Riboflavin and amotosalen photochemical treatments of platelet concentrates reduce thrombus formation kinetics in vitro

Background: Photochemical treatment (PCT) of platelet concentrates using photosensitizers and ultraviolet light illumination reduces the proliferation potential of pathogens by damaging biomolecules. Materials and Methods: The impact of riboflavin (RF-PRT)- and amotosalen (AS-PCT)-based pathogen inactivation on platelets was studied using microfluidic flow chambers on immobilized collagen using standard platelet concentrates prepared from buffy coats in additive solution. Flow cytometry, metabolic parameters and light transmission aggregometry with thrombin-related peptide, collagen and ristocetin were determined concurrently. Results: Both PCTs significantly decreased the platelet surface coverage kinetics in flow chambers over the course of the 7-day study. Platelet aggregation was affected following RF-PRT in response to all agonists, while AS-PCT mainly impacted low-dose ristocetin agglutination. RF-PRT induces premature platelet activation because integrin (IIb3) was spontaneously activated, and -degranulation, phosphatidylserine/-ethanolamine exposure and anaerobic metabolism significantly increased following treatment, which was not the case for AS-PCT. On the other hand, AS-PCT significantly diminished thrombus growth onto von Willebrand factor under shear flow. This defect was caused by fewer integrin (IIb3) interactions, not by defective GPIb-VWF binding as shown by adhesion experiments in the presence of tirofiban. Moreover, integrin (IIb3) activation was also affected following the activation of platelets via GPVI-FcRIIa or PAR1. Finally, amotosalen illumination as such is sufficient to induce platelet damage, with no additional measurable effect of the chemical adsorption step. Gamma irradiation caused no significant difference compared to controls on any time-point or for any parameter. Conclusion: Both PCTs significantly reduce thrombus formation rate but by different biochemical mechanisms