Pharmacokinetics and Pharmacodynamics of an Oral Formulation of Apixaban in Horses After Oral and Intravenous Administration

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

A novel canine model of immune thrombocytopenia: has immune thrombocytopenia (ITP) gone to the dogs?

Canine immune thrombocytopenia (ITP) is analogous to human ITP, with similar platelet counts and heterogeneity in bleeding phenotype among affected individuals. With a goal of ultimately investigating this bleeding heterogeneity, a canine model of antibody-mediated ITP was developed. Infusion of healthy dogs with 2F9, a murine IgG2a monoclonal antibody to the canine platelet glycoprotein GPIIb (a common target of autoantibodies in ITP) resulted in profound, dose-dependent thrombocytopenia. Model dogs developed variable bleeding phenotypes, e.g. petechiae and haematuria, despite similar degrees of thrombocytopenia. 2F9 infusion was not associated with systemic inflammation, consumptive coagulopathy, or impairment of platelet function. Unexpectedly however, evaluation of cytokine profiles led to the identification of platelets as a potential source of serum interleukin-8 (IL8) in dogs. This finding was confirmed in humans with ITP, suggesting that platelet IL8 may be a previously unrecognized modulator of platelet-neutrophil crosstalk. The utility of this model will allow future study of bleeding phenotypic heterogeneity including the role of neutrophils and endothelial cells in ITP

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

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 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 (LMWH) heparin 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 to 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

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

Influence of canine donor plasma hemostatic protein concentration on quality of cryoprecipitate

Background Cryoprecipitate (CRYO) is a plasma component containing high concentrations of factor VIII (FVIII), von Willebrand factor (VWF), and fibrinogen. Because Greyhounds are reported to have lower plasma VWF and fibrinogen concentrations, their plasma may not yield high potency CRYO. Objectives To determine if plasma hemostatic protein concentration is a good predictor of CRYO potency and if a difference exists in quality of CRYO prepared from Greyhounds versus non‐Greyhounds. Animals Twenty Greyhounds and 20 non‐Greyhounds. Methods A 450 mL unit of blood was collected from each donor, centrifuged to prepare fresh frozen plasma (FFP), and processed to CRYO. Aliquots of FFP and CRYO were analyzed for FVIII, VWF, and fibrinogen content and factor recovery. Results A positive correlation was found among donor plasma FVIII, VWF and fibrinogen concentration, and CRYO factor content (P < .001). Mean recovery was highest for VWF (67%), followed by fibrinogen (47%), and FVIII (37%). No breed difference was found in mean CRYO FVIII content, but CRYO VWF and fibrinogen were lower in Greyhounds (P = .004 and P < .001, respectively). No difference was found between Greyhounds and non‐Greyhounds for the number of CRYO units meeting human blood banking standards. Conclusions and Clinical Importance Factor concentration in FFP is associated with CRYO potency, suggesting that prescreening of blood donors may enhance CRYO quality. Despite lower VWF and fibrinogen content, CRYO prepared from Greyhounds is acceptable based on blood banking standards for humans, indicating that Greyhound FFP does not need to be excluded from CRYO production

A Common Missense Variant Causing Factor XI Deficiency and Increased Bleeding Tendency in Maine Coon Cats

Hereditary factor XI (FXI) deficiency is characterized as an autosomal mild to moderate coagulopathy in humans and domestic animals. Coagulation testing revealed FXI deficiency in a core family of Maine Coon cats (MCCs) in the United States. Factor XI-deficient MCCs were homozygous for a guanine to adenine transition resulting in a methionine substitution for the highly conserved valine-516 in the FXI catalytic domain. Immunoblots detected FXI of normal size and quantity in plasmas of MCCs homozygous for V516M. Some FXI-deficient MCCs experienced excessive post-operative/traumatic bleeding. Screening of 263 MCCs in Europe revealed a mutant allele frequency of 0.232 (23.2%). However, V516M was not found among 100 cats of other breeds. Recombinant feline FXI-M516 (fFXI-M516) expressed ~4% of the activity of wild-type fFXI-V516 in plasma clotting assays. Furthermore, fFXIa-M516 cleaved the chromogenic substrate S-2366 with ~4.3-fold lower catalytic efficacy (kcat/Km) than fFXIa-V516, supporting a conformational alteration of the protease active site. The rate of FIX activation by fFXIa-M516 was reduced &gt;3-fold compared with fFXIa-V516. The common missense variant FXI-V516M causes a cross-reactive material positive FXI deficiency in MCCs that is associated with mild-moderate bleeding tendencies. Given the prevalence of the variant in MCCs, genotyping is recommended prior to invasive procedures or breeding

Proteomic profiling of the thrombin-activated canine platelet secretome (CAPS).

Domestic dogs share the same environment as humans, and they represent a valuable animal model to study naturally-occurring human disease. Platelet proteomics holds promise for the discovery of biomarkers that capture the contribution of platelets to the pathophysiology of many disease states, however, canine platelet proteomic studies are lacking. Our study objectives were to establish a protocol for proteomic identification and quantification of the thrombin-activated canine platelet secretome (CAPS), and to compare the CAPS proteins to human and murine platelet proteomic data. Washed platelets were isolated from healthy dogs, and stimulated with saline (control) or gamma-thrombin (releasate). Proteins were separated by SDS-page, trypsin-digested and analyzed by liquid chromatography and tandem mass spectrometry (MS). CAPS proteins were defined as those with a MS1-abundance ratio of two or more for releasate vs. unstimulated saline control. A total of 1,918 proteins were identified, with 908 proteins common to all dogs and 693 characterized as CAPS proteins. CAPS proteins were similar to human and murine platelet secretomes and were highly represented in hemostatic pathways. Differences unique to CAPS included replacement of platelet factor 4 with other cleavage products of platelet basic protein (e.g. interleukin-8), novel proteins (e.g. C-C motif chemokine 14), and proteins in relatively high (e.g. protease nexin-1) or low (e.g. von Willebrand factor) abundance. This study establishes the first in-depth platelet releasate proteome from healthy dogs with a reference database of 693 CAPS proteins. Similarities between CAPS and the human secretome confirm the utility of dogs as translational models of human disease, but we also identify differences unique to canine platelets. Our findings provide a resource for further investigations into disease-related CAPS profiles, and for comparative pathway analyses of platelet activation among species