Poster T19 - Plasma Osmolarity as a Determinant of Platelet Morphology and Function in Heart Failure

Introduction: Heart failure (HF) is associated with increased sodium and water retention, though disproportionately, leading to hypervolemic hyponatremia and reduced plasma osmolality ( < 275 mOsm/kg). HF is also strongly associated with thrombosis and thromboembolic adverse advents, either in the native heart, or in mechanical circulatory support (MCS) devices implanted to restore hemodynamics. Platelets, critical cells in thrombotic processes, are mechanosensitive, responding to altered shear flow and turbulence associated with HF or MCS devices. Excess plasma water and hypo-osmolarity can induce a gradient via which cells become swollen, edematous and stiff as water is forced into cells. It is recognized in cell biology that volume regulation and cell swelling can impact cell mechanics and function. However, it is not well understood how these processes are regulated or affect platelet function. Here, we investigate the effect of altered tonicity on platelet morphology and geometry, and its corresponding effect on platelet activation and aggregation.

Materials and

Methods: Human gel-filtered platelets (GFP; 20,000/µL) or platelet rich plasma (PRP; 100,000/µL) were obtained from fresh ACD-A anticoagulated human whole blood. Modified Tyrode’s buffer osmolality was measured (Advanced Instruments Osmo1), mixed with either GFP or PRP, and adjusted to a final concentration of either 250 mOsm/kg (“Hypotonic”), 285 mOsm/kg (“Isotonic”), or 325 mOsm/kg (“Hypertonic”). Scanning ion conductance microscopy (SICM) (Park Systems NX12) was utilized to quantify platelet morphology and volume. Platelet activation was measured as thrombin generation rate utilizing a previously described modified prothrombinase assay. Platelet aggregation was quantified from agonist-mediated aggregation using a light-transmission aggregometer (BIO/DATA Corp; PAP-8E). All experiments were performed in duplicate with N ≥ 3 donors.

Results, Conclusions, and Discussions: Overall, platelet geometry and function were altered in an osmolarity-dependent fashion. Hypotonic conditions led to an average 2.1µm3 (17.7%) increase in platelet volume, compared to isotonic conditions (Fig 1A). Interestingly, despite this large increase in platelet volume, there was a non-significant increase in thrombin generation for platelets in hypotonic conditions; however, a hypertonic environment led to a significant increase in platelet thrombin generation rate (0.14 min-1 average increase; Fig 1B). Collagen-mediated aggregation had a negative correlation to osmolarity, with significantly higher aggregation under hypotonic conditions (average 11.7% increase; Fig 1C).
Hypotonicity leads to increased platelet volume and increased aggregability to collagen; however, platelet thrombin generation remains unaffected. The results of our study provide insight into operative mechanisms of thrombogenicity and altered platelet function under heart failure hypo-osmotic conditions. Further, these studies are hypothesis-generating as to the potential role of water, ion and membrane mechanisms operative, providing the foundation for definition of the impact of platelet volume regulation on shear-mediated platelet dysfunction in future studies.

Acknowledgements (Optional):