Platelet-Dependent Killing of C. albicans

There are around 150,000-400,000 platelets per microliter of blood in a healthy human being (Williams, n.d.), each which are around two microns in size (“Platelets on the Web,” n.d.). Platelets are blood cells which assist in the clotting of blood vessels, a process also known as hemostasis.

These platelets originate from megakaryocytes, which are precursor cells from the bone marrow that circulate in the blood to recognize any and all damaged vessels (Williams, n.d).

The process of clotting the blood consists of a series of reactions. A rupture first occurs on the endothelium, or the tissue that forms a single layer of cell lining around the blood vessels (Ariëns et al., 2002). Platelets then adhere, or stick, to the exposed endothelial cells mediated by von Willebrand factor (vWF), a large glycoprotein-clotting factor (Ariëns et al., 2002). For the platelets to properly adhere to the endothelial cells, they must release the contents of three main granules: the dense (δ-), alpha (α-), and lysosomal (λ-) granules (Blair & Flaumenhaft, 2009; Campbell et al., 2008). To ultimately result in platelet aggregation, thrombosis, or coagulation of the blood, must occur, leading to the formation of fibrin, a non-globular protein involved in the clotting of blood (Geraldo et al., 2014). This occurs through two different pathways: the contact activation pathway and the tissue factor pathway. The contact activation pathway has a minor role in thrombosis as it forms certain proteins that help in coagulation such as FXII, prekallikrein and HMWK (Geraldo et al., 2014). The tissue factor pathway generates thrombin, an enzyme essential to the coagulation cascade, which eventually leads to the release of fibrin to plug the break in the vessel wall (Polgar et al., 2005). Finally, a temporary clot is created that is eventually dissolved with the help of a plasmin, an enzyme that destroys fibrin (Polgar et al., 2005).

Although the main function of platelets is hemostasis, studies have shown that they have more roles. Platelets are also part of the innate immune system, or the defense systems, and the adaptive immune system, or the specialized defense system (“Phagocytosis,” n.d.). In innate immunity, platelets have shown to use their surface receptors to sense pathogens and then, consequently, attach to them (Saboor et al., 2013). The platelets attach to the pathogen and release the contents of the granules, which assist in host defense (Saboor et al., 2013). The pathogen is then weakened in its ability to harm the body (Speth, Loffler, Krappman, Lass-Florl & Rambach, n.d.).

Each surface receptor on the platelets assists in a different manner. GPIIb/IIIa is the most abundant platelet receptor on the plasma membrane (Saboor et al., 2013). When this receptor binds to a pathogen, it mediates the adhesion between the platelet and pathogen. A second type of platelet receptor, GPIbα, binds to bacteria but does not always result in platelet activation. The primary platelet activation surface receptor, the third type, is glycoprotein GPIa/IIa, which is also capable of binding to viruses. Fc receptor FcγRIIa, the fourth type of surface receptor, binds to IgG, the most abundant type of antibody that protects against bacterial and viral infections and fights off pathogens (Saboor et al., 2013; Arman & Krauel, 2015). This receptor also helps in the activation of platelets and in enhancing GPIIb/IIIa and GPIbα receptors. Lastly, the toll-like receptors, a class of proteins present in the immune system, aid in the activation of platelets and phagocytosis, the engulfing of foreign particles, and the pathogens (Saboor et al., 2013). The use of these receptors is the reason platelets work together to fight off infections the immune system encounters.

These properties of platelets have encouraged researchers to find any correlations between the diagnosis of thrombocytopenia, or the deficiency of platelets in the blood, and increased susceptibility to infections (Elkstrand et al., 2016). After an analysis of patient records that the researchers diagnosed with thrombocytopenia, they found that these patients had an increased risk of viral infections, fungal infections, skin infections, sexually transmitted diseases, urinary tract infections and gastrointestinal infections. These results show that there was an increased risk of infections for patients who had been diagnosed with thrombocytopenia (Elkstrand et al., 2016).

Further studies tested platelets against several infections. One experiment was conducted to test the ability of platelets to kill a bacterial infection known as Escherichia coli, or E. coli (Riaz et al., 2012). Healthy human blood was prepared until solely the platelets of the blood were left.  After adding the solution of activated platelets (using thrombin) and infection onto an agar plate, they found that the platelets had effectively killed E.coli (Riaz et al., 2012). Based on these results, another similar experiment was conducted with a bacterial infection known as Staphylococcus aureus, or S. aureus (Ali et al., 2016). The results showed that activated platelets (also using thrombin) participate in host defense against S. aureus and also showed that rather than directly killing the colonies, platelets may also be important for the function of other phagocytic cells that are responsible for killing the bacteria (Ali et al., 2016). The results of platelets against E. coli and S. aureus has sparked an interest in the effect of platelets against a fungus disease known as Candida albicans, or C. albicans.

C. albicans is a dimorphic fungus that grows in two forms: as yeast and as filamentous cells (Erdogan & Rao, 2015). This fungus is responsible for 50-90% of all cases of candidiasis in humans (Martins et al., 2014). A group of researchers conducted an experiment in vivo. After infecting mice, with and without platelets, with oropharyngeal candidiasis, the tongue was removed to count the colonies of C. albicans formed (Conti & Hunter, 2017). The results showed that mice without platelets had a significantly higher number of colonies found on the tongue compared to the mice with platelets in their blood (Figure 1).

Figure 1

 

compared to the mice with platelets in their blood (Figure 1).                                                          

As a continuation of this experiment, the kidneys of the same mice were extracted to observe the differences for the mice that contained platelets and the ones that lacked platelets. After counting the colonies of C. albicans, they found that the colonies found in the kidneys of mice who lacked platelets significantly exceeded the mice who contained platelets (Figure 2) (Conti & Hunter, 2017).      

The problem is that the reason why an abundant amount of C. albicans colonies had reached the kidneys of the mice without platelets in their blood is unknown. It is possible that the lack of platelets caused an abundance of C. albicans, thus infecting the kidneys. However, it is also possible that the lack of platelets damaged the kidneys, and caused the abundance of C. albicans.Therefore, the purpose of this experiment was to examine the effectiveness of platelets in whole blood on reducingC. albicans colonies in vitroin order to provide additional support for the original experiment.  It was expected that platelets would participate in host defense against C. albicans in whole blood. The rationale stemmed from first, the past studies which show that there was a correlation between patients who contained a low number of platelets in their blood and a high occurrence of infections. Second, from the experiments showing the effectiveness of platelets killing E. coli and S. aureus. And third, the study showing that there were C. albicans colonies on the kidney of the mice directly following the infection of the mice with oropharyngeal candidiasis.

Methods:

The first experimental procedure was to create the media used for plating. In a two liter Erlenmeyer flask, 50 grams of yeast extract peptone dextrose (YPD) medium, 15 grams of agar, and 950 mL of deionized water were all mixed together on a stir plate until a homogenous mixture was created. The mixture was then autoclaved for 40 minutes at 121℃. After retrieving the media from the autoclave, 13 mL of the media was placed in 75 plates.

Secondly, the original 107 inoculum of C. albicans was diluted down to 105 and 104 to serve as the control. This was done three times to result in three 105 inoculums and three 104 inoculums. Then, around 500 µL of blood was extracted from three different mice using cardiac puncture, all containing platelets in their bloodstream. 100 µL of blood from one mouse was placed into two different microcentrifuge tubes, one labeled “+” for the addition of thrombin (a potent platelet activator) and the other labeled “-” for the lack of thrombin. This was done for all three mice, resulting in six tubes. Then, 10 µL of C. albicans with an optical density reading of 0.81 at 600nm was added to all six tubes. Following this, 10 µL of thrombin was put into the three tubes labeled “+” and 10 µL of phosphate-buffered saline (PBS) solution was put into the other three tubes labeled “-.” These six tubes incubated for an hour at 37℃.

After one hour, serial dilutions were performed in duplicate using PBS solution. Next, 24 YPD plates were obtained and 10 plating beads were set on each plate. Then, 100 µL of each dilution was put on each plate, shaken sufficiently with the plating beads inside, and the beads were emptied from the plates. The plates were then incubated for 48 hours. After this period, the number of C. albicans colonies on each plate was counted.

The same experiment was repeated with the blood of six more mice, three of which contained platelets in the blood stream and three which were platelet-depleted. After extracting the blood to confirm that the blood did not in fact contain any platelets, flow cytometry was used (Figure 3). The Figure shows that in the blood without platelets, there are no data points in the circled area. Additionally, a marker for platelets can be detected in control mice while the marker is absent in depleted mice showing that platelets are undetectable.

Figure 3

The procedure was identical to the one preceding for the rest of the experiment       

Results:

To test the hypothesis that platelets can kill C. albicans, two experiments were performed: one to test the ability of activated platelets to effectively kill C. albicans versus unactivated platelets, and second to test the ability of activated platelets versus no platelets at all to kill C. albicans. There was a clear difference between activated platelets (+ thrombin) and unactivated platelets (- thrombin) and the killing of the C. albicans (p=0.0182) (Figure 4).

Figure 4

The second experiment was conducted to ensure that the thrombin was not the sole cause of the decrease in the colonies, but rather the platelets themselves. When the experiment was conducted with blood containing platelets and blood without platelets, the results once again showed a significant decrease in the number of colonies when faced with blood containing activated platelets (p=0.0427) (Figure 5).

Figure 6 shows the visual decrease in colonies when faced with activated platelets.

Discussion:

The results supported the original hypothesis that platelets participate in host defense against C. albicans in whole blood. The results confirm that there is a direct correlation between the presence of platelets itself in whole blood and the decrease of the C. albicans colonies.  The implication of the results relates to the original problem. The original problem was that it was unknown if the presence of platelets in the bloodstream affects the number of C. albicans colonies present. Since the results show that the one does affect the other, it answers the question of why there was such a great abundance of C. albicans colonies on kidney of the mice.

The internal validity concerns the fact that since the experiment was done using whole blood, it cannot be fully confirmed that platelets were the only acting antibodies that caused the killing of C. albicans. The only way to confirm this would be to test C. albicans against blood which contains one specific antibody and the other blood sample does not, until the acting antibody is pinpointed. However, a good future experiment would be to determine if platelets enhance macrophage killing of C. albicans in whole blood.

Future research should focus on what properties of platelets cause infections to decrease in their size significantly. Once the specific properties of the platelets which cause the killing are found, researchers may be able to apply the findings to humans with candidiasis infections. The findings of the properties of platelets will better explain why some patients have more aggressive forms of candidiasis or are more resistant to any treatments.

References:

  1. Ali R., Wuescher L., Dona K., & Worth R. (2016). Platelets mediate host-defense against S. aureus through direct bactericidal activity and by enhancing macrophage activities. Manuscript submitted for publication.

  2. Ariëns, R. A., Lai, T. S., Weisel, J. W., Greenberg, C. S., & Grant, P. J. (2002). Role of factor XIII in fibrin clot formation and effects of genetic polymorphisms. Blood, 100(3), 743- 754.

  3. Arman, M., & Krauel, K. (2015). Human platelet IgG Fc receptor FcγRIIA in immunity and thrombosis. Journal of Thrombosis and Haemostasis, 13(6), 893-908.

  4. Blair, P., & Flaumenhaft, R. (2009). Platelet α–granules: Basic biology and clinica correlates.Blood Reviews, 23(4), 177–189. http://doi.org/10.1016/j.blre.2009.04.001.

  5. Conti, H., & Hunter, J. (2017). [Untitled work]. Unpublished raw data.

  6. Ekstrand, C., Linder, M., Cherif, H., Kieler, H., & Bahmanyar, S. (2016). Increased susceptibility to infections before the diagnosis of immune thrombocytopenia. Journal of  Thrombosis and Haemostasis, 14(4), 807-814.

  7. Erdogan, A., & Rao, S. S. (2015). Small intestinal fungal overgrowth. Current gastroenterology  reports, 17(4), 1-7.

  8. Geraldo, R. B., Sathler, P. C., Lourenço, A. L., Saito, M. S., Cabral, L. M., Rampelotto, P. H., & Castro, H. C. (2014). Platelets: still a therapeutical target for haemostatic disorders. International journal of molecular sciences, 15(10), 17901-17919.

  9. Martins, N., Ferreira, I. C., Barros, L., Silva, S., & Henriques, M. (2014). Candidiasis: predisposing factors, prevention, diagnosis and alternative treatment. Mycopathologia,  177(5-6), 223-240.

  10. Phagocytosis | definition of phagocytosis by Medical dictionary. (n.d.). Retrieved from http://medical-dictionary.thefreedictionary.com/phagocytosis

  11. Platelets on the Web. (n.d.). Retrieved from https://www.ouhsc.edu/platelets/platelets/platelets intro.html

  12. Polgar, J., Matuskova, J., & Wagner, D. D. (2005). The P‐selectin, tissue factor, coagulation triad. Journal of Thrombosis and Haemostasis, 3(8), 1590-1596.

  13. Riaz, A. H., Tasma, B. E., Woodman, M. E., Wooten, R. M., & Worth, R. G. (2012). Human platelets efficiently kill IgG-opsonized E. coli. FEMS Immunology & Medical Microbiology, 65(1), 78-83.

  14. Saboor, M., Ayub, Q., & Samina Ilyas, M. (2013). Platelet receptors; an instrumental of platelet physiology. Pakistan journal of medical sciences, 29(3), 891.

  15. Smith, S. A., Travers, R. J., & Morrissey, J. H. (2015). How it all starts: Initiation of the clotting cascade. Critical Reviews In Biochemistry & Molecular Biology, 50(4), 326-336. doi:10.3109/10409238.2015.1050550

  16. Speth, C., Löffler, J., Krappmann, S., Lass-Flörl, C., & Rambach, G. (2013). Platelets as immune cells in infectious diseases. Future microbiology, 8(11), 1431-1451.

  17. Williams, M. (n.d.). What are Platelets and Why They are Important: Johns Hopkins Women’s Cardiovascular Health Center. Retrieved from

    http://www.hopkinsmedicine.org/heart_vascular_institute/clinical_services/centers_excell  ence/womens_cardiovascular_health_center/patient_information/health_topics/platelets.html

 

 

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Platelet-Dependent Killing of C. albicans

There are around 150,000-400,000 platelets per microliter of blood in a healthy human being (Williams, n.d.), each which are around two microns in size (“Platelets on the Web,” n.d.). Platelets are blood cells which assist in the clotting of blood vessels, a process also known as hemostasis.

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