Abstract

Equid herpes virus type-1 (EHV-1) is a major pathogen of horses, causing abortion storms and outbreaks of herpes virus myeloencephalopathy. These clinical syndromes are partly attributed to ischemic injury from thrombosis in placental and spinal vessels. The mechanism of thrombosis in affected horses is unknown. We have previously shown that EHV-1 activates platelets through virus-associated tissue factor-initiated thrombin generation. Activated platelets participate in thrombus formation by providing a surface to localize coagulation factor complexes that amplify and propagate thrombin generation. We hypothesized that coagulation inhibitors that suppress thrombin generation (heparins) or platelet inhibitors that impede post-receptor thrombin signaling [phosphodiesterase (PDE) antagonists] would inhibit EHV-1-induced platelet activation ex vivo. We exposed platelet-rich plasma (PRP) collected from healthy horses to the RacL11 abortigenic and Ab4 neuropathogenic strains of EHV-1 at 1 plaque-forming unit/cell in the presence or absence of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH) or the PDE inhibitors, 3-isobutyl-1methylxanthine (IBMX), and cilostazol. We assessed platelet activation status in flow cytometric assays by measuring P-selectin expression. We found that all of the inhibitors blocked EHV-1- and thrombin-induced platelet activation in a dose-dependent manner. Platelet activation in PRP was maximally inhibited at concentrations of 0.05 U/mL UFH and 2.5 μg/mL LMWH. These concentrations represented 0.1–0.2 U/mL anti-factor Xa activity measured in chromogenic assays. Both IBMX and cilostazol showed maximal inhibition of platelet activation at the highest tested concentration of 50 μM, but inhibition was lower than that seen with UFH and LMWH. Our results indicate that heparin anticoagulants and strong non-selective (IBMX) or isoenzyme-3 selective (cilostazol) PDE antagonists inhibit ex vivo EHV-1-induced platelet activation. These drugs have potential as adjunctive therapy to reduce the serious complications associated with EHV-1-induced thrombosis. Treatment trials are warranted to determine whether these drugs yield clinical benefit when administered to horses infected with EHV-1.

Highlights

  • Equid herpesvirus type-1 (EHV-1) is a major contagious pathogen of horses, causing individual cases and outbreaks of respiratory disease worldwide

  • We found that unfractionated heparin (UFH) at a concentration of 2 U/mL spiked into platelet-rich plasma (PRP) inhibited platelet activation induced by both strains of EHV-1 at 1 plaqueforming unit (PFU)/cell and by the positive control treatment of 0.15 U/mL of thrombin

  • We found that EHV-1-induced platelet activation was inhibited by UFH in PRP of all tested horses at a concentration of 0.05 U/mL, with variable inhibition among individuals at UFH concentrations of 0.02 U/mL

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Summary

Introduction

Equid herpesvirus type-1 (EHV-1) is a major contagious pathogen of horses, causing individual cases and outbreaks of respiratory disease worldwide. The most severe clinical consequences of EHV-1 infection are abortion and a neurologic syndrome, called equine herpes myeloencephalopathy (EHM). Horses suffering from EHM are frequently euthanized due to the severity of neurologic symptoms and performance horses affected with milder neurologic disease often have reduced performance [1]. Abortion and EHM are thought to result from EHV-1 infection of placental and neural tissue, with the virus reaching these tissues through a cell-associated viremia and subsequent endothelial infection, vasculitis, and tissue invasion [2,3,4,5]. Pathologic studies in experimentally and naturally infected horses have revealed the presence of thrombi in placental and neural vessels [2, 5, 6].

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