1) implicated in antimony resis... [more] Resistance to antimonials has become a clinical threat in the treatment of visceral leishmaniasis (VL). Unravelling the resistance mechanism needs attention to circumvent the problem of drug resistance. In one of the resistant isolates, we earlier identified a gene (PG1) implicated in antimony resistance whose localization in the present study was confirmed on the pellicular plasma membrane of the parasite thereby indicating towards membrane modification as a mechanism of resistance in this resistant isolate.
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Overexpression in Escherichia coli and purification of pteridine reductase (PTR1) from a clinical isolate of Leishmania donovani
Protein Expression and Purification.
Pteridine reductase 1 (PTR1) is part of a novel metabolic pathway in Leishmania associated with folate metabolism. Its main function is to salvage pterins but a second one is to reduce folates. The novelty and possible uniqueness of the pathway in which PTR1 is involved opens the possibility of deve... [more] Pteridine reductase 1 (PTR1) is part of a novel metabolic pathway in Leishmania associated with folate metabolism. Its main function is to salvage pterins but a second one is to reduce folates. The novelty and possible uniqueness of the pathway in which PTR1 is involved opens the possibility of developing specific inhibitors, which in combination with dihydrofolate reductase inhibitors could be highly effective against Leishmania. In order to increase our understanding of this putative important chemotherapeutic target, we present here the cloning, overexpression and purification of this enzyme from a clinical isolate of Leishmania donovani causing kala azar in India. This recombinant enzyme will set the basis for inhibition studies as well as for structure–function relationships.
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