Measurement of platelet aggregation in ovine blood using a new impedance aggregometer

Background: Whole blood platelet aggregometry (impedance) is an important method to investigate platelet function disorders. Examination of hemostatic function in sheep is important with respect to their role as an animal model of human disease. Objective: The aim of this study was to evaluate and optimize selected methodological aspects (anticoagulant, agonist concentration) of impedance aggregometry in ovine blood using the new Multiplate 5.0 analyzer. Methods: Blood samples were collected in hirudin anticoagulant from 40 clinically healthy sheep. Samples from selected sheep were collected in citrate, with or without the addition of calcium chloride. The agonists adenosine diphosphate (ADP), collagen, ristocetin, arachidonic acid, and thrombin receptor‐activating peptide (TRAP) were added in several concentrations to induce aggregation. Results: Based on maximum aggregation values and internal precision, no significant difference was found between ADP concentrations of 3–10 μmol/L and collagen concentrations of 3–5 μg/mL (P>.05). The lowest interindividual variation of approximately 3–4‐fold was seen with 4 and 5 μmol/L ADP and 4 and 5 μg/mL collagen. Ristocetin, arachidonic acid, and TRAP did not induce significant aggregation at any concentration. Aggregation results were significantly lower when measured in citrate‐ vs hirudin‐anticoagulated blood, regardless of the presence of calcium chloride. Conclusions: Our results indicate that the multiplate impedance aggregometer is suitable for the measurement of platelet aggregation in sheep using optimal agonist concentrations of 4–5 μmol/L ADP and 4–5 μg/mL collagen. Hirudin‐anticoagulated blood is the preferred sample material.     

Effects of aspirin dose escalation on platelet function and urinary thromboxane and prostacyclin levels in normal dogs

Established “low” aspirin dosages inconsistently inhibit platelet function in dogs. Higher aspirin dosages consistently inhibit platelet function, but are associated with adverse effects. The objectives of this study were to use an escalation in dosage to determine the lowest aspirin dosage that consistently inhibited platelet function without inhibiting prostacyclin synthesis. Eight dogs were treated with five aspirin dosages: 0.5 mg/kg q24h, 1 mg/kg q24h, 2 mg/kg q24h, 4 mg/kg q24h and 10 mg/kg q12h for 7 days. Utilizing aggregometry and a whole‐blood platelet function analyzer (PFA‐100), platelet function was evaluated before and after treatment. Urine 11‐dehydro‐thromboxane‐B₂ (11‐dTXB₂) and 6‐keto‐prostaglandin‐F_1α (6‐keto‐PGF_1α), were measured. Compared to pretreatment, there were significant post‐treatment decreases in the maximum aggregometry amplitude and increases in the PFA‐100 closure times for all dosages expect 0.5 mg/kg q24h. There was no difference in amplitude or closure time among the 2 mg/kg q24h, 4 mg/kg q24h, and 10 mg/kg q12h dosages. Compared to pretreatment values, there was a significant decrease in urinary 11‐dTXB₂‐to‐creatinine and 6‐keto‐PGF_1α‐to‐creatinine ratios, but there was no dose‐dependent decrease for either metabolite. An aspirin dosage of 2 mg/kg q24h consistently inhibits platelet function without decreasing prostacyclin synthesis significantly more than lower aspirin dosages.     

Suspected aspirin resistance in individual healthy adult warmblood horses

The reasons for this prospective experimental study were to determine a dosing scheme with loading and maintenance dose of aspirin inducing inhibition of platelet function measured by whole blood impedance aggregometry. Ten horses received aspirin orally in the morning with one loading dose of 4.7–5 mg/kg and maintenance doses of 1–1.3 mg/kg daily the following 4 days. Aggregometries (COLtest, ASPItest, ADPtest) and serum salicylic acid were measured. ASPItest showed significant difference in inhibition at 24 and 48 hr (p < .05) and 96 hr (p < .01). Significant change for ADPtest and COLtest couldn't be detected. Serum salicylic acid concentrations were significantly (p < .01) increased at 6 and 12 hr. Despite this, three horses failed any inhibitory effect of platelet function, suspecting an aspirin resistance. Regarding the other seven horses platelet aggregation induced by ASPItest was reduced between 37% and 100% from baseline at 6 and 12 hr and between 0 and 98% during the next 4 days. Correlations of serum concentration of salicylic acid and aggregometries couldn't be detected. It can be presumed that equine platelets are less susceptible to aspirin what may compromise eventually the anticoagulatory effects and efficacy in preventing and treating diseases with increased platelet activation as endotoxaemia or laminitis.     

Temporal aspects of onion-induced antiplatelet activity

Organosulfur compounds in onion extracts are formed following thelysis of the S-alk(en)yl-L-cysteine sulfoxides by alliinase. Thesecompounds inhibit the aggregation of human blood platelets and offer thepotential for positive cardiovascular health benefits. An experiment wasdesigned to examine temporal and temperature effects on onion-inducedantiplatelet activity. Platelet aggregation is induced by various agonists,including ADP, collagen, and thrombin. Unexpectedly, all freshly-juicedonion extracts (ca. 5 minutes post-juicing) appeared to exhibit both anagonist-free aggregation peak (AFP) and a platelet inhibitory peak (PIP)characteristic of inhibition of platelet aggregation. The AFP was minimalby 30 minutes and dissipated in all treatments by 120 minutes, whilethe PIP increased as onion extracts aged and did not change after 30minutes at 25 ^°C. This finding confirms the observation that the in vitro platelet inhibitory activity of onion organosulfur compounds istime dependent. Freshly-prepared onion extracts were incubated with theADP scavenger enzyme apyrase (E.C. 3.6.1.5). AFPs were abolished inapyrase-treated extracts, suggesting that this response may have been dueto free ADP in onion extracts, although an amount of ADP required togenerate such a response would be unexpected in onion extracts. Inaddition, platelet aggregates were not observed in the AFP, suggesting thisresponse may be associated with changes in light transmission throughplatelet rich plasma that are not associated with platelet aggregation.Artifacts of analysis are, therefore, possible when assessing onion-inducedantiplatelet activity with freshly-juiced extracts. Temporal formation ofplatelet-inhibiting organosulfur compounds should be taken into accountduring both in vitro and in vivo assessment of onion-inducedantiplatelet activity.