Just like the body regulates various processes through homeostasis; it minimizes blood loss when blood vessels are injured through the blood clotting mechanism. If blood was not able to clot after accidents that cause injury to blood vessels, an individual would end up losing his/her entire blood volume and subsequently cause death. Also, the body has mechanisms of regulating blood clotting so as to prevent over-clotting and blockage of blood vessels by the clots. It is also through such mechanisms that blood clotting is not initiated in blood vessels that have not been damaged.
Platelets are also known thrombocytes, and they are a component of the blood that is essential for blood clotting. They limit blood loss after injury by clumping together in a mesh-like structure formed by fibrin strands hence preventing further oozing of blood from infected vessels. Platelets are non-nucleated cells that arise from cells in the bone marrow known as megakaryocytes (Geddis, 2010). After an injury has occurred and there is bleeding, activation of platelets occurs, and it results in a conformational change that differentiates them from inactivated platelets. With the profound role of platelets in blood coagulation, their numbers need to be kept within the normal ranges that would prevent thrombocytopenia, and this is accomplished through the continued synthesis of the cells from stem cells.
Process of Platelet Formation
Platelets are formed in the bone marrow through a stepwise process that beings with the embryo cell. Once they divide, stem cells are differentiated into different types of other cells of the brain, blood, and other body organs. The cell that forms the embryo is usually known as the totipotent cell, and it usually has the capability to divide and form various cells that will form the organs of the entire body. From these cell results the hematopoietic cells that further divide to form blood cells. The hematopoietic stem cells then keep on dividing to keep the totipotent cells alive, and they become the first line of blood cells. These cells have the feature of pleuripotency, meaning that they can divide and form any blood cell ranging from RBCs, WBCs, and even platelets (Geddis, 2010).
Progenitor cells are then produced from the hematopoietic cells through the effect of colony stimulating factors. Production of progenitor cells then means that only specific cells will be produced by a particular cell hence the aspect of pleuropotency becomes impossible (Geddis, 2010). The specific cell lines, which were previously formed from a single cell, pool in different places and separate each other depending on their similarities and function. Under the effect of colony stimulating factors, progenitor cells give rise to megakaryoblasts which form the basis of platelet formation. Megakaryoblasts are undeveloped cells that are only found in the bone marrow, hence the essence of the bone marrow in platelet synthesis. Upon maturation, megakaryoblasts form megakaryocytes through the effect of colony stimulating factors. It is up to this point that colony stimulating factors have a significant role in the process of platelet synthesis (Geddis, 2010).
After the formation of megakaryocytes, erythropoietin takes control of the downstream synthesis process. Erythropoietin is a protein produced in the liver and kidneys after which it is released into the bloodstream. It gets into the bones and bone marrow through the network of blood vessels that reach these areas. The effect of this protein in the bone marrow is seen with the maturation of megakaryocytes and their extrusion from the bone. These cells are the precursors of the platelets and are, therefore, transported outside the bone and bone marrow through capillary vessels. As aforementioned, platelets contain no nucleus (Geddis, 2010). Therefore, the nucleus in the fully developed megakaryocytes is eradicated through a process referred to as nuclear death. The cell membrane then breaks to release the dead nucleus. During this phase, the cells have outgrowths known as pro platelet extensions which are essential in forming the proteins required by the cell. The final formation of refined platelets occurs when the pro platelets further disintegrate to form smaller fragments within the blood. It is these fragments that are counted during a platelet count to diagnose thrombocytopenia (Geddis, 2010).
It is a condition where an individual’s number of platelets is found to fall below the normal ranges of 150000 to 450000/µL of blood (Kistanguri & McCrae, 2014). Such a diagnosis may be explained by an impaired bone marrow synthesis pathway or due to a genetic defect. For instance, when one has an impaired liver or kidney, erythropoietin will not be produced, and the platelets formation pathway will stop at megakaryocytes. The hemolytic uremic syndrome impairs the kidney through the infection with bacteria such as E. coli. The pathway may also be inhibited by some drugs as a result of all these causes, the number of circulating platelets decreases when they are trapped in the spleen, or when their production is low, and destruction is high. Platelets may be trapped in the spleen which is an organ that eliminates unwanted substances from blood (Kistanguri & McCrae, 2014). Also, platelet production in the bone can be decreased due to factors such as leukemia, chemotherapeutic drugs or even infection by viruses. Uncontrolled breakdown of platelets may be activated by various conditions unto which the body is exposed such as childbirth. Autoimmune infections may also cause platelet destruction when they are recognized by antibodies as foreign materials. The presence of bacteria in blood is likely to lead to platelet destruction if the whole infected cell has to be destroyed. In the end, the bacteria is killed together with the blood cells as a body mechanism of creating an immune response against the disease-causing microorganism (Kistanguri & McCrae, 2014). Thrombocytopenia can also result when there is thrombotic thrombocytopenic purpura, a situation whereby many minute blood clots are formed throughout the body. When this situation arises, very many platelets are used up, and their numbers drop rapidly. Lastly, Thrombocytopenia can be caused by some medications which have an effect on the immune system. Such an instance can occur if the mode of action of the drug exerts a mechanism that confuses the immune system which further destroys platelets as an immune response. Such drugs include heparin, quinine, and antibiotics that contain sulfur. Due to these varied causes of thrombocytopenia, various scientists have been investigating the various factors that could be significant in causing the condition in a large number of diagnosed cases (Kistanguri & McCrae, 2014). Through the analysis of the sequence of platelet production, it becomes evident that a variety of factors may cause thrombocytopenia through targeting various steps of the sequence. To clarify some of these factors and their relationship to the condition, two articles addressing the same were evaluated, and their effectiveness established through the validity of methods used and results obtained. Both studies involved the in vitro effect of platelet formation in sera and plasma samples with autoantibodies targeting pro platelets. The general study design posed by this commentary is the direct comparison of the two studies and the combined implications of their results in establishing the main causes of thrombocytopenia in most patients.
Materials and Methods
The first study aimed at investigating the effect of autoantibodies against megakaryocytes, making them unable to form pro platelets and subsequently platelets in vitro. 19 patients who had been previously diagnosed with immune thrombocytopenia (ITP) were asked to willingly donate blood samples that would be used for the research (Iraqi et al., 2015). To establish a control for the experiment, blood samples were also obtained from nine healthy people. This study was a standard one, taking into consideration essentials of a study like this one. Hence it obtained authorization by the Human Rights Committee. Centrifugation of the blood samples was then performed at 188rpm to obtain serum that was used in subsequent steps. Both test and control sera were treated with protein-G agarose beads to isolate and purify IgG that would then be subjected to the culture cells. The purified fraction of the immunoglobulin was then treated with 1× saline solution containing phosphate buffer and stored appropriately to prevent degradation (Iraqi et al., 2015). Hematopoietic cells, from which blood cells originate, were isolated and cultured. These cells were obtained from umbilical cord blood that had been prior donated by healthy individuals, denoting the competitiveness of the cells.
Successful isolation of CD34-positive cells from the umbilical cord blood involved the use of the CD34 MicroBead kit. Culturing proceeded with the use of Stemline II media which had been enriched with recombinant thrombopoietin whose role was to activate the differentiation process of megakaryocytes. After approximately a week of uninterrupted cell growth, the cells were counted and their megakaryocytes assessed. After that, isolated IgG samples from both test and control sera were added into the wells that had been prior made in the culture. This addition was done in 1:10 dilutions and the steps repeated to ensure reproducibility. The culture was then allowed to grow for five more days, after which the megakaryocytes that had borne pro platelets were counted using microscopy (Iraqi et al., 2015).
The second study also aimed at proving the contribution of autoantibodies towards thrombocytopenia due to the autoimmune reaction with megakaryocytes. To achieve this, the researchers also obtained blood samples from patients who had been diagnosed with chronic ITP. The procedures used to obtain the samples also satisfied the Ethics Committee which is concerned with factors such informed consent from the patients. As opposed to the first study, this study involved collection of the blood samples in EDTA a similar portion was also collected in sodium citrate (Lev et al., 2014). As opposed to the first experiment, the researchers of this experiment were more interested in studying the plasma and not the serum. It is for this reason that they chose to use EDTA in blood sample collection so as to prevent coagulation of the blood. This aspect marked the major difference between the two studies. The essence of sodium citrate was to enable the production of platelet-poor plasma after a series of two centrifugation processes. The plasma was then recalcified using calcium chloride and was centrifuged after the clot had been removed. Also, while the first study obtained its cord blood from a cord blood bank, this one did actual collection from pregnancies and deliveries at designed hospitals (Lev et al., 2014).
The anticoagulated plasma was used to test the specificity of the autoantibodies that would be used for the experiment. This step was an essential one in ensuring that the selected antibodies were specific to the mutations that would be studied, a step that was not performed by the first researchers. The recalcified plasma from both infected and control groups was incubated with washed platelets and platelet-plasma adsorption analyzed. This analysis was done in the form of autoantibodies that had bound to the plasma (Lev et al., 2014). As opposed to immunoglobulin purification using agarose beads in study one, this study purified its IgG through affinity chromatography. A further ultracentrifugation was done to the purified immunoglobulin so as to concentrate it before being resuspended in PBS. Just like study one, CD34 + cells from the umbilical cord blood were cultured, and pro platelet count was done on them. This study had main aims of monitoring the process of platelet formation and how it is affected by recalcification and platelet-adsorption plasma, and IgG. It made a further effort as compared to study one which only established the effect of autoantibodies from IgG on platelet synthesis (Lev et al., 2014).
Both studies showed a significant effect on autoantibodies by IgG and other immunoglobulins which were found in some of the patients in the process of platelet synthesis. Since the serum used in the experiments were obtained from infected patients, it was considered immune thrombocytopenia. Study one specifically also found out that autoantibodies had no direct effect on the number of megakaryocytes and their maturation processes (Iraqi et al., 2015). It went ahead to establish that immune thrombocytopenia does not affect megakaryocyte ploidy, the size of the cell nor activation of caspases. This decision was arrived at after comparison of the ploidy levels of CD41+ in the test and control sera. It goes further to suggest a corrective measure which is the treatment of the megakaryocytes with agonists of the thrombopoietin receptor to prevent its binding by the autoantibodies (Iraqi et al., 2015).
Also, study two found out that not only ITP serum was capable of inhibiting platelet formation, but also plasma. This effect is observed due to the observed ability of ITP plasma to inhibit the formation of pro platelets which was contrary to normal plasma from the control group.
The study further established that ITP plasma had an effect on the morphology of pro platelets obtained from normal megakaryocytes in such plasma. The pro platelets were found to exhibit differences from normal ones since they were shorter, thicker, and had swellings, among other differences. By studying chromatin condensation, it was also evident in the study that apoptosis of normal megakaryocytes in ITP plasma was a cause of reduced platelet production. However, ELISA was performed to establish the role autoantibodies in pro platelet inhibition (Lev et al., 2014).
The process of platelet formation entails division and transformation of various cells to ensure full maturity. A defect in this process can be observed at the various stages and further cause thrombocytopenia. Various studies have been done to prove the various reasons that could be the cause of this condition. From the results of the two studies, it can be assumed that most of the cases occur as a result of autoimmune reactions which target various precursor particles that lead to full maturation of platelets. As megakaryocytes mature, they express certain molecules on their surfaces which may become targets of autoantibodies hence tempering with the subsequent formation of pro platelets. The results of the studies show that autoantibodies have varied effects on the formation of platelets, with profound effect observed in obscuring pro platelet formation.
Iraqi, M., Perdomo, J., Yan, F., Choi, P. and Chong, B.H., 2015. Immune Thrombocytopenia: Antiplatelet Autoantibodies Inhibit Proplatelet Formation by Megakaryocytes and Impair Platelet production in vitro. Haematological, 100(5), pp. 623-632.
Lev, P.R, Grodzielski, M., Goette, N., Glembotsky, A.C., Espasandin, Y.R., Pierdominici, M.S., Contfrufo, G., Montero, V.S., Ferrari, L., Molinas, F.C., Heller, P.G. and Marta, R. 2014. Impaired Proplatelet Formation in Immune Thrombocytopenia: a Novel Mechanism Contributing to Decreased Platelet Count. British Journal of Haematology 165, pp. 854-864.
Geddis, A.E. 2010. Megakaryopoiesis. NIH Public Access, 47(3), pp. 1-5.
Kistanguri, G. and McCrae, K.R. 2014. Immune Thrombocytopenia. HHS Public Access, 27(3), pp. 1-16.