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\begin{document}

\title{Impaired In-vitro Platelet Aggregation and Secretion Tests in Children
with Henoch-sch\selectlanguage{ngerman}öenlein Purpura\selectlanguage{english}}



\author[1]{Esra Yazarlı}%
\author[2]{Pınar  Işık Ağras}%
\author[3]{Yildiz Dallar}%
\author[3]{Bulent Alioglu}%
\affil[1]{Mustafa Kemal Universitesi Tayfur Ata Sokmen Tip Fakultesi}%
\affil[2]{Ankara Güven Hospital}%
\affil[3]{Ankara Training and Research Hospital}%


\vspace{-1em}



  
  \date{May 19, 2024}


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\selectlanguage{english}
\begin{abstract}
Abstract Aim: This study aimed to investigate the in-vitro platelet
aggregation and secretion tests in children patients with
Henoch-sch\selectlanguage{ngerman}öenlein Purpura that recently referred to as Ig A vasculitis
Methods: This is a cross-section study that included 55 patients with
Henoch-schöenlein Purpura and 31 healthy children as a control group.
Children who have a history of drug use, chronic diseases, and bleeding
diseases were excluded from the study. Complete blood count, thrombocyte
aggregation, and secretion tests were studied in both groups. These
tests were re-evaluated in remission of the disease. Results: It was
found that epinephrine-stimulated platelet aggregation and collagen,
epinephrine, ristocetin, arachidonic acid, standard dose thrombin, and
high dose thrombin-stimulated platelet secretion results were lower in
the patients with Henoch-schöenlein Purpura compared to the healthy
control group in the acute period (respectively P=0.014, 0,003; 0,003;
0,027; 0,034; 0,010; 0,049). When the values of patients with
Henoch-schöenlein Purpura in the acute period and the remission of the
disease were compared, collagen-stimulated platelet aggregation and
epinephrine-stimulated platelet secretion values were found to be lower
in patients with patients in the acute period (P= 0.016; 0.039)
Conclusion: Impairment in vitro platelet aggregation and secretion tests
in the patients with Henoch-schöenlein Purpura suggest that the tendency
to bleeding in these patients may be due to platelet impairment
function. Key Words: Henoch-schöenlein Purpura, platelet aggregation
tests, platelet secretion tests, children, Ig A vasculitis. What's
already known about this topic? There is a tendency to bleeding in
Henoch Shcöenlein Purpura patients, such as gastrointestinal bleeding,
nonthrombocytopenic system purpura. What does this article add? It was
found that impairment in-vitro platelet aggregation and secretion tests
in Henoch Shcöenlein Purpura patients.%
\end{abstract}\selectlanguage{ngerman}%



\sloppy


\textbf{Introduction}

Henoch-sch\selectlanguage{ngerman}öenlein Purpura (HSP), recently more commonly referred to as
Ig A vasculitis, is the most common leukocytoclastic vasculitis of small
vessels in childhood ({[}1{]}. It affects the especially skin and
gastrointestinal system, joints, kidneys, rarely other organs.
Nonthrombocytopenic purpura, gastrointestinal system bleeding, abdominal
pain, arthritis, nephritis, and testicular involvement may occur during
the disease {[}2{]}. Most cases occur after upper respiratory tract
infection. Drugs and vaccines also have been reported to be triggered
{[}3-5{]}.

Although Ig A accumulation has been demonstrated in the affected organs'
small vessels, the etiopathogenesis is still elucidated {[}6, 7{]}. It
is thought that triggering factors cause endothelial dysfunction by
stimulating the vascular endothelium. This situation plays an important
role in pathogenesis {[}8, 9{]}. Platelets have multiple functions,
including hemostasis, inflammation, host defenses, vascular tone. The
endothelium has an important role in the regulation of hemostasis. When
vascular endothelium is damaged, vasocontraction occurs in vascular
smooth muscle, and platelets come together to form the platelets plug
{[}10{]}. Purpuric rashes, hematuria, hematemesis, and melena in
patients with HSP suggest that coagulation disorders may occur {[}11{]}.
The fact that platelet count, bleeding time, clotting time, prothrombin
time, and activated partial thromboplastin time were normal in patients
suggest that it is due to vascular cause rather than coagulation
disorders {[}12{]}. However, in some studies, it has been reported that
platelet functions are impaired in patients with HSP {[}13, 14{]}. This
study aimed to evaluate in vitro platelet aggregation and secretion
tests in patients with HSP, an essential vasculitis of childhood.

\textbf{Methods}

The study included 55 patients with HSP and 31 healthy children as a
control group. EULAR/PRES classification criteria were used for the
diagnosis of HSP {[}15{]}. The control group with no health problems
that were admitted for routine check-ups enrolled at pediatric
outpatient clinics. Children with known chronic diseases and bleeding
diseases were excluded from the study. Patients with a history of drug
use were not included in the study.

The study was approved by the local ethics committee. Informed parental
consent was obtained for all children.

Complete blood count, thrombocyte aggregation, and thrombocyte secretion
tests were studied in the acute phase of the disease. 24 patients with
HSP did not continue the study for various reasons. Thrombocyte function
tests of 21 continuing to study patients with HSP were re-evaluated in
remission of the disease.

For complete blood count tests, two cc of blood was taken from the
antecubital vein for each patient and healthy control groups into one
standard tube containing 7.5\% K3- ethylenediaminetetraacetic acid
solution was used. White blood cell, absolute neutrophil count, absolute
lymphocyte counts, absolute monocyte count, platelet count, mean
platelet volüme, absolute neutrophil count, absolute lymphocyte, and
absolute monocyte counts hemoglobin were measured. For platelet
aggregation and secretion tests, four cc of blood was taken from the
antecubital vein for each patient and healthy control groups into two
standard tubes containing 0.5 ml (1 volume) of 0.109 M trisodium citrate
solution. Platelet aggregation tests were carried out with the American
origin Chronolog Corporation (model 700) brand device using the optical
aggregometer method. Platelet secretion tests were also studied with the
American Chronolog Corporation (model 700) device using the
lumiaggregometry method. Collagen (2 \selectlanguage{greek}μ\selectlanguage{english}g/mL), epinephrine (5 \selectlanguage{greek}μ\selectlanguage{english}M),
standard dose of ADP (5 \selectlanguage{greek}μ\selectlanguage{english}M), high dose of ADP (10 \selectlanguage{greek}μ\selectlanguage{english}M), standard dose of
thrombin {[}1 unit (30 \selectlanguage{greek}μ\selectlanguage{english}L){]}, high dose of thrombin {[}4 units (30
\selectlanguage{greek}μ\selectlanguage{english}L){]}, ristocetin (1.25 mg/mL), and arachidonic acid (0.5 mM) were used
as agonists to test for platelet aggregation and secretion

Data analysis was performed using SPSS for Windows 11.5 package program.
Shapiro Wilk test was used to determine whether the distribution of
continuous variables was normal. Descriptive statistics were shown as
mean ~\selectlanguage{ngerman}± standard deviation for continuous variables and nominal
variables as the number of cases (percentages \%). Continuous variables
with normal distribution between the groups were investigated by the
student's t-test, and continuous variables were not normally distributed
with the Mann-Whitney U test. Wilcoxon test was used to determine a
statistically significant change between the values measured in the
acute period and the remission of the disease.

Results

Mean age of the patient and healthy control group (patient group 8.3±3.2
years and healthy control group 7.7± 3.7 years; p=0.265) and gender
distribution (patient group 31 girls (56.4\%) and 25 boys (43.6\%);
healthy control group 15 girls (48.4\%) and 16 boys (51.6\%); p=0.476)
were statistically similar.

When the clinical features of the patient group were evaluated; in 55
(100\%) patients skin involvement, in 13 (25\%) patients arthritis, in
10 (19.2\%) patients gastrointestinal involvement, in 2(3.8\%) patients
renal involvement and in 2 (3.8\%) patients genitourinary system
involvement were detected. Steroid treatment was administered to 14
(25.4\%) patients, including gastrointestinal involvement 10, severe
skin involvement 2, renal involvement 2.

In the acute period, the white blood cell count, absolute neutrophil
count, absolute lymphocyte, and absolute monocyte counts were found to
be higher in patients with HSP compared to the control group (
respectively \emph{P} \textless{}0,001; 0,001; \emph{P} = 0,010 ve
0,044) (Table 1). When the platelet aggregation tests were examined,
epinephrine-stimulated platelet aggregation test results were lower in
patients with HSP (P=0.014) (table 2). However, there were no
differences between the two groups in terms of collagen, standard dose
ADP, arachidonic acid, standard dose thrombin, high dose thrombin,
ristocetin cofactor, high dose ADP, and ristocetin-stimulated platelet
aggregation results (table2). When compared in term of platelet
secretion tests, collagen, epinephrine, ristocetin, arachidonic acid,
standard dose thrombin and high dose thrombin-stimulated platelet
secretion results were found to be lower in patients with HSP (
respectively \emph{P} = 0,003; 0,003; 0,027; 0,034; 0,010; 0,049) (Table
2). However, there was no difference between the two groups in terms of
standard-dose ADP and high dose ADP-stimulated platelet secretion
results (table2).

When the values of 21 patients with HSP in the acute period and the
remission of the disease were compared, it was found that the white
blood cell count, platelet count, absolute neutrophil count, and
absolute monocyte count were higher in the acute period
(respectively\emph{P} = 0,011; 0,001; 0,011; 0,044) (Tablo3). When
platelet aggregation tests were examined, collagen-stimulated platelet
aggregation values were found to be lower in patients with HSP in the
acute period (\emph{P} = 0.016) (Tablo 4). However, no difference was
found in epinephrine, standard dose ADP, high dose ADP, standard dose
thrombin, high dose thrombin, ristocetin cofactor, and arachidonic acid-
stimulated platelet aggregation results between the values measured in
the acute period and the remission of the disease. When platelet
secretion tests were examined, epinephrine-stimulated platelet secretion
value was lower in patients with HSP in the acute period (\emph{P} =
0.039) (Tablo 4). There was no difference between the values of
arachidonic acid, standard dose thrombin, high dose thrombin, standard
dose ADP, high dose ADP, collagen, and ristocetin-stimulated platelet
secretion results in the acute period and the remission of the disease.

\subsection*{DISCUSSION}

{\label{discussion}}

Although the presence of clinical conditions such as purpuric rash,
hematuria, hematemesis, and melena without thrombocytopenia in patients
with HSP has been attributed to the vasculitis state, there are also
various publications suggesting that coagulation disorders may be
present {[}13, 14{]}.

Studies on mean platelet volüme showing platelet function and activation
have also increased in HSP patients in recent years. Xiang Shi et
al.{[}16{]} conducted a study with 97 patients with HSP and 120 healthy
children as a control group. The mean platelet volüme was significantly
lower in patients with HSP than in the control group. It was found that
the mean platelet volüme of the patients in the acute phase was also
lower than the recovery period.

In some past publications, impaired coagulation system has also been
reported patients with HSP. A study of 54 patients with HSP, prolonged
platelet aggregation, decreased plasminogen, decreased antithrombin III,
and increased fibrin monomers {[}14{]}. In another study conducted with
23 adult patients with HSP, impairment platelet aggregation with ADP was
shown in patients with HSP {[}17{]}.

ADP and epinephrine stimulated platelet aggregation tests were evaluated
in another study conducted with 11 male and 13 female patients aged 4-12
years with HSP. It was found that block in ADP release in 58.3\% of
patients, severe deterioration in aggregation in 29.2 \%, whereas ADP
--stimulated aggregation tests were reported as normal in 8.3\% of
patients {[}13{]}.

In our study, when platelet aggregation and platelet secretion tests
were examined in patients with HSP, the results of
epinephrine-stimulated platelet aggregation and collagen, epinephrine,
ristocetin, arachidonic acid, standard dose thrombin, and high dose
thrombin-stimulated platelet secretion results in children with HSP, we
found that lower than the control group. 24 of 55 patients were excluded
from to study for various reasons. Thrombocyte function tests of 21
patients with HSP who were continuing to study were rechecked to
remission of the disease. In 21 patients with HSP, platelet aggregation
results stimulated with collagen and ristocetin were found to be lower
in the acute period than the value measured in the remission of the
disease. When platelet secretion tests were examined,
epinephrine-stimulated platelet secretion results were found to be lower
than the value measured in the acute period compared to the value
measured in the remission of the disease. These results show that HSP
children have impaired in-vitro platelet aggregation and secretion in
the acute phase.

As a result of our study, impairment in-vitro platelet aggregation and
secretion tests in the patients with HSP suggest that the tendency to
bleeding in these patients may be due to the impairment of platelet
function. There are a few studies in the literatüre on platelet function
tests in patients with HSP. We believe that to elucidate better the
pathogenesis of bleeding tendency in patients with HSP, more extensive
and further studies are needed on this subject.

Limitations: 24 of the 55 patients participating were excluded from our
study. The number of cases examined in vitro platelet aggregation and
secretion tests during the remission period decreased.

\textbf{References}

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3. Aktas B TP, Kurt OK, Ensari A, Demirer T. Severe Henoch-Schönlein
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