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Homology modeling and structural validation of tissue factor pathway inhibitor.

ABSTRACT: Blood coagulation is a cascade of complex enzymatic reactions which involves specific proteins and cellular components to interact and prevent blood loss. The coagulation process begins by either "Tissue Dependent Pathway" (also known as extrinsic pathway) or by "contact activation pathway" (also known as intrinsic pathway). TFPI is an endogenous multivalent Kunitz type protease inhibitor which inhibits Tissue factor dependent pathway by inhibiting Tissue Factor:Factor VIIa (TF:FVIIa) complex and Factor Xa. TFPI is one of the most studied coagulation pathway inhibitor which has various clinical and potential therapeutic applications, however, its exact mechanism of inhibition is still unknown. Structure based mechanism elucidation is commonly employed technique in such cases. Therefore, in the current study the generated a complete TFPI structural model so as to understand the mechanistic details of it's functioning. The model was checked for stereochemical quality by PROCHECK-NMR, WHATIF, ProSA, and QMEAN servers. The model was selected, energy minimized and simulated for 1.5ns. The result of the study may be a guiding point for further investigations on TFPI and its role in coagulation mechanism.

SUBMITTER: Agrawal P

PROVIDER: S-EPMC3796881 | biostudies-literature | 2013

REPOSITORIES: biostudies-literature

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Homology modeling and structural validation of tissue factor pathway inhibitor.

Agrawal Piyush P Thakur Zoozeal Z Kulharia Mahesh M

Bioinformation 20130923 16

Blood coagulation is a cascade of complex enzymatic reactions which involves specific proteins and cellular components to interact and prevent blood loss. The coagulation process begins by either "Tissue Dependent Pathway" (also known as extrinsic pathway) or by "contact activation pathway" (also known as intrinsic pathway). TFPI is an endogenous multivalent Kunitz type protease inhibitor which inhibits Tissue factor dependent pathway by inhibiting Tissue Factor:Factor VIIa (TF:FVIIa) complex an ...[more]

PMID: 24143050

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Project description:Vascular injury results in the activation of coagulation and the release of proteolytic enzymes from neutrophils and connective- tissue cells. High concentrations of these inflammatory proteinases may destroy blood coagulation proteins, contributing to coagulation and bleeding disorders associated with severe inflammation. Matrix metalloproteinase-8 (MMP-8) is released from neutrophils at sites of inflammation and vascular disease. We have investigated the effect of MMP-8 degradation on the anticoagulant function of tissue factor pathway inhibitor (TFPI) as a potential pathological mechanism contributing to coagulation disorders. MMP-8 cleaves TFPI following Ser(174) within the connecting region between the second and third Kunitz domains ( k (cat)/ K (m) approximately 75 M(-1).s(-1)) as well as following Lys(20) within the NH(2)-terminal region. MMP-8 cleavage of TFPI decreases the anticoagulant activity of TFPI in factor Xa initiated clotting assays as well as the ability of TFPI to inhibit factor Xa in amidolytic assays. Yet, MMP-8 cleavage does not alter the ability of TFPI to inhibit trypsin. Since the inhibition of both factor Xa and trypsin is mediated by binding to the second Kunitz domain, these results suggest that regions of TFPI other than the second Kunitz domain may directly interact with factor Xa. (125)I-factor Xa ligand blots of TFPI fragments generated following MMP-8 degradation were used for probing binding interactions between factor Xa and regions of TFPI, other than the second Kunitz domain. In experiments performed under reducing conditions that disrupt the Kunitz domain structure, (125)I-factor Xa binds to the C-terminal fragment of MMP-8-degraded TFPI. This fragment contains portions of TFPI distal to Ser(174), which include the third Kunitz domain and the basic C-terminal region. An altered form of TFPI lacking the third Kunitz domain, but containing the C-terminal region, was used to demonstrate that the C-terminal region directly interacts with factor Xa.

Project description:The activated mammalian CAPN-structures, the CAPN/CAST complex in particular, have become an invaluable target model using the structure-based virtual screening of drug candidates from the discovery phase to development for over-activated CAPN linked to several diseases, such as post-ischemic injury and cataract formation. The effect of Ca²?-binding to the enzyme is thought to include activation, as well as the dissociation, aggregation, and autolysis of small regular subunits. Unfortunately, the Ca²?-activated enzyme tends to aggregate when provided as a divalent ion at the high-concentration required for the protease crystallization. This is also makes it very difficult to crystallize the whole-length enzyme itself, as well as the enzyme-inhibitor complex. Several parameters that influence CAPN activity have been investigated to determine its roles in Ca²?-modulation, autoproteolysis, phosphorylation, and intracellular distribution and inhibition by its endogenous inhibitor CAST. CAST binds and inhibits CAPN via its CAPN-inhibitor domains (four repeating domains 1-4; CAST1-4) when CAPN is activated by Ca²?-binding. An important key to understanding CAPN1 inhibition by CAST is to determine how CAST interacts at the molecular level with CAPN1 to inhibit its protease activity. In this study, a 3D structure model of a CAPN1 bound bovine CAST4 complex was built by comparative modeling based on the only known template structure of a rat CAPN2/CAST4 complex. The complex model suggests certain residues of bovine CAST4, notably, the TIPPKYQ motif sequence, and the structural elements of these residues, which are important for CAPN1 inhibition. In particular, as CAST4 docks near the flexible active site of CAPN1, conformational changes at the interaction site after binding could be directly related to CAST4 inhibitory activity. These functional interfaces can serve as a guide to the site-mutagenesis in research on bovine CAPN1 structure-function relationships for the design of small molecules inhibitors to prevent uncontrolled and unspecific degradation in the proteolysis of key protease substrates.

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Homology modeling and structural validation of tissue factor pathway inhibitor.

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Homology modeling and structural validation of tissue factor pathway inhibitor.

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