Assessment of Platelet Function in Horses: Ultrastructure, Flow Cytometry, and Perfusion Techniques

Abstract

We studied equine platelet function and activation using ultrastructural examination, flow cytometry, and perfusion. The main aim of the study was to evaluate hemostatic mechanisms in horses using these techniques. Ultrastructural observations were done on resting and activated platelets. Flow cytometry was used to evaluate binding of antibodies to major platelet glycoproteins (GPIIb-IIIa, GPIV, and GPIb) and activation-dependent antigens (P-selectin and lysosomal integral membrane protein [LIMP]). Perfusion techniques were used to evaluate the interaction between platelets and damaged subendothelium. Aggregation experiments were done to identify the best agonists for flow cytometry. Ultrastructural observations confirmed that equine platelets lack a developed open canalicular system and that release of granule contents occurs by fusion of adjacent granule membranes that ultimately connect with external membranes. Flow cytometry identified a 2-fold increase in binding of antibodies against GPIIb-IIIa and GPIV after activation. Binding of antibodies against P-selectin and LIMP increased from 2.12 and 1.74% to 15.5 and 11.6%, respectively, in response to thrombin and to 21.86 and 10.50%, respectively, in response to collagen. Annexin V binding increased moderately after activation. Perfusion experiments with citrated blood indicated that equine platelets react more strongly to subendothelium than do human platelets. When blood was anticoagulated with low molecular weight heparin, a marked impairment of platelet interactions was observed. In conclusion, although some differences were observed between human and equine platelet function, some techniques currently used to assess human platelet function may be useful to assess equine platelets.

Protein expression in platelets from six species that differ in their open canalicular system

“…Finally, the platelet responses to thrombin (0.1 U/mL), as measured by aggregometry, were different. Consistent with previous studies [7, 27–32], OCS − platelets showed markedly altered aggregation kinetics as compared to OCS + platelets.…”

“…Previous studies of the structure of platelets from different species show that platelets from some species lack recognizable OCS structures and do not have the distinctive pattern of internal membrane staining when membrane-impermeant tannic acid is used as a stain [3–7, 22]. Based on previous reports and observations from our laboratory, we separated the platelets from six different species into OCS + (human, mouse and dog) and OCS − (horse, cow and camel) groups (Table III).…”

“…Comparative ultrastructural studies of platelets show that certain species lack the invaginated tubular membrane structures defined as OCS [5–7]. The present study seeks to delineate some of the biochemical differences associated with the presence or absence of the OCS by examining the expression of 18 different proteins in platelets from three species that have obvious OCS (OCS + ; human, mouse and dog) and three that do not (OCS − ; cow, camel and horse) [3–7, 22].…”

“…The present study seeks to delineate some of the biochemical differences associated with the presence or absence of the OCS by examining the expression of 18 different proteins in platelets from three species that have obvious OCS (OCS + ; human, mouse and dog) and three that do not (OCS − ; cow, camel and horse) [3–7, 22]. Specific focus was placed on the small GTPases (Rho, Ras and Arf families) and their regulators, which are known to play roles in cytoskeleton and membrane dynamics.…”

Effects of Firocoxib, Flunixin Meglumine, and Phenylbutazone on Platelet Function and Thromboxane Synthesis in Healthy Horses

“…However, when compared to the gold standard and more specific indicator of platelet aggregation, turbidimetric aggregometry, platelet function was not inhibited during drug administration, suggesting that the collagen/epinephrine cartridge was not an accurate representation of platelet function in horse. In horses, epinephrine is not a consistent and potent platelet agonist; therefore, the collagen/epinephrine cartridge in the platelet function analyzer does not consistently stimulate platelet aggregation, limiting the clinical application of the collagen/epinephrine cartridge …”