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

Abstract

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.

Keywords: EHV-1; P selectin; heparin; thrombin generation; unfractionated heparin.

Figure 1

Experimental design for a randomized heparin placebo-controlled anticoagulant drug trial in 9 horses. The order of drug administration in individual horses was block-randomized by personnel not involved in drug administration or data acquisition or analysis. Drugs or placebo were administered for 2 days at 12 h intervals with blood samples being taken for testing before treatment (T0) and 36, 40 and 60 h after the start of treatment. A 10 day washout period separated drug treatments. The 36, 40 and 60 h time points corresponded to times just before, 4 h after and 24 h after the last subcutaneous injection of the drug or placebo. For UFH, the drug was given as a loading dose of 100 U/kg, then 3 maintenance doses of 80 U/kg were given at 12 h intervals (4 total doses). For LMWH, the drug was given at a dose of 80 U/kg every 24 h (2 doses), with placebo administration at the intervening 12 h intervals (2 doses). The timing of the initiation of UFH and LMWH treatment was staggered to ensure that samples for blood collection and subsequent measurements were consistent in relation to the last dose. Placebo was given every 12 h (4 doses). Blood samples were collected at 0 and 60 h for flow cytometric evaluation of platelet activation (measurement of P selectin expression after exposure to agonists ex vivo; Flow), measurement of drug inhibitory activity with an anti-Xa bioassay (anti-Xa), automated hemogram analysis (CBC), evaluation for red blood cell agglutination (RBC agg) and measurement of select biochemical analytes (Chem). Additional blood samples were collected at 36 and 40 h for flow cytometric evaluation of platelet activation and anti-Xa inhibitory activity.

Figure 2

Changes in the median percentage of platelets expressing P selectin in platelet-rich plasma of 9 horses before treatment (0 h), 36 h (before the last dose), 40 h (4 h after the last dose) and 60 h (24 h after the last dose) of placebo (red), unfractionated heparin (UFH, green) or low-molecular-weight heparin (LMWH, blue). Platelets were exposed to PBS (A) as a negative control, ADP (B) as a positive control, low (C), 0.15 U/ml) and high (D), 0.5 U/ml) concentrations of thrombin, or low (1 × 10⁶ PFU/ml) and high (5 × 10⁶ PFU/ml) concentrations of the EHV-1 strains, Ab4 (E), low concentration; (F), high concentration and RacL11 (G), low concentration; (H), high concentration for 10 min ex vivo. Note that the Y-axis scale bar is different for PBS and low and high concentrations of Ab4. *Colored stars represent significant changes for the relevant drug at each time point compared to 0 h (p < 0.05).

Figure 3

Changes in the percentage of platelets expressing P selectin in platelet-rich plasma of individual horses at 0 h (baseline) and 40 h after starting treatment (4 h after the last dose) with placebo (left column), unfractionated heparin (UFH, middle column) or low-molecular-weight heparin (LMWH, right column). Platelets were stimulated with low concentrations of thrombin (A), 0.15 U/ml, n = 4) or the EHV-1 strains (1 × 10⁶ PFU/ml), Ab4 (B) and RacL11 (C), for 10 min ex vivo (n = 9). Significant changes in the median percentage of platelets expressing P selectin at 40 h compared to 0 h are shown. Each horse has the same unique symbol at both time points for all treatments (horse 1: -□-; horse 2: -○-; horse 3: -⋆-; horse 4: -⧫-; horse 5: -X-; horse 6: -▾-; horse 7: -·-; horse 8: -▪-; horse 9: -▴-).

Figure 4

Changes in the percentage of platelets expressing P selectin in platelet-rich plasma of 9 individual horses at 0 h (baseline) and 40 h after starting treatment (4 h after the last dose) with placebo (left column), unfractionated heparin (UFH, middle column) or low-molecular-weight heparin (LMWH, right column). Platelets were stimulated with high concentrations of thrombin (A), 0.5 U/ml) or the EHV-1 strains (5 × 10⁶ PFU/ml), Ab4 (B) and RacL11 (C), for 10 min ex vivo. Significant changes in the median percentage of platelets expressing P selectin at 40 h compared to 0 h are shown. Each horse has the same unique symbol at both time points for all treatments (horse 1: -□-; horse 2: -○-; horse 3: -⋆-; horse 4: -⧫-; horse 5: -X-; horse 6: -▾-; horse 7: -·-; horse 8: -▪-; horse 9: -▴-).

Figure 5

Changes in the percentage of platelets expressing P selectin in platelet-rich plasma of 9 individual horses at 0 h (baseline) and 40 h after starting treatment (4 h after the last dose) with placebo (left column), unfractionated heparin (UFH, middle column) or low-molecular-weight heparin (LMWH, right column). Platelets were exposed to the PBS negative control (A) or the ADP positive control (B), 40 µM) for 10 min ex vivo. Significant changes in the median percentage of platelets expressing P selectin at 40 h compared to baseline (0 h) are shown. Each horse has the same unique symbol at both time points for all treatments (horse 1: -□-; horse 2: -○-; horse 3: -⋆-; horse 4: -⧫-; horse 5: -X-; horse 6: -▾-; horse 7: -·-; horse 8: -▪-; horse 9: -▴-). Note the Y-axis scale for PBS differs from that of ADP.

Figure 6

Changes in the median percentage of platelets expressing P selectin (with individual data points) in platelet-rich plasma (PRP) diluted 1:100, 1:20 and 1:5 in binding buffer. The diluted PRP was then exposed to low (A) or high (B) concentrations of thrombin (low concentration, 0.15 U/ml; high concentration 0.5 U/ml) and the EHV-1 strains (low concentration, 1 × 10⁶ PFU/ml; high concentration 5 × 10⁶ PFU/ml), Ab4 and RacL11, for 10 min ex vivo. Phosphate-buffered saline (PBS) served as a negative control for platelet stimulation. Blood was collected from the same horse on 3 different occasions for these experiments. Significant changes in the median percentage of P selectin-positive platelets between dilutions are shown.

Figure 7

Representative curves showing the variation in baseline thrombin generation (nm thrombin generated over time in minutes) in platelet-poor plasma (PPP) from a single horse (horse 2) exposed ex vivo to high concentrations (5 × 10⁶ PFU/ml) of the EHV-1 viral strains, Ab4 and RacL11. Baseline samples were obtained before horse was given placebo, unfractionated heparin (UFH) or low-molecular-weight heparin (LMWH). The RacL11 strain had a shorter lag time and faster rate of thrombin generation than the Ab4 strain. In this particular horse, the RacL11 strain generated more thrombin than the Ab4 strain.