Protein disulfide isomerase uses thrombin-antithrombin complex as a template to bind its target protein and alter the blood coagulation rates.

During inflammation and situations of cellular stress protein disulfide isomerase (PDI) is released in the blood plasma from the platelet and endothelial cells to influence thrombosis. The addition of exogenous PDI makes the environment pro-thrombotic by inducing disulfide bond formation in specific plasma protein targets like vitronectin, factor V, and factor XI. However, the mechanistic details of PDI interaction with its target remain largely unknown. A decrease in the coagulation time was detected in activated partial thromboplastin time (APTT), prothrombin time (PT), and thrombin time (TT) on addition of the purified recombinant PDI (175 nM). The coagulation time can be controlled using an activator (quercetin penta sulfate, QPS) or an inhibitor (quercetin 3-rutinoside, Q3R) of PDI activity. Likewise, the PDI variants that increase the PDI activity (H399R) decrease, and the variant with low activity (C53A) increases the blood coagulation time. An SDS-PAGE and Western blot analysis showed that the PDI does not form a stable complex with either thrombin or antithrombin (ATIII) but it uses the ATIII-thrombin complex as a template to bind and maintain its activity. A complete inhibition of thrombin activity on the formation of ATIII-thrombin-PDI complex, and the complex-bound PDI-catalyzed disulfide bond formation of the target proteins may control the pro- and anti-thrombotic role of PDI.

A common protein C inhibitor exosite partially controls the heparin induced activation and inhibition of serine proteases.

Protein C inhibitor (PCI) maintains hemostasis by inhibiting both procoagulant and anticoagulant serine proteases, and plays important roles in coagulation, fibrinolysis, reproduction, and anti-angiogenesis. The reactive site loop of PCI traps and irreversibly inhibits the proteases like APC (activating protein C), thrombin (FIIa) and factor Xa (FXa). Previous studies on antithrombin (ATIII) had identified Tyr253 and Glu255 as functional exosites that interact and aid in the inhibition of factor IXa and FXa. Presence of exosite in PCI is not known, however a sequence comparison with the PCI from different vertebrate species and ATIII identified Glu239 to be absolutely conserved. PCI residues analogous to ATIII exosite residues were mutated to R238A and E239A. Purified variant PCI in the presence of heparin (10 µg/ml) showed a 2-4 fold decrease in the rate of inhibition of the proteases. However, the stoichiometry of inhibition of FIIa, APC, and FXa by native PCI, R238A and E239A variants were found to be close to 1.0, which also indicated the formation of stable complexes based on SDS-PAGE and western blot analysis with thrombin and APC. Our findings revealed the possible presence of an exosite in PCI that influences the protease inhibition rates.

Naringin binds to protein disulfide isomerase to inhibit its activity and modulate the blood coagulation rates: Implications in controlling thrombosis.

Currently used antithrombotic drugs are beset with several drawbacks which necessitates the need for new and cheaper alternatives. Protein disulfide isomerase (PDI) is secreted in the blood plasma in cellular stress conditions and initiates the thrombus formation. A screening of library of natural compounds revealed that naringin had a high binding affinity for the PDI (-8.2 kcal/mol). Recombinant PDI was purified using the affinity chromatography. Incubation of purified PDI (3 µM) with naringin (0-100 µM, pH 7.4, 25 °C) partially modulated its conformation. Consequently, the fluorescence emission spectra of the PDI binding to naringin were assessed using the Stern-Volmer equation, which indicated an association constant of 2.78 × 104 M-1 suggesting an appreciable affinity for the naringin, with a unique binding site. An insulin turbidity assay showed that PDI activity is decreased in the presence of naringin indicating inhibition. Molecular dynamic simulation studies showed the changes in the PDI structure on binding to the naringin. Incubation of naringin (80 µM) in fresh human plasma along with exogenous PDI (175 nM) showed a significant delay in the intrinsic and extrinsic coagulation pathways. We show that naringin is able to modulate the PDI conformation and activity resulting in altered blood coagulation rates.

Mannose 2, 3, 4, 5, 6-O-pentasulfate (MPS): a partial activator of human heparin cofactor II with anticoagulation potential.

Thromboembolic diseases are a major cause of mortality in human and the currently available anticoagulants are associated with various drawbacks, therefore the search for anticoagulants that have better safety profile is highly desirable. Compounds that are part of the dietary routine can be modified to possibly increase their anticoagulant potential. We show mannose 2,3,4,5,6-O-pentasulfate (MPS) as a synthetically modified form of mannose that has appreciable anticoagulation properties. An in silico study identified that mannose in sulfated form can bind effectively to the heparin-binding site of antithrombin (ATIII) and heparin cofactor II (HCII). Mannose was sulfated using a simple sulfation strategy-involving triethylamine-sulfur trioxide adduct. HCII and ATIII were purified from human plasma and the binding analysis using fluorometer and isothermal calorimetry showed that MPS binds at a unique site. A thrombin inhibition analysis using the chromogenic substrate showed that MPS partially enhances the activity of HCII. Further an assessment of in vitro blood coagulation assays using human plasma showed that the activated partial thromboplastin time (APTT) and prothrombin time (PT) were prolonged in the presence of MPS. A molecular dynamics simulation analysis of the HCII-MPS complex showed fluctuations in a N-terminal loop and the cofactor binding site of HCII. The results indicate that MPS is a promising lead due to its effect on the in vitro coagulation rate.Communicated by Ramaswamy H. Sarma.

Strand 1A variant in neuroserpin shows increased aggregation and no loss of inhibition: implication in ameliorating polymerization to retain activity.

Neuroserpin (NS) is predominantly expressed in the brain and is the primary inhibitor of tissue plasminogen activator (tPA). NS variants are associated with the neurogenerative disease termed familial encephalopathy with neuroserpin inclusion bodies (FENIB). The disease is characterized by variable age of onset and severity. The reactive center loop (RCL) insertion-based inhibitory mechanism of NS requires a coordinated conformational change leading to a shift in the strands of the ß-sheet A and movement of helix F. Strand 1A is connected to the helix F at its C terminal end and with the strand 2A at its N terminal, both these domain move for accommodating the inserting loop; therefore, a variant that influences their movement may alter the inhibition rates. A molecular dynamic simulation analysis of a H138C NS variant from strand 1A showed a large decrease in conformational fluctuations as compared with wild-type NS. H138 was mutated, expressed, purified and a native-PAGE and transmission electron microscopy (TEM) analysis showed that this variant forms large molecular weight aggregates on a slight increase in temperature. However, a circular dichroism analysis showed its secondary structure to be largely conserved. Surprisingly, its tPA inhibition activity and complex formation remain unhindered even after the site-specific labeling of H138C with Alexa fluor C5 maleimide. Further, a helix F-strand 1A (W154C-H138C) double variant still shows appreciable inhibitory activity. Increasingly, it appears that aggregation and not loss of inhibition is the more likely cause of shutter region-based variants phenotypes, indicating that hindering polymer formation using small molecules may retain inhibitory activity in pathological variants of NS.

Identification and validation of two alternatively spliced novel isoforms of human α-1-antichymotrypsin.

α-1-antichymotrypsin (ACT) is a serine proteinase inhibitor that controls the activity of proteases like chymotrypsin, cathepsin G and mast cell chymase. Familial variants of ACT results in liver and lung diseases, but it is also reported to be associated with several other disease conditions. ACT is mainly synthesized in the liver using four coding exons, namely E1, E2, E3 and E4 encoding a 423 amino acid protein that also includes a 23 amino acid signal peptide. It is found to be associated with amyloid plaques and is elevated during inflammatory response and modulates cytokine based signal transduction pathways, independent of its anti-protease activity. Therefore, the multispecificity of ACT and its non-inhibitory roles in diseased conditions warrants an assessment of possible existence of the other isoforms. Consequently, scanning of introns, 5' and 3' region of the ACT gene using computational tools like FGENESH and FEX did indicate the presence of coding regions. Using a combined approach of bioinformatics and molecular biology, we have found one novel exon located in the intronic region between exons E1 and E2, that splices with exon E2 and replaces N-terminal exon E1, generating an ACT isoform with a novel 151 base pair N-terminus. This isoform was found to lack the signal sequence and is smaller in size but its reactive centre loop remains intact. A truncated transcript was also confirmed with an extension of the E3 by a 12 nucleotide intronic region including a stop codon. Modelling studies show that due to removal of E4 this isoform lacks the RCL. Novel isoform ACT-N lacks E1 but has a conserved RCL. However, due to loss of strands of ß-sheet A, it may also be inactive, but with ability to bind the target proteases. The novel truncated ACT-T isoform lacks the RCL and may have a non-inhibitory role. These hypothesis will need further work for functional validation.

Detection of truncated isoforms of human neuroserpin lacking the reactive center loop: Implications in noninhibitory role.

Neuroserpin is a serine protease inhibitor expressed mainly in the brain and at low levels in other tissues like the kidney, testis, heart, and spinal cord. It is involved in the inhibition of tissue plasminogen activator (tPA), plasmin, and to a lesser extent, urokinase-type plasminogen (uPA). Neuroserpin has also been shown to plays noninhibitory roles in the regulation of N-cadherin-mediated cell adhesion. It is involved in neuroprotection from seizure and stroke through tPA-mediated inhibition and also through its other protease targets. Mutations in critical domains of neuroserpin lead to its polymerization and neuronal death. In this study, a novel truncated isoform of human neuroserpin was identified in the brain and liver, which was confirmed by reverse transcriptase-PCR and DNA sequencing using exon-specific primers. Structural characterization of novel isoform using MD simulations studies indicated that it lacks the reactive center loop (RCL) but largely maintains its secondary structure fold. The novel truncated variant was cloned, expressed, and purified. A comparative intrinsic fluorescence and 4,4'-bis-1-anilino naphthalene 8-sulfonate studies revealed a decrease in fluorescence emission intensity and a more exposed hydrophobic surface as compared to the reported isoform. However, the novel isoform has lost its ability for tPA inhibition and complex formation. The absence of RCL indicates a noninhibitory role for the truncated isoform, prompting a detailed search and identification of two smaller isoforms in the human brain. With indications of the noninhibitory role of neuroserpin, identifying novel isoforms that appear to be without the tPA recognition domain is significant.

Contrasting conformational dynamics of ß-sheet A and helix F with implications in neuroserpin inhibition and aggregation.

Neuroserpin (NS) is an inhibitory protein of serpin super family, its shutter region variants have high propensity to aggregate leading to pathological disorders like familial encephalopathy with NS inclusion bodies (FENIB). Helix F and ß-sheet A of NS participate in the tissue plasminogen activator (tPA) inhibition but the mechanism is not yet completely understood. A microsecond (µs) molecular dynamics simulation of the helix F and strand 3A variants showed predominant fluctuations in the loop connecting the strands of ß-sheet A. Therefore to understand the role of helix F and strand 3A of ß-sheet A, cysteine was incorporated at the position N182 in stand 3A (N182C) and position W154 (W154C) in the helix F using site-directed mutagenesis. Purified variants were further labeled with Alexa Fluor488 C5 maleimide dye. Temperature dependent study using non-denaturing PAGE showed the formation of large aggregates of helix F variant W154C but not the strand 3A N182C variant. Interestingly tPA inhibition was found to be decreased in the labeled N182C with decreased tPA-complex formation as compared to labeled W154C NS variant. The fluorescence emission intensity of the labeled helix F variant W154C decreased in the presence of an increasing concentration of tPA, whereas an increase in emission intensity was observed in labeled strand 3A variant N182C, indicating more exposure of strand 3A and shielding of helix F. Taken together the data shows that helix F has a predominant role in the aggregation but a minor role in the inhibition mechanism.

Using serpins cysteine protease cross-specificity to possibly trap SARS-CoV-2 Mpro with reactive center loop chimera.

Human serine protease inhibitors (serpins) are the main inhibitors of serine proteases, but some of them also have the capability to effectively inhibit cysteine proteases. Severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) main protease (Mpro) is a chymotrypsin-type cysteine protease that is needed to produce functional proteins essential for virus replication and transcription. Serpin traps its target proteases by presenting a reactive center loop (RCL) as protease-specific cleavage site, resulting in protease inactivation. Mpro target sites with its active site serine and other flanking residues can possibly interact with serpins. Alternatively, RCL cleavage site of serpins with known evidence of inhibition of cysteine proteases can be replaced by Mpro target site to make chimeric proteins. Purified chimeric serpin can possibly inhibit Mpro that can be assessed indirectly by observing the decrease in ability of Mpro to cleave its chromogenic substrate. Chimeric serpins with best interaction and active site binding and with ability to form 1:1 serpin-Mpro complex in human plasma can be assessed by using SDS/PAGE and Western blot analysis with serpin antibody. Trapping SARS-CoV-2 Mpro cysteine protease using cross-class serpin cysteine protease inhibition activity is a novel idea with significant therapeutic potential.

Identification and characterization of a novel isoform of heparin cofactor II in human liver.

Heparin cofactor II (HCII) is predominantly expressed in the liver and inhibits thrombin in blood plasma to influence the blood coagulation cascade. Its deficiency is associated with arterial thrombosis. Its cleavage by neutrophil elastase produces fragment that helps in neutrophil chemotaxis in the acute inflammatory response in human. In the present study, we have identified a novel alternatively spliced transcript of the HCII gene in human liver. This novel transcript includes an additional novel region in continuation with exon 3 called exon 3b. Exon 3b acts like an alternate last exon, and hence its inclusion in the transcript due to alternative splicing removes exon 4 and encodes for a different C-terminal region to give a novel protein, HCII-N. MD simulations of HCII-N and three-dimensional structure showed a unique 51 amino acid sequence at the C-terminal having unique RCL-like structure. The HCII-N protein purified from bacterial culture showed a protein migrating at lower molecular weight (MW 55 kDa) as compared to native HCII (MW 66 kDa). A fluorescence-based analysis revealed a more compact structure of HCII-N that was in a more hydrophilic environment. The HCII-N protein, however, showed no inhibitory activity against thrombin. Due to large conformational variation observed in comparison with native HCII, HCII-N may have alternate protease specificity or a non-inhibitory role. Western blot of HCII purified from large plasma volume showed the presence of a low MW 59 kDa band with no thrombin activity. This study provides the first evidence of alternatively spliced novel isoform of the HCII gene.

Structural alteration in hypochlorous acid modified antithrombin indicates generation of neo-epitopes.

Increased tendency of cancer patients to develop venous thromboembolism (VTE) is associated with high rates of mortality. Elevation of procoagulant proteins and down regulation of naturally occurring coagulation inhibitors appears to form the basis of high risk of VTE in malignancy. A reduced level of anticoagulant protein like antithrombin (AT) will influence both coagulation and angiogenesis, as its cleaved and latent conformations show potent antiangiogenic activity. We show a concentration dependent perturbation in the secondary and tertiary structures of AT conformers exposed to hypochlorous acid (HOCl). Modulated under a very narrow concentration range of HOCl, native AT undergoes oligomerization, aggregation and fragmentation based on spectroscopic, SDS and native-PAGE studies. Factor Xa inhibition assay demonstrated a progressive decrease in inhibition activity of AT on modification by HOCl. Bis-ANS result showed that hydrophobic patches were more exposed in the case of HOCl-modified AT when assessed fluorometrically. Dosage of HOCl-modified AT in experimental animals induced high titer antibodies showing more specificity towards modified forms in comparison to unmodified forms. Auto-antibodies isolated from cancer patients also showed enhanced binding with HOCl-modified AT in comparison to native counterpart. Compared to normal AT, structurally and functionally altered conformation of HOCl-modified AT showed increased immunogenic sensitivity. HOCl modified AT can contribute to prothrombotic and angiogenic environment during cancer progression/development.

Identification and characterization of a novel variant in C-terminal region of Antithrombin (Ala427Thr) associated with type II AT deficiency leading to polymer formation.

Antithrombin (AT) deficiency is a rare but strong risk factor for the thrombosis development. Mutations in gene encoding AT (SERPINC1) have provided a detailed understanding of AT deficiency and subsequent development of thrombotic complications. In the present study, we describe a case of thrombotic patient with reduced AT activity and normal AT antigen levels. AT deficiency in the patient was explained by the presence of heterozygous mutation g.13397A>G (Ala427Thr) in exon 6 of SERPINC1. Reduced APTT and TT with normal PT were observed. The mutation was found to be absent in healthy controls (n = 62). In vitro purification and characterization of variant AT showed significant decrease in fluorescence emission intensity, decreased bis-ANS fluorescence emission, changes in secondary structure and presence of polymerized AT in patient's plasma as assessed by fluorescence, circular dichroism and transmission electron microscopy respectively. Furthermore, molecular dynamics simulation studies showed altered conformation due to Ala427Thr substitution. Our study shows that genetic screening should be carried out in AT deficient patients in addition to the routinely used functional assays to understand the molecular basis of disease development.

Changes in strand 6B and helix B during neuroserpin inhibition: Implication in severity of clinical phenotype.

Neuroserpin (NS) is predominantly expressed in brain and inhibits tissue-type plasminogen activator (tPA) with implications in brain development and memory. Nature of conformational change in pathological variants in strand 6B and helix B of NS that cause a relatively mild to severe epilepsy (and/or dementia) remains largely elusive. MD simulation with wild type (WT) NS, strand 6B and helix B variants indicated that substitution in this region affects the conformation of the strands 5B, 5A and reactive centre loop. Therefore, we designed variants of NS in strand 6B (I46D and F48S) and helix B (A54F, L55A and L55P) to investigate their role in tPA inhibition mechanism and propensity to aggregate. An interaction analysis showed disturbance of a hydrophobic patch centered at strands 5B, 6B and helix B in I46D and F48S but not in A54F, L55A, L55P and WT NS. Purified I46D, F48S and L55P variants showed decrease in fluorescence emission intensity but have similar α-helical content, however results of A54F and L55A were comparable to WT NS. Analysis of tPA inhibition showed marginal effect on A54F and L55A variant with tPA-NS complex formation. In contrast, I46D, F48S and L55P variants showed massive decrease in tPA inhibition, with no tPA-NS complex formation. Analysis of native PAGE under under polymerization condition showed prompt conversion of I46D, F48S and L55P to latent conformation but not A54F and L55A variants. Identification of these novel conformational changes will aid in the understanding of variable clinical phenotype of shutter region NS variants and other serpins.

Genetically encoded FRET-based optical sensor for Hg2+ detection and intracellular imaging in living cells.

Due to the potential toxicity of mercury, there is an immediate need to understand its uptake, transport and flux within living cells. Conventional techniques used to analyze Hg2+ are invasive, involve high cost and are less sensitive. In the present study, a highly efficient genetically encoded mercury FRET sensor (MerFS) was developed to measure the cellular dynamics of Hg2+ at trace level in real time. To construct MerFS, the periplasmic mercury-binding protein MerP was sandwiched between enhanced cyan fluorescent protein (ECFP) and venus. MerFS is pH stable, offers a measurable fluorescent signal and binds to Hg2+ with high sensitivity and selectivity. Mutant MerFS-51 binds with an apparent affinity (Kd) of 5.09 × 10-7 M, thus providing a detection range for Hg2+ quantification between 0.210 µM and 1.196 µM. Furthermore, MerFS-51 was targeted to Escherichia coli (E. coli), yeast and human embryonic kidney (HEK)-293T cells that allowed dynamic measurement of intracellular Hg2+ concentration with a highly responsive saturation curve, proving its potential application in cellular systems.

Identification of a novel alternatively spliced isoform of antithrombin containing an additional RCL-like loop.

Antithrombin (AT3) is one of the most important inhibitors of blood coagulation proteases that belong to the serpin family of protease inhibitors. In this study, a novel alternatively spliced isoform of AT3 was identified, both at transcript and protein level. This novel transcript contains an additional region in the continuation of exon 3b that was included in the transcript due to use of an alternate 5' splice site. The existence of the novel transcript was confirmed in human brain and liver through RT-PCR. An analysis of the complete transcript indicated that the native reactive centre loop (RCL) of AT3 is maintained; however the novel amino acid sequence projects out as an additional loop as evident from MD simulation studies. A unique amino acid sequence present in the novel isoform was used for the development of polyclonal antibody. The expression of novel isoform was confirmed in human brain and liver tissue using Western blot analysis. Interestingly an alignment of RCL like domain with other inhibitory serpins showed significant similarity with the neuroserpin RCL. To the best of our knowledge, this is the first evidence of alternatively spliced AT3 sequence containing an additional loop and could have physiological relevance.

Deciphering the role of trehalose in hindering antithrombin polymerization.

Serine protease inhibitors (serpins) family have a complex mechanism of inhibition that requires a large scale conformational change. Antithrombin (AT), a member of serpin superfamily serves as a key regulator of the blood coagulation cascade, deficiency of which leads to thrombosis. In recent years, a handful of studies have identified small compounds that retard serpin polymerization but abrogated the normal activity. Here, we screened small molecules to find potential leads that can reduce AT polymer formation. We identified simple sugar molecules that successfully blocked polymer formation without a significant loss of normal activity of AT under specific buffer and temperature conditions. Of these, trehalose proved to be most promising as it showed a marked decrease in the bead like polymeric structures of AT shown by electron microscopic analysis. A circular dichroism (CD) analysis indicated alteration in the secondary structure profile and an increased thermal stability of AT in the presence of trehalose. Guanidine hydrochloride (GdnHCl)-based unfolding studies of AT show the formation of a different intermediate in the presence of trehalose. A time-dependent fluorescence study using 1,1'-bi(4-anilino)naphthalene-5,5'-disulfonic acid (Bis-ANS) shows that trehalose affects the initial conformational change step in transition from native to polymer state through its binding to exposed hydrophobic residues on AT thus making AT less polymerogenic. In conclusion, trehalose holds promise by acting as an initial scaffold that can be modified to design similar compounds with polymer retarding propensity.

Antithrombotic potential of esculin 7, 3', 4', 5', 6'-O-pentasulfate (EPS) for its role in thrombus reduction using rat thrombosis model.

Currently available anticoagulants for prevention and treatment of thrombosis have several limitations, thus, small organic scaffolds that can dissolve clots in vivo in a dose dependent manner with lesser side effects are highly desirable. Here we report the synthesis of esculin pentasulfate (EPS) and assessment of its in vitro, in vivo and ex vivo anticoagulant and antithrombotic potential. Assessment of in vitro clotting times showed prolonged activated partial thromboplastin time (APTT), prothrombin time (PT) and thrombin time (TT) in the presence of EPS. EPS also showed remarkable reduction in thrombus formation when administered in occlusion induced thrombotic rats at a low dose (2.5 mg/kg). Further, assessment of clot rate with plasma isolated from EPS treated rats confirmed its anticoagulation potential. EPS at varying concentrations showed no significant cytotoxic effect on HEK293 cell line. Further, molecular docking analysis of EPS with known anticoagulant proteins [(antithrombin (ATIII) and heparin cofactor II (HCF II)] that require heparin revealed good binding affinity (-7.9 kcal/mol) with ATIII but not with HCF II. ATIII when incubated with EPS showed increased fluorescence intensity, with no change in secondary structure. Overall, our results clearly show the in vivo modulation of thrombus formation using a modified natural scaffold EPS.

A hydrophobic patch surrounding Trp154 in human neuroserpin controls the helix F dynamics with implications in inhibition and aggregation.

Neuroserpin (NS) mediated inhibition of tissue-type plasminogen activator (tPA) is important for brain development, synapse formation and memory. Aberrations in helix F and ß-sheet A movement during inhibition can directly lead to epilepsy or dementia. Conserved W154 residue in a hydrophobic patch between helix F and ß-sheet A is ideally placed to control their movement during inhibition. Molecular Dynamics (MD) simulation on wild type (WT) NS and its two variants (W154A and W154P) demonstrated partial deformation in helix F and conformational differences in strands 1A and 2A only in W154P. A fluorescence and Circular Dichroism (CD) analysis with purified W154 variants revealed a significant red-shift and an increase in α-helical content in W154P as compared to W154A and WT NS. Kinetics of tPA inhibition showed a decline in association rates (ka) for W154A as compared to WT NS with indication of complex formation. Appearance of cleaved without complex formation in W154P indicates that the variant acts as substrate due to conformational misfolding around helix F. Both the variants however showed increased rate of aggregation as compared to WT NS. The hydrophobic patch identified in this study may have importance in helix F dynamics of NS.

Prevalence of Factor V Genetic Variants Associated With Indian APCR Contributing to Thrombotic Risk.

Phenotypic resistance to activated protein C (APC) is a complex mechanism associated with increased thrombosis risk. Activated protein C resistance (APCR) is mainly influenced by FVLeiden mutation, and various other single nucleotide polymorphisms (SNPs) in FV gene are known to be associated with APCR. The aim of present study was to investigate the incidence and assess possible mechanisms of APCR in Indian patients with deep vein thrombosis (DVT). Three hundred and ten Doppler-proven patients with DVT were screened for APCR, and 50 APCR positive patients and 50 controls were typed for FVLeiden, Hong Kong, Cambridge, HR2 haplotype, Glu666Asp, Ala485Lys, and Liverpool using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or allele specific PCR. FVLeiden was commonest cause of APCR (50%) in Indian patients with DVT being statistically significant ( P = .001) compared to controls. FV Liverpool, FV Glu666Asp and FV Ala485Lys were studied for the first time in Indian population. FV Liverpool, FV Glu666Asp, Hong Kong, and Cambridge were found to be absent. High frequency of Ala485Lys in patients shows that it might be a risk factor contributing to APCR in Indian patients with DVT. HR2 haplotype was not associated with APCR; however, presence of homozygous HR2 haplotype in patients only indicates the role it might play in Indian APCR population. In conclusion, contribution of FVLeiden causing APCR in Indian population is not as strong as previously reported in Western countries. The presence of other SNPs observed in the present study requires such studies on larger sample size to understand the molecular basis of defect.

Identification of 2 Novel Polymorphisms and rs3138521 in 5' Untranslated Region of SERPINC1 Gene in North Indian Population With Deep Vein Thrombosis.

Antithrombin III (AT) is the most important endogenous anticoagulant, and genetic variability in SERPINC1, gene encoding AT, is low. Mutations leading to AT deficiency and increased thrombotic risk are well known; however, only 2 studies have reported mutations in regulatory region of SERPINC1 gene till date. Aim of the present study was to identify genetic variations in SERPINC1 5' untranslated region (UTR) in Indian patients with deep vein thrombosis (DVT) having AT deficiency. DNA sequencing was used to identify underlying genetic defects in SERPINC1 regulatory region. In silico tools TFBIND and PROMO were used to identify transcription factor binding sites in the promoter region. We have identified 2 novel polymorphisms, g.25G>A and g.-1A>T, and 2 known g.67G>A and rs3138521 5' UTR polymorphisms in SERPINC1 regulatory region in Indian patients with DVT for the first time. In present study, allele frequencies of rs3138521 (S: 0.37 and F: 0.63) were similar to that reported in Western population and were not associated with low plasma AT levels ( P value .5). This is the first report of regulatory region polymorphisms in SERPINC1 gene in Indian population. Our results strongly suggest that similar studies should be included when ever no mutation is detected in protein-coding region of AT gene.