This blog is about topics in clinical research. This blog will include comments on recent research articles of my interest (and hopefully, of your interest), as well as on research methodology. Of course, other topics that interest me and are important enough in my view to be shared on this blog will be discussed. I will appreciate comments, suggestions, and remarks.
Saturday, August 28, 2010
Platelets and Apoptosis
Various in vivo and in vitro methods can be used to determine platelet lifespan. One often recommended is the use of 111indium-labelled platelets. In this method, labeled platelets are injected into the patient and samples are collected at various time intervals (at 45 min, 2, 3, 4 hours) after injection and then daily for up to 10 days. Recovered platelets are calculated from each sample, data is plotted on an arithmetic graph paper and survival time is calculated.
What Determines Platelet Lifespan?
The lifespan of a platelet in circulation is about 8-10 days and dying platelets are continuously replaced by new platelets formed in the bone marrow. Platelets are removed from circulation either by consumption during hemostasis or through uptake by reticuloendothelial (RE) system. For the later process, initially, it was thought that platelets die due to usual ‘wear and tear’ while they are in circulation. Various lines of evidence suggested this possibility. For example, older platelets are less responsive to physiological agonists as compared to younger platelets. More recently, there is increasing evidence that platelets undergo programmed cell death through apoptosis and during this process express certain receptors on their surface that lead to uptake by RE system.
There are two pathways through which apoptosis can be triggered; extrinsic and intrinsic. Extrinsic pathway is activated by the stimulation of cell-surface receptors (death receptors) which leads to the formation of Death Inducing Signaling Complex (DISC) and ultimately of caspases. On the other hand, intrinsic pathway is activated when the intracellular balance of pro-apoptotic and anti-apoptotic proteins tilt in the favor of the pro-apoptotic proteins. The pro-apoptotic proteins then trigger mitochondrial damage, which initiates the apoptosis cascade.
BCL-2 Family of Proteins
BCL-2 family of proteins is mainly responsible for intrinsic pathway of apoptosis (also called programmed cell death). There are about 25 proteins in this family. Some of these are pro-apoptotic while others are anti-apoptotic. A third subgroup within this family (BH3-only) may be involved in sensing signals that trigger programmed cell death. Anti-apoptotic proteins include Bcl-2, Bcl-w, Bcl-XL, Mcl-1 and A1 while pro-apoptotic proteins are mainly Bak and Bax. . BH3-only proteins include Bim, Bad, Bmf, Hrk, Bik, Noxa, and Puma. Together these proteins regulate programmed cell death.
In a series of elegant experiments Mason et al has shown that the main anti-apoptotic protein in platelets is Bcl-XL . They showed that mutations in Bcl-XL gene resulted in dose dependent reductions in platelet lifespan. The found that the major pro-apoptotic protein in platelets is Bak and to a minor extent Bax. Experiments in which the genes for these proteins were deleted result in doubling of the platelet lifespan. BH3-only proteins also appear to play important pro-apoptotic role in platelet lifespan as BH3-only mimetic compounds trigger apoptosis and rapid development of thrombocytopenia. In contrast, platelets from double-null mice (mice knocked out for both Bak and bax) are refractory to BH3-only mimetic compounds suggesting that the effect of BH3-only proteins is mediated through Bak and Bax.
In a study recently published study Kelly et al  showed that Bad-deficient mice had elevated platelets in their blood. They further showed that Bad is present in platelets and that bone marrow of Bad deficient mice showed normal number of megakaryocytic. They further studied the half life of platelets from Bad-deficient mice and found it to be modestly increased. To be certain that this was the intrinsic property of the platelets and not due to other host factors, they injected platelets from Bad-deficient mice into wild-type mice and found increased platelet lifespan. These series of experiments suggest that Bad acts by increasing the lifespan of platelets. On the other hand, mice deficient of another BH3-only protein, Bim, has mild thrombocytopenia. Bim appears to be involved in platelet formation by megakaryocytic and therefore, its deficiency is associated with impaired platelet formation. Interestingly, mice deficient in both Bad and Bim have normal number of platelets in their blood.
Another recent study shed some more light on the role of Bad in platelet lifespan. Catani et al  showed that activation of type-1 cannabinoid receptor by either endocannabinoid anandamide or methanandamide result in prolongation of platelet lifespan. This effect appears to be mediated though activation of Akt which, in turn, phosphorylates Bad (a BH3-only protein). A phosphorylated Bad cannot enter mitochondria and thus unable to bind and inactivate the anti-apoptotic Bcl-XL protein. Thus cytosolic sequestration of Bad results in prolongation of platelet lifespan in this model.