Effects of riboflavin and ultraviolet light treatment on platelet thrombus formation and thrombus stability on collagen.

BACKGROUND: Pathogen reduction technologies (PRTs) are considered for the implementation of safer platelet (PLT) transfusion. PRT treatment involves the addition of a photosensitizer to a blood component followed by ultraviolet (UV) light irradiation. However, the effects of PRT treatment on PLT thrombus formation and thrombus stability have not been satisfactorily clarified. STUDY DESIGN AND METHODS: Leukoreduced PLT concentrates (PCs) were treated with riboflavin and UV light (Mirasol PRT). PLT thrombus formation on collagen was evaluated by the microchannel method, by which the total amount of PLTs deposited was measured as indices of thrombus formation and thrombus stability. Using a cone-plate shear-induced PLT aggregometer, PLT reactivity in blood flow was examined in a wide range of shear stresses of 6 to 108 dyn/cm2 . RESULTS: There was no significant difference in surface coverage between PRT-treated PLTs and control PLTs on collagen. On the other hand, the total amount of PRT-treated PLTs deposited was higher than that of control PLTs. The promotive effect of PRT treatment on PLT deposition completely disappeared in the presence of tirofiban, a potent integrin αIIbß3 inhibitor. The percentage of the dissociation of PRT-treated PLTs on collagen was lower than that of control PLTs after flushing with phosphate-buffered saline. PRT treatment significantly inhibited PLT aggregation under high-shear-stress conditions. CONCLUSION: Riboflavin-based PRT treatment of PCs leads to the enhancement of PLT thrombus formation and thrombus stability on collagen. However, it does not enhance the reactivity of PLTs not in contact with collagen under high-shear-stress conditions.

Real-time measurement of platelet shape change by light scattering under riboflavin and ultraviolet light treatment.

BACKGROUND: The adoption of pathogen reduction technologies (PRTs) is considered for the implementation of safer platelet (PLT) transfusion. However, the effects of PRT treatment including irradiation with ultraviolet (UV) light on PLT shape have not yet been fully clarified. STUDY DESIGN AND METHODS: Leukoreduced PLT concentrates (PCs) were treated with riboflavin and UV light (Mirasol PRT, TerumoBCT). PLT shape and adenosine diphosphate (ADP)-induced shape change were evaluated by a light scattering method where the amplitude of the scattered signal intensity was measured as the indicator of the proportion of discoid PLTs. Using a modified fluorometer, the real-time effects of different wavelengths of UV light on PLT shape were examined over the range of 300 to 360 nm at the same dose. RESULTS: The proportion of discoid PLTs in the Mirasol PRT-treated PCs decreased immediately after treatment. The difference in the proportion between PRT-treated and untreated PLTs became larger with storage. Although this modification correlated significantly with the pH decrease and P-selectin expression, the Mirasol PRT-treated PLTs retained sufficient ability to undergo an ADP-induced shape change. In the study using the modified fluorometer, the proportion of discoid PLTs significantly decreased with the wavelength (< 320 nm) of irradiated UV light. CONCLUSION: Mirasol PRT treatment of PCs decreases the proportion of discoid PLTs, which seemed to be caused by the irradiation with UV light of short wavelengths (< 320 nm), not that of long wavelengths (≥ 320 nm) in the Mirasol PRT system. Modification of UV light wavelength may improve the quality of PRT-treated PCs.

Effects of riboflavin and ultraviolet light treatment on platelet thrombus formation on collagen via integrin αIIbß3 activation.

BACKGROUND: The adoption of pathogen reduction technology (PRT) is considered for the implementation of safer platelet (PLT) transfusion. However, the effects of PRT treatment on PLT thrombus formation under blood flow have not yet been fully clarified. STUDY DESIGN AND METHODS: Leukoreduced PLT concentrates (PCs) obtained by plateletpheresis were treated with riboflavin and ultraviolet light (Mirasol PRT). PC samples were passed through a column filled with collagen-coated beads at a fixed shear rate after 1, 3, and 5 days of storage. The thrombus formation ability was evaluated by measuring collagen column retention rate. The change in the activation state of integrin αIIbß3 on PLTs during storage was examined by flow cytometry. RESULTS: The retention rate of the PRT-treated PLTs was significantly higher than that of the control PLTs on the day of treatment and decreased with storage but remained higher than those of the control during storage. This modification did not correlate with the total αIIbß3 or fibrinogen binding on the PLTs but correlated significantly with PAC-1 binding. Mn(2+) -induced αIIbß3 activation also fully restored the retention rate in the Day 5 PRT-treated PLTs along with the increase in PAC-1 binding. CONCLUSION: Riboflavin-based PRT treatment of PCs leads to the enhancement of thrombus formation on collagen, which is related to the activation status of αIIbß3, which does not bind to fibrinogen but binds to PAC-1. The impact of this finding on the hemostatic or even thrombogenic potential in vivo must await clinical evaluation.

A Leu55 to Pro substitution in the integrin alphaIIb is responsible for a case of Glanzmann's thrombasthenia.

Glanzmann's thrombasthenia (GT) is a hereditary bleeding disorder caused by a quantitative or qualitative defect in the integrin alphaIIbbeta3. A new mutation, a T to C substitution at base 258 in the alphaIIb gene, leading to the replacement of Leu55 with Pro, was found by sequence analysis of a patient's alphaIIb cDNA. In transfection experiments using COS7 cells, the cells co-transfected with the mutated alphaIIb cDNA containing C258 and wild-type beta3 cDNA scarcely expressed the alphaIIbbeta3 complex. The Leu55 to Pro substitution in the alphaIIb gene was found to be responsible for this case of Glanzmann's thrombasthenia.