Original ResearchEffect of Different Storage Conditions on Platelet Aggregation in Horse
Introduction
The two main components of the thrombotic process are platelet aggregation and coagulation. In particular, platelet aggregation is a crucial step in the clot-forming process that involves cell-cell and cell-substrate adhesion, and cell signaling and response, all of which occur within the moving blood.1 The role of platelets in hemostasis and arterial thrombosis involves their adherence to sites of vessel injury or ruptured atherosclerotic plaques, aggregation to form hemostatic plugs or thrombi, and acceleration of the coagulation cascade leading to the formation of thrombin.2 This phenomenon, most accurately described as platelet cohesion although more commonly referred to as platelet aggregation, was quickly identified as the most important event in hemostatic plug formation.3, 4 Blood platelets can be considered as a component of the hemostatic system whose response to an agonist can be altered by compounds formed by or stored within platelets.5 A variety of substances are significant mediators in hemostasis and thrombosis, and they promote platelet aggregation in vitro, including adenosine diphosphate (ADP), adrenaline, collagen, and arachidonic acid.6, 7, 8 It has been well established that equine platelets aggregate in response to platelet-activating factor and ADP,9 but the potential effect of pre-analytical factors, such as storage temperature and time, on the formation of platelet aggregates (platelet clumping) is largely elusive in horses. The effect of anticoagulant, temperature of storage, and blood storage time on platelet response has been evaluated in healthy and ill dogs10, 11 and in human beings.12, 13, 14, 15, 16, 17 The loss of hemostatic function with storage at 4°C for 24 hours or longer has been well documented by autologous radiolabeled studies as well as by studies performed on thrombocytopenic patients.18, 19, 20 In many clinical conditions, alterations of platelet function occur in animal diseases such as epistaxis, hematuria, and hemorrhagic diarrhea.21 In equine medicine, verminous arteritis, systemic thromboembolism associated with endotoxemia, acute laminitis, ischemic gastrointestinal disorders,22 and several drugs used, such as acetylsalicylic acid, paracetamol, dextran,23, 24, 25, 26 are capable of modifying equine platelet aggregation. Therefore, studies on platelet function in horse, as previously reported in human beings and dogs, are essential for understanding the process of hemostasis. The platelet aggregation tests allow the assessment of primary hemostasis,27 and previously, it was used in horses to evaluate the effect of physical exercise on the daily rhythm of platelet aggregation.28
The aim of the present study was to examine the effects of short-term incubation, after storage for 6 hours at room temperature (RT), and after 6 and 24 hours storage at 8°C, and the effects of freezing at −20°C for 24 and 48 hours on platelet aggregatory responses in comparison with freshly isolated platelets.
Section snippets
Materials and Methods
Blood samples were obtained from 58 healthy horses of varying breeds and gender, ranging in age from 4 to 12 years, with a mean body weight of 480 ± 50 kg. Before starting, the study horses were subjected to a complete clinical examination, including change in hemostatic mechanisms, rectal temperature, respiratory and heart rates, as well as packed cell volume (reference range, 32%–53%) and platelet count (reference range, 120–400 K/μL), and all were considered within normal limits. Only
Results
All hematological values and clotting parameters obtained from 58 healthy horses were found to be within the normal physiological range for horses29 (Table 1).
Table 2 shows the average values of the maximum degree of aggregation and the slope of platelet aggregation, expressed in their conventional units of measurement, with SD and statistical significances, obtained in the different experimental conditions. Average values of the percentage of platelet aggregation and the slope of platelet
Discussion
The results of this study suggest that the storage of equine plasma for more than 6 hours after collection at RT and at 8°C has a significant effect on platelet aggregation, and that the storage of plasma for 24 and 48 hours at −20°C alters platelet aggregation.
Many clinicians use frozen plasma for identification of disease states or other characterization of patient health. However, there is very little information available on the effect of freezing on plasma samples. Until recently, most
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2013, Research in Veterinary ScienceCitation Excerpt :Evaluation of platelet response, or aggregation to agonists in vitro is used to extrapolate species-specific platelet responses in vivo. Platelet aggregation studies using adenosine diphosphate (ADP), collagen, arachidonic acid, and epinephrine have been performed in many domestic and nondomestic species in vitro (Pelagalli et al., 2002; Soloview et al., 1999; Gader et al., 2006; Piccione et al., 2010). Particularly, it is was well established that equine platelets aggregate in response to ADP (Rickards et al., 2003) and its appearance in the media surrounding the platelet as a consequences of stimulus-induced secretion may constitute a positive feedback mechanism of platelet activation (Clemmons and Meyers, 1984).
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