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A Matter of Life & Death

Platelets are produced in the bone marrow by cells called megakaryocytes.	iStock

Platelets are produced in the bone marrow by cells called megakaryocytes. iStockphoto

By Vanessa Solomon, Benjamin Kile & Emma Josefsson

Discovering the factors that control the lifespan of the cells that form blood clots could improve cancer treatments and extend the shelf-life of blood donations.

Mammals have evolved a blood-clotting system that stops bleeding by plugging damaged blood vessels. This blood-clotting system involves platelets and must be tightly regulated. Low platelet numbers in the blood or clotting factor deficiencies like haemophilia can cause fatal blood loss. Conversely, pathological platelet activation can lead to heart attack or stroke when clots block blood vessels in the heart or brain.

Our research is unravelling which factors control platelet numbers. The platelet volume in blood is dependent on how quickly new platelets can be formed from their mother cells in the bone marrow and how promptly platelets are lost from the circulation.

We have previously discovered quite a bit about the proteins regulating platelet survival, and hope that medications can be designed to control these proteins’ function. In the longer term, medications that increase platelet numbers may be useful for treating dangerously low platelet numbers, which can be a side-effect of anti-cancer treatments. Furthermore, combination therapy during chemotherapy treatments could be selected to avoid targeting the specific molecules that keep platelets and their mother cells alive. Compounds that can extend platelet lifespan would also have applications in prolonging the storage of platelets, enabling more efficient use of precious blood donations.

Humans normally maintain between 150 and 450 billion platelets in each litre of blood, with approximately 100 billion new platelets being formed every day. The platelets are produced by cells in the bone marrow called megakaryocytes. These are massive cells, often as large as a 100 µm, which is roughly the diameter of a human hair and 10 times larger than other blood cells such as white blood cells.

While white blood cells, and indeed most other types of cells in the human body, contain two copies of each of the 23 chromosomes, during megakaryocyte development the chromosomes are replicated without cell division, resulting in cells that contain as many as 64 copies of each chromosome.

Although megakaryocytes are the largest type of bone marrow cell, their platelet progeny are the smallest blood cell, at only 2-4 µm in size. To form platelets, mega­karyocytes extend long, fingerlike extensions into the bone marrow’s blood vessels, which are broken by the shear forces of the blood flow (in a process called “shedding”) to release tiny fragments of the mother cell – which are now the platelets – into the blood.

Ultimately the megakaryocyte will be diminished to the point where it has no more cell body to form into platelets. It is thought that the spent megakaryocyte’s remnant nucleus is engulfed and destroyed by other cells in the bone marrow.

The average time that a human platelet spends in the blood is 10 days. Usually only a small fraction of the circulating platelets will form blood clots – as the remainder of platelets get older they are removed from the blood as they travel through the spleen and liver.

Regulation of Cell Survival in Platelets and Megakaryocytes
Within the body, the number of cells in any organ is affected by the balance of new cell production and the death of cells. The number of platelets in the blood stream depends on the lifespan of the platelets in circulation as well as the rate of new platelet formation. Platelet formation is influenced, in part, by the number of megakaryocytes in the bone marrow.

Platelet deficiencies (thrombocytopaenia) can occur when too few platelets are formed or when too many platelets are lost from the circulation. Thrombocytopaenia is a side-effect of many chemotherapeutic medications and radiation therapy used to treat cancer.

Untreated, thrombocytopaenia can be fatal as low numbers of platelets hinder blood clot formation, resulting in uncontrolled bleeding. If a cancer patient develops severe thrombocytopaenia it is unsafe to proceed with anti-cancer treatments that will continue to diminish platelet numbers. Thus, thrombocytopaenia can prevent cancer patients from receiving their full dose of chemotherapy or radiotherapy, meaning their cancer is not optimally treated.

Thrombocytopaenia can be treated either by stimulating the body to make more platelets or by transfusing a patient with platelets obtained from a blood donation. By knowing which molecules are important for regulating how many new platelets are formed and for controlling megakaryocyte and platelet lifespan, our research group hopes to identify new ways to adjust platelet numbers in the blood.

The Bcl-2 Protein Family
The survival of many cells in our body are governed by programmed cell death, which is otherwise known as apoptosis. This type of cell death is not a passive process, but instead occurs through a controlled disassembly of the cells.

Apoptosis is important in mammals, both to delete superfluous cells during development and to remove damaged or unnecessary cells in the adult. In the past 20 years there has been much evidence linking defective regulation of apoptosis to human diseases.

Members of the Bcl-2 family of proteins tightly regulate this process. Some Bcl-2 family proteins are “pro-survival” – they allow cells to survive by blocking apoptosis. The critical function of the pro-survival molecule Bcl-2 was discovered more than 20 years ago by Professors David Vaux, Suzanne Cory and Jerry Adams of the Walter and Eliza Hall Institute.

Other Bcl-2-like proteins are “pro-death”, allowing the apoptosis machinery to switch on. When this occurs, proteins and DNA within the cell are rapidly destroyed, and the cell breaks up into small “apoptotic bodies” that are soon swallowed by other cells. A pair of closely related pro-death proteins called Bax and Bak are essential triggers for the apoptotic self-destruction machinery.

Animals lacking pro-survival proteins (such as Bcl-2 or its close relative Bcl-xL) develop degenerative diseases (or do not survive gestation) because of excessive death of tissues in vital organs such as the brain or blood.

Conversely, because apoptosis is a way that the body removes defective cells, including potentially cancerous cells, defects in apoptosis are linked to cancer.

In the immune system, apoptosis is also important for removing cells that attack the body’s own tissues, and the survival of these “self-reactive” cells can lead to auto­immune conditions such as type 1 diabetes, lupus and rheumatoid arthritis. Cells that express high levels of pro-survival proteins such as Bcl-2 or Bcl-xL are predisposed to develop into cancerous or autoimmune cells, as are cells lacking particular pro-death proteins.

A few years ago, our research group discovered that platelet lifespan in the blood is controlled by the Bcl-2 family of proteins. Platelets that have lower than normal levels of the pro-survival protein Bcl-xL have a reduced lifespan, while platelets that lack the pro-death protein Bak are extremely long-lived and accumulate in the blood to cause an excess of circulating platelets (thrombocytosis).

Our experiments suggested that Bcl-xL and Bak function as part of a “molecular clock” to control platelet lifespan. In a young platelet, adequate levels of Bcl-xL allow the platelet to survive, but after a few days) Bcl-xL levels decay while the levels of the more durable Bak remain stable. Without Bcl-xL to keep the platelet alive, the unrestrained Bak activates the platelet’s apoptotic machinery, killing the platelet and resulting in its removal from the blood.

Platelets are an important component of donated blood, and platelet transfusions are used to treat thrombocytopaenia in many patients, including cancer patients. Unfortunately, due to their fragile nature, platelets have a shelf-life of only 5 days, so platelet supplies are highly susceptible to shortages and require regular replenishment from new donations. In contrast, red blood cells and plasma can be stored for weeks or months.

Treatments that increase platelet lifespan could be used to extend the shelf-life of platelets obtained from blood donations, reducing the wastage of this limited resource. Early evidence suggests that the death of platelets in storage involves the Bcl-2 protein family, so agents that block apoptotic cell death could be developed into a “preservative” that keeps donated platelets alive for longer.

Modifying Megakaryocyte Survival in the Clinic
Many chemotherapeutic medicines cause thrombocytopaenia because they trigger cell death in megakaryocytes and their progenitors, reducing the number of platelets in the blood and hindering the body’s ability to make new platelets.

Paradoxically the apoptotic machinery, including Bcl-2 family proteins, has been linked to the formation of new platelets from megakaryocytes. For decades, researchers have commented on the similar appearances of a megakaryocyte shedding platelets in culture and a blebbing apoptotic cell. This led to the hypothesis that perhaps platelets bud off megakaryocytes through an apoptosis-like process.

We decided to address this intriguing question by studying megakaryocytes that are incapable of dying by apoptosis because they lack the genes encoding pro-death Bak and Bax. We discovered that these megakaryocytes could still form platelets normally, which suggests that platelet shedding from megakaryocytes does not require an apoptosis-like process.

To more conclusively address the question of whether platelet shedding involved pro-survival or pro-death Bcl-2 family proteins, we switched our focus from megakaryocytes that are resistant to apoptosis (because they lack pro-death Bak and Bax) to megakaryocytes that lack pro-survival Bcl-xL. These megakaryocytes were more prone to dying, and could not form platelets normally. Rather, pro-survival Bcl-xL is required to keep megakaryocytes alive during the process of platelet shedding. Thus, like platelets, megakaryocytes need Bcl-xL to survive and do their job.

Megakaryocytes lacking pro-death Bak and Bax were protected from apoptosis when they were treated with certain chemotherapy. This effect was enough to reduce the severity of chemotherapy-induced thrombocytopaenia.

While these studies are in their early days, it is possible that future medications that could specifically promote megakaryocyte survival – perhaps by blocking pro-death Bak and Bax – could form a treatment for thrombocytopaenia in some settings. There are already medications in clinical trials that inhibit specific pro-survival proteins, which kill certain types of cancer cells. In theory, new medications that promote cell survival by blocking pro-death proteins such as Bak and Bax could be developed.

Our research team is continuing to dissect the machinery that governs the lifespan of platelets and megakaryocytes. We want to find out whether there are pro-death or pro-survival molecules other than Bcl-xL, Bak and Bax that are important for the control of platelet numbers.

We hope that thrombocytopaenia can become a more manageable condition through the development of new strategies that keep platelets and megakaryocytes alive.

Vanessa Solomon, Benjamin Kile and Emma Josefsson are based at The Walter & Eliza Hall Institute of Medical Research.