Recent studies showed that clotting factor FVIIa (FVIIa), which initiates the coagulation cascade upon binding to procoagulant cofactor tissue factor (TF) following vascular injury, also binds to anticoagulant cofactor endothelial cell protei C receptor (EPCR). EPCR plays a critical role in the protein C anticoagulant pathway by promoting the activation of protein C. EPCR also promotes activated protein C (APC)-induced cytoprotective signaling. Studies conducted in the last funding cycle revealed that FVIIa binding to EPCR on the endothelium facilitates the transport of FVII/FVIIa from the circulation to extravascular sites and FVIIa bound to EPCR activates the protease-activated receptor 1 (PAR1)-mediated cell signaling and provides the barrier protective effect. Pharmacological concentrations of rFVIIa were found to displace endogenous protein C from EPCR. rFVIIa is routinely used to treat hemophilia patients with inhibitory antibodies against FVIII and FIX. It is also being used as an emergency hemostatic agent in both pediatric and adult patients. Despite its widespread and successful use, the understanding of the mechanism of rFVIIa action in bleeding disorders is incomplete. A clear understanding of the mechanistic action of rFVIIa in therapy is essential to improve the clinical efficacy of rFVIIa, and to better manage patients and develop a new generation of FVIIa-based therapeutics. We hypothesize that pharmacological concentrations of rFVIIa compete with endogenous protein C for the limited EPCR sites on the endothelium, which diminishes EPCR-dependent APC generation, and thus the down-regulation of the APC anticoagulant pathway. The down-regulation of the APC anticoagulant pathway would enhance FVIIa-induced thrombin generation as factor Va is not readily inhibited by the reduced APC levels. We also hypothesize that EPCR-mediated FVIIa transport and FVIIa-EPCR-mediated barrier protective and anti-inflammatory effects play crucial roles in preventing the joint damage and bone loss in rFVIIa prophylaxis. The following aims will test the above hypotheses.
Aim 1 : Investigate the importance of FVIIa:EPCR interaction in the hemostatic effect of rFVIIa in treating bleeding disorders and elucidate its mode of action;
Aim 2 : Characterize FVIIa bio-distribution and tissue retention in prophylaxis and evaluate the contribution of FVIIa-EPCR-mediated hemostatic, barrier protective and anti-inflammatory effects to the prophylactic effect of preventing hemophilic arthropathy;
and Aim 3 : Elucidate the mechanism by which EPCR ligation with EPCR mAb, in the absence of rFVIIa treatment, provides the vascular barrier protective effect in hemophilia. To perform these studies, we will generate novel mFVIIa variants and unique transgenic mice and employ saphenous vein bleeding, the needle puncture knee injury, and VEGF-and LPS- induced vascular leakage model systems. The outcome of the proposed studies will introduce a paradigm shift in our understanding of how the pharmacological concentration of rFVIIa provides the hemostatic effect in hemophilia patients and patient with other bleeding disorders.

Public Health Relevance

The proposed studies will provide valuable and novel information on how the interaction of clotting factor VIIa with endothelial cell protein C receptor (EPCR) modulates hemostasis and inflammation. Data obtained from the proposed studies will add new dimension to our existing knowledge on how recombinant factor VIIa functions as an effective hemostatic agent in treating hemophiliacs with inhibitors, and how prophylactic use of FVIIa can reduce joint bleeds and prevent joint destruction in hemophilia patients. The data from the proposed studies is likely to help in improving the treatment options for hemophilia patients and development of new drugs to treat bleeding disorders that will be more effective, safe, and easy to administer and cost less.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
2R01HL107483-05
Application #
9103795
Study Section
Hemostasis and Thrombosis Study Section (HT)
Program Officer
Chang, Henry
Project Start
2011-04-01
Project End
2020-08-31
Budget Start
2016-09-01
Budget End
2017-08-31
Support Year
5
Fiscal Year
2016
Total Cost
Indirect Cost
Name
University of Texas Health Center at Tyler
Department
Type
Overall Medical
DUNS #
800772337
City
Tyler
State
TX
Country
United States
Zip Code
75708
Keshava, Shiva; Sundaram, Jagan; Rajulapati, Anuradha et al. (2017) Factor VIIa interaction with EPCR modulates the hemostatic effect of rFVIIa in hemophilia therapy: Mode of its action. Blood Adv 1:1206-1214
Keshava, Shiva; Rao, L Vijaya Mohan; Pendurthi, Usha R (2016) Intrapleural Adenoviral-mediated Endothelial Cell Protein C Receptor Gene Transfer Suppresses the Progression of Malignant Pleural Mesothelioma in a Mouse Model. Sci Rep 6:36829
Keshava, S; Sundaram, J; Rajulapati, A et al. (2016) Pharmacological concentrations of recombinant factor VIIa restore hemostasis independent of tissue factor in antibody-induced hemophilia mice. J Thromb Haemost 14:546-50
Tucker, Torry A; Jeffers, Ann; Alvarez, Alexia et al. (2014) Plasminogen activator inhibitor-1 deficiency augments visceral mesothelial organization, intrapleural coagulation, and lung restriction in mice with carbon black/bleomycin-induced pleural injury. Am J Respir Cell Mol Biol 50:316-27
Mohan Rao, L Vijaya; Esmon, Charles T; Pendurthi, Usha R (2014) Endothelial cell protein C receptor: a multiliganded and multifunctional receptor. Blood 124:1553-62
Vatsyayan, Rit; Kothari, Hema; Mackman, Nigel et al. (2014) Inactivation of factor VIIa by antithrombin in vitro, ex vivo and in vivo: role of tissue factor and endothelial cell protein C receptor. PLoS One 9:e103505
Sundaram, Jagan; Pendurthi, Usha R; Esmon, Charles T et al. (2014) Blockade of endothelial cell protein C receptor augments factor VIIa hemostatic effect in hemophilia treatment. Blood 124:3031-3
Vijaya Mohan Rao, L (2014) Secretory group V phospholipase A2 : a new player in thrombosis? J Thromb Haemost 12:1918-20
Sundaram, J; Keshava, S; Gopalakrishnan, R et al. (2014) Factor VIIa binding to endothelial cell protein C receptor protects vascular barrier integrity in vivo. J Thromb Haemost 12:690-700
Nayak, Ramesh C; Keshava, Shiva; Esmon, Charles T et al. (2013) Rab GTPases regulate endothelial cell protein C receptor-mediated endocytosis and trafficking of factor VIIa. PLoS One 8:e59304

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