Hemostatic function of cold-stored platelets in a thrombocytopenic rabbit bleeding model.

BACKGROUND: Transfusion of cold-stored platelet concentrates (CS-PCs) appears effective in massively bleeding patients. However, few studies have evaluated their in vivo hemostatic function in severe thrombocytopenia. STUDY DESIGN AND METHODS: The in vivo function of plasma-depleted human PCs was evaluated in rabbits with a blocked reticuloendothelial system and busulfan-induced thrombocytopenia. On day 1, a human apheresis PC was processed in a platelet additive solution (PAS-PC) and split evenly for cold or room temperature storage (RTS). On days 3, 6, or 9, RTS- or CS-PAS-PCs were transfused (4.0 × 109 platelets/kg) after plasma depletion into two to four rabbits that developed adequate thrombocytopenia (<25 × 109 /L). Ear bleeding time was measured by two incisions in small veins. The hemostatic rate was defined as the percentage of rabbits achieving bleeding cessation within 600 s at either incision. The experiment was repeated using five different PCs on each storage day. RESULTS: The mean pre-transfusion rabbit platelet count was 8.6 ± 5.2 × 109 /L. The hemostatic rates with RTS- and CS-PAS-PCs were both 100% on day 3, 93 ± 15% and 73 ± 15% on day 6 (p = .07), and 65 ± 36% and 73 ± 37% on day 9 (p = .27), respectively, with no statistical differences. Total platelet counts were significantly lower after CS-PAS-PC than RTS-PAS-PC transfusion on all days (e.g., 58.7 ± 5.7 vs. 42.4 ± 14.7 × 109 /L, p = .0007, day 9), and did not reach 50 × 109 /L in several experiments. Platelet count increments correlated significantly with hemostatic efficacy for CS-PAS-PC transfusion only. DISCUSSION: CS-PAS-PCs might achieve similar hemostasis as RTS-PAS-PCs in thrombocytopenic patients with mild bleeding. Hemostatic efficacy could be improved by transfusing more CS-PAS-PCs.

Production and nonclinical evaluation of an autologous iPSC-derived platelet product for the iPLAT1 clinical trial.

Donor-derived platelets are used to treat or prevent hemorrhage in patients with thrombocytopenia. However, ∼5% or more of these patients are complicated with alloimmune platelet transfusion refractoriness (allo-PTR) due to alloantibodies against HLA-I or human platelet antigens (HPA). In these cases, platelets from compatible donors are necessary, but it is difficult to find such donors for patients with rare HLA-I or HPA. To produce platelet products for patients with aplastic anemia with allo-PTR due to rare HPA-1 mismatch in Japan, we developed an ex vivo good manufacturing process (GMP)-based production system for an induced pluripotent stem cell-derived platelet product (iPSC-PLTs). Immortalized megakaryocyte progenitor cell lines (imMKCLs) were established from patient iPSCs, and a competent imMKCL clone was selected for the master cell bank (MCB) and confirmed for safety, including negativity of pathogens. From this MCB, iPSC-PLTs were produced using turbulent flow bioreactors and new drugs. In extensive nonclinical studies, iPSC-PLTs were confirmed for quality, safety, and efficacy, including hemostasis in a rabbit model. This report presents a complete system for the GMP-based production of iPSC-PLTs and the required nonclinical studies and thus supports the iPLAT1 study, the first-in-human clinical trial of iPSC-PLTs in a patient with allo-PTR and no compatible donor using the autologous product. It also serves as a comprehensive reference for the development of widely applicable allogeneic iPSC-PLTs and other cell products that use iPSC-derived progenitor cells as MCB.

In vitro thrombus formation and in vivo hemostasis mediated by platelets irradiated with bactericidal ultraviolet C from xenon flash under flow conditions.

BACKGROUND: We previously reported a flow path-ultraviolet C (UVC) irradiation system for platelet concentrates (PCs) with platelet additive solution (PAS) to minimize contamination by bacteria. Here, we investigated functionalities of irradiated platelets (PLTs) in in vitro thrombus formation and in vivo hemostasis. STUDY DESIGN AND METHODS: PAS-PCs were irradiated with flash UVC using the flow path system. Their variables (PLT count, mean platelet volume, pH, glucose, lactate, glycoprotein [GP] Ib, and activated integrin αIIbß3) were evaluated. Static adhesion to collagen or fibrinogen was analyzed using fluorescent microscopy. Thrombus formation under flow conditions was assessed using a collagen-coated bead column. Adenosine diphosphate (ADP)-induced Akt phosphorylation was determined by western blot. In vivo hemostasis and circulatory survival of PLTs were assessed with a rabbit bleeding model. RESULTS: All variables, except for GPIb expression, were slightly, but significantly, impaired after flash UVC irradiation throughout the 6-day storage period. No difference was observed in static adhesion to either collagen or fibrinogen between irradiated and nonirradiated PAS-PCs. In vitro thrombus formation of flash UVC-irradiated PAS-PCs was significantly greater than that of nonirradiated PAS-PCs. ADP-induced Akt phosphorylation was enhanced in irradiated PAS-PCs. In vivo hemostatic efficacy was comparable between the groups on Day 1. The efficacy declined in nonirradiated PAS-PCs on Day 5, while it was retained in flash UVC-irradiated PAS-PCs. Circulatory survival of PLTs was lower in irradiated PAS-PCs. CONCLUSIONS: PAS-PCs irradiated with UVC from xenon flash have favorable properties to achieve hemostasis compared with nonirradiated PAS-PCs.

Designing a Bioinert Surface by Simple Coating with Cholesterol End-Modified Poly(ethylene glycol).

A bioinert surface has been designed by simple coating with cholesterol end-modified poly(ethylene glycol), Chol-U-Pr-mPEG, using a cholesterol anchor. A poly(propylene) (PP) surface was immersed into the Chol-U-Pr-mPEG aqueous solution, where control mPEGs without cholesterol were not suitable for the design of bioinert surfaces. The resulting surfaces coated with Chol-U-Pr-mPEG above and below its critical micelle concentration were swollen and less swollen, respectively. Chol-U-Pr-mPEG with a molecular weight of 2000, Chol-U-Pr-mPEG (2k), formed a swollen layer with a thickness of 10-15 nm and adhered to the PP surface with an estimated Kd value of 4.4×10-7 M. The resulting Chol-U-Pr-mPEG (2k)-coated surface with the swollen layer suppressed the adsorption of γ-globulin proteins and the adhesion of platelets in plasma. Although the PP surface coated with Chol-U-Pr-mPEG with a molecular weight of 5000, Chol-U-Pr-mPEG (5k), also suppressed the adsorption of γ-globulin proteins, the Chol-U-Pr-mPEG (5k)-coated PP surface did not suppress the adhesion of the platelets in plasma despite the existence of a swollen layer with a thickness of 20-25 nm. These results suggest that the Chol-U-Pr-mPEG-coated PP surface with an optimized swollen layer has been established as a bioinert surface by a facile method.

Turbulence Activates Platelet Biogenesis to Enable Clinical Scale Ex Vivo Production.

The ex vivo generation of platelets from human-induced pluripotent cells (hiPSCs) is expected to compensate donor-dependent transfusion systems. However, manufacturing the clinically required number of platelets remains unachieved due to the low platelet release from hiPSC-derived megakaryocytes (hiPSC-MKs). Here, we report turbulence as a physical regulator in thrombopoiesis in vivo and its application to turbulence-controllable bioreactors. The identification of turbulent energy as a determinant parameter allowed scale-up to 8 L for the generation of 100 billion-order platelets from hiPSC-MKs, which satisfies clinical requirements. Turbulent flow promoted the release from megakaryocytes of IGFBP2, MIF, and Nardilysin to facilitate platelet shedding. hiPSC-platelets showed properties of bona fide human platelets, including circulation and hemostasis capacities upon transfusion in two animal models. This study provides a concept in which a coordinated physico-chemical mechanism promotes platelet biogenesis and an innovative strategy for ex vivo platelet manufacturing.

Refined methods to evaluate the in vivo hemostatic function and viability of transfused human platelets in rabbit models.

BACKGROUND: To bridge the gap between in vitro function and clinical efficacy of platelet (PLT) transfusion products, reliable in vivo PLT functional assays for hemostasis and survival in animal models are required. However, there are no standardized methods for assessing the in vivo quality of transfused human PLTs. STUDY DESIGN AND METHODS: Plasma-depleted human PLT concentrates (PCs; Day 3, Day 5, Day 7, Day 10, and damaged) were transfused into busulfan-induced rabbits with thrombocytopenia with prolonged bleeding times 1 day after treatment with ethyl palmitate (EP) to block their reticuloendothelial systems. The hemostatic effect of PC transfusion was evaluated by the ear fine vein bleeding time. For the in vivo survival assay, splenectomized EP-treated rabbits were transfused with human PCs, and viability of the human PLTs in the rabbits was determined by flow cytometry using human PLT-specific antibodies and Trucount tubes. RESULTS: The hemostatic effect of PCs was slightly reduced with increasing storage periods for early time points, but more dramatically reduced for later time points. PLT survival was similar after 3 and 7 days of storage, but PLTs stored for 10 days showed significantly poorer survival than those stored only 3 days. CONCLUSION: Our new and improved protocol for in vivo assessment of transfused PLTs is sufficiently sensitive to detect subtle changes in hemostatic function and viability of human PLTs transfused into rabbit models. This protocol could contribute to preclinical in vivo functional assessment and clinical quality assurance of emerging novel PLT products such as cultured cell-derived human PLTs.

Comparison between in vitro properties of washed platelet concentrates suspended in M-sol and those in BRS-A, both of which were prepared with an automated cell processor.

BACKGROUND: Washed platelet concentrate (WPC) is prepared manually in general, but automated preparation is desirable to minimize variation in the WPC quality and enhance WPC production. Recently, the software was improved for an automated cell processor (ACP) to control all processes of WPC preparation. M-sol and BRS-A, which are mixtures of medical solutions, are widely used for WPC preparation with a manual method in Japan. In this study, we prepared WPC suspended in M-sol (WPC-M) or BRS-A (WPC-B) with the ACP, and compared their in vitro properties during 7-day storage. STUDY DESIGN AND METHODS: PC was divided into two equal aliquots for WPC-M and WPC-B. A divided PC, medical solutions and disposable materials were set in the ACP, and it was started to prepare WPC-M or WPC-B on Day 0. Prepared WPC was stored on a flatbed shaker until Day 7. RESULTS: The pH of WPC-M and WPC-B was maintained above 6.8 during the 7-day storage. The differences in aggregation (%), HSR (%), P-selectin expression, GPIbα expression, and phosphatidylserine expression between WPC-M and WPC-B were minimal until Day 3. CONCLUSION: The in vitro properties of WPC-B are not markedly different from those of WPC-M until Day 3.

Photo-reactive polyvinylalcohol for photo-immobilized microarray.

A new photo-reactive polymer, polyvinylalcohol modified with phenylazido groups, was synthesized as a microarray matrix. The polymer is soluble in water and spin-coated onto glass plate. Aqueous solutions of proteins were micro-spotted onto the coated glass and were fixed by ultraviolet light irradiation. Subsequently, cell adhesion on the photo-immobilized protein microarray was investigated. Non-specific adhesion of cells onto non-protein-spotted regions was reduced in comparison with the previously prepared microarray chip (Biomaterials 24 (2003) 3021). The adhesion behavior of cells depended on the kind of immobilized proteins and the type of cells. The microarray will be useful for cell diagnosis and for the selection of biomaterials to regulate cell behavior.

Targeted gene delivery to sinusoidal endothelial cells: DNA nanoassociate bearing hyaluronan-glycocalyx.

Liver sinusoidal endothelial cells (SECs) possess unique receptors that recognize and internalize hyaluronic acid (HA). To develop a system for targeting foreign DNA to SECs, comb-type polycations having HA side chains were prepared by coupling HA to poly(L-lysine) (PLL). The HA-grafted-PLL copolymer (PLL-g-HA) thus formed was mixed with DNA in 154 mM NaCl to form soluble nanoassociates bearing hydrated hyaluronate shells. Agarose gel retardation assays revealed selective interaction of the PLL backbone with DNA despite the presence of polyanionic HA side chains. To determine whether the PLL-g-HA/DNA complexes were recognized by SEC HA receptors in vivo, we injected Wistar rats i.v. via the tail vein with PLL-g-HA complexed to a beta-galactosidase expression plasmid (pSV beta-Gal) labeled with 32P. One hour postinjection, >90% of the injected radioactivity remained in the liver. Administration of the PLL-g-HA complexed to an FITC-labeled DNA revealed that the carrier-DNA complex was distributed exclusively in SECs. A large number of SECs expressing beta-galactosidase was detected along the sinusoidal lining after transfection with PLL-g-HA/pSV beta-Gal. Moreover, PLL-g-HA effectively stabilized DNA triplex formation. In conclusion, the new PLL-g-HA/DNA carrier system permits targeted transfer of exogenous genes selectively to the SECs.

Preparation of a protein micro-array using a photo-reactive polymer for a cell-adhesion assay.

A protein micro-array, called a "cell chip" was constructed by using a photo-reactive polymer for a cell-adhesion assay. Various amounts of albumin or fibronectin were covalently immobilized on a polystyrene dish using a micro-spotter with a dip pen. First, poly(acrylic acid) carrying azidophenyl groups was synthesized as the photo-reactive polymer. Secondly, the aqueous solution of a photo-reactive polymer (several nanoliters) was cast using the dip pen of the micro-spotter and dried in air. Subsequently, aqueous solutions of proteins were cast on the same place using the micro-spotter. After drying, the dish was irradiated with ultraviolet light. Finally, the immobilization was confirmed by staining with a dye. The immobilization was stable even after washing with Tween-20. The protein-immobilized area depended on the manipulation of the micro-spotter and the size of the dip pen. Subsequently, cell adhesion on the photo-immobilized protein micro-array was investigated. The adhesion behavior of cells depended on the kind of immobilized proteins and the kind of cells. The protein micro-array will be useful for cell diagnosis and for the selection of biomaterials to regulate cell behavior.