FDPOs enhanced wound healing in db/db mice as well as FDPIs and RT-PLTs. Wound closure was obtained 6 days earlier than untreated wounds and histologic examination revealed reduced granulation and increased cellular angiogenesis.
The use of fresh platelets has gained value in medicine as an essential part of wound treatments. This is not surprising since platelets contain a number of bioactive factors that contribute to the process of wound healing, such as platelet‐derived growth factor (PDGF) and transforming growth factor (TGF). Fresh platelets’ short shelf life limits platelet‐based therapies. If platelets can be stabilized in freeze‐dried form (FDP) then long‐term storage as well as pathogen inactivation methods become possible. Adlyfe and Oregon Freeze‐Dry have been developing technology to stabilize freeze‐dried human platelets that can be subjected to gamma irradiation and stored for a long duration. Upon reconstitution, irradiated FDP retained growth factors PDGF‐AB, PDGF‐BB, and TGF‐B1 in quantities similar to fresh platelets as judged by capture ELISA. The rehydrated FDP promoted new DNA synthesis and cellular proliferation of primary human dermal fibroblasts and endothelial cells (HUVECs) similar to fresh platelets. The FDP also promoted remodeling of extracellular matrix by accelerating fibroblast‐mediated contraction of collagen gels and stimulated HUVECs to undergo angiogenesis and form capillary structures in vitro. Therefore, we conclude that FDP and fresh platelets have comparable in vitro wound healing potential. Preclinical wound healing stud"ies in diabetic mice are under way and further development will allow FDP to be a safe and well‐suited alternative to fresh platelets for wound healing applications.
Availability of platelet concentrates for treatment of bleeding associated with thrombocytopenia, trauma, or drug-induced coagulopathies is problematic due to the short 5 day platelet storage time and because platelets require controlled shaking at ambient temperature in order to remain viable, a condition which augments bacterial growth. To address the platelet availability problem we expanded upon trehalose cryo-preservation technology to create a lyophilized hemostatic platelet derivative. Washed platelets were stabilized by accumulation of 5–10 mM intracellular trehalose via fluid phase endocytosis then formulated with excipients and lyophilized. Lyophilized platelets were instantaneously rehydrated with > 90% recovery and were stable for at least 3–6 months at ambient temperatures. Rehydrated (RH) platelets responded quantitatively to α-and γ-thrombin and ristocetin by transmittance aggregometry and were partially agglutinated by collagen as judged by aggregometry and single cell counting using the Platelet Works® system. RH platelets co-aggregated in a dose dependent manner when mixed with fresh autologous platelets during collagen-induced activation. Aggregation response to low-dose thrombin and collagen was inhibited by the GPIIb/IIIa antagonist RGDS and by EGTA. RH platelets were quantitatively incorporated into fibrin clots and elicited platelet-dependent fibrin-clot retraction ~ 60% as well as fresh platelets. RH platelets were similar in size to fresh and had less than 25% submicron particles as judged by electronic particle counting and flow cytometry scatter profiles. RH platelets were partially activated upon rehydration as judged by anti P-selectin and anti-LAMP-3 binding, yet GPIIb/IIIa remained in a resting conformation, as judged by a lack of PAC-1 binding. GPIIb/IIIa receptors were present as judged by the binding of complex-dependent (clone 5B12) and function-blocking (clone P2) antibodies. RH platelets also contained intact GPIbα as judged by binding of the function-blocking MoAb AN51. Function of GPIIb/IIIa and collagen receptors on RH platelets was further demonstrated as RH platelets adhered to immobilized fibrinogen and collagen in the absence of added agonists and in a dose-dependent manner. Moreover, RH platelets exhibited a two-fold increase in platelet procoagulant activity in the presence of thrombin receptor agonist peptide SFLLRN as judged by Annexin-V binding. Procoagulant and hemostatic activity was further demonstrated as RH platelets accelerated the clotting of recalcified whole thrombocytopenic blood in a dose-dependent manner similarly to fresh platelets. Lastly, RH platelets corrected the coagulopathy induced by contact pathway inhibition with aprotinin during the recalcification of citrated whole blood. The technology has been scaled to single donor platelet aphaeresis units, equivalent to a standard transfusion dose. Preclinical animal models of safety, efficacy, and circulation persistence are currently being evaluated. In summary, trehalose- stabilized lyophilized platelet derivatives contain numerous in vitro hemostatic properties and may offer an attractive alternative to fresh platelet transfusions when the latter are indicated yet unavailable.
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