Unfractionated and Low-Molecular-Weight Heparin and the Phosphodiesterase Inhibitors, IBMX and Cilostazol, Block Ex Vivo Equid Herpesvirus Type-1-Induced Platelet Activation

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.

Subcutaneous Administration of Low-Molecular-Weight Heparin to Horses Inhibits Ex Vivo Equine Herpesvirus Type 1-Induced Platelet Activation

“…In addition to within-horse variability in baseline platelet reactivity to EHV-1, we noted that the percentage of platelets expressing P selectin after exposure to low or high concentrations of the Ab4 strain were lower than what we have published previously ( 30 ). There are several possible explanations for this, including the use of different horses (with intrinsic variation in coagulation factor or inhibitor concentrations), the use of different lots of purified virus, or an effect of the change in protocol with decreased sample dilution.…”

“…The reaction was quenched with 0.6 ml of platelet buffer containing 0.2 mM supplemental GPRP and 2.5 mM calcium chloride and the samples were immediately analyzed with a flow cytometer (BD FACSCalibur, BD Biosciences, San Jose, CA, USA). For analysis, platelets were gated on their forward and side scatter characteristics and the percentage of P selectin-positive platelets determined from histogram plots, as we have previously described ( 30 ).…”

“…We have recently reported that UFH and LMWH can inhibit EHV-1-induced platelet activation when spiked in equine platelet-rich plasma (PRP) ex vivo ( 30 ). Activated platelets were quantified by the flow cytometric-based detection of surface P selectin, as a marker of α-granule release.…”

Equine Herpesvirus-1 Infection, Clinical Features, Treatment and Control

“…It was reported that, EHV-1 activates platelets through virus-associated tissue factor-initiated thrombin generation that participate in thrombus formation by providing a surface to localize coagulation factor complexes. Accordingly, using of anticoagulants that suppress thrombin generation or platelet inhibitors that impede post-receptor thrombin signaling (phosphodiesterase antagonists) would inhibit EHV-1-induced platelet activation (Stokol et al, 2016). Interestingly, subcutaneous injection of 25000 IU heparin to horses during EHV-1 outbreak resulted in lower EHM incidence (3.2%) than untreated horses (23.3%) (Walter et al, 2016).…”

Activated platelets and platelet-leukocyte aggregates in the equine systemic inflammatory response syndrome

“… 25 , 50 , 53 Increased numbers of activated platelets and/or PLAs measured by flow cytometry have been described in horses after near-maximal treadmill exercise, 72 after induced infection with equine infectious anemia virus, 49 in the course of carbohydrate-induced laminitis, 71 and in vitro after incubation with lipopolysaccharides 9 and equid herpesvirus 1 ( Equid alphaherpesvirus 1 ). 60 – 62 Activation markers in horses with equine asthma after antigen challenge were expressed inconsistently, with increased activation in one study 29 and no difference to the control groups in 2 other studies. 20 , 30 In experimentally induced equine endotoxemia, platelet activation was not increased at the time of leukocyte nadir.…”