Platelet stabilization

Details Platelet stabilization Platelet stabilization

2002-04-30

2004-12-21

Saucier, Sandra (Department: 1651)

Chemistry: molecular biology and microbiology

Maintaining blood or sperm in a physiologically active state...

Reexamination Certificate

active

06833236

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to methods for incorporating carbohydrates into platelets, to stabilise the platelets during storage.
BACKGROUND TO THE INVENTION
Blood platelets are one of the complex components involved in maintaining haemostasis. When the vessel wall is damaged, platelets adhere to exposed surfaces composed of collagen, microfibrils, and basement membrane. Adherent platelets promote the recruitment of other platelets to form an aggregated mass called a haemostatic platelet plug. The result is to activate coagulation proteins which provide the network to stabilize the platelet plug and reduce bleeding, allowing tissue repairs to occur.
Platelets are transfused to patients for many clinical indications. For instance, platelet infusions are used to correct deficiencies or dysfunctions of circulating platelets as a result of trauma, disease, or drug induced dysfunction. Patients suffering from idiopathic thrombocytopenia and those undergoing ablative chemotherapy are treated with platelet infusions. The increasing use of ablative chemotherapy for a wide variety of malignancies has resulted in an increased need for replacement platelet therapy.
A major difficulty in using isolated platelets is their short shelf-life. Platelets are only approved by the Food and Drug Administration (FDA) for storage in a liquid state for up to five days at room temperature, during which time the functional properties rapidly deteriorate. This causes many logistic problems in both civilian and military medicine.
Further drawbacks of storing platelets in a liquid state include the necessity of considerable storage space and constant agitation within bags of specially developed gas permeable plastics. Typically, single buffy-coat-derived platelets are stored in a suspending plasma volume of 45 to 65 ml. Recently, a study reported liquid storage establishing a minimum plasma volume of 30-50 ml (Home et al. (1994) Transfusion 34:39; and Ali et al. (1994) Transfusion 34:44). This storage method still requires considerable space, however, and the shelf life is not extended beyond approximately five days. The major problem with liquid storage is that the platelets need to be stored at approximately 20° C., as even short periods of exposure to lower temperatures during storage result in substantial changes in their in viva and in vitro properties (Moroff G. et al. (1994) Transfusion 34:317). As this storage usually also requires agitation of the platelets during storage at about 20° C. often in the presence of glucose, this presents optimal conditions for bacterial growth and this is a major problem with the storage of liquid platelets.
To minimise the problems of bacterial growth, refrigerated storage at 4° C. or frozen storage at −80° C. has been proposed. However, this requires methods to prevent the cold-activation of the platelets to be stored and the possible use of cryo-protectants. This method would however, require extensive washing of the platelets, to remove the cryo-protectants, before therapeutic use.
In addition, platelets are activated during the process of freeze drying and can only be used as a heamatology standard. The electrokinetic properties of the preserved platelets are different from those of fresh matched platelets. Other attempts at lyophilizing platelets have met with suboptimal results. Fixing the platelet prior to freeze-drying improves their function, but these freeze-dried platelets need to be stored frozen at −80° C. (Read et al. (1995) Proc. Natl. Acad. Sci. 92:397).
WO-A-98/34478 discloses methods for stabilising platelets by incorporating the carbohydrate trehalose into the platelets, and then drying in the presence of a stabilising carbohydrate. The preferred method for incorporating the trehalose into the platelets is electropermeabilisation. Although this technique does result in high uptake of trehalose, the technique has the disadvantage of activating the platelets, and it can only be applied on a small scale unless expensive equipment is used.
SUMMARY OF THE INVENTION
The present invention is based on the realisation that platelets can be loaded with high concentrations of a carbohydrate sugar by pre-activating, but not fully activating, the platelets. Without wishing to be bound by theory, this may involve inducing the platelets to form microvesicles which incorporate carbohydrates on their formation, and then allowing uptake of the microvesicles by the platelets.
According to the present invention, a method for the production of stabilized platelets comprises the steps of:
(i) pre-activating platelets, to induce the formation of microvesicles;
(ii) contacting the pre-activated platelets with a carbohydrate, whereby the carbohydrate is incorporated into the platelets; and
(iii) drying the thus-loaded platelets.
The present invention provides a suitable method for, high efficient loading of platelets with a suitable carbohydrate, which can be carried out on a large scale and is sufficiently gentle to prevent activation of the platelets. After incorporation, the platelets may be dried by any suitable means, preferably in the presence of an external carbohydrate, for storage and transport.


REFERENCES:
patent: 5653999 (1997-08-01), Gaudreault et al.
patent: WO 98/34478 (1998-08-01), None
Nakano et al., “Retinol induces platelet aggregation via activation of phospholipase A2”, BBRC 154 (3): 1075-80 (1988).*
Lewis, D.A. “Erythrocytes as microvasicles” retrieved from STN-International, Accession No. 121:212705 CA, XP002160827, abstract, 1992.

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