Inhibition of Factor IXa by Protein S: Critical Function of the Protein S SHBG-like domain

Location

Medical Education Building, LSUHSC-NO

Presentation Date

10-10-2019 10:00 AM

End Date

10-10-2019 12:00 PM

Description

Coagulation is the process of fibrin clot formation to reduce or halt blood flow, a process that is critical in the prevention of excessive bleeding from vascular injuries. A steady balance of coagulants and anticoagulants is needed to maintain hemostasis. An excess of one or more coagulants causes thrombosis, whereas an excess of anticoagulants causes hemophilia. Protein S (PS), a vitamin K-dependent glycoprotein, acts as an anticoagulant by a variety of mechanisms, including serving as cofactors for activated protein C (APC) and tissue factor pathway inhibitor (TFPI)1–3 . PS deficiency can result in severe symptoms, including deep vein thrombosis, arterial thrombosis (stroke, heart attack), and pulmonary embolism4–7 . Perhaps the most critical anticoagulant activity of PS is its ability to inhibit coagulation factor IXa (fIXa)8,9. Factor IXa is an essential component of the coagulation cascade. Factor IXa interacts with factor VIIIa (fVIIIa) to form the “Xase” complex that converts factor X (fX) into Xa (fXa), the downstream effects of which lead to fibrin clot formation. Currently, we do not know the exact binding site(s) for fIXa within PS. Previous work in the lab indicated an involvement of the PS sex-hormone-binding globulin (SHBG)-like domains in binding fIXa. The SHBG is primarily composed of two laminin G (LG)- type domains (LG1 and LG2)9 . The human PS LG1, LG2, and LG1+2 domains have been cloned into the GST tagging pGEXGO1 expression vector, and the expressed polypeptides were purified by affinity chromatography using glutathione Sepharose. The goal of this project was to determine whether the PS LG domains retain the ability to bind and inhibit fIXa. To investigate the activity of the LG domains, we measured the inhibitory effect of the LG1, LG2, and LG1+2 recombinant polypeptides toward fIXa activation of fX. Compared with controls, fX activation was decreased by ~47% in the presence of LG1+2. LG1 alone reduced fX activation by ~21%, and LG2 alone had no significant effect on fIXa activation of fX. Next, we measured the kinetic parameter (KM) of fIXa by varying fX concentrations and at a fixed concentration of fIXa. We observed that the KM of fIXa for fX was ~750 nM in the presence of LG1+2, an affinity similar to the affinity in the presence of intact PS (~790 nM)8 . The KMs in the presence of separate LG1 and LG2 were higher (2100 nM and 4200 nM), suggesting that LG1+2 is indeed needed to mimic the function of PS. Next, we performed activated partial thromboplastin time (aPTT) assays to measure the activities of these domains on clotting time of PS-deficient plasma. Each LG domain polypeptide caused prolonged clotting time compared with the control, with LG1+2 producing the largest increase of 10 seconds when added at a concentration of 450 nM. Preliminary data from the lab showed that the thrombin generation assay, considered the most important physiological assay in coagulation, showed a decrease in thrombin generation of PS-deficient plasma in the presence of the LG domains. The LG1+2 polypeptide produced the greatest decrease in thrombin generation, mimicking the activity of PS in normal pooled plasma. In sum, these results delineate the domains of PS that contain specific binding sites for fIXa. Identification and analysis of these binding sites is critical for our ultimate goal of making a short polypeptide of PS to be used as a therapeutic to treat patients prone to thrombosis.

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Mentor: Rinku Majumder, PhD (Department of Biochemistry and Molecular Biology)

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Oct 10th, 10:00 AM Oct 10th, 12:00 PM

Inhibition of Factor IXa by Protein S: Critical Function of the Protein S SHBG-like domain

Medical Education Building, LSUHSC-NO

Coagulation is the process of fibrin clot formation to reduce or halt blood flow, a process that is critical in the prevention of excessive bleeding from vascular injuries. A steady balance of coagulants and anticoagulants is needed to maintain hemostasis. An excess of one or more coagulants causes thrombosis, whereas an excess of anticoagulants causes hemophilia. Protein S (PS), a vitamin K-dependent glycoprotein, acts as an anticoagulant by a variety of mechanisms, including serving as cofactors for activated protein C (APC) and tissue factor pathway inhibitor (TFPI)1–3 . PS deficiency can result in severe symptoms, including deep vein thrombosis, arterial thrombosis (stroke, heart attack), and pulmonary embolism4–7 . Perhaps the most critical anticoagulant activity of PS is its ability to inhibit coagulation factor IXa (fIXa)8,9. Factor IXa is an essential component of the coagulation cascade. Factor IXa interacts with factor VIIIa (fVIIIa) to form the “Xase” complex that converts factor X (fX) into Xa (fXa), the downstream effects of which lead to fibrin clot formation. Currently, we do not know the exact binding site(s) for fIXa within PS. Previous work in the lab indicated an involvement of the PS sex-hormone-binding globulin (SHBG)-like domains in binding fIXa. The SHBG is primarily composed of two laminin G (LG)- type domains (LG1 and LG2)9 . The human PS LG1, LG2, and LG1+2 domains have been cloned into the GST tagging pGEXGO1 expression vector, and the expressed polypeptides were purified by affinity chromatography using glutathione Sepharose. The goal of this project was to determine whether the PS LG domains retain the ability to bind and inhibit fIXa. To investigate the activity of the LG domains, we measured the inhibitory effect of the LG1, LG2, and LG1+2 recombinant polypeptides toward fIXa activation of fX. Compared with controls, fX activation was decreased by ~47% in the presence of LG1+2. LG1 alone reduced fX activation by ~21%, and LG2 alone had no significant effect on fIXa activation of fX. Next, we measured the kinetic parameter (KM) of fIXa by varying fX concentrations and at a fixed concentration of fIXa. We observed that the KM of fIXa for fX was ~750 nM in the presence of LG1+2, an affinity similar to the affinity in the presence of intact PS (~790 nM)8 . The KMs in the presence of separate LG1 and LG2 were higher (2100 nM and 4200 nM), suggesting that LG1+2 is indeed needed to mimic the function of PS. Next, we performed activated partial thromboplastin time (aPTT) assays to measure the activities of these domains on clotting time of PS-deficient plasma. Each LG domain polypeptide caused prolonged clotting time compared with the control, with LG1+2 producing the largest increase of 10 seconds when added at a concentration of 450 nM. Preliminary data from the lab showed that the thrombin generation assay, considered the most important physiological assay in coagulation, showed a decrease in thrombin generation of PS-deficient plasma in the presence of the LG domains. The LG1+2 polypeptide produced the greatest decrease in thrombin generation, mimicking the activity of PS in normal pooled plasma. In sum, these results delineate the domains of PS that contain specific binding sites for fIXa. Identification and analysis of these binding sites is critical for our ultimate goal of making a short polypeptide of PS to be used as a therapeutic to treat patients prone to thrombosis.