1. What are platelets for?
2. Why platelet substitutes?
3. Properties of an ideal platelet substitute
4. Platelet derived products
5. Platelet substitutes
6. Other approaches to treating thrombocytopenia
7. Future prospects

What are Platelets for?

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Platelets are different than oxygen carrying substitutes like PFCEs and HBOCs. They are an essential component of the thrombogenic response to bleeding. When platelets in the circulation encounter a damaged endothelial blood vessel lining, they adhere to the damaged lining and to each other, and release several coagulation factors. Bleeding patients with thrombocytopenia may be given platelet transfusions to aid hemostasis.

Why Platelet Substitutes?

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The risk of HIV infection from a red blood cell transfusion has dropped to about 1 in 2 million, but the risk of bacterial contamination of platelet products is about 1 in 2,000. Platelets are much more difficult to deal with than red blood cells. Platelets must be stored in plastic bags that are permeable to oxygen and carbon dioxide, at 22° C, with gentle continuous agitation, and have a shelf life of 5 days. Platelets can be collected by separating whole blood donations, or by apheresis. Platelets separated from whole blood give only about one fifth of a “dose” of platelets, so a patient would need platelets from 5 donors. Apheresis can collect a whole “dose” of platelets from a single donor. Another disadvantage of fresh platelets is that they contain leukocytes, an immunologically active type of cell that may contribute to adverse reactions to transfusion. After transfusion, patients can become alloimmunized to the HLA antigens of the donor platelets, necessitating treatment with HLA-matched platelets.

Properties of an Ideal Platelet Substitute

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An ideal platelet substitute would provide effective in-vivo hemostasis, would not generate an immune reaction or have immunosuppressive effects, would be sterile, have a long duration of action, a long shelf life, have simple storage requirements and be easy to administer.

Platelet-Derived Products

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Frozen platelets

Platelets can be cryopreserved in 6% dimethylsulfoxide (DMSO) for up to 10 years when stored at -80° C. The disadvantages are that they are cumbersome to use, much more expensive to produce than fresh platelets, and the freezing process appears to cause some morphological defects. Cryopreserved platelets are the only alternative to fresh platelets in clinical use, but their use is generally limited to storing autologous platelets for transfusion in alloimmunized patients with acute leukemia. Recently, a cryoprotectant agent called ThromboSol has been produced by LifeCell. The components of ThromboSol inhibit the cold-induced platelet activation pathways, limiting the detrimental effects of cold storage. This decreases the requirement for DMSO in the freezing process and has been shown to increase the in vivo recovery of the cryopreserved platelets.

Cold liquid-stored platelets

In the 1960's, platelets were stored at 4° C in an attempt to slow bacterial proliferation. But the cold conditions were unfavorable for platelet survival, so the product had a shelf life of only 24 hours. In the 1970's, it was shown that platelets had a greater survival and hemostatic efficacy when stored at room temperature (22° C). By the mid 1980's, platelets could be stored for up to 5 days at 22° C. Recently there has been renewed interest in finding an effective process of cold storage. Three general approaches have been utilized: inhibiting cytoskeleton actin assembly, using ThromboSol as a cryoprotectant, and using antifreeze glycoproteins. Using inhibitors of cytoskeleton actin assembly prevents the disc-to-sphere-shape change seen in chilled platelets. ThromboSol has been reported to give greater retention of in vitro function, but in vivo findings have not been reported. Antifreeze glycoproteins are found in the circulation of polar fish. The use of these glycoproteins in cold storage of platelets has been shown to inhibit platelet shape change and cold-induced activation, but no in vivo findings have been reported.

Photochemically treated platelets

Photochemical treatment of liquid-stored platelets with psoralen and long-wavelength ultraviolet radiation has been explored as a means to inactivate bacteria, viruses and protozoa by disrupting their DNA. The treatment does not appear to affect in vitro platelet function. Three phase III clinical trials have been completed. The results indicate that the photochemically treated platelets have normal hemostatic function, but have slightly reduced in vivo recovery and survival. This preparation method has been approved for clinical use in Europe .

Lyophilized platelets

Lyophilization was first explored as a platelet preparation method in the 1950's, but animal studies failed to show any hemostatic efficacy in vivo. But a recent preparation method may be more effective: platelets are prepared using 1.8% paraformaldehyde and freeze-dried with 5% albumin. Rehydrated platelets have a similar appearance to fresh platelets and have most of the same surface proteins. They have been shown to be hemostatically effective in animals, although their duration of action appears to be short.

Platelet-derived microparticles

Platelet microparticles are microvesicles of platelet membranes which are formed spontaneously during storage. The microparticles have similar hemostatic properties as intact platelets. They can be produced from outdated platelets. Cypress Bioscience has developed an infusible platelet membranes (IPM) product called Cyplex using outdated platelets from blood banks. With a shelf life of only 5 days, a considerable amount of outdated platelets are available. Cyplex is produced by lysing the platelets by freeze-thawing, heating the product to 60° C to inactivate viruses, and lyophilization. The product has a shelf life of over 2 years when stored at 4° C. Phase I and II trials of IPMs have been conducted, and the product has been shown to be safe and effective so far.

Culture-derived platelets

In 1995, the process of growing platelets in the laboratory from megakaryocyte progenitors was described. The resulting platelets appeared to be functionally and morphologically similar to fresh donated platelets in vitro. Little work has been done to follow up on this research, but this may represent a promising method in the future.

Platelet Substitutes

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Red cells with surface-bound fibrinogen or RGD peptides

Fibrinogen is a molecule in the circulation that binds activated platelets together. Specifically, it is the multiple Arginine-Glycine-Aspartine (RGD) sequences in fibrinogen which interact with surface proteins of platelets. Autologous erythrocytes (red blood cells) can be prepared with fibrinogen bound to the surface, or with just the RGD fragments bound to the surface. These have been called “thromboerythrocytes.” They have been shown to be hemostatically effective in some animal models of thrombocytopenia.

Fibrinogen-coated albumin microcapsules/microspheres

Fibrinogen has also been used to coat microspheres or microcapsules of the human protein albumin. Two preparations have been evaluated in preclinical trials: Synthocytes (developed by Andaris , UK ) and Thrombospheres (developed by Hemosphere). Thrombospheres have a smaller mean diameter, and in vivo studies suggest that they may have a longer duration of action.

Liposome-based agents

Two liposome-based agents have been studied: plateletsomes and factor Xa with phospholipid vesicles. Plateletsomes are lipid vesicles with platelet glycoproteins on their surface. Both have been shown to be hemostatically effective in vitro and in some animal models, but the latter approach was associated with high toxicity.

Other Approaches to Treating Thrombocytopenia

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HLA-reduced platelets

To reduce the incidence of HLA-alloimunization of transfused patients, efforts have been made to inactivate the HLA antigens of platelets. Methods have used chloroquin-treatment and citric acid-treatment, but both of these can severely affect the quality of the platelets.

Activated factor VII

Factor VII is a protein component of the coagulation cascade. Preliminary observations indicate that recombinant activated factor VII may be effective in treating bleeding patients with thrombocytopenia.

Future Prospects

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One hurdle to the development of new platelet products and platelet substitutes is to define a way to quantify the effects of platelets and related products on bleeding. Several platelet substitutes look promising, but we will probably see photochemically treated platelets and cold-stored platelets in clinical use much sooner. Given the demand for platelets and the extremely short shelf life of fresh platelets, there is real incentive to produce a safe alternative.