Aggregation of erythrocytes in burn disease

Grigory Y Levin and Marpha N Egorihina

Abstract

The manuscript describes experiments designed to examine factors that influence

erythrocytes aggregation within the blood of burn patients. Results showed that the rate and
degree of erythrocytes aggregation increased significantly in burn patients, and what is
especially unfavorable for microcirculation, erythrocytes disaggregation decreased. We show
that normalization of blood plasma contents completely restores erythrocytes aggregation and
disaggregation of burn patients. The rate and degree of aggregation was also increased as the
fibrinogen concentration increased. It is found that fibrinogen-induced aggregation of
erythrocytes is accompanied by “saturation” effect. The aggregation was not affected by
monoclonal antibodies against platelet GPIIb/IIIa receptors. The level of oxidized fibrinogen
in blood plasma of burn patients increased by about two fold. However, correlation between
the level of oxidized fibrinogen and erythrocytes aggregation was not found. The level of
medium molecular peptides increased sharply in blood plasma from burn patients. This
increase was accompanied by a dose-dependent increase in erythrocytes aggregation. Based
on these results the authors conclude that in burn patients erythrocytes aggregation is affected
by changes in the contents of blood plasma, specifically fibrinogen and the product of its
transformation - fibrin fibrin monomer.

Keywords: thermal trauma, erythrocyte aggregation, fibrinogen, fibrin monomer

Introduction

Microcirculatory disorders always occur after thermal trauma and determine the development
of such complications as acute renal insufficiency, respiratory distress, multiple organ
dysfunction syndrome [1-4]. Reduction of oxygen

service
of vitals is always revealed after a major burn injury. Generalized vasoconstriction of
adducting
micro vessels is the first, though a very unstable response to the trauma. Vasoconstriction
passes soon and other factors, mainly hemorheological, determine microcirculatory disorders.
First of all, it’s refers to erythrocyte aggregation, as its increase in combination with decrease
in disaggregation causes the development of the syndrome of hyper viscosity of blood [5,6].
The increase in erythrocyte aggregation may be connected with the influence of plasma
factors (increase in proteolysis, free radical oxidation, concentration of high molecular weight
proteins, etc.) as well as with cellular factors (transmembrane redistribution of phospholipids,
change of neuraminic acid content in membranes, change of level of endocellular Ca2+,
etc)[7-10].

The mechanism of blood cell hyperaggregation in burn disease is still unclear. It has not even
been determined which factors, plasma or cellular, cause the disorders of red blood cell
aggregation in burn disease, or whether they are reversible.

It is important to research these issues as it determines the therapy tactics aimed at correction
of microcirculatory disorders in thermal trauma.

Materials and methods

The study was performed on 48 blood samples from patients in the acute period of burn
disease (a second- or third-degree burn >20% of total body surface area) and 81 blood
samples from healthy volunteers. The blood was stabilized with 3.8% sodium citrate solution
in 9:1 ratio and centrifuged for 20 min at 3000 rpm. Plasma was separated from erythrocyte
mass, leukocytes and platelets removed, and after that plasma and erythrocyte mass were
mixed in 2:1 ratio respectively.

Erythrocyte aggregation was studied by a rheoscope constructed using the method of

H.Schmid-Schönbein et al. [11], a modification of G.Y.Levin et al. [12]. The process of
aggregation was recorded during hydrodynamic mixing and upon its stop. The process of
erythrocyte disaggregation was recorded when creating sheer stress, set at the exact rate.
Erythrocyte aggregation and disaggregation were assessed using the following parameters:

1. Degree of aggregation according to the maximum amplitude of aggregatogram (mm) - Ma.

2. Amplitude of aggregatogram at 40 sec after the start of the aggregation process (mm) - A40.
3. Degree of erythrocyte disaggregation in percentage of Ma at 10 sec-1, 15 sec-1, 20 sec-
1
sheer rate - D10, D15, D20.
During the study of the influence of plasma factors on erythrocytes aggregation the

main attention was paid to concentration of fibrinogen, products of its transformation - fibrin
monomers and oxidized fibrinogen and also to the possibility of blocking fibrinogen
receptors of erythrocytes membranes by monoclonal antibodies of fibrinogen receptors of
platelets. When studying the effect of fibrinogen concentration on the aggregation of
erythrocytes, dry fibrinogen (Bio Chemika) was added to their suspension. The final
concentration of fibrinogen was 5 to 9 g/l in different series of experiments. Each series of
experiments with given concentrations of fibrinogen was performed on blood from one
donor. As control, aggregation parameters of erythrocytes from the same donor were used.

The level of oxidized fibrinogen in blood plasma from burn patients and healthy donors was
determined using the method of R.L. Levine et al. [13], a modification of Y.I.Ragino et al.
[14]. The effect of oxidized fibrinogen on erythrocytes aggregation was studied using donor
blood. For an oxidative modification, the solution of fibrinogen (2.5 g/l) was exposed to UV
irradiation. Degree of the oxidative modification of the fibrinogen solution was monitored
using the method of R.L. Levine et al., a modification of E.E.Dubinina et al. [15]. Each series
of experiments with fibrinogen of various degrees of oxidation was performed on blood of
one donor.

For studying the effect of fibrin monomer (FM) on aggregation of blood cells, FM was
provided by the Altai branch of the Hematologic Scientific Centre of the Russian Academy of
Medical Sciences [16], in the final concentration of 41.66, 31.25 and 22.72 mg/100 ml.

To block fibrinogen receptors of erythrocytes, we used monoclonal antibodies FRaMon

(CRC64), which are antagonists of the platelet membrane receptor of fibrinogen - IIb-IIIa
glycoprotein complex [17]. Their concentration in plasma was 0.0011 mg/ml.

Besides, the influence of medium molecular peptides (MMP) on erythrocytes aggregation

was studied. MMP concentration in blood increases greatly as a result of proteolysis in acute
period of burn disease.

Medium molecular peptides, which are peptides with molecular weight less than 5000
daltons, were detected using the method of N.I.Gabrielyan, V.I.Lipatova [18]. For their
extraction from blood of burn patients we used microcentrifuge tubes Amicon Ultra-4,
Ultracel-10k (“MILLIPORE”- USA, Ireland). Extracted MMPs were added to erythrocyte
suspension from a healthy donor.
The results of the study were processed with methods of non-parametric statistics using
Mann-Whitney criteria, Wilcoxon matched pairs test.

Results

The results of the study show, that aggregation rate and degree increase significantly in the
acute period of burn disease. Disaggregation of erythrocytes in burn patients at all sheer rates
was decreased greatly (Table 1).

Table 1
Changes in erythrocyte aggregation and disaggregation in the acute period of burn disease

To estimate the role of plasma and erythrocyte factors in change of aggregation in burn
disease, several series of experiments were performed, in which blood plasma from burn
patients was added to erythrocytes from healthy donors and vice versa - donor plasma was
added to erythrocytes from burn patients. Group, rhesus and individual compatibility were
preliminary tested. It was shown that burn blood plasma, added to intact erythrocytes, caused
sharp increase in aggregation and decrease in disaggregation of erythrocytes (Table 2). When
blood plasma from healthy donors was added to erythrocytes from burn patients, erythrocyte
aggregation returned to completely normal.

Table 2
Changes in erythrocyte aggregation and disaggregation in healthy donors under the influence
of blood plasma from burn patients
The study of fibrinogen concentration in blood plasma from burn patients demonstrated its
considerable increase - up to 5.54 ± 0.21 g/l on average. The study of hyperfibrinogenemia
effect on erythrocyte aggregation showed a progressive increase in its degree and rate at
increased fibrinogen concentration. Only at fibrinogen concentration over 7.5 g/l the rate and
degree of aggregation decreased in comparison to maximum. However, even in this case the
aggregation was significantly higher than in control group. Disaggregation of erythrocytes at
increased fibrinogen concentration decreased significantly. To add to this, the most
considerable changes were seen at low sheer rate (Table 3). It is to be noted, that even at
maximum fibrinogen concentration (9 g/l), against the background of already beginning
decrease in erythrocyte

aggregation, the strength of aggregates was still high and did not decrease (Table 3).

Table 3
Effect of hyperfibrinogenemia on erythrocyte aggregation and disaggregation

The study showed that incubation of erythrocyte suspension with monoclonal antibodies
against platelet GPIIb/IIIa receptors did not change their aggregation. Moreover, we detected
decrease in erythrocyte disaggregation under the influence of FRaMon (Table 4).

Table 4
Effect of monoclonal antibodies against GPIIb/IIIa on aggregational properties of
erythrocytes

The study of oxidized fibrinogen level in blood plasma from severely burned patients
demonstrated its practically twofold rise (p<0,001) compared to norm (from 11.53 ± 0.94 to
21.06 ± 1.59 U / 1 mg of fibrinogen / 1 ml of plasma). We found out, that at low level of
fibrinogen oxidation the degree of erythrocyte aggregation increases, though not much (Table
5). There was no significant change in erythrocyte aggregation degree at further increase of
fibrinogen oxidation. The rate of aggregation stayed practically the same at all studied levels
of oxidized fibrinogen. Disaggregation of erythrocytes decreases at low sheer rate (15 sec-1)
at the insignificant level of fibrinogen oxidation, and increases to the initial level when its
oxidation degree rises (Table 5).

Table 5
Effect of fibrinogen oxidation degree on erythrocyte aggregation and disaggregation

The study showed that the increase in fibrin monomer concentration in erythrocyte
suspension from healthy donors lead to a progressive dose-dependent increase in their
aggregation. At the same time erythrocyte disaggregation under the effect of fibrin monomer
did not demonstrate a statistically significant change, though it tended to decrease at low
sheer rate (Table 6).

Table 6
Effect of fibrin monomer on erythrocyte aggregation and disaggregation

The level of medium molecular peptides increased sharply in blood plasma from severely
burned patients compared to healthy donors (from 0.53±0.02 г/Λ to 0.97±0.13 g/l; p<0,001).
It was shown that a rise in MMP concentration caused a dose-dependent increase of
erythrocyte aggregation (Table 7).
Table 7
Effect of different concentrations of medium molecular peptides on erythrocyte aggregation

Discussion

The study of erythrocyte aggregation has shown that severe thermal burns are accompanied
by a significant increase in erythrocyte aggregation. It is known that an important role in
hemorheological impairment is played by not only erythrocyte aggregation itself but the
degree of its reversibility - disaggregation. If usually disaggregation occurs at sheer rate that
exists in venules at normal blood flow, than the growth of strength of erythrocyte aggregates
demands considerably higher sheer rate for them to disaggregate. In burn trauma the most
unfavorable conditions for microcirculation are created: blood-flow rate decreases and critical
sheer rate increases. That is what we show in our study.

We found that blood plasma in burn patients sharply increases aggregation of intact
erythrocytes and improves the strength of their aggregates. When blood plasma from healthy
donors is added to erythrocytes, they regain normal aggregation properties. This indicates that
the main reason of increased erythrocyte aggregation in burn disease is change of properties
in blood plasma. Dysfunction of red blood cells is secondary and, what is most important,
practically fully reversible.

According to references and our data, burn disease is usually accompanied by severe
hyperfibrinogenemia. It is known that the most widely accepted theory of erythrocyte
aggregation is the “bridge” theory [19-20]. According to it, fibrinogen creates “bridges”
between adjacent cells. It is proven that human erythrocytes express a large

number of adhesive receptors [21-24]. D. Lominadze and W.L.Dean [25] showed presence of
a fibrinogen specific integrin-like receptor on the surface of erythrocytes. We have proven
that increase of fibrinogen concentration in blood plasma from healthy donors causes a
progressive increase in erythrocyte aggregation and growth of strength of their aggregates.
Thus, hyperfibrinogenemia, observed in burn disease, plays an important role in changing
erythrocyte aggregation properties. We have detected an effect of “saturation” in erythrocyte
aggregation that consists in the following: when the threshold fibrinogen concentration is
achieved, its further increase is accompanied by decrease in erythrocyte aggregation. It is to
be noted that we found similar data when studying the effect of hyperfibrinogenemia on
platelet aggregation [26]. This allows us to conclude that platelet and erythrocyte aggregation
mechanisms have much in common, and the defying role in their

interaction
is played by fibrinogen.

Despite the presence of fibrinogen specific integrin-like receptors on erythrocyte membranes,

the question about identity of these receptors to platelet receptors is still open. F.A. Carvalho
et al. (2010) [27] suggest similarity of fibrinogen receptors of erythrocytes to platelet integrin
αIIbβ3. To explore the identity of the given receptors, studies were performed with monoclonal
antibodies FRaMon, where the active principle are F (ab') 2 fragments. Principle of action of
these monoclonal antibodies is based on selective non-competitive inhibition of platelet
fibrinogen receptors [18]. We found that erythrocyte aggregation does not change with use of
FRaMon. Received data indicate that fibrinogen receptors of erythrocytes and platelets are
not identical. It is not clear why erythrocyte disaggregation decreases after their incubation
with antibodies. We may suggest that FRaMon is able to change the character and binding
strength of fibrinogen and erythrocyte receptors. Similar data, received by F.A. Carvalho et
al. [27], indicate that low-molecular weight inhibitor of interaction between fibrinogen and
GPIIb/IIIa platelet receptors (eptifibatide) changes binding strength between fibrinogen
molecules and erythrocyte receptors.

When studying the effect of oxidized fibrinogen on erythrocyte aggregation properties, we

found that there was no significant change of rate and degree of erythrocyte aggregation. This
could be related to the following. Oxidized fibrinogen causes significant changes in
erythrocyte membrane [28]. And we may observe migration of phospholipids (first of all,
phosphatidylserin), localized on the inner side of membrane, to the outer side of membrane.
Phosphatidylserin binds to fibrinogen [29], causing development of the so-called “non-
specific” erythrocyte aggregation. However,

pecific aggregation, caused by interaction between fibrinogen and selective receptors on

erythrocyte membrane due to conformational changes of fibrinogen oxidation should
decrease. Multidirectional action of these factors may be the reason why there are no
significant changes in erythrocyte aggregation under the influence of oxidized fibrinogen.
The strength of erythrocyte aggregates grew at a low degree of fibrinogen oxidation and went
down at its increase. Most probably it can be explained by a less strong bond between
fibrinogen and erythrocyte membrane receptors due to conformational changes of fibrinogen
molecules in oxidation.

One of the main products of fibrinogen degradation is fibrin monomer. Its level in blood
increases significantly in severe thrombinemia observed after thermal trauma. We did not
find any data about the role of fibrin monomers in erythrocyte aggregation in the sources
available. We have shown that FM expresses significant proaggregating activity. The
mechanism of influence of fibrin monomers on erythrocyte aggregation properties is still
unclear. It is possible that fibrin monomer, having molecules of approximately same size and
form as fibrinogen, also builds “bridges” between cells. However, it is also possible that the
bonds between cells, formed under the influence of FM, despite its significant proaggregating
activity, are less strong compared to “fibrinogenic”, which is the reason for no significant
increase in strength of erythrocyte aggregates. Diminishing strength of bonds, formed by
fibrin monomer, may be the result of its negative charge decrease compared to fibrinogen.
Thus, due to proteolysis, 14 negative charges with fibrin peptides detach from fibrinogen
molecule and the isoelectric point of fibrin monomer increases [30]. Suggested hypothesis
agrees with the “bridge” theory, where dependence of erythrocyte aggregation on the protein
nature is foreseen.

It is known that sharp activation of proteolysis, which occurs after thermal trauma, causes
increased number of medium molecular peptides in blood of severely burned patients [31-
32]. Medium molecular peptides are one of the main groups of endotoxins responsible for
development of toxemia in burn disease. They affect activity of all systems and organs
because in their structure they are similar to regulatory peptides. But it is still unclear what
role medium molecular peptides play in erythrocyte aggregation in burn disease. We have
found that medium molecular peptides in burn patients have a proaggregating effect on
erythrocytes. The mechanism of this activity is not clear. It is known that MMP can change
the resting potential and electrical resistance of erythrocytes. Probably, it leads to an increase
in their aggregation at high level of medium mass molecules. It agrees with the aggregation
model based on the change of zeta-potential of erythrocytes [33].

Conclusion
Dysfunctions of erythrocyte aggregation properties in burn disease are mainly defined by
change of blood plasma contents and are reversible.

The most important factor causing change of erythrocyte aggregation properties in burn
disease is an increase in fibrinogen concentration in blood plasma. It is found that fibrinogen-
induced aggregation of erythrocytes is accompanied by “saturation” effect.

Antibodies to platelet receptors (FRaMon) do not affect erythrocyte aggregation. It indicates

that „fibrinogenic“ receptors of platelets and erythrocytes are not identical.

An important role of fibrin monomer and medium molecular peptides as agonists of

erythrocyte aggregation in burn disease is shown.

Int J Burns Trauma. 2011; 1(1): 34–41.

Published online Sep 3, 2011.
PMCID: PMC3415941

Grigory Y Levin and Marpha N Egorihina

Federal State Institution “Nizhny Novgorod Research Institute of Traumatology and

Orthopedics” of Ministry of Health,
and Social Development of Russian Federation, Nizhny Novgorod, Russia
Address correspondence to: Dr. Grigory Y. Levin, Federal State Institution “Nizhny
Novgorod Research Institute of Traumatology and Orthopedics” of Ministry of Health and
Social Development of Russian Federation, Nizhny Novgorod, Russia. E-
mail: ur.ca.nnu@nivel

Received July 10, 2011; Accepted August 18, 2011.

IJBT Copyright © 2011

Articles from International Journal of Burns and Trauma are provided here courtesy of e-
Century Publishing Corporation

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Sumber:

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3415941/ Internasional journal of burn and

trauma