Correspondence Michelle Kutzler, College of Veterinary Medicine, Oregon State University, 105 Magruder Hall, Corvallis, OR 97331 USA E-mail: firstname.lastname@example.org
Background: Limited information exists regarding hemostasis in camelids despite the importance of platelet function testing in the accurate identification of platelet disorders. As further importation of llamas to North America is restricted, variability in breeding stock will continue to decrease, potentially leading to an increase in heritable bleeding disorders.
Objective: The objective of this study was to measure platelet aggregation responses in clinically healthy llamas and provide baseline data to which abnormal platelet function may be compared in the future.
Methods: Blood samples were collected from 39 healthy adult llamas, citrated, and centrifuged to produce platelet-rich plasma (PRP). Within 4 hours of the blood draw, 20 μL of each agonist reagent were added to 180 μL of PRP. Final concentrations of agonists were 2 × 10−5 M ADP, 0.19 mg collagen/mL PRP, 1 × 10−4 M epinephrine, and 500 μg arachidonic acid/mL PRP.
Results: Llama platelets were most responsive to ADP and collagen, with a maximum percent aggregation (mean±SD) of 71.3±18.6% and 55.8±19% and aggregation rates of 9.5±3.9 and 6.7±3.7 cm/min, respectively. Llama platelet aggregation in response to epinephrine and arachidonic acid was minimal to absent.
Conclusions: This study is the first of its kind to establish baseline values for platelet aggregation in healthy adult llamas.
Life-threatening bleeding disorders occur when platelet aggregation, a crucial step in the clot-forming process, does not proceed as expected. When a vessel wall is damaged, endogenous factors recruit and activate platelets from circulation, causing them to adhere to the subendothelium and form an initial platelet plug. If any step in the platelet activation pathway fails, bleeding will occur.
Injury to the vascular endothelium exposes the subendothelial collagen layer. Collagen adherence triggers platelet activation. The cytoplasmic granules within platelets are secreted into the extracellular space, releasing ADP, serotonin, and other constituents, which propagate adhesion and aggregation of other platelets in the area of compromised endothelium. ADP results in a conformational change of the major platelet integrin alpha2b beta3a (αIIbβ3), which allows for the binding of its ligand fibrinogen.1 Fibrinogen and von Willebrand factor are then able to form bridges between adjacent platelets and the platelet plug is finally stabilized by fibrin.2
Epinephrine potentiates platelet aggregation by binding adenylate cyclase G-protein–coupled receptors, which results in inhibition of adenylate cyclase (via Gi) and reduction of cAMP.3 Additionally, epinephrine potentiates platelet aggregation induced by collagen, ADP, and arachidonic acid.3,4 Activated platelets also synthesize thromboxane, which stimulates additional platelet activation and aggregation, as well as vasoconstriction.5,6 Arachidonic acid is an endogenous precursor of thromboxane, and is also used in in vitro models of platelet aggregation.5,6
Evaluation of platelet response, or aggregation, to agonists in vitro is used to extrapolate species-specific platelet responses in vivo. In vitro platelet aggregation studies using ADP, collagen, arachidonic acid, and epinephrine have been performed in many domestic and nondomestic species.7–14 Llamas and other camelids are evolutionarily distinct from other ungulates due to the arid and high-altitude environments in which they evolved. An adaptive platelet response, like other hematologic adaptations of camelidae (eg, elliptical-shaped erythrocytes), may be present in llamas.15,16
Platelet aggregation studies characterize abnormalities in platelet shape change, signal recognition, signaling pathways, interplatelet adhesion, and secretory responses.17 They have been used to identify animals with abnormal phenotypes leading to hereditary thrombopathies, such as Glanzmann thrombasthenia, as well as identifying carriers with mild functional abnormalities.17
Little information is available regarding normal platelet function in camelids. An extensive search of the literature revealed only 1 report on platelet aggregation in 1 llama and 1 guanaco of unspecified age and sex.18 Because importation of South American llamas is severely restricted by the USDA,19 genetic variability in North American breeding stock will decrease, potentially leading to heritable platelet disorders. The information provided in this study will serve as a baseline to which abnormal platelet function may be compared.
Materials and Methods
Whole blood samples were collected by jugular venipuncture from 39 healthy adult llamas owned by Oregon State University or local llama producers. The sample population included 9 geldings, 1 intact male, and 29 females. The llama's age ranged from 3 to 20 years (mean=10.6 years). A solution of 3.8% sodium citrate was added postcollection to the blood in the syringe at a ratio of 9:1 (blood:citrate). None of the animals used in the study had received acetylsalicylic acid-containing compounds (aspirin) or other nonsteroidal anti-inflammatory drugs within the previous 12 months. A physical examination that included body weight, body condition score, rectal temperature, heart and respiratory rates, and general physical appearance was performed. A PCV, total protein concentration, and platelet count were determined for each animal to ensure no preexisting conditions existed that could alter platelet function. All experimental procedures were approved by the Oregon State University Institutional Animal Care and Use Committee.
The citrated whole blood samples were centrifuged at 220 g for 10 minutes to produce platelet-rich plasma (PRP) and at 1500 g for 15 minutes to produce platelet-poor plasma (PPP).20 The PRP and PPP were used to calibrate a dual channel optical density aggregometer (Sienco Dual Sample Aggregation Meter, Model DP-247-F, Sienco Inc., Arvada, CO, USA) to 5% and 95% light transmittance, respectively, on an attached flatbed recorder (Pharmacia Aggregometer Chart Recorder, Model 482, Uppsala, Sweden), with a tracing rate of 0.5 cm/min. Within 4 hours of blood collection, platelet aggregation was induced using agonists (Cat. No. 101310, Lot No. 06800083, Bio/Data, Horsham, PA, USA) added to 180 μL PRP. Final concentrations were 2 × 10−5 M ADP, 0.19 mg collagen/mL PRP, 1 × 10−4 M epinephrine, and 500 μg arachidonic acid/mL PRP. Each reaction was stirred at a rate of 600 rpm and maintained at 37°C under normal atmospheric conditions. Samples were processed in duplicate.
Aggregation was recorded until maximum aggregation occurred. Maximum percent aggregation as a function of optical density was calculated by dividing the number of divisions between baseline and maximum amplitude by the number of divisions between baseline and 100% on the chart paper.20 The rate of aggregation as a function of the slope was calculated by drawing a line tangent to the aggregation curve, and determining the slope from 2 points along that line using the formula y2–y1/x2–x1=slope.20 The mean of the duplicates was used for these calculations. Data to establish normal llama platelet aggregation response to agonists were analyzed using Microsoft Excel (Microsoft Corporation, Seattle, WA, USA) and Statview software (Version 5.0.1, SAS Institute, Cary, NC, USA). Results were reported as mean±SD and minimum–maximum values.
All llamas were clinically healthy based on physical examination. PCV and plasma protein concentrations were 28.6±3.6% (22–36%) and 5.7±0.4 g/dL (5.0–6.8 g/dL), respectively. Mean platelet count was 230,000±57,000/μL (143,000–366,000/μL). Platelet aggregation tracings using ADP, collagen, epinephrine, and arachidonic acid for a representative llama are shown in Figure 1. Platelet aggregation from arachidonic acid was minimal and testing was discontinued after 20 animals failed to respond to the agonist. Maximum percent aggregation in response to ADP, collagen, and epinephrine was 71.3±18.6% (18.9–98.3%), 55.8±19% (20.3–94%), and 5.5±1.6% (2.2–9.4%), respectively. Rate of aggregation in response to ADP and collagen was 9.5±3.9 (2.4–18.9) cm/min and 6.7±3.7 (1.2–16.6) cm/min, respectively. No rate could be determined for epinephrine, because the slope was undefined.
The platelet aggregation response to ADP, collagen, and epinephrine reported in the current study was in agreement with results from the 1 llama that were reported previously.18 A study of platelet aggregation similar to ours was conducted on 103 Arabian camels (Camelus dromedarius).12 Similar to llamas, camel platelets were unresponsive to arachidonic acid (1.64 mmol/L) and epinephrine (100 μmol/L). ADP and collagen at the same concentrations used in our study yielded positive aggregation responses in camels, but the maximum percent aggregation in camels (42±24% and 35±28%, respectively) was lower than in the llamas. However, the slope of the aggregation curve in the llamas was not as steep for ADP and collagen as it was for camels (12±7 and 10±6 cm/min, respectively).
In cattle, elephants, miniature pigs, mink, and rats, arachidonic acid fails to stimulate platelet aggregation.1,6,7,14 In the present study, arachidonic acid failed to stimulate llama platelet aggregation even when the concentration was doubled from 0.5 to 1 mg/mL (data not shown). One possibility for this response is that llama platelets are unable to produce thromboxane through the mechanism tested in vitro. However, as thromboxane was not measured in this study, it cannot be concluded that this was the reason for the failed response. Another possibility is that llama platelets are insensitive to thromboxane. Platelets from rats, cattle, and elephants have been shown to be insensitive to thromboxane, while platelets of guinea pigs, horses, humans, rabbits, and about 30% of dogs react especially well.8 Testing the llama platelet aggregation response to exogenous thromboxane would aid in determining whether this species is also insensitive; however, this was beyond the scope of the current study.
It has been suggested that platelets exposed to a low concentration of epinephrine in the blood during periods of stress may be prepared for a more rapid response to subendothelial collagen exposed during a “fight or flight” situation.4 Epinephrine in vitro was found to synergize with other agonists (eg, ADP) and exhibit a strong aggregation response by increasing the affinity of these agonists for their receptors.9,13 However, except for humans and other primates, epinephrine alone, even at supraphysiologic concentrations, induces a weak to absent response when added to mammalian platelet suspensions.8,14 It is not known if epinephrine would have the same in vitro additive effect with ADP or collagen in llamas as this was not examined in the present study.
In conclusion, this study is the first of its kind to establish baseline values for platelet aggregation in healthy, adult llamas. Multiple concentrations of agonists should be used to fully evaluate platelet response in llamas. If comparative studies were to be done between llamas and other species, ADP and collagen would be the agonists of choice. Comparative studies of platelet aggregation among other camelids, particularly alpacas, or in neonates, would also be of interest.
Funding for this project was through the Merck–Merial Summer Scholars Program, 2005.