Print version ISSN 1413-8670
Braz J Infect Dis vol.7 no.6 Salvador Dec. 2003
Effect of pathogenic yeasts on human platelet aggregation
Teresinha de Jesus Carvalho Neiva; Jairo Ivo dos Santos
Federal University of Santa Catarina, Centre of Health Science, Florianópolis, SC, Brazil
We investigated the effects of Candida albicans, Cryptococcus neoformans and the respective culture supernatants on human platelet aggregation (PA). Both yeasts were unable to aggregate the platelets directly. On the other hand, cells of these yeasts significantly (P < 0.01) inhibited PA at concentrations equal to or higher than 1 x 106 cells/mL for C. albicans and equal to or higher than 1 x 105 cells/mL for C. neoformans. When the supernatants of one-week broth cultures were added to the activated platelets no inhibition in aggregation was observed. Apparently somatic components of these yeasts, but not their metabolic products, exert an antagonistic effect on the aggregation of human platelets, possibly aiding the fungi in their evasion of the microbicidal defense system during vascular dissemination.
Key Word: Candida albicans, Cryptococcus neoformans, platelet aggregation.
Platelets are blood components that participate in physiological as well as pathological processes, such as homeostasis, thrombosis, atherosclerosis and infections [1-4]. In platelet aggregation (PA), the platelets interact with each other to form a hemostatic plug or thrombus. This process can be triggered in vitro by mixing fresh platelets with agonists, such as collagen or adenosine diphosphate (ADP), which activate the resting platelets .
Many fungal species, or their products, can interfere directly in platelet functions. The inhibition of PA, induced by components present in extracts of moulds and mushrooms, is well known [6-10]. In addition, pathogenic fungi, like the yeasts Candida albicans and Cryptococus neoformans, can cause infections that are particularly severe in immunocompromised individuals [11, 12]. During fungal infection, these patients are under increased risk for fungal proliferation in their blood and further vascular dissemination [13,14].
The mechanisms by which fungal pathogens initiate vascular infections are believed to be complex, and they involve interactions of fungal cells with plasma components, e.g., fibronectin , endothelial cells  and platelets , and avoidance of plasma host defenses, such as complement and phagocytosis. Platelet activation also releases inflammatory mediators, e.g., arachidonic acid metabolites and platelet microbicidal proteins [5,18]. Hence, fungal cells can interact with blood components, including those associated with hemostasis processes.
Therefore, we evaluated the effect of C. albicans and C. neoformans cells, as well as their culture supernatants on human PA, in vitro.
Materials and Methods
Reagents. Collagen and ADP were purchased from Chronolo. Corp., USA, while the remaining reagents were purchased from Sigma (USA).
Fungal preparation. Candida albicans (strain 3153 from the London School of Hygiene and Tropical Medicine Collection) and C. neoformans (strain ACL-68, previously isolated from the cerebrospinal fluid of an AIDS patient) were cultured in Sabouraud's dextrose agar during five days at 25oC. Afterwards, the yeast cells were suspended in sterile saline, counted in a haemocytometer and were diluted to various concentrations of 1 x 104 to 1 x 107 cells/mL. Supernatants were also obtained from one-week cultures in Sabouraud broth. These cultures yielded approximately 1 x 107 cells/mL after one week of incubation. To obtain the supernatant, the broth was centrifuged at 500 g for 5 min, and then the supernatant was collected and centrifuged again at 8,000 g for an additional 5 min. The supernatant was then collected and used for the platelet aggregation test. Sterile Sabouraud's broth was used as a control.
Subjects. The study included 12 healthy volunteers, who gave informed consent before participating in this study.
Isolation of platelets. Human blood platelets were obtained from healthy, drug-free individuals and were collected in tubes containing 3.8% sodium citrate. Platelet-rich plasma was prepared by centrifugation of citrated blood at room temperature for 6 min at 180 g. Platelets were adjusted to 2.5 x 108 cells/mL with sterile saline.
Measurement of PA. PA was determined by the turbidimetric method  using a Net Lab aggregometer. Aliquots of 450 µL of a platelet suspension were transferred into small cuvettes and stirred at a constant speed of 180g at 37ºC. The platelets were pre-incubated for 5 min at 37oC with different yeast concentrations, or with the supernatants of the cultures, and were stimulated with 6 µM ADP and 2 µg collagen/mL. The extent of aggregation (%) was recorded continually for 5 min after addition of the agonist. To evaluate the ability of C. albicans and C. neoformans to aggregate platelets, fungal cells (1 x 107 cells/mL) or culture supernatants were mixed with platelets (2.5 x 108 cells/mL) and incubated at 37ºC during 10 min.
Statistical analysis. Data were expressed as mean ± SD. The Student's t-test was employed to determine if there were differences between the groups. Differences were considered significant when the probability was P < 0.05. The statistical program INSTAT-2 was utilized.
Results and Discussion
We observed that C. albicans and C. neoformans cells were unable to aggregate platelets directly (Figure 1). Studies on interactions of yeasts and platelets have demonstrated a direct agonistic effect on platelet aggregation. The yeast forms of the dimorphic fungi Histoplasma capsulatum can induce platelet aggregation in vitro . Likewise, a Saccharomyces cerevisiae mannan fraction was found to be lethal when injected intravenously into mice, resembling the effect induced by the administration of platelet-activating factor [21,22]. In addition, somatic fractions of this yeast, which were chemically defined as being composed of phosphocholines, were also capable of activating platelets . Willcox et al.  found that human PA is induced by all Candida species, except for C. albicans. The aggregation of platelets releases microbicidal or microbistatic substances that are active against Candida spp. [24,25]. Therefore, the ability of C. albicans to inhibit the aggregation of platelets could contribute to its survival in the blood during vascular infections.
High cell counts of both C. albicans and C. neoformans were capable of preventing PA in the presence of collagen and ADP (Table 1). The inhibitory effect was always concentration dependant. A significant inhibition was observed after incubation of platelets with 1 x 106 cells/mL of C. albicans. The maximum inhibitory effect was obtained when 1 x 107 cells/mL was used. On the other hand, C. neoformans produced a more intense inhibitory effect on PA than C. albicans. We found that as low as 1 x 105 cells/mL were capable of inhibiting PA (Table 2). This antagonistic effect could also explain the capacity of this fungus to evade the microbicidal mechanisms triggered by PA. Numerous moulds and mushrooms, or their products, can easily inhibit PA in vitro [5,6,8,26,27]. In these cases, the antagonistic effects derive from substances, such as proteins, hydrazine derivatives, peptides, nucleosides, nucleotides, lecithins, saponins and sterols, which are present in the fungal extracts and can directly inhibit the aggregating agents .
Finally, to determine if soluble by-products from yeast cultures have any effect on PA, centrifuged supernatants from one-week cultures containing 1 x 107 cells/mL were added to the platelets before being activated by collagen and ADP. No change in the intensity of PA was observed for the supernatants of the two yeasts (Table 3). It is known from in vivo studies, that the capsule prevents the platelets from adhering to C. neoformans . However, the polysaccharide capsule does not necessarily play a role in the inhibition of PA, since we observed that the supernatant of one-week cultures of C. neoformans, which should contain soluble capsular antigens, did not inhibit PA.
In conclusion, unidentified C. albicans and C. neoformans cell component(s) can directly inhibit platelet activation, playing a role in fungal evasion from host defenses, during vascular dissemination.
1. McCrae K.R., Bussel J.B., Mannucci P.M., et al. Platelets: an update on diagnosis and management of thrombocytopenic disorders. Hematology (Am. Soc. Hematol. Educ. Program.) 2001;1:282-305. [ Links ]
2. Theilmeier G., Michiels C., Spaepen E., et al. Endothelial von Willebrand factor recruits platelets to atherosclerosis-prone sites in response to hypercholesterolemia. Blood 2002;99:4486-93. [ Links ]
3. Roux W., Pieper C., Cotton M. Thrombocytopoenia as marker for HIV exposure in the neonate. J Trop Pediatr 2001;47:208-10. [ Links ]
4. Yeaman M.R. The role of platelets in antimicrobial host defense. Clin Infec Dis 1997;25:951-70. [ Links ]
5. Colman R.W., Hirsh J., Marder J.V., Salzman E.W. Hemostasis and thrombosis. Basic principles and clinical practice. Philadelphia, USA: J.B. Lippincott Company, 3rd edition, 1994. [ Links ]
6. Ando T., Tsurumi Y., Ohata N., et al. Vinigrol, a novel antihypertensive and platelet aggregation inhibitor agent produced by a fungus, Virgaria nigra. I. Taxonomy, fermentation, isolation, physicochemical and biological properties. J Antibiot (Tokyo) 1988;41:25-30. [ Links ]
7. Chu M., Truumees I., Gunnarsson I., et al. A novel class of platelet activating factor antagonists from Phoma sp. J Antibiot (Tokyo) 1993;46:554-63. [ Links ]
8. Erkel G., Lorenzen K., Anke T., et al. Kuehneromycins A and B, two new biological active compounds from a Tasmanian Kuehneromyces sp (Strophariaceae, Basidiomycetes). Z Naturforsch 1995;50:1-9. [ Links ]
9. Kagamizono T., Nishino E., Matsumoto K., et al. Bassiatin, a new platelet aggregation inhibitor produced by Beauveria bassiana K-717. J Antibiot (Tokyo) 1995;48:1407-12. [ Links ]
10. Fabian K., Lorenzen K., Anke T., et al. Five new bioactive sesquiterpenes from the fungus Radulomyces confluens (Fr.) Christ. Z Naturforsch 1998;53:939-45. [ Links ]
11. Zuger A., Louie E., Holzman S.R., et al. Cryptococcal disease in patients with the acquired immunodeficiency syndrome. An Int Med 1986;104:234-40. [ Links ]
12. Walsh T.J., Groll A.H. Emerging fungal pathogens: evolving challenges to immunocompromised patients for the twenty-first century. Transpl Infect Dis 1999;1:247-61. [ Links ]
13. Kobza K., Steenblock V. Demonstration of Candida in blood smears. Br Med J 1977;1:1640-1. [ Links ]
14. Kates M.M., Phair J.B., Yungbluth M., Weil S.L. Demonstration of Candida in blood smears. Lab Med 1988;19:25. [ Links ]
15. Jakab E., Paulsson M., Ascenio F., Ljungh, A. Expression of vitronectin and fibronectin binding by Candida albicans yeast cells. APMIS 1993;101:187-93. [ Links ]
16. Filler S.G., Pfunder A.S., Spellberg J.P., Edwards J.E. Candida albicans stimulates cytokine production and leukocyte adhesion molecule expression by endothelial cells. Infec Immun 1996;64:2609-17. [ Links ]
17. Maisch P.A., Calderone R.A. Role of surface mannan in adherence of Candida albicans to fibrin-platelet clots formed in vitro. Infect Immun 1981;32: 92-7. [ Links ]
18.Caccese D., Pratico D., Ghiselli A., et al. Superoxide anion and hydroxyl radical release by collagen-induced platelet aggregation: role of arachidonic metabolism. Thromb Haemost 2000;83:485-90. [ Links ]
19.Neiva T.J.C., Benedetti A.L., Tanaka S.M.C.N., et al. Determination of serum aluminum, platelet aggregation and lipid peroxidation in hemodialyzed patients. Braz J Biol Med Res 2002;35:345-50. [ Links ]
20. DesPrez R.M., Steckley S., Stroud R.M., Hawiger J. Interaction of Histoplasma capsulatum with human platelets. J Infec Dis 1980;142:32-9. [ Links ]
21. Nagase T., Mikami T., Suzuki S., et al. Lethal effect of neutral mannan fraction of baker's yeast in mice. Microbiol Immunol 1984; 28:997-1007. [ Links ]
22. Mikami T., Fukushi K., Ishitani M., et al. Induction of platelet activating-factor in mice by intravenous administration of a neutral fraction of baker's yeast mannan. Lipids 1991;26:1404-7. [ Links ]
23. Nakayama R., Kumagai H., Saito K. Evidence for production of platelet-activating factor by yeast Saccharomyces cerevisae cells. Biochim Biophys Acta 1994;1199:137-42. [ Links ]
24. Willcox M.D., Webb B.C., Thakur A., Harty D.W. Interactions between Candida species and platelets. J Med Microbiol 1998;47:103-10. [ Links ]
25. Robert R., Nail S., Marot-Leblond A., et al. Adherence of platelets to Candida species in vivo. Infect Immun 2000;68:570-6. [ Links ]
26. Gentry P.A., Ross M.L., Bondy G.S. Inhibitory effect of trichothecene mycotoxins on bovine platelets stimulated by platelet activating factor. Can J Vet Res 1987;51:490-4. [ Links ]
27. Pinto A., Cirino G., Meli R., et al. Inhibitory effects of fungi on aggregation of rabbit platelets and rat polymorphonuclear leucocytes in vitro. J Ethnopharmacol 1998;22:91-9. [ Links ]
28. Hammerschmidt D.E. Szechwan purpura, N Engl J Med 1980;302:1191-3. [ Links ]
29. Nail S., Robert R., Dromer F., et al. Susceptibilities of Cryptococcus neoformans strains to platelet biding In vivo and to the fungicidal activity of thrombin-induced platelet microbicidal proteins in vitro. Infec Immun 2001;69:1221-5. [ Links ]
Dr..Teresinha de Jesus Carvalho Neiva
Departamento de Análises Clínicas, Centro de Ciências da Saúde, Universidade Federal de Santa Catarina, Campus Universitário
Phone: 0xx48 331 9712
Fax: 0xx48 331 9542
Received on 15 May 2003
revised 08 August 2003