(P-CB-12) Emergence of Various Platelet Subpopulations with Specific Hemostatic Properties in Cold-Stored Amotosalen-UVA Pathogen-Reduced Platelet Concentrates

Background/Case Studies: Current increase in the need for therapeutic versus prophylactic platelet transfusions leads to growing interest in cold-stored platelets (CSP), owing to their potentially advantageous characteristics as compared to standard room temperature platelets (RTP). We assessed the emergence of various platelet subpopulations and related functional properties in platelet concentrates (PCs) treated with amotosalen-UVA pathogen reduction and stored at 22°C or 4°C for 21 days (D).

Study

Design/Methods: A pool-and-split strategy was used to obtain double-dose buffy-coat (BC)-PCs collected into PAS-C/plasma (55/45%) treated with amotosalen-UVA and stored at 22°C with constant agitation or at 4°C without agitation. Eight platelet subpopulations were identified using a combination of markers (P-selectin and phosphatidylserine (PhtdSer) exposure, DYm, PAC-1 binding for activated GPIIbIIIa) analyzed by multicolor flow cytometry (FCM). Functional testing included thrombin generation by calibrated automated thrombography, aggregometry, thrombus formation on a collagen-coated surface under flow conditions (1500 s^-1) and clot viscoelasticity of reconstituted whole blood (Quantra QPlus, HemoSonics LLC, Stago). Statistical comparisons were done by Pearson's correlation and two-way ANOVA followed by Tukey’s post-hoc test (n=4).

Results/Findings: Multicolor FCM analysis indicated that the subpopulation of resting platelets decreased from 78±3% to 45±2% at D7 at 22°C and to 8±1% at 4°C. In CSP, procoagulant (30±4% from D7 to D21) and apoptotic (53±3% by D21) platelets, both exposing PhtdSer, supplanted resting platelets. This activated pattern remained unaltered upon TRAP + convulxin stimulation as of D7, indicating maximally activated platelets in PCs. CSP displayed enhanced thrombin generation capacity as of D7 compared to RTP (p< 0.01), consistent with increased proportion of procoagulant platelets (r=-0.7776 p< 0.01). Aggregation to various agonists (collagen, TRAP, arachidonic acid) decreased progressively during storage and was lower in CSP vs. 7-day RTP (D21 p< 0.01), consistent with high procoagulant platelet content (r=-0.6829 p< 0.05) characterized by loss of aggregation capacity. Thrombus formation on collagen under flow with reconstituted hirudinated whole blood was best preserved at 4°C at least up to D14, compared to only D7 at 22°C. Platelet contribution to clot stiffness was lower with CSP as of D7 when compared with 7-day RTP (p< 0.01), consistent with a high proportion of procoagulant platelets (r=-0.7227 p< 0.01), lacking the ability to aggregate, contract and contribute to clot strengthening.

Conclusions: CSP is a promising strategy to prolong platelet storage, leading to phenotypic changes with resting platelets replaced by PhtdSer-bearing procoagulant and apoptotic platelet subpopulations with increased thrombin production, reduced aggregation and clot firmness.