The Protein S LG1+2 domain Contributes Significantly to Inhibition of Factor Ixa
Publication Date
July 2019
Location
LSU Health Medical Education Building
Document Type
Abstract
Start Date
26-7-2019 9:00 AM
End Date
26-7-2019 12:00 PM
Description
Protein S (PS) is a key vitamin K-dependent, anticoagulant protein. Homozygous PS deficiency causes fatal purpura fulminans in neonates, a dramatic phenotype that emphasizes the critical function of PS in the regulation of human blood coagulation. Similarly, heterozygous PS deficiency is associated with increased risk of venous thromboembolism. Consistent with these clinical manifestations, gene ablation of PS in mice results in an embryonic lethal phenotype related to consumptive coagulopathy and intracranial hemorrhage. Recently, the Majumder lab identified an important function of PS, i.e., PS inhibits Factor IXa(FIXa) in the coagulation pathway. Coagulation occurs by a cascade of reactions, culminating in the formation of a fibrin clot. A key event in the coagulation cascade is the activation of Factor X(FX). The activation of FX relies on FIXa binding to its cofactor, Factor VIIIa(FVIIIa), forming a complex that activates FX to FXa. FXa, along with its cofactor, FactorVa(FVa), activates prothrombin to thrombin, which in turn generates fibrin. The binding of PS to FIXa inhibits formation of the FIXa/FVIIIa complex, thereby inhibiting FX generation. This newly recognized inhibitory function of PS presents a novel possibility of using PS as a therapeutic agent to treat hypercoagulation disorders. PS is composed of a GLA domain, a thrombin sensitive region, four EGF domains, and the LamninG-1/G-2 domains (LG1+LG2). LG1 and LG2 were cloned and overexpressed in E.coli. The LG1+2 protein reduced the rate of FX activation by 69% in the presence of the FIXa/FVIIIa complex; this reduced rate agreed with previous measurements that used intact PS. However, the separate LG1 and LG2 domains reduced the rate of FX activation by FIXa by only 41% and 28%. In this project, we used circular dichroism spectroscopy and isothermal titration calorimetry to determine structural changes that accompanied binding of the PS LG1 and LG2 domains to FIXa. In addition, we used ex vivo plasma assays such as activated partial thromboplastin times (aPTT) and thrombin generation assays (TGA) to identify the interactions between the LG domains and FIXa in physiological conditions. We observed that the binding of the LG1+2 and LG1 domains with FIXa were exothermic with a 1:1 stoichiometry. The binding affinities LG1+2 and LG1 to FIXa were 1.2x107 M-1 and 5.16x106 M-1 , respectively. The calculated ΔG values of LG1+2 and LG1 binding to FIXa were -9.46 Kcal/mol and -8.90 Kcal/mol, respectively. These parameters agree well with the parameters for intact PS binding to FIXa. The circular dichroism data revealed significant conformational changes that accompanied binding of LG1+2 domain to FIXa. We also observed conformational changes with binding of LG1 domain to FIXa; however, the change was greater with LG1+2 domain. The LG1+2 domain decreased thrombin generation in PS-deficient plasma by ~50%, whereas the LG1 domain reduced thrombin generation by only ~25%. The clotting time of PS-deficient plasma (~30 s) was prolonged by 10 s by addition of 300 nM LG1+2 domain. On the basis of these results, we conclude that the LG1+2 domain is needed to bind and inhibit FIXa . However, LG1or LG2 alone is not sufficient to recapitulate the inhibitory effects of PS on FIXa .
Recommended Citation
Sylvain, Amber E., "The Protein S LG1+2 domain Contributes Significantly to Inhibition of Factor Ixa" (2019). Summer Research Internship Program. 33.
https://digitalscholar.lsuhsc.edu/srip/2019/undergrad/33
The Protein S LG1+2 domain Contributes Significantly to Inhibition of Factor Ixa
LSU Health Medical Education Building
Protein S (PS) is a key vitamin K-dependent, anticoagulant protein. Homozygous PS deficiency causes fatal purpura fulminans in neonates, a dramatic phenotype that emphasizes the critical function of PS in the regulation of human blood coagulation. Similarly, heterozygous PS deficiency is associated with increased risk of venous thromboembolism. Consistent with these clinical manifestations, gene ablation of PS in mice results in an embryonic lethal phenotype related to consumptive coagulopathy and intracranial hemorrhage. Recently, the Majumder lab identified an important function of PS, i.e., PS inhibits Factor IXa(FIXa) in the coagulation pathway. Coagulation occurs by a cascade of reactions, culminating in the formation of a fibrin clot. A key event in the coagulation cascade is the activation of Factor X(FX). The activation of FX relies on FIXa binding to its cofactor, Factor VIIIa(FVIIIa), forming a complex that activates FX to FXa. FXa, along with its cofactor, FactorVa(FVa), activates prothrombin to thrombin, which in turn generates fibrin. The binding of PS to FIXa inhibits formation of the FIXa/FVIIIa complex, thereby inhibiting FX generation. This newly recognized inhibitory function of PS presents a novel possibility of using PS as a therapeutic agent to treat hypercoagulation disorders. PS is composed of a GLA domain, a thrombin sensitive region, four EGF domains, and the LamninG-1/G-2 domains (LG1+LG2). LG1 and LG2 were cloned and overexpressed in E.coli. The LG1+2 protein reduced the rate of FX activation by 69% in the presence of the FIXa/FVIIIa complex; this reduced rate agreed with previous measurements that used intact PS. However, the separate LG1 and LG2 domains reduced the rate of FX activation by FIXa by only 41% and 28%. In this project, we used circular dichroism spectroscopy and isothermal titration calorimetry to determine structural changes that accompanied binding of the PS LG1 and LG2 domains to FIXa. In addition, we used ex vivo plasma assays such as activated partial thromboplastin times (aPTT) and thrombin generation assays (TGA) to identify the interactions between the LG domains and FIXa in physiological conditions. We observed that the binding of the LG1+2 and LG1 domains with FIXa were exothermic with a 1:1 stoichiometry. The binding affinities LG1+2 and LG1 to FIXa were 1.2x107 M-1 and 5.16x106 M-1 , respectively. The calculated ΔG values of LG1+2 and LG1 binding to FIXa were -9.46 Kcal/mol and -8.90 Kcal/mol, respectively. These parameters agree well with the parameters for intact PS binding to FIXa. The circular dichroism data revealed significant conformational changes that accompanied binding of LG1+2 domain to FIXa. We also observed conformational changes with binding of LG1 domain to FIXa; however, the change was greater with LG1+2 domain. The LG1+2 domain decreased thrombin generation in PS-deficient plasma by ~50%, whereas the LG1 domain reduced thrombin generation by only ~25%. The clotting time of PS-deficient plasma (~30 s) was prolonged by 10 s by addition of 300 nM LG1+2 domain. On the basis of these results, we conclude that the LG1+2 domain is needed to bind and inhibit FIXa . However, LG1or LG2 alone is not sufficient to recapitulate the inhibitory effects of PS on FIXa .
Comments
Mentor: Dr. Rinku Majumder, Department of Biochemistry