Horses with inflammatory and infectious disorders are often treated with injectable heparin anticoagulants to prevent thrombotic complications. In humans, a new class of direct oral acting anticoagulants (DOAC) appear as effective as heparin, while eliminating the need for daily injections. Our study in horses evaluated apixaban, a newly approved DOAC for human thromboprophylaxis targeting activated factor X (Xa). Our goals were to: (1) Determine pharmacokinetics and pharmacodynamics of apixaban after oral (PO) and intravenous (IV) administration in horses; (2) Detect any inhibitory effects of apixaban on ex vivo Equid herpesvirus type 1 (EHV-1)-induced platelet activation, and (3) Compare an anti-Xa bioactivity assay with ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) for measuring apixaban concentrations. In a blinded placebo-controlled cross-over study, five horses received a single dose (0.2 mg/kg) of apixaban or placebo PO or IV. Blood was collected before and at 3 (IV) or 15 (PO) min, 30 and 45 min, and 1, 2, 3, 4, 6, 8, and 24 h after dosing for measuring apixaban UPLC-MS concentrations and anti-Xa activity. Pharmacodynamic response was measured in a dilute prothrombin time (dPT) assay. Flow cytometric EHV-1-induced platelet P-selectin expression and clinical pathologic safety testing were performed at baseline, 2 and 24 h and baseline and 24 h, respectively. We found no detectable apixaban in plasma PO administration. After IV administration, plasma apixaban levels followed a two-compartment model, with concentrations peaking at 3 min and decreasing to undetectable levels by 8 h. The elimination half-life was 1.3 ± 0.2 h, with high protein binding (92–99%). The dPT showed no relationship to apixaban UPLC-MS concentration and apixaban did not inhibit EHV-1-induced platelet activation after IV dosing. Apixaban anti-Xa activity showed excellent correlation to UPLC-MS (r2 = 0.9997). Our results demonstrate that apixaban has no apparent clinical utility as an anticoagulant for horses due to poor oral availability.

Equine herpesvirus type 1 (EHV-1) is a major cause of infectious respiratory disease, abortion and neurologic disease. Thrombosis in placental and spinal vessels and subsequent ischemic injury in EHV-1-infected horses manifests clinically as abortion and myeloencephalopathy. We have previously shown that addition of heparin anticoagulants to equine platelet-rich plasma (PRP) can abolish ex vivo EHV-1-induced platelet activation. The goal of this study was to test whether platelets isolated from horses treated with unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH) were resistant to ex vivo EHV-1-induced activation. In a masked, block-randomized placebo-controlled cross-over trial, 9 healthy adult horses received 4 subcutaneous injections at q. 12 h intervals of one of the following treatments: UFH (100 U/kg loading dose, 3 maintenance doses of 80 U/kg), 2 doses of LMWH (enoxaparin) 80 U/kg 24 h apart with saline at the intervening 12 h intervals, or 4 doses of saline. Blood samples were collected before treatment and after 36 h, 40 h (4 h after the last injection) and 60 h (24 h after the last injection). Two strains of EHV-1, Ab4 and RacL11, were added to PRP ex vivo and platelet membrane expression of P selectin was measured as a marker of platelet activation. Drug concentrations were monitored in a Factor Xa inhibition (anti-Xa) bioassay. We found that LMWH, but not UFH, inhibited platelet activation induced by low concentrations (1 × 106 plaque forming units/mL) of both EHV-1 strains at 40 h. At this time point, all horses had anti-Xa activities above 0.1 U/ml (range 0.15–0.48 U/ml) with LMWH, but not UFH. By 60 h, a platelet inhibitory effect was no longer detected and anti-Xa activity had decreased (range 0.03 to 0.07 U/ml) in LMWH-treated horses. Neither heparin inhibited platelet activation induced by high concentrations (5 × 106 plaque forming units/mL) of the RacL11 strain. We found substantial between horse variability in EHV-1-induced platelet activation at baseline and after treatment. Minor injection site reactions developed in horses given either heparin. These results suggest that LMWH therapy may prevent thrombotic sequelae of EHV-1, however further evaluation of dosage regimens is required.

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