The Harry M. Zweig Memorial Fund for Equine Research

Platelets are a Trojan Horse that Deliver Equine Herpes Virus to Endothelial Cells

Principal Investigator: Tracy Stokol
Contact Information: Email:; Phone: 607-253-3255
Project Costs: $163,131
Project Period: 01/01/15-12/31/16

StokolDr. Tracy Stokol

Equine herpesvirus type I (EHV-1) is a highly contagious pathogen, causing outbreaks of disease worldwide.
The most severe clinical manifestations of EHV-1 are abortion and equine herpesvirus myeloencephalopathy
(EHM), both of which are caused by infection of endothelial cells (EC) with subsequent vasculitis, thrombosis
and tissue infection. We still do not clearly understand how the virus infects ECs, the portal of entry for the
virus to underlying tissues. Currently, it is thought that ECs acquire infection from peripheral blood
mononuclear cells (PBMCs), however PBMC binding to ECs requires pre-activation of both PBMCs and ECs
and this may not be a common route of infection. We propose that platelets are an overlooked reservoir of
infection for ECs. We have found that EHV-1 can be retrieved from platelets isolated from EHV-1-infected
horses and that EHV-1 activates platelets in vitro and in vivo. EHV-1-activated platelets bind to and infect
unactivated ECs under low flow conditions in our in vitro microfluidic model of the vasculature. We have also
found that platelet binding under flow is mediated by P selectin, an adhesion molecule expressed on activated
platelet surfaces. We hypothesize that activated platelets transfer virus to ECs in two ways: 1) Binding directly
to ECs via P selectin and 2) Binding to, infecting and activating PBMCs, via P selectin. Once activated, infected
PMBCs would be able to bind to and infect ECs. Since there are 25-50 fold more platelets than PBMCs in blood
and platelets interact with unactivated ECs, platelets are likely to be effective at delivering virus to ECs. We
also propose that inhibitors of EHV-1-induced platelet activation and P selectin expression will prevent
platelets from binding to and infecting ECs or PBMCs. If we do find a drug that can stop platelets from acting as “Trojan horses”, we could potentially prevent abortion and EHM by stopping activated platelets from both offloading their lethal cargo to ECs supplying the placenta and spinal cord and participating in clot formation.
Aim 1: Platelet infection of ECs: Here we will determine the efficiency of platelet-mediated EC infection under physiological shear. We will use washed platelets and fucoidan to assess the role of activated platelets and P selectin, respectively. We will use electron microscopy to see where the virus is located in the platelet.

Aim 2: Platelet infection of PBMCs: Here we will assess whether platelets can transfer infection to and activate immunopurified PBMC subsets (monocytes and T cells) in a P selectin-dependent manner.

Aim 3: Inhibitory drug screen: Here, we will screen a phosphodiesterase-3 inhibitor and two factor X inhibitors for their ability to inhibit EHV-1-induced platelet activation and P selectin upregulation and platelet-mediated transfer of virus to ECs and PBMC subsets. An effective drug could potentially be used to prevent platelets from infecting these cells and would be useful adjunctive treatment of infected or exposed horses to prevent abortion and EHM.