P74: Plasma Osmolarity as a Determinant of Platelet Morphology and Function in Heart Failure

Purpose: Heart failure (HF) is associated with increased sodium and water retention, though disproportionately, leading to hypervolemia and reduced plasma osmolality (<275 mOsm/kg). HF is also associated with thrombosis/thromboembolic adverse advents, either in the native heart, or in mechanical circulatory support (MCS) devices implanted to restore hemodynamics. Platelets, critical cells in thrombosis, are mechanosensitive, responding to altered shear flow associated with HF or MCS devices. Excess plasma water and hypo-osmolarity can induce a gradient via which cells become swollen and stiff as water is forced into cells. It is recognized in cell biology that volume regulation and 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 affect of altered tonicity on platelet morphology/geometry and corresponding affect on platelet activation and aggregation. Methods: Human gel-filtered platelets (GFP; 20,000/µL) or platelet rich plasma (PRP; 100,000/µL) were obtained from fresh ACD-A anticoagulated whole blood. Modified Tyrode’s buffer osmolality was measured (Advanced Instruments; Osmo1), mixed with platelets and adjusted to a final concentration of either 250mOsm/kg (“Hypotonic”), 285mOsm/kg (“Isotonic”), or 325mOsm/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 light-transmission aggregometry (BIO/DATA Corp; PAP-8E). All experiments were performed in duplicate with N ≥ 3 donors. Results: 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). Despite this large increase in volume, there was a non-significant increase in thrombin generation rate in hypotonic conditions; however, a hypertonic environment led to a significant increase (0.14 min-1 average increase; Fig 1B). Collagen-mediated aggregation had a negative correlation to osmolarity, with significantly higher aggregation in hypotonic conditions (average 11.7% increase; Fig 1C). Conclusions: 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, providing the foundation for defining the impact of platelet volume regulation on shear-mediated platelet dysfunction in future studies.