Heterogeneity in Agonist-induced Activation Responses and Thrombus Formation: Differences between Large and Small Platelets

Background: Heterogeneity between the platelets of a subject exist in response to collagen and thrombin, for instance in Ca²⁺-dependent exposure of procoagulant phosphatidylserine (PS). The reasons for these differences are largely unknown. Here, we investigated if platelet size is a contributor to this functional heterogeneity.

Methods: Fractions of large and small platelets were separated by differential centrifugation, using blood from healthy blood donors. Aggregation of the isolated platelets was measured by light transmission aggregometry. In platelets loaded with fluorescent ratioed Ca²⁺ dye, cytosolic Ca²⁺ rises were measured in response to thapsigargin, 2-t-butylhydrochinone (TBHQ), or TRAP-6. Static adhesion of isolated platelets was followed in time by microscopy. Autologous blood, depleted from platelets and leukocytes, was reconstituted with large or small platelets at similar amounts or similar plateletcrits, and recalcified. Collagen-dependent thrombus formation was assessed under flow at defined wall-shear rate, using multicolor fluorescence microscopic imaging. Formed thrombi were analyzed in time for platelet adhesion, aggregation, P-selectin expression, and PS exposure.

Results: The mean volumes of large and small platelets fractions were 12.1 fL and 7.7 fL (p < 0.0001, n = 6), respectively. Intracellular Ca²⁺ stores of large platelets were 85 ±5% higher than those of small platelets, although their volume was only 55±4% bigger. TRAP-6-induced mobilization of cytosolic Ca²⁺ was significantly higher in large than in small platelets (p=0.015). Collagen-induced maximum aggregation was similar (83 vs. 88%), although aggregation was delayed for small platelets (Lag time 40 vs. 56s). In the static system, large platelets showed enhanced adhesion and spreading on a collagen surface (< 120 s), in comparison to small platelets (>300 s) and upon full spreading, mean surface area coverage (SAC) was higher for large (63.8 µm²) than for small (42.3 µm²; p < 0.0001) platelets. In reconstituted blood samples with the same plateletcrits, collagen-induced adhesion at high-shear flow rate was accelerated and enhanced for large platelets (26.2% SAC), in comparison to plateletcrit adjusted small platelets (15.9%, p < 0.05). In addition, platelet aggregate size, P-selectin expression and PS exposure were significantly (p < 0.05) increased in the blood samples with large platelets. This indicates that within the same individual large platelets show increased adhesion and activation at the collagen surface compared to small platelets.

Conclusion: Within the same subject, large platelets show higher Ca²⁺ signaling, activation responses, and incorporation into aggregates and thrombi, in comparison to small platelets. Both higher receptors levels and higher Ca²⁺ rises can explain this increased functionality.

No conflict of interest has been declared by the author(s).