ZWEIGBANNER  

The Harry M. Zweig Memorial Fund for Equine Research


The Role of Platelets in the Pathogenesis of Equid Herpes Virus Type-1 Infection

Principal Investigator: Tracy Stokol
Contact Information: Email: tracy.stokol@cornell.edu; Phone: 607-253-3255
Project Costs: $133,798
Project Period: 01/01/12-12/31/13

StokolDr. Tracy Stokol

Equid herpes virus-1 (EHV-1) is an important infectious cause of neonatal deaths, abortion and neurological disease in horses. Both abortion and neurological disease (the syndrome called equine herpes virus myeloencephalopathy or EHM) are caused by thrombosis of blood vessels supplying the placenta and spinal cord. The mechanisms underlying the development of thrombi in infected horses are unknown. Platelets are intimately involved in thrombus formation. Once activated, platelets adhere to blood vessels, form aggregates and amplify thrombin generation through exposure of the anionic membrane phospholipid, phosphatidylserine (PS), and release of PS-enriched membrane-derived microparticles. It is currently unknown if platelets are involved in the pathogenesis of thrombosis in horses with EHV-1 infection. We hypothesize that EHV-1 activates platelets, either directly or indirectly through infected monocytes or endothelial cells, causing them to become procoagulant through exposure of PS and release of PS-enriched microparticles. We will test our hypothesis using flow cytometry and novel in vitro microfluidic assay as outlined in our aims below, with appropriate positive and negative controls. If our hypothesis is correct, it would indicate that platelets are likely involved in the development of vascular thrombi in horses with EHV-1 infection and that platelets are a hitherto unexplored therapeutic target for preventing or treating the thrombotic sequelae of abortion and EHM in affected horses. Platelets are a highly attractive target for antithrombotic therapy. There are several commercially available platelet inhibitors, such as clopidogrel and aspirin, which could be readily tested for their activity in preventing platelet activation in EHV-1 infection and easily administered to infected horses if proven efficacious with minimal side effects.

Aim 1: Direct activation of equine platelets by EHV-1: Here we will test whether a neurologic (Ab4) or abortiefacient strain (NY03) of EHV-1 binds to and activates equine platelets. Viral binding and platelet activation will be examined with flow cytometry using fluorescent-labeled viruses and markers of platelet activation, specifically exposure of PS and P-selectin and release of platelet-derived microparticles. This aim will be completed in the first 6-9 months.

Aim 2: Indirect activation of equine platelets by EHV-1-infected monocytes: Here we will determine if platelets (that have not been exposed to EHV-1) are activated by indirect or direct contact with monocytes previously infected with EHV-1. We will incubate platelets with EHV-1-infected monocytes and determine if platelets are activated by direct cell contact, soluble mediators or microparticles released from EHV-1-infected monocytes, using similar flow cytometric procedures as described in aim 1. This aim will begin in year 1 and be completed by the middle of year 2.

Aim 3: Indirect activation of equine platelets by EHV-1-infected endothelial cells: Here we will use a novel microfluidic device consisting of a microfluidic channel in which equine carotid endothelial cells will be grown to confluence. Endothelial cells will be infected in situ with EHV-1, then fluorescent-labeled platelets will be infused into the device and their interactions with the infected endothelium (rolling, adhesion, aggregate formation) will be monitored in real-time using digital video microscopy, then quantified post-acquisition with dedicated imaging software. This aim will begin in the early part of year 2 and be completed within the requested 2-year funding period.