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Experimental and Toxicologic Pathology 59 (2007) 105–114 www.elsevier.de/etp

Comparative ultrastructural analyses of platelets and fibrin networks using the murine model of asthma
E. PretoriusÃ, O.E. Ekpo, E. Smit
Department of Anatomy, School of Health Sciences, Faculty of Health Sciences, University of Pretoria, BMW Building, P.O. Box 2034, Pretoria 0001, South Africa Received 10 October 2006; accepted 13 February 2007

Abstract
The murine Balb/c asthma model has been used successfully for a number of in vivo immunological applications and for testing novel therapeutics, and it is a reliable, clinically relevant facsimile of the human disease. Here we investigate whether this model can be used to study other components of the human body, e.g. ultrastrucure. In particular, we investigate the effect of the phytomedicine Euphorbia hirta (used to treat asthma), on the ultrastructure of fibrin as well as platelets, cellular structures that both play an important role in the coagulation process. Hydrocortisone is used as positive control. Ultrastructure of the fibrin networks and platelets of control mice were compared to mice that were asthmatic, treated with two concentrations of hydrocortisone and one concentration of the plant material. Results indicate control mice possess major, thick fibers and minor thin fibers as well as tight round platelet aggregates with typical pseudopodia formation. Minor fibers of asthmatic mice have a netlike appearance covering the major fibers, while the platelets seem to form loosely connected, granular aggregates. Both concentrations of hydrocortisone make the fibrin more fragile and that platelet morphology changes form a tight platelet aggregate to a more granular aggregate not closely fused to each other. We conclude that E. hirta does not impact on the fragility of the fibrin and that it prevents the minor fibers to form the dense netlike layer over the major fibers, as is seen in untreated asthmatic mice. This ultrastructural morphology might give us better insight into asthma and the possible new treatment regimes. r 2007 Elsevier GmbH. All rights reserved.
Keywords: Scanning electron microscopy; Platelets; Fibrin; Balb/c murine asthma model; Euphorbia hirta

Introduction
In vivo animal models have been used successfully during the past few years to study diseases like asthma (Epstein, 2004a, b). The murine model in particular, is used successfully because mice allow for a variety of in vivo immunological applications (Bice et al., 2000).
ÃCorresponding author. Tel.: +27 12 319 2533; fax: +27 12 319 2240. E-mail address: resia.pretorius@up.ac.za (E. Pretorius).

According to Epstein (2006), the allergic asthma as observed in an experimental mouse model is a reliable, clinically relevant facsimile of the human disease. Furthermore, antigen-induced mouse allergic asthma is a useful model for testing novel therapeutics (Epstein, 2006) and has been used for testing many novel agents aimed at reducing lung inflammation, mucus hypersecretion, airway hyperresponsiveness and IgE profiles. However, the question that arises is whether the murine model like the asthma Balb/c model can be used to study other components of the human body other

0940-2993/$ - see front matter r 2007 Elsevier GmbH. All rights reserved. doi:10.1016/j.etp.2007.02.011

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than the typical immunological processes. The coagulation process and particularly ultrastructure of cellular components involved in haemostasis-like platelets and fibrin networks, might be studied successfully using this model. Comparing the effects of hydrocortisone and phytomedicine on, e.g., platelet and fibrin formation and morphology using the murine model might give researchers insight into how these products affect the coagulation system. Platelets have traditionally been associated with disorders of the cardiovascular system, where they are involved in the maintenance of haemostasis as well as the initiation of repair following tissue injury (Herd and Page, 1994). However, they also play an important physiological role in allergic processes and immunological mechanisms. In conditions like allergic asthma, platelets participate by acting as inflammatory cells, by releasing mediators, spasmogens and/or by interacting with other inflammatory cell types. These mediators include enzymes active in the coagulation cascade. Platelets are activated by a number of stimuli resulting in the expression and/or activation of surface receptors, secretion of vaso-active substances, adhesion, aggregation, and finally thrombus formation (Lazarus et al., 2003). The activation may be due to damage of the vessel wall or activation of the endothelium by chemicals, cytokines, and also inflammatory processes (Camera et al., 1999; Butenas and Mann, 2002) typically involved in conditions like allergic asthma. Over the past years, the interest in phytomedicine use particularly in the treatment of diseases like asthma has greatly increased. One such plant which is used for asthma is Euphorbia hirta (Euphorbiacea); which is a plant with great anti-inflammatory potential (Dickshit, 1943; Hazleton and Hellerman, 1954; Watt and BreyerBrandwijk, 1962; Le Strange, 1977; Wong, 1980; Lanhers et al., 1990, 1991; Skidmore-Roth, 2001; Lindsey et al., 2002). Although the cytotoxic potential of the plant has been studied, little is known of the effect of the plant on cellular function and morphology and no report is available of the effect of the plant on the coagulation system morphology. Therefore, because it is known that the plant is used for asthma, the use of the Balb/c mouse asthma model seems to be a good model to investigate the effect of phytomedicine on the coagulation process, in the presence of asthma. E. hirta is found worldwide but is also indigenous to Africa. In East and West Africa, extracts of the decoction of the flowering and fruiting plants are used in the treatment of asthma and respiratory-tract infections and are sometimes combined with bronchial sedatives like Grindelia robusta in preparations for inhalation (Oliver, 1959; Kokwaro, 1976). The plant is also used in the treatment of coughs, chronic bronchitis and pulmonary disorders; for relieving hay fever and catarrh; as an anti-hypertensive agent, analgesic, antipyretic and sedative; and the diuretic properties of the

plant have also been reported (Dickshit, 1943; Hazleton and Hellerman, 1954; Watt and Breyer-Brandwijk, 1962; Le Strange, 1977; Wong, 1980; Lanhers et al., 1990, 1991). E. hirta contains a great number of active ingredients including alkaloids, flavonoids, glycosides, sterols, tannins and triterpenoids (Gupta and Garg, 1966; Atallah and Nicholas, 1972; Sofowora, 1984; Galvez et al., 1993). The exact mechanisms by which E. hirta relieves asthma are not clear, but significant and dosedependent anti-inflammatory effects have been observed. Research also showed that aqueous extracts of E. hirta strongly reduced the release of prostaglandins I2, E2, and D2 (Hiermann and Bucar, 1994). Despite the array of chemical compounds characterized from E. hirta and the diverse local medicinal uses of the plant, very little pharmacological evaluations have been carried out to ascertain the rationale behind most of the folkloric claims of its efficacy (Johnson et al., 1999). However, it seems as if the flavonoid Quercitrin (3-rhamnosylquercetin) converted to Quercetin (3-O-alphaL-rhamnopyranoside – Quercetrin) in the alimentary canal, possesses great potential. Quercitrin is a bioflavonoid with anti-oxidant properties, as well as antiinflammatory activities and is therefore the glycosylated form of Quercetin (Comalada et al., 2005). The other flavonoid, Myricitrin also seems to be a powerful anti-oxidant, inhibiting NOS. The sterols 24-methylene-cycloartenol and b-sitosterol exert significant and dose-dependent anti-inflammatory activity (Martinez-Vazquez et al., 1999). The triterpene b-amyrin also seems to have anti-inflammatory effects. The combined effects of b-amyrin, 24-methylene-cycloartenol and b-sitosterol, may therefore account for the potent dose-dependent anti-inflammatory activity of E. hirta. Unfortunately, many of the other components of E. hirta extracts have not been studied sufficiently to know if they also might have anti-inflammatory effects. However, its effectiveness in treating asthma probably lies in the synergistic relationships between particularly the flavonoids, sterols and triterpenoids. The current research therefore investigates the effect of E. hirta on the ultrastructure of platelets and fibrin using the Balb/c murine model. Hydrocortisone is used here as positive control for treatment of asthma.

Materials and methods
Preparing water extracts of E. hirta
Plant material was collected in the Pretoria region of South Africa. A herbarium specimen was prepared and compared to an authentic specimen in the HGJW Sweikerdt herbarium at the University of Pretoria.

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The above-ground parts of the plant were allowed to dry at room temperature for 1 week. The material was grounded into a fine powder and subdivided into four 50 g samples. The samples were subsequently extracted with 500 ml of water, after which it was filtered and dried on a rotary evaporator at 40 1C. The water extract was freeze-dried. This procedure was repeated three times for each sample. A stock solution of 62.5 mg/kg was prepared that was administered orally to the mice. This implied that each animal (average weight 20 g) received 0.01 ml of plant material. This dose was decided upon after studying literature that mentioned physiological doses suggested by herbalists. Typically, a teaspoon of the herb is added to a teacup volume of water and allowed to simmer for 20 min (Lindsey et al., 2002). Alternatively, an extract of the plant could be prepared and the recommended adult dose range of the fluid extract is 0.2–0.3 ml, taken three times daily and of the infusion, 120–300 mg three times daily (Skidmore-Roth, 2001).

Implementing the Balb/c asthma model
Six-week-old (male) Balb/c mice each of average weight 20 g maintained in the Onderstepoort Animal Care facility and provided OVA-free food and water ad libitum, were used. All experimental protocols complied with the requirements of the University of Pretoria’s Animal Use and Care Committee. Mice were divided in the following groups (6 animals/ group):

groups and 11 ml citrate for every 100 ml of blood, was added. Blood was then centrifuged at 1000 rpm for 2 min to obtain platelet rich plasma (PRP). Human thrombin (provided by The South African National Blood Services) was used to prepare fibrin clots (Pretorius et al., 2006). The thrombin is 20 U/ml and is made up in biological buffer containing 0.2% human serum albumin. When thrombin is added to PRP, fibrinogen is converted to fibrin and intracellular platelet components, e.g., transforming growth factor, platelet derived growth factor and fibroblastic growth factor are released into the coagulum. Ten microliters of mouse PRP was mixed with 10 ml of human thrombin. The PRP and thrombin mix was immediately transferred with a pipette tip to a 0.2 mm millipore membrane to form the coagulum (fibrin clot) on the membrane. This millipore membrane was then placed in a Petri dish on filter paper dampened with PBS to create a humid environment and placed at 37 1C for 10 min. This was followed by a washing process where the millipore membranes with the coagula were placed in PBS and magnetically stirred for 120 min. This was done to remove any blood proteins trapped within the fibrin network (Pretorius et al., 2006).

Preparation of washed fibrin clot for SEM
Washed fibrin clots were fixed in 2.5% glutaraldehyde in Dulbecco’s phosphate buffered saline (DPBS) buffer with a pH of 7.4 for 1 h. Each fibrin clot was rinsed thrice in phosphate buffer for 5 min before being fixed for 1 h with 1% osmium tetraoxide (OsO4). The samples were rinsed thrice with distilled water for 5 min and were dehydrated serially in 30%, 50%, 70%, 90% and three times with 100% ethanol. The SEM procedures were completed by critical point drying of the material, mounting and examining the tissue with a JEOL 6000F FEGSEM.

    

Control mice. Asthmatic mice. Mice exposed to low dose hydrocortisone (100 mg/kg). Mice exposed to higher dose hydrocortisone (125 mg/kg). Mice exposed to physiologically comparable levels of E. hirta (0.01 ml of 62.5 mg/kg plant material).

Sensitization (on day 0 and day 5) of mice was via intraperitoneal injection of 25 mg OVA (grade V; SigmaAldrich) and 2 mg Al(OH)3 that was dissolved in 0.5 ml of 0.9% saline solution. All mice except the control mice (which were left untreated), were sensitized. Nebulization with 1% OVA in phosphate-buffered saline (PBS) (1 mg OVA in 100 ml PBS) was performed twice daily, for 1 h on days 13–15. E. hirta as well as hydrocortisone (low and high dose) were administrated on days 15–18, again on day 22, 25, 29 and 32. Animals were again nebulized on days 34–36 and treated daily with E. hirta from day 39 to day 45 when the animals were terminated.

White blood cell count to determine asthmatic status of mice
Blood samples of mice from each group were collected on the day the mice were terminated and histological blood smears prepared. Under a 100 Â magnification 5 fields in each blood smear were evaluated and all leucocytes counted.

Results
Platelet and fibrin structure morphology of each of the mice in each group were investigated. Morphology was found to be constant for a particular group and morphology was homogenous for each sample and for

Preparation of fibrin clots
On day 45 during termination, 100–500 ml of blood was drawn from each of the Balb/c mice in each of the

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each test field. SEM stubs were analyzed systematically to cover the whole fibrin network area for each animal. Figs. 1(a) and (b) show fibrin networks and platelet aggregates from control mice. Both thick, major fibers (label A) and a thin network of minor fibers (label B) are present in the controls. Platelet aggregates form round, dense groupings with pseudopodia extending from the aggregates. Figs. 2(a) and (b) are fibrin and platelet aggregates from asthmatic mice that were not treated with plant material or hydrocortisone. Fibrin fibers in the asthmatic mice also consist of thick, major fibers and thin minor fiber networks; however, major fibers seem to have a matted appearance (Fig. 2(a) label A) and seem to be fused longitudinally. Minor fiber networks are more prominent (Fig. 2(a) label B) and cover the major fiber network. Fig. 2(b) shows an aggregation of platelets. However, they differ from the control aggregates in that they look more granular and do not clump together closely. Furthermore, although pseudopodia are visible (Fig. 2(b) label A), they are much smaller and less bulbous than pseudopodia of the control. Figs. 3(a) and (b) show fibrin and platelet aggregates of the mice treated with the lower hydrocortisone concentration. Fibrin fibers appeared flimsy and breakages are present and can be seen (Fig. 3(a) label B). When viewing the networks using the SEM, the fibers tend to break just with the electron bundle moving over a region. This suggests that these fibers are much more prone to breakages than those of the controls or even those found in the asthmatic mice. Platelet aggregates where also granular (similar to the untreated asthmatic mice) and the aggregates did not form the tight round, dense aggregates that were seen in the controls. Rather, a loosely associated aggregate with small pseudopodia is seen (Fig. 3(b) label B). The higher hydrocortisone dose (Figs. 4(a) and (b)) also showed a flimsy fiber network easily prone to breakages and both major and minor fibers are seen. These fiber networks have a similar appearance as the untreated asthmatic mice, where the fine minor fibers form a much thicker covering than is found in the controls. Platelet aggregates also did not have a tight round appearance and appeared granular and not attached closely to each other as seen in the controls (Fig. 4(b)). Figs. 5(a) and (b) shows a fibrin network and platelet aggregate from mice that were treated with E. hirta. Both major and minor fibers are present (Fig. 5(a) labels A and B); however, fibers were much more stable and did not break as easily as the hydrocortisone groups and the minor fibers are much less prominent than those of the asthmatic mice group. Fig. 5(b) shows a platelet aggregate. Aggregates from E. hirta look similar to that of the controls. The aggregate is much more condensed and round while the pseudopodia are more bulbous and also more similar to that of the controls.

Fig. 1. (a) Control fibrin network with thick, major fibers as well as thin, minor fibers. Label A ¼ thick, major fibers; Label B ¼ thin, minor fibers. (b) Control platelet aggregate forming dense, round aggregate with pseudopodia.

Blood smear results
The results of the mean values obtained from the different fields are represented in Table 1. Fig. 6 shows the microscope fields counted.

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Fig. 2. (a) Fibrin network from asthmatic Balb/c mice showing thick, major fibers as well as thin, minor fibers. Label A ¼ thick, matted, major fibers; Label B ¼ thin, minor fiber forming a dense network. (b) Platelet aggregate from asthmatic Balb/c mice forming course, granular aggregate.

Fig. 3. (a) Fibrin network from low dose hydrocortisonetreated (100 mg/kg) asthmatic Balb/c mice, forming flimsy fibrin network. Label A ¼ flimsy fibers with breakages. (b) Platelet aggregate from low dose hydrocortisone-treated (100 mg/kg) asthmatic Balb/c mice. Label A ¼ granular platelet aggregate.

From these results it can clearly be seen that the mice that were in the positive control group (made asthmatic and not treated with any product) were indeed asthmatic. Both doses of hydrocortisone lowered the white blood counts in the mice, while the plant extract at both doses also lowered the white blood cell counts. Specifically the neutrophils, eosinophils and basophils were lowered, and these cells are known to be active in inflammation. Fig. 7 shows a column graph illustrating the results from the blood smears.

Discussion
Results from the white blood cell counts of the mice, indicated that asthma was indeed induced in this study. Furthermore, E. hirta reduced the neutrophils, eosino-

phils and basophils counts, and these cells are known to be active in inflammation. Although platelets have traditionally been associated with disorders of the cardiovascular system, they also play an important physiological role in allergic processes and immunological mechanisms. Platelets play an important and fundamental part in asthma, as they are activated by inflammatory processes, typically involved in asthma. Furthermore, platelet-activating factor (PAF) as well as platelet factor 4 (PF4) and also thrombin itself, fibrinogen, fibrin, are all known to be involved in asthma. Importantly, platelets contribute to the adhesion of eosinophils to inflamed endothelium of patients with allergic asthma. Platelet depletion is also known to reduce PAF, suggesting that PAF plays a central role in the processes by which platelets facilitate

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Fig. 4. (a) Fibrin network from high dose hydrocortisonetreated (125 mg/kg) asthmatic Balb/c mice network. Label A ¼ Flimsy fiber network. (b) Platelet aggregate from high dose hydrocortisone-treated (125 mg/kg) asthmatic Balb/c mice forming granular aggregate.

Fig. 5. (a) Fibrin network from asthmatic Balb/c mice treated with E. hirta (0.01 ml of 62.5 mg/kg plant material) showing fibrin network with thick, major fibers as well as thin, minor fibers. Label A ¼ thick, major fibers; Label B ¼ thin, minor fibers. (b) Platelet aggregate from asthmatic Balb/c mice treated with E. hirta (0.01 ml of 62.5 mg/kg plant material), round aggregate with pseudopodia. Label A ¼ pseudopodia. Table 1. Summary of the mean values obtained from white blood cell counts for every exposure group Control Asthma Low Low E. High High HC hirta HC E.hirta Neutrophil Eosinophil Basophil Lymphocyte Monocyte 39 13 7 144 203 336 104 58 1203 737 36 10 3 41 32 24 8 3 70 89 91 3 2 81 63 31 4 1 82 113

the induction of eosinophil accumulation, which is central in asthma. Also, PAF is involved in increased vascular permeability and platelets have been shown to be in contact with the vasculature of the bronchi of patients with asthma and in this way, platelets evoke contraction of smooth muscles of the respiratory passages that directly leads to an asthma attack. Fibrin and platelets are therefore two important factors in the disease. Fibrinogen itself is also widely recognized as a marker for systemic inflammation as it is considered an acute phase protein. Pitchford et al. (2004) mentioned that there is evidence of platelet recruitment to the lungs of asthmatics after allergen exposure, suggesting that platelets participate in various aspects of asthma.

Morley et al. (1984) has earlier suggested that platelet activation may contribute to airway remodeling in asthma.

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Furthermore, the association between thrombin, fibrin and asthma seems to be the following:

 

 

   

Thrombin is known to increase airway smooth muscle contraction ex vivo (Panettieri et al., 1995). Increased thrombin generation occurs in the airway of patients with asthma (Gabazza et al., 1999). Thrombin may play a role in the pathogenesis of airway remodeling. Human platelets can produce PAF upon thrombin stimulation in the lungs (Touqui et al., 1985). Fibrinogen can be produced by lung epithelia as a result of inflammatory stimulus (Lawrence and Simpson-Haidaris, 2004). Fibrin degradation products have also been found to increase pulmonary vascular smooth muscle contraction (Kern et al., 1986). Airway fibrin deposition occurs in inflammatory disorders of the lung, and it is known that fibrin inhibits surfactant function (Wagers et al., 2003).



 

Fibrin is typically formed at sites of vascular damage (Touqui et al., 1985). Extra-vascular thrombin, fibrinogen, and fibrin have been found in the sputum of patients with asthma (Pizzichini et al., 1996; Banach-Wawrzenczyk et al., 2000; Wagers et al., 2004). Wagner and co-workers in 2004 hypothesized that airway hyperresponsiveness seen in asthma is largely the result of decreased stability of airways and subsequent airway closure secondary to the formation of fibrin on the distal airway surface. According to the authors the coagulation system and fibrinolytic system proteins is associated with the pathogenesis of airway hyperresponsiveness in asthma. There is decrease in plasminogen activator (PA) activity in asthma (Wagers et al., 2004). Activity of PAIact (PA inhibitor) is increased in homogenates of lung tissue of mice with allergic airway inflammation, thereby potentially promoting the accumulation of fibrin by suppressing fibrinolysis (Wagers et al., 2004).

Fig. 6. Illustrating the five different areas of microscopic fields evaluated on the blood smear.

In control Balb/c mice we find major and minor fibrin networks (Fig. 1(a)); this is similar to previous findings in humans (Pretorius et al., 2006). Also, the morphology of platelet aggregates appears as a collection of platelets that has a dense, round shape. This is also similar to human platelet aggregates (Pretorius et al., 2006). However, in the asthmatic mice the minor fibers seem to be more dense and covering the major fibers (as seen in Fig. 2(a)). This might be the reason why in asthma, an accumulation of fibrin is seen in airways (Wagers et al., 2004); perhaps because the fibrin mass is more dense and

1400

1200

Amount of Leucocytes

1000

800
Neutrophil Eosinophil Basophil Lymphocyte Monocyte

600

400

200

0 Control Asthma Low HC Low E.Hirta High HC High E.Hirta Treatment Group

Fig. 7. Column graph illustrating the results from the blood smears.

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that the fibrin networks are not disaggregated so quickly (fibrinolysis) and stay in the airways for longer periods, forming fibrin plugs. Also, surfactant function is inhibited in asthma (Wagers et al., 2003), possibly because of the denser fibrin network. Also, degradation products of fibrin have been found to increase smooth muscle contraction. This might be because the fine minor fibrin network inhibits the surfactant to function optimally and interferes with smooth muscle function. It is well-known that glucocorticosteroids are the most useful class of drugs employed in the treatment of patients with allergic asthma (Lantero et al., 1997; Pretorius, 2005). There is also increasing evidence that fibrin(ogen) physiology is affected by glucocorticoids. After fibrin is produced via the thrombin (intrinsic and extrinsic) pathway, it undergoes fibrinolysis. This process is under the control of PAs, which are serine proteases that convert the proenzyme plasminogen to active plasmin, a broad-spectrum proteolytic enzyme that readily degrades fibrin as well as extracellular matrix glycoproteins including laminin, vitronectin, fibronectin, and proteoglycans (Bator et al., 1998). Platelets also seem to be affected by glucocorticoid treatment (Tutluoglu et al., 2005). Platelets have the capacity to release mediators with potent inflammatory or anaphylactic properties; these mediators include PF4 and beta-thromboglobulin (BTG) (Tutluoglu et al., 2005). BTGs are also chemokines that play an important role in mediating cell recruitment and activation necessary for inflammation and the repair of tissue damage. Plasma levels of PF4 and BTG also show changes in chronic inflammatory diseases such as asthma; and Tutluoglu et al. (2005) found that plasma levels of PF4 among patients with an asthma attack were significantly higher than those of controls and a further increase in plasma PF4 levels was detected after steroid therapy. From our results it seems as if both low and high hydrocortisone dosages produce flimsy fibrin networks that break easily and that are not as stable as was found in the control fibrin networks (Figs. 3(a) and 4(a)). Also the platelet aggregates do not have the typical round, compact shape; rather the platelet aggregates are widely spread, granular, and not tightly associated with each other (Figs. 3(b) and 4(b)); also aggregates appear more like those found in the asthmatic mouse. An interesting observation was that the extract of E. hirta, did not cause the fibrin networks to be as flimsy and fragile (Fig. 5(a)). However, the fine minor fibers seem to be more prominent than in the controls and more similar to those found in the asthmatic mice. Platelet aggregates showed the same morphology as those of the control mice, without the widely spread granular appearance found in hydrocortisone-treated mice (Figs. 3(b) and 4(b)). However, further studies need

to be done to determine the effect of the plant on lymphocytic lung infiltrates, Ag-specific production of IL-4 and IL-5 from spleen and lung cells in vitro, elevated levels of IgG1 as well as expression of Th2 cytokine RNA in lungs.

Conclusion
Asthma is a very complex condition, with many physiological factors playing a role in its presentation. However, it seems as if platelets and products of the coagulation cascade form an intricate and important part of asthma and not only affect the presentation of the condition itself but also interact with the typical choice of treatment, namely glucocorticoid therapy. The question that arises is how the platelet activation process and the coagulation cascade contribute to asthma in the presence of other pharmaceutical products or phytomedicine. From previous research it is know that the phytomedicine E. hirta possess products that are antiinflammatory and it is widely used in the treatment of asthma. However, very little information is available of the effect of the plant on cellular systems of the body and how exactly it plays a role in the treatment of asthma. From the current research it seems as if E. hirta does not make the fibrin fibers as fragile as is found with the hydrocortisone-treated mice and it also does not change the integrity and morphology of the platelets as was found in hydrocortisone-treated mice. However, this research only touched on a small part of the process and probably now leaves more questions regarding the effect of hydrocortisone on the coagulation process as well as the exact mechanisms and effects of E. hirta on asthma in general and on other cellular systems of the body. It is also suggested that further ultrastructural studies, e.g., transmission electron microscopy might give additional information regarding the morphology of the platelets in particular. We conclude that E. hirta does not impact on the fragility of the fibrin and that it prevents the minor fibers to form the dense netlike layer over the major fibers, as is seen in untreated asthmatic mice. This ultrastructural morphology might give us better insight into asthma and the possible new treatment regimes.

Acknowledgments
We thank the National Research Foundation of South Africa (NRF) for funding E. Pretorius (Indigenous Knowledge Systems (FA2004033100004)). We would like to acknowledge the South African National Blood Service for providing the Thrombostim preparation which included the LPRP and the human thrombin.

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