Sodium bicarbonate as a local adjunctive agent for limiting platelet activation, aggregation, and adhesion within cardiovascular therapeutic devices

Fig. 1. Effect of Sodium Bicarbonate on pH. Human platelets derived from ACD-A anticoagulated whole blood, were incubated (60:40) with purge solution. Purge solutions were formulated in base solution of 5% dextrose in water (“D5W”) with additives 50U/mL heparin (“D5W + Heparin”), 25 mEq/L sodium bicarbonate (“D5W + 25 Bicarb”), 50 mEq/L sodium bicarbonate (“D5W + 50 Bicarb”), or 100 mEq/L sodium bicarbonate (“D5W + 100 Bicarb”). pH was measured after 15 min of incubation with gel-A gel-filtered platelets (GFP) or B platelet rich plasma (PRP) utilizing micro pH electrode (Orion; PerpHecT ROSS). Values are reported as displayed as mean ± standard error. Data were collected from N ≥ 3 human donors; **indicates p-value < 0.01 in comparison to D5W

Fig. 2. Effect of sodium bicarbonateon platelet morphology. Humangel-filtered platelets (GFP) derived from ACD-A anticoagulated whole blood, were incubated (60:40) with purge solution. Purge solutions were formulated in base solution of 5% dextrose in water (“D5W”) with additives 50 U/mL heparin (“D5W + Heparin”), 25 mEq/L sodium bicarbonate (“D5W + 25Bicarb”), 50 mEq/L sodium bicarbonate (“D5W + 50Bicarb”), or 100 mEq/L sodium bicarbonate (“D5W + 100Bicarb”). Platelets were then pelleted via high-speed centrifugation and prepared for transmission electronmicroscopy (JEOL1200X). Images were captured at 10000× magnification. Scale bars (bottom right) in each image represent 1 μm

Fig. 3. Effect of sodium bicarbonate on platelet activation. Human gel-filtered platelets (GFP) derived from ACD-A anticoagulated whole blood, were incubated (60:40) with purge solution. Purge solutions were formulated in base solution of 5% dextrose in water (“D5W”) with additives 50 U/mL heparin (“D5W-Heparin”), 25 mEq/L sodium bicarbonate (“D5W-25 Bicarb”), 50 mEq/L sodium bicarbonate (“D5W-50 Bicarb”), or 100 mEq/L sodium bicarbonate (“D5W-100 Bicarb”). Flow cytometric detection (FACScantoII, BD BioSciences) of fluorescent platelet activation markers A P-selectin (CD62P); and B Annexin V binding were measured from the platelet surface. GFP samples were simultaneously measured for C thrombin generation rate via colorimetric detection in a modified prothrombinase assay. Values are reported as % of corresponding measurement in D5W solution for each donor, displayed as mean ± standard error. Data were collected from N ≥ 3 human donors, performed in duplicate (n ≥ 6). *Indicates p-value < 0.05; **Indicates p-value < 0.01 in comparison to D5W

Fig. 4. Effect of purge solution on agonist-mediated platelet aggregation. Human platelet rich plasma (PRP), derived from ACD-A anticoagulated whole blood, was incubated (60:40) with purge solution. Purge solutions were formulated in base solution of 5% dextrose in water (“D5W”) with additives 50U/mL heparin (“D5W-Heparin”), 25 mEq/L sodium bicarbonate (“D5W-25Bicarb”), 50 mEq/L sodium bicarbonate(“D5W-50Bicarb”), or 100 mEq/L sodium bicarbonate (“D5W-100Bicarb”). PRP samples were stimulated to aggregate via addition of agonists A 32 μM TRAP-6; B 10 μM calcium ionophore (A23187); C 20 μM ADP; or D 5 μg/mL collagen. Aggregation was measured over 10 min with light-transmission aggregometer (PAP-8E; Bio/Data Corporation) traces of aggregation over time are displayed into prow; quantitative data are reported as area under the curve (bottom row). Values are reported as % of aggregation measured in D5W solution for each donor, displayed as mean ± standard error. Data were collected from N ≥ 4 human donors; *Indicates p-value < 0.05;**indicates p-value < 0.01 in comparison to D5W

Fig. 5. Effect of sodium bicarbonate on platelet adhesion. Human gel-filtered platelets (GFP), derived from ACD-A anticoagulated whole blood, were incubated (60:40) with purge solution. Purge solutions were formulated in base solution of 5% dextrose in water (“D5W”) with additives 50 U/mL heparin (“D5W-Heparin”), 25 mEq/L sodium bicarbonate (“D5W-25 Bicarb”), 50 mEq/L sodium bicarbonate (“D5W-50 Bicarb”), or 100 mEq/L sodium bicarbonate (“D5W-100 Bicarb”). Samples were incubated with cytoplasmic membrane stain (BioTracker490, Invitrogen) and platelets were allowed to adhere to glass microscope slides. Images of slides were taken at 100× magnification and digitally analyzed for coverage area of platelets adhered to the surface. Data are reported as % area of platelet coverage of the image field of view. Scale bars (bottom right) in each image represent 25 μm. Digitally magnified images of platelet singlets are displayed as insets (upper right) with scale bars representing 3 μm. Values in graph are displayed as mean ± standard error; *Indicates p < 0.05 in comparison to D5W