Purpose: Circulating platelets in the mammalian bloodstream rapidly adhere to vessel surfaces, other cell types and to each other in response to a variety of physiological stimuli such as the extracellular matrix protein collagen and the coagulation protease thrombin. Platelets make extensive cell-cell contacts that enable the formation of multicellular particles known as aggregates, which are essential for the normal physiological process of preventing blood loss (hemostasis). Upon reaching a critical size, aggregates can also become life-threatening thrombi that restrict blood flow and initiate a variety of ischemic cardiovascular pathologies including myocardial infarction and stroke, the leading causes of death and morbidity worldwide.
Platelet aggregation is routinely measured ex vivo with light transmission aggregometry (LTA), which detects aggregate formation by measuring the turbidity of a platelet solution stimulated with an agonist such as collagen or thrombin. Although popular and clinically useful, LTA measures platelet reactivity indirectly and does not report aggregate sizes or population distributions. Here, we hypothesize that particle size analysis using laser defraction directly captures critical aspects of platelet reactivity such as aggregate size and population distribution, which potentially complements and enhances data generated by LTA.
Methods: To test this hypothesis, we purified platelets from rat blood, stimulated them with agonists and measured aggregation via two methods: 1) LTA with a Chrono-log Model 700 lumi-aggregometer or 2) laser defraction with a Malvern Mastersizer 3000 particle size analyzer. LTA samples were immediately fixed in formalin and remained on ice for 24 hours before particle size analysis.
Results: The rate and extent of platelet aggregation measured by LTA was dependent on agonist concentration and platelet number as expected. Aggregation was also completely abolished by pretreatment with the known platelet inhibitor prostaglandin I2 (PGI2), which validated our platelet preparations. Similar to LTA, laser defraction particle analysis clearly distinguished unstimulated from agonist-stimulated or inhibitor-treated platelets. Unlike LTA, however, laser defraction revealed distinct aggregate sizes associated with various platelet populations. Platelets not treated with agonist (unstimulated) resolved into one major population corresponding in size to individual, unaggregated platelets 1 – 5 microns in diameter, whereas platelets stimulated with collagen were concentrated in additional platelet populations ranging from 100 – 916 microns in diameter (see Figure 1). These collagen-dependent populations were dramatically larger than the unstimulated platelet population and were completely eliminated by PGI2, indicating they consisted entirely of multicellular aggregates. Aggregate size was dependent on the total number of platelets in the reaction; increasing platelet concentration up to 2 x 10e8/mL correlated with enlarged aggregates in the presence of collagen (see Figure 1). Furthermore, aggregate size positively correlated with agonist concentration as increasing doses of collagen generated more populations with larger particle diameters.
Conclusion: In conclusion, laser defraction particle analysis accurately measures shifting levels of platelet activation under various conditions much like LTA, but with the additional advantages of absolute particle size resolution and population recognition. Laser defraction combined with LTA, therefore, represents a promising new ex vivo platform for determining platelet reactivity, which is an essential first step for determining therapeutic efficacies of potential antiplatelet agents.
Paul Johnson– Adjunct Faculty and Manager - QC/R&D Analytical Labs, Campbell University College of Pharmacy and Health Sciences, North Carolina