Please note! This essay has been submitted by a student.
Blood vessel injury and bleeding are reduced by specialised cells that reside in the blood, called platelets. Platelets are made by cells in the bone marrow, called megakaryocytes (MKs) which release long pseudopodial extensions called proplatelets.(Patel, Hartwig and Italiano, 2005; Machlus and Italiano, 2013) Proplatelet formation occurs when megakaryocytes remodel their cytoplasm into long extensions, which are used as lines of assembly for platelets.(Thon et al., 2010) Platelets are important in many physiological processes, and therefore understanding their mechanisms is critical for research.(Machlus and Italiano, 2013) One-way platelets are being studied is regarding how CCL5, which is released from platelets, increases the formation of proplatelets by megakaryocytes, which is discussed in a paper written by Machlus et al.
When the body undergoes physical stress, platelet count increases impermanently in short bursts, however the mechanism behind this is unknown. Activated platelets release various proteins into circulation, which is called a releasate.(Wijten et al., 2013) Proplatelet production increases by almost half if megakaryocytes (MKs) are treated with this releasate. (Machlus et al., 2016)
One of the cytokines released is chemokine ligand 5 (CCL5), also known as RANTES (Murray, 2013) which is a cytokine chemoattractant for various cells involved in immunity, including monocytes, memory T helper cells and eosinophils. CCL5 is involved in HIV suppression and acts on chemokine receptor 5 (CCR5) which is present on MKs (CCL5 C-C motif chemokine ligand 5 [Homo sapiens (human)] – Gene – NCBI, no date). More than 25ng/mL of CCL5 is released after platelets are activated by TRAP (thrombin-receptor activating peptide). Machlus et al hypothesized that CCL5 could regulate production of platelets by binding to CCR5 on MKs.
In order to confirm that CCL5 increases proplatelet production, MKs were treated with maraviroc which is a CCR5 antagonist and works by inhibiting CCL5 and stopping it from activating.(Singh et al., 2018) When either maraviroc or immunodeficient CCL5, which has been neutralized using an antibody, was added to MKs, the effect of CCL5 on proplatelet production decreased, this reduction suggests that CCL5 does in fact increase proplatelet production.(Machlus et al., 2016)
CCR5 expression on MKs and platelets was measured using a technique called flow cytometry. This is a method of analysing characteristics of cells and uses the principle that fluorescent labelled antibodies emit light to identify cell surface and cytoplasmic antigens.(Brown and Wittwer, 2000) Propidium iodide is a DNA binding stain that was used to determine ploidy.(Crowley et al., 2016)(Machlus et al., 2016)
CCL5/CCR5 participation in platelet production during inflammation was measured in a study. Mice were injected with dextran sodium sulfate (DSS) which is a chemical that induces the disease acute colitis. (Chassaing et al., 2014) Maraviroc and saline vehicle control were injected daily for 7 days.
One overall limitation of the research conducted in this paper is that it was conducted on mice/rodents. This means that any conclusions found are not conclusive and cannot be relied upon. It cannot be presumed that mechanisms seen and understood in murine will be entirely the same in humans. Which also means that any therapeutic therapy or treatment ideas may cause more detrimental effects in humans compared to in mice.
Throughout Machlus et al’s paper it was clear that a conscious effort was made when planning the experiments to ensure validated results. It wasn’t just one factor tested against platelet count for example, a control and also an antagonist would be used. Saline vehicle control groups were used to ensure that the vehicle alone has no effect on the results. As well as incorporating antagonists to see if the effect therefore was reversed. This allowed to gain a better insight into the overall mechanisms underlying the results and ensured that any results obtained could be as accurate and reliable as can be.
[image: ]Although other papers have been used to contribute to the details of the methodology in this paper, the report makes little reference to previous studies or other researchers work and conclusions. Including these could have been a useful tool to either support or criticise findings and increase the analysis of the data.
As shown in figure 1, DSS lead to a positive correlation between white blood cell (WBC) count and platelet count. This correlation was not present in the mice treated with saline or maraviroc. This suggests that the component of WBC’s that increase platelet count is linked to CCR5 due to the absence of correlation when CCR5 antagonist, maraviroc was present. The methodology of this experiment is strong due to the fact that a saline vehicle control was used to ensure that the saline alone wasn’t causing an effect.Figure 1: Mice either were treated with saline vehicle control, injected with maraviroc or Colitis was induced via DSS. After 7 days, blood was analysed, and platelet count was correlated against WBC count.
Consistent throughout previous work including megakaryocyte cultures, on embryonic day 13.5, murine foetal livers were collected, and single cell suspensions were prepared. (Lecine et al., 2000; Italiano et al., 2008; Thon et al., 2010; Machlus et al., 2016) In order to acquire top quality megakaryocytes, cells were implanted on a 1.5%-3.5% albumin step gradient and sedimented and then proplatelets were isolated.(Schwer et al., 2001; Italiano et al., 2008; Thon et al., 2010) This consistency throughout previous work conveys a message of reliability. However, the stages of platelet release and maturation is a complex process and isn’t fully understood. Megakaryocyte cultures usually end up a mix of hematopoietic cells.(Thon et al., 2010) This means that the results in Machlus et al’s report cannot be considered 100% accurate due to the fact that the cells used may not be entirely isolated platelets/proplatelets and actually be made up of a mix other hemopoietic cells. Research into the terminal stages of proplatelet maturation and platelet release would allow a more in-depth knowledge of how to specifically isolate platelets at the desired stage which would increase the accuracy of research around megakaryocytes and platelets.
Immunofluorescent microscopy is a technique used in a variety of papers concerning platelets. Overall, the method found included fixing the platelet sample with 4% formaldehyde and centrifuging the sample onto a coverslip which is coated in poly-l-lysine. Trixton-X-100 was used to permeabilize the platelets and then they were blocked overnight in phosphate-buffered-saline. (Italiano et al., 2008; Patel-Hett et al., 2008; Thon et al., 2010; Machlus et al., 2016) This method was almost entirely the same in all methods read, meaning it has high reliability and the accuracy of the results can be trusted. The results of this microscopy enabled a confirmation that platelets and MK contain CCL5.
Following on from this, further research using flow cytometry found that both human and mouse MKs had high CCR5 expression compared to human and mouse platelets. As it is known that platelets release CCL5, we can confirm that they act on MKs through the CCR5 receptor. It is suggested that CCR5 is purposely removed from the platelet surface as it is a rare phenomenon for a receptor not to be present on both.
Cellular ploidy is the number of sets of chromosomes inside the cell.(Crespel and Meynet, 2017) Even though the principles behind proplatelet formation and ploidy is complicated, it is known that increased proplatelet formation and ploidy are correlated. CCL5’s role in this was studied via endomitosis. 65% fewer MKs were 2N and 76% more were 16N. Maraviroc inhibited the increased ploidy which further supports the conclusion that CCL5 increases ploidy. Nonetheless, it isn’t really made clear what the relevance of the correlation between ploidy and proplatelet formation is. It is made clear how ploidy is determined and the fact that there’s a positive correlation but there isn’t any background on how increased ploidy is useful in the mechanism of proplatelet formation.
Due to the fact that a correlation has been found between CCL5 and elevated platelet count, we could use this as a basis to try and therapeutically treat thrombocytopenia. Thrombocytopenia is a condition where you express low platelet counts.(Seifoleslami, 2017)As we know CCL5 acts on the receptor CCR5, stimulation of the CCR5 receptor could increase platelet counts and eliminate thrombocytopenia. In an attempt to find a stimulatory substance for CCR5 that initiates proplatelet formation without causing other physiological responses, I would find molecules that are known to bind to CCR5 and test them in multicellular and animal models that express thrombocytopenia. One idea being, heat shock protein 70 (HSP70) is a known molecule that interacts with CCR5(Whittall et al., 2006). HSP70 could be injected daily into mice with thrombocytopenia and after 2 weeks, the blood could be analysed to see if platelet levels have risen or not. A saline vehicle control would also be administered to a control group to enable us to make sure the vehicle alone did not cause any difference. In another group, TAK-779 which is a CCR5 antagonist, would also be administered in addition to allow us to see if it was in fact interaction with CCR5 that caused the platelet increase.(Esté, 2001)This research would be very beneficial as thrombocytopenia is common to experience under certain conditions and there has been a rise in the increase of platelet transfusions needed. In the United States, over 10 million units are dedicated to platelet transfusions.(Thon et al., 2010) If we can better understand ways to increase platelet count and combat thrombocytopenia, there would be less demand on blood banks.
One of the main HIV co receptors is CCR5. CCR5 is involved in HIV entry and cell to cell spreading.(Lecine et al., 2000) If there was a way to block CCR5, HIV would be unable to enter the cell and spread, and therefore HIV would be prevented/limited. There are several different approaches that can be taken to do this. One of these is concerning natural anti-CCR5 antibodies that have been found in some individuals. HIV binds to CCR5 on the HIV binding domain, which is the second external loop. Anti-CCR5 antibodies bind to the first external loop, which decreases virus infectivity. If these antibodies could be modified to bind to a specific sequence on the second external loop of a CCR5 receptor, then HIV could ultimately be prevented.(Lopalco, 2010) Another perspective to be considered is the effect of rapamycin on CCR5. Rapamycin is a drug that reduces the density and reduce surface expression of CCR5.(Heredia et al., 2007)
To conclude, the research into how CCL5 increases megakaryocyte proplatelet formation is a topic that has nontrivial significance. CCL5 was found to increase proplatelet formation. The conclusions made from the research can enable further studies into possible therapeutic targets for thrombocytopenia, which would in turn reduce the need for platelet transfusions and blood bank demands would decrease. A broader impact of this research is involving HIV treatment. Further research into how CCL5 can be used to treat HIV will potentially lead to a treatment for HIV. HIV which leads to AIDS, causes millions of deaths each year and finding a treatment for HIV would be life-saving. The CCL5/CCR5 axis is a contributing factor to many different physiological problems. CCL5 and increased angiogenesis is another topic that is being researched which would be of great use if we could use as a therapeutic treatment. As you can see already, there are various mechanisms that CCL5 is already known to affect, and if it was studied even more, the possibilities of possible treatments are not limited.