Plasma Level of Tissue Factor Pathway Inhibitor in Children With Idiopathic Recurrent Epistaxis (Nosebleeds)
Eitedal M. Daoud,
Reda M. Daoud ,
Lobna M. Saber
Maha M. Saber
Previously TFPI has never been studied in childhood nosebleeds. In this study, we determined the free plasma level of TFPI in children with recurrent attacks of idiopathic epistaxis (nosebleeds), during active attacks of nasal bleeding and 24-48 h after treatments (remission state). We investigated 50 children. They were divided into 2 groups. Group 1, 20 children during active attack of nosebleeds. Group 2, 30 children during remission state. All patients enrolled in this study were selected from ENT-Out Patient Clinics Al-Azhar University Hospitals Cairo, Egypt (El-Hossain Hospital). Results were compared to 30 healthy controls age and sex matched. Free plasma TFPI was measured by Enzyme-Linked Immunosorbent Assay (ELISA) in all patients and healthy control child. There was high significant increase of free plasma TFPI levels in children during acute nosebleeds and in remission state (group 1 and group 2) in comparison to healthy control children (group 3) (p< 0.0001, p< 0.0001), respectively. The highest level of free plasma TFPI was recorded in group 1, followed by children in group 2. In the same time, TFPI level in group 1 of children was highly significantly increased as compared to the level in children in group 2 (p< 0.001) As regard gender, there were highly significant increased in free plasma levels of TFPI in both groups (group 1 and 2) as compared to the same genders of the control children (group 3) (p< 0.001 and p< 0.001), respectively. Comparing hemoglobin levels, there was significant decrease in its level in children in group 1 and II when compared to children in group 3 (control) (p< 0.01 and p< 0.01). From the present study we concluded that, free plasma level of TFPI is increased in children with idiopathic recurrent nosebleeds, comparable to healthy control children matched for age and sex. The highest level of free TFPI was recorded in children with active attacks of nosebleeds. This high level of TFPI may reflects the degree of endothelial cells dysfunction in this group of children and the state of vascular wall cells in microvasculature of their Little`s areas. Further studies are recommended to clarify more the role of the increased free TFPI in pediatric cases with recurrent idiopathic epistaxis and its effect on endothelial cells function as well as vascular wall cells integrity in microvascular circulation.
Epistaxis is defined as acute bleeding from the nostril nasal cavity or nasopharynx. It is well know that, in about 90% of all pediatric epistaxis, no underlying systemic cause, but recurrent episodes are a source of significant distress and anxiety in children, their carers and clinicians. Such episodes of nosebleeds are common cause of morbidity and hospital referral (Stankiewicz, 2004).
Recurrent idiopathic epistaxis in children is repeated, self-limiting nasal bleeding up to the age of 16 years for which no specific cause has been identified. Although it is very common in children, it is rare before the age of two years (Burton and Doree, 2005). In the majority of these children, the bleeding is from the anterior septum and is usually attributed to crusting, vestibulitis and digital trauma (nose picking) (Kubba et al., 2001).
Anterior bleeds are responsible for about 80% of epistaxis however; most (80-90%) are actually idiopathic. They occur at an anastomosis called Kiesselbachs plexus on the lower part of the anterior septum known as Littles area (Pope and Hobbs, 2005). In most cases the bleeding stops spontaneously, but severe or recurrent cases may need hospital treatment (Vaiman et al., 2002). The most common interventions for these cases are cautery of visible vessels and the application of an antiseptic nasal cream to reduce vestibulitis and crusting (Burton and Doree, 2005).
TFPI is a multivalent kunitz-type protease inhibitor of 276 amino acids and approximately 42 kDa that has three domains (Bajaj et al., 2001). It is synthesized mainly by the vascular endothelium (80-85%) and has also been detected in at least four intravascular pools, e.g., bound to the endothelial surface, associated with lipoproteins (10%), carrier-free within the plasma and sequestered in platelets (3%) (Hackeng et al., 2006). TFPI interact with blood coagulation factor VIIa/tissue factor complex and activated factor X via its kunitz-type domains 1 and 2, whereas the K3 domain appears to lack protease inhibitory activity (Piro and Broze, 2004). Furthermore, it plays an important role in inhibiting Tissue Factor (TF)-induced coagulation by factor Xa-dependent pathway of the activated tissue-factor VIIa complex, preventing formation of microvascular thrombosis (Hackeng et al., 2006). A major pool of TFPI is the form associated with the surface of endothelial cells, which is speculated to play an important role in regulating the function of vascular wall cells and modulation of cell proliferation (Kato, 2002). It is cleared from circulation primarily by the liver and the kidney (Van Dreden et al., 2001).
The aim of the present study was to evaluate the free plasma levels of TFPI in childhood idiopathic recurrent nosebleeds (epistaxis), during active bleeding and in remission state (within 24-48 h after treatment), as it may be used as a marker for endothelial cells dysfunction in these cases or to reflect the state of functioning vascular wall cells in microvasculature circulations.
MATERIALS AND METHODS
The present study included three groups of children. All were enrolled from E.N.T Out-Patient Clinics in Al-Azhar University Hospitals Cairo. Egypt (El-Hossin Hospital) from May to Aug, 2005.
Group 1: Comprised 20 children during active attacks of nosebleeds, they were 9 males and 11 females. Their age range was (4-12) years with a mean (6.9±2.45) years.
Group 2: Comprised 30 children in remission state (within 24-48 h) they were 16 males and 14 females. Their age range (3-11) years and a mean (6.37±2.4) years. They were received medication in the form of vitamin C, cautery and an application of nasal antiseptic cream.
Group 3: Comprised 30 healthy control children. They were 16 males and 14 females. Their age range (3-8) years with a mean (5.5±1.53) years.
Exclusion criteria: Patients older than 16 years, children with any bleeding disorders (coagulopathy) were excluded. Patients with renal insufficiency, liver disease or malignancy, subject on heparin therapy or patients without parental consent, were not enrolled in the present study. Patients with thromboembolic disorders and stroke were also excluded. All patients and controls were subjected to the following:
||Full clinical history, age of onset of epistaxis, its frequency,
amount, duration, unilateral or bilateral. Family history of mucocutaneous
bleeding, history of anticoagulant drug intake. For assessment severity
of bleeding, we used epistaxis scoring system developed by Katsanis et
al. (1988). Scores of (0-6) were classified as mild, scores of (7-10)
||Thorough pediatrics examination and, E.N.T evaluation using
head-light and anterior rhinoscopy to evaluate the nasal cavity in children
with nosebleeds. As well as, to exclude local causes of epistaxis.
||Three milliliters of venous blood were collected in pyrogen
free-tubes (contain Na citrate) citrated blood with 1:9 ratio (1 Na citrate-9
blood) centrifuged at 3000 rpm for 10 min. Plasma was immediately separated
and kept at -20°C until assayed to measure free plasma TFPI.
||Blood samples: Venous blood, for complete blood picture and
total platelets count.
||Bleeding time, prothrombine time and activated partial thromboplastine
time were done to all patients and controls.
||Complete blood picture using Automated coulter counter T-660
||Free plasma TFPI was measured by immunosorbent assay (ELISA)
IMUSBIND for assay of total TFPI using a sandwitch ELISA employing a rabbit anti-human TFPI native polyclonal antibody as the capture antibody. Specificity of the capture antibody for native, complexed and truncated TFPI was conformed by Westem blot. Analysis visualizing a single band at 34 KD, corresponding to the mobility of intact native TFPI and visualizing a single band at 21 KD, corresponding to the mobility of a truncated form of TFPI. Diluted plasma samples incubate in microtest wells pre-coated with this capture antibody. TFPI is detected using a biotinytated monoclonal antibody specific for the kunitz-domain 1 of TFPI the subsequent binding of the streptavidine conjugated Hoarse Redish Peroxidase (HRP) complete the formation of the antibody enzyme detection complex. The addition of TMB substrate and its subsequent reaction with HRP provides a blue colour. Sensitivity is increased by addition 0.5 M sulphoric acid stop-solution yielding a yellow colour. TFPI levels are determined by measuring sample solution absorbance at 450 nm and comparison against those of a standard curve developed using native TFPI.
Statistical analysis: Statistical analysis was done using Statistical Package for Social Sciences (SPSS) software version 12 (Chicago, USA). Student t-test was applied for comparison of two groups of quantitative data. ANOVA-test used for comparison of more than two parameters. In addition, Spearman's correlation coefficient (r-value) was used to evaluate the correlation between TFPI and others clinical and laboratory parameters. p-value <0.05 considered significant.
Children with active nosebleeds (group 1) and children in group 2 (in remission)
had statistically significant decrease in their haemoglobin levels as compared
to control group (group 3) (p<0.01).
||Clinical and Laboratory data of all patients and controls
|*: Significant p<0.01 as compared to control group
||Free plasma levels of TFPI in the three groups of children
enrolled in the study with their Genders (Mean, Range±SD)
|P1 is p value when group 1 and II and group 3 were compared
by ANOVA test in all patients variables; P2 is p-value when group 1 and
II were compared to group 3; P3 is p-value when group 1 was compared to
As regard to the epistaxis scores in group 1 and 2 no statistically significant
difference between them (Table 1 as % severe) because most
of patients recording mild score of epistaxis.
In Table 2 the free plasma levels of Tissue Factor Pathway Inhibitor (TFPI) in all studied groups. In group 1 and 2, there were an increase in their plasma levels of free TFPI, which was statistically highly significant as compared to the control group (p<0.0001, p<0.001). Also, male and female patients in the same groups (group 1 and 2) had an increase level of plasma free TFPI, which was statistically highly significant as compared to males and female patients in control group (p<0.0001, p<0.001), respectively. As regard gender in group 1 and 2, there was no statistically significant difference between them. Furthermore, there was statistically significant difference between free TFPI in plasma of children in group 1 (active nosebleeds) when compared to children in group 2 (in remission) (p<0.001) Table 2. The highest level of TFPI was recorded in group 1 and its gender as compared to children in both groups (group 2 and 3) Table 2. No correlation was found between neither age nor sex in all the enrolled cases or controls in the present study (data not shown).
Epistaxis (nosebleeds) is a common childhood symptom that may prompt a referral to a pediatric hematologist or otorhinolaryngologist (Claudio Sandoval et al., 2002) and is usually minor and self-limiting (Burton and Doree, 2005). Nosebleeds affect 30% of children aged 0-5 years, 56% of those aged 6-10 years and 64% of those aged 11-15 years. There is very little in the published literature on recurrent idiopathic epistaxis in childhood and the spontaneous resolution rate is unknown (Kubba et al., 2001).
In the present study, there was significant decrease in hemoglobin level in children with nosebleeds (group 1) and those in remission (group 2) as compared to normal healthy control children (group 3). Commonly anemia may be one of presenting clinical and/or laboratory investigations in children with recurrent epistaxis due to repeated bleeding or severity of bleeding. This agrees with present study and in the work done by Callejo et al. (1998), who reported that; anemia is one of the clinical and laboratory manifestation of recurrent attacks of nosebleeds in children. Also, Dai and Jurges (2005) illustrate an atypical presentation of epistaxis with anemia and hypovolaemic shock in a girl 10 months old. Controversially, Claudio et al. (2002), reported, no change in hemoglobin level in children attending ENT department with recurrent idiopathic nosebleeds without coagulopathy diseases.
TFPI is expressed by different cell types, such as platelets, monocytes, macrophages
and smooth muscle cells, but endothelial cells are thought to represent the
principal source of this protein (Morange et al., 2004). TFPI is a 42-kDa
protein that consists of three closely linked kunitz domains (Hack, 2000). These
domains allow TFPI to function by a unique mechanism. The dynamics of TFPI in
the microvasculature are rather complex (Opal and Esmon, 2003) its releases
inhibits and prevents microvescular thrombus formation (Fuster et al.,
2005) and administration of exogenous recombinant TFPI has been claimed to protect
against intravascular coagulation and venous thrombosis in animals (Huynh et
al., 2001). In the present study, free plasma TFPI was significantly elevated
in children with idiopathic recurrent epistaxis during acute attacks of bleeding
and in between attacks (within 24-48 h) when compared to control healthy children.
The highest levels of free form of TFPI were recorded in the children with active
attacks of nosebleeds (group 1). Present suggestion is that, this high level
of free TFPI may be due to disturbed blood vessels function and integrity in
this Littles area, which reflects the endothelial cells state mainly found
in the microcirculation as the main source of this free TFPI is the endothelial
cells lining the blood vessels. While the decreased level of it in remission
state can be attributed to partially improving in the function of vascular wall
cells with vitamin C treatment and/or degradation of this protein in liver and
Previously TFPI has never been studied in childhood nosebleeds. But many studies have been recorded its level in some diseases, as a risk factor for an occurrence or as a marker of endothelial cell dysfunction. Abdel Gader et al. (2005) has reported. A significant elevation of TFPI in children with nephrotic syndrome comparable to healthy control children.
Furthermore, increased levels of TFPI were reported also, in diabetic patients (Leurs et al., 1995) and in patients suffering from cancer or ischemic heart disease (Fuster et al., 2005; Iversen et al., 1998). Meanwhile, decreased level of TFPI value have been found in patients with venous thrombosis or ischemic stroke as well as in coronary circulation of patients with acute coronary syndromes (Dahm et al., 2003; Golino et al., 2003).
In present study, no correlation was found between age or sex of children and their free plasma level of TFPI (not shown). Controversially, in the study done by Duering et al. (2004) they; found a negative correlation between level of TFPI and age of the patients. This can be due to small sample of cases enrolled in our study and narrow age range of cases and controls.
Normally, TFPI has very low plasma concentration; about 20% of TFPI circulate in plasma associated with lipoprotein. The majority remains associated with the endothelial surface bound to the cell-surface glycolsaminolgycans (Bashir and Bass, 2006). The free portion of TFPI presents high anticoagulant activity and its plasma level strongly correlates with endothelial cell markers (Morange et al., 2004; Pinotti et al., 2005; Blann et al., 2000). So, in the present study idiopathic recurrent nosebleeds can be explained by or attributed to the high levels of plasma free TFPI, assayed in the group of children investigated during acute attacks of nosebleeds. TFPI could be a cause of idiopathic nosebleeds partially due to its high anticoagulant activity of its free portion, as well as due to capillary endothelial dysfunction in microvasculature of Little's area.
From this study, we concluded that free plasma level of TFPI is increased in
children with idiopathic recurrent nosebleeds, comparable to healthy control
children matched for age and sex. The highest levels of free TFPI were recorded
in children with active attacks of nosebleeds. Its high level may reflects the
degree of endothelial cells dysfunction in this group of children and the state
of vascular wall cells in microvasculature of their Little`s areas. Further
studies and investigations are recommended to clarify more the role of TFPI
in pediatric cases with recurrent idiopathic epistaxis and its effect on endothelial
cells function as well as vascular wall cells integrity in microvascular circulation.
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