Abstract
Background:
Refrigerated platelets are rapidly cleared upon transfusion. The mechanism of this fast clearance is not fully understood. It was recently reported that refrigeration-induced binding of von Willebrand factor (VWF) to platelets contributed to the rapid clearance of platelet after transfusion. Adding a peptide that blocks the binding of VWF to platelet GPIbα during refrigeration can improve the post-transfusion recovery and survival of refrigerated murine platelets in vivo. However, since the interaction of VWF with GPIbα is required for primary hemostasis, adding a long-acting inhibitory peptide to stored platelets may impede the hemostatic function of these platelets after transfusion.
DNA aptamer ARC1779 binds VWF and inhibits its binding to GPIbα. It has previously been developed as an anti-thrombotic agent. Its half-life is relatively fast, at a reported 2 hours in human.
Objective:
We aimed to test whether ARC1779 can improve the post-transfusion recovery and survival of refrigerated platelets, and, due to its fast clearance rate, without significantly impeding the hemostatic functions of these platelets in vivo.
Methods:
Human or murine platelets were stored at 4°C with or without ARC1779 for 48 hours. VWF binding was measured by flow cytometry. After refrigeration, human platelets were retro-orbitally transfused into NOD-SCID mice, while murine platelets into wild-type mice. Post-transfusion, periodic blood draw was performed, and the percentage of infused platelets in the total platelet population was measured by flow cytometry to calculate the recovery and survival over time. To monitor the hemostatic function, refrigerated murine platelets were transfused into transgenic IL4R-IbαTg mice, and a tail bleeding time assay was performed at four hours after transfusion. IL4R-IbαTg platelets lack the extracellular domain of GPIbα and cannot bind VWF and other GPIbα ligands. As a result, hemostasis in IL4R-IbαTg mice is severely impaired.
Results and conclusion:
Treatment of ARC1779 during 48-hour refrigeration efficiently inhibited VWF binding to platelets. ARC1779-treated refrigerated murine platelets exhibit increased post-transfusion recovery and survival rates compared to untreated refrigerated ones. (Recovery of ARC1779-treated platelets: 76.7±5.5%; Untreated: 63.7±0.8%, N=6, p<0.01. Half-life time of ARC1779-treated platelets: 31.4±2.36 hr; Untreated: 28.1±0.86 hr, N=6, p<0.05). A similar increase was also observed for refrigerated human platelets (Recovery: 49.4±4.4% vs. 36.8±2.1%, N=6, p<0.01; Half-life time: 9.2±1.5 hr vs. 8.7±0.9 hr, N=6, n.s.). Additionally, the tail bleeding time in ARC1779-treated group was significantly shorter than no ARC1779-treated group (160±65 vs. 373±96 seconds, N=7, p<0.01). For comparison, un-transfused IL4R-IbαTg mice have a tail bleeding time longer than 1200 s, and those transfused with freshly prepared platelets 155±80 s. Overall, these results demonstrate that inhibiting the GPIbα-VWF interaction by ARC1779 during cold storage of platelets improves platelet post-transfusion recovery. The treatment also helps to preserve the hemostatic function of refrigerated platelets. These results suggest that an inhibitor of the GPIbα-VWF interaction but with a limited half-life may be a potential therapeutic option to enable or improve the refrigeration of platelets for transfusion treatment.
Disclosures
Li: Neoletix: Consultancy, Equity Ownership.
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