Lyophilized platelets inhibit platelet aggregation with simultaneous paradoxical promotion of platelet adhesion

Abstract

Lyophilized platelets have been explored as a potential hemostatic agent due to their long-term ambient storage capabilities that make them readily available in various scenarios. Additionally, their high biocompatibility and the key role of platelet interactions in various clinical conditions make them a promising platform for drug delivery. To explore these applications and for wider clinical deployment, the interactions between lyophilized platelets and fresh platelets must be examined. This project characterized receptor expression on the lyophilized platelet surface and their ability to bind fibrinogen using flow cytometry. The effect of lyophilized platelets on aggregation of unaltered platelets was assessed using light transmission aggregometry while the effect on adhesion was evaluated using static and microfluidic assays. Lyophilized platelets maintained significant levels of GPIIb and GPVI receptors on their surface, though the expression was reduced from fresh platelets. Additionally, lyophilized platelets maintained GPIb expression similar to fresh platelets. Furthermore, 15.8% of the lyophilized platelets exhibited the active conformation of the GPIIb/IIIa receptor, indicating a significant increase over fresh platelets. Lyophilized platelets also exhibited an increase in exposed phosphatidylserine and fibrinogen binding. Despite the effect of lyophilized platelets in promoting the adhesion of fresh platelets on a collagen-coated surface, their net effect was inhibitory on platelet aggregation. This study demonstrates that lyophilized platelets can have paradoxical effects on platelet adhesion and aggregation, which could have an impact for clinical applications. Detailed characterization and engineering of these effects will be important for their continued development as a drug delivery platform.

Keywords: blood product; lyophilized platelets; platelet adhesion; platelet aggregation; platelets.

FIGURE 1

Characterization of Key Receptor Surface Expression on Lyophilized and Fresh Human Platelets by Flow Cytometry. Quantification of expression level (left), percent positive events (middle), and representative histograms (right), for (A,B,C) GPIIb [LP: n = 14, PLT: donors = 5, n = 12], (D,E,F) active GPIIb/IIIa [LP: n = 6, PLT: donors = 4, n = 12], (G,H,I) GPIb [LP: n = 6, PLT: donors = 5, n = 12], (J,K,L) GPVI [LP: n = 13, PLT: donors = 3, n = 6].

FIGURE 2

Characterization of Phosphatidylserine Exposure and Fibrinogen Binding on Lyophilized and Fresh Human Platelets. Quantification of expression level (left), percent positive events (middle), and representative histograms (right), for (A,B,C) phosphatidylserine [LP: n = 6, PLT: donors = 2, n = 4], and (D,E,F) fibrinogen binding [LP: n = 12, PLT: donors = 2, n = 5].

FIGURE 3

Aggregation of Platelets in the presence of LPs at Different Concentrations and Ratios. Quantification (A,B) and representative LTA curves (C–F) for the aggregation of platelets compared to platelets mixed with LPs at ratios of 5:1 and 5:2 platelets to LPs using ADP agonist at concentrations of 20 uM (A; shown in representative figures (C–F) and 50 uM (B) using healthy platelet levels of (C) 200,000 platelets/uL [donors = 4, n = 8] and (D) 150,000 platelets/uL [donors = 4, n = 8], as well as thrombocytopenic platelet levels of (E) 100,000 platelets/uL [donors = 4, n = 8] and (F) 75,000 platelets/uL [donors = 3, n = 6] for all ratios. LP alone conditions are included for each platelet condition as a control.

FIGURE 4

Adhesion of Platelets Mixed with Lyophilized Platelets on a Collagen Surface. Normalized quantification of the adhesion of platelets in comparison to platelets mixed with LPs (at ratios of 5:1 and 5:2 platelets to LPs) in static conditions using and LDH assay (A,B) and under flow using calcein AM live-cell staining (C,D). Both assays were conducted at a (A,C) normal platelet concentration of 200,000 platelets/uL [Static: donors = 2, n = 4, Flow: donors = 3, n = 12], and a (B,D) thrombocytopenic platelet concentration of 75,000 platelets/uL [Static: donors = 2, n = 4, Flow: donors = 3, n = 12]. Note that the calcein AM live-cell staining (C,D) does not stain the inert LPs and thus the LP only condition does not represent their adhesion and is used as a control to ensure that there is no off target staining.