Oral presentations O1.1 O1.2 Session 1: Biochemistry of hemostasis

Session 1: Biochemistry of hemostasis
Oral presentations
O1.2
O1.1
Molecular structure and biological
function of von Willebrand factor
Contact of blood and endithelial cells with
surface-modified metallic biomaterials
Bogdan Walkowiak1,2*
1Institute
of Materials Engineering, Technical University of
Łódź, Łódź, Poland, 2Department of Molecular and Medical
Biophysics, Medical University of Łódź, Łódź, Poland
*e-mail: Bogdan Walkowiak < bogdan.walkowiak@csk.
umed.lodz.pl>
Significant progress made in materials science, mainly in
the technology of surface, allows for intentional modifications of metallic surfaces. Thin layers of modified surfaces separate metallic substrates from environment and
prevent them from corrosion. However, a real opportunity to control the interaction of surface with surrounding
tissues and body fluids, with respect to the anticipated
application, seems to be even more important. This way,
one can produce a metallic implant with strictly defined
properties for specific applications.
A long term contact of blood components or endothelial cells with biomaterials can be crucial for a variety
of biological processes. Response of blood platelets and
endothelial cells to this contact may be a highly important factor in the case of such processes as inflammation,
blood coagulation, angiogenesis, osteointegration and
many others, in which endothelial cells and blood platelets play a key role. In the case of reconstructive arterial
or cardiac surgery the cells are in direct contact with the
implant surface.
Our practical experience with surface modifications concerns medical steel 316L and titanium alloy Ti6Al4V. Both
substrates were coated with thin layers of carbon (DLC
or NCD films) by RF PCVD method elaborated by Prof.
Mitura team (1). The titanium alloy was also subjected to
glow discharge procedure. This process was carried out
by Prof. Wierzchon group (2), and resulted in the production of thin layers of TiN or TiCN on titanium alloy
surface. Both modifications prevented the base materials
from corrosion and made alloy surface more durable. We
have found that carbon layers generally made metallic
surface more biocompatible and more resistant to biofilm production. Introducing titanium nitride or titanium
carbonitride layers allows for the modulation of thrombogenity degree of the surface. Our investigations employed fluorescence and electron microscopy techniques,
SPR-biosensor technology, 2D-electrophoresis and flow
cytofluorymetry.
Keywords: surface modification, metallic materials, biocompatibility, blood platelets, endothelial cells
References:
1. Mitura S et al. (1999) J Chaos, Solitons Fractals 10: 2165.
2. Czarnowska E et al. (2000) J Mater Sci Mater Med 11: 73.
Ksenia Bykowska*
Institute of Hematology and Blood Transfusion, Warszawa,
Poland
*e-mail: Ksenia Bykowska < [email protected]>
In 1926, Eric von Willebrand a medical doctor from Helsinki described for the first time a family with prolonged
bleeding time. The predominant symptoms in this family
were nose and gum bleeding, bleeding after tooth extraction, menorrhagia and bleeding from trivial wounds. After him this disorder was named called von Willebrand
disease and the protein deficient from the blood in this
family was called — the von Willebrand factor. This factor plays two major hemostatic roles. It mediates platelet attachment, through Ib/IX/V glikoprotein complex to
subendothelial tissue at the site of vascular injury under
condition of high shear rate. It is the carrier protein of
coagulation factor VIII, an essential cofactor in coagulation, thats protects factor VIII from proteolytic degradation. Von Willebrand factor (vWF) is a large, single chain
adhesive glikoprotein synthesized exclusively in endothelial cells and megakariocytes under the control of a
gene located on chromosome 12. The translation product
contains an 22 amino acid signal peptide, large propeptide sequence (741 amino acids) and mature vWf protein
(2050 amino acids) composed of many regions differ in
their structure and function. In the endoplasmic reticulum the signal peptide is cleaved, the precursors form
dimers by disulfide bonding at the C-terminal ends of
monomers. Then dimers multimerize in Golgi apparatus
by forming of additional disulfide bonds at the N-terminal ends of dimers. After multimeryzation propeptide is
removed by intra-celular proteolysis. Molecules are then
subjected to further modification, glycosylation and sulfation. The multimers are constitutively secreted to blood
stream after stimulation of respective cells by a variety
of stimuli. A portion of them is stored in Weibel-Palade
bodies of endothelial cells and also in platelets α granules. The newly synthesized vWF and stored in endothelial cells is ultra large (ultralarge, UvWF) and hyperreactive in hemostasis. Ultralarge vWf forms spontaneously
bonds with the platelet GP Ib/IX/V complex resulting in
platelet adhesion, aggregation and thrombus formation
In healthy persons this is prevented by metalloprotease
cleaving ultralarge vWf to less active multimers with
lower molecular weight, present in human plasma.
Synthesis reduction, defect in amino acid composition of
vWF as well as defects in dimerization or multimerization
may lead to bleeding disorder. The increase of vWF concentration in plasma to level above normal as well as presence of ultralarge multimers may lead to thrombosis.
42nd Meeting of the Polish Biochemical Society
Vol. 54 O1.3
O1.4
The role of plasminogen activation
system in cancer cell biology
Thrombin activatable fibrinolysis
inhibitor (TAFI)
Ewa B. Żekanowska*
Janusz Kłoczko*
Department of Pathophysiology, Collegium Medicum,
Uniwersity of Mikołaj Kopernik, Bydgoszcz, Poland
*e-mail: Ewa B. Żekanowska < [email protected]>
Department of Hematological, Medical Academy of Bialystok,
Białystok, Poland
*e-mail: Janusz Kłoczko < [email protected]>
The plasminogen activation system is composed of plasminogen activators, tissue- type (t-PA) and urokinasetype (u-PA) which convert the zymogen plasminogen
to active plasmin. Plasmin is a broad specificity enzyme
that degrades fibrin and several proteins of the extracellular matrix, and is able to activate pro-metalloproteinases and growth factors (TGF- β, bFGF, VEGF). T-PA and
u-PA activity is controlled by plasminogen activator inhibitor type-1 (PAI-1) and type-2 (PAI-2) belonging to the
serpin family. T-PA plays a crucial role in intravascular
fibrinolysis, u-PA is involved in cell mediated proteolysis.
U-PA binds a specific, high affinity cell-surface receptor
(u-PAR) increasing u-PA activity and directing plasmin
activity to the cell surface.
Recent studies showed that the agressive, invasive phenotype of cancer cells is strongly tied to plasminogen activation system. Many clinical and experimental studies
have proved that the high expression of u-PA, u-PAR and
PAI-1 was closely related to the poor prognosis for different cancers (brest, lung, colon, ovarian, gastric, kidney,
liver, colorectal)
Several functions of u-PA, u-PAR and PAI-1 have been
shown to be involved in the invasive behaviour of cancer
cell. U-PA/u-PAR influence cell migration via proteolytic
mechanism i.e. plasmin generation and non-proteolytic
mechanism i.e. chemotaxis, adhesion, proliferation. The
u-PA/u-PAR complex is strongly involved in the attachment/detachment process of cancer cells, a mechanism
obviously essential for cell migration. Interaction of uPA/u-PAR with PAI-1 induces internalization of the ternary complex which subsequently results in intracellular
degradation of u-PA and PAI-1, while u-PAR is recycled
to the cell surface. By this the proteolytic activity is efficiently reorganized on the cell surface enabling pericellular proteolysis and degradation of the extracellular
matrix. Binding of u-PA to cell surface associated u-PAR
leads to activation of various intracellular signaling molecules such as tyrosine-, serine-protein kinases, stimulates
carcinoma cell migration by enhancing integrin-mediated signal transduction. PAI-1 may control tumor angiogenesis by regulating proteolytic and non-proteolytic
events in endothelial cell migration that may depend on
the expression by these cells of integrins, u-PA uPAR,
on endocytosis of u-PA/u-PAR complex, as well on the
composition of the extracelllar matrix. PAI-1 could inhibit
apoptosis in cancer cell. Recent observations indicate a
much more complex role of PAI-1 in tumor progression
than initially expected.
The plasminogen activation system represents a novel
target for tumor- biology based therapy by interfering
with the expression of u-PA, u-PAR and PAI-1 at gene or
protein level.
A delicate balance between coagulation and fibrinolysis
determines the stability of the fibrin clot. A Thrombin
Activatable Fibrinolysis Inhibitor (TAFI) plays important
role in this process. Activated by thrombin TAFI (TAFIa)
also described as plasma carboxypeptidase B, carboxipeptidase U and carboxipeptidase R, inhibits fibrinolysis by removing carboxy-terminal lysine residues from
fibrin.
Elimination of these lysines abrogates the fibrin cofactor
function of t-PA-mediated plasminogen activation resulting in a decreased rate of plasmin generation and thus
down regulation of fibrinolysis. The role of TAFI in bleeding and thrombotic disorders is discussed as well as its
novel emerging role in inflammation.
Abstracts
O1.5
O1.6
The influence of E-NTPDase 1 (apyrase EC
3.6.1.5) and adenylate kinase (EC 2.7.4.3)
on pig blood’s platelet aggregation
Coagulation and fibrinolysis in parietal
thrombus of abdominal aortic aneurysm
Studzińska1,
Bożena
Anna Seroka, Małgorzata
Rochnowska, Michał A. Komoszyński2*
1Department
of Biochemistry, Nicolaus Copernicus
University, Toruń, Poland, 2Department of Biochemistry,
Nicolaus Copernicus University, Toruń, Poland
*e-mail: Michał A. Komoszyński < [email protected].
torun.pl>
Adenosine 5’-diphosphate (ADP) is the most important
signaling molecule and it plays a key role in hemostasis
and the development and extension of arterial thrombosis. ADP was the first low molecular weight agent recognized to cause platelet aggregation.
NTPDases and adenylate kinase (AK) are the main enzymes involved in metabolism of extracellular adenine
nucleotides. The majority of studies was concentrated on
the role of apyrase in the inhibition of platelets activation.
Up to now, there are no experiments on the role of adenylate kinase in this process.
We studied the influence of aggregation agents: ADP (20
μM) and collagen (7.5 μg/ml) on platelet aggregation
from pig blood. Both compounds activate aggregation of
platelets in the citrated platelet-rich plasma (PRP). 1U of
apyrase and AK activity added to PRP before ADP or collagen, inhibits the platelet aggregation. One minute after
adding of ADP or collagen as much as 5U of apyrase and
adenylate kinase is necessary for stopping of the platelet
aggregation.
The infuence of apyrase and adenylate kinase on metabolism ADP was analysed by high-performace liquid chromatography (HPLC) metod. Products of degradation of
purines by apyrase and AK are different. In the presence
of apyrase activity ADP was converted to AMP but we
also observed increased level of adenosine compared to
control (PRP + 20 μM ADP). This may be due the fact that
apyrase from Sigma Corporation has got a lot of 5’-nucleotidase activity. In the presence of adenylate kinase activity, products of the reaction were AMP, ATP and IMP.
These data suggest that investigated enzymes play an
important role in the pathogenesis of cardiovascular
system. Anticoagulation role of apyrase and adenylate
kinase indicate the possibility to using these enzymes as
antithrombotic drugs in the treatment of arteriosclerosis
and heart diseases.
2007
Maria Jastrzębska1*, Miłosław Cnotliwy2
1Chair
of Laboratory Diagnostics and Molecular Medicine,
of Vascular and General Surgery and Angiology,
Pomeranian Medical University, Szczecin, Poland
*e-mail: Maria Jastrzębska < [email protected].
pl>
2Department
Background: Development of intraluminal thrombus
(ILT) in the abdominal aortic aneurysm (AAA) is a natural reaction to lesion of the arterial wall. Local hemostatic
processes and secondary fibrinolysis in the intraluminal
thrombus, through the activation of proteolysis in the extracellular matrix may constitute one of the factors that
lead to an aneurysm rupture. In our study we compared
some coagulation and fibrinolysis parameters in the layer
of the thrombus directly adjacent to the aneurysmal wall,
between the thinner and thicker part of the intraluminal
thrombus.
Methods: The sections sampled for the study were harvested from the thick (more than 25 mm) and thin (up
to 10 mm) slices of the ILT obtained from the same 32
abdominal aortic aneurysms, namely from the layer directly adjacent to the aneurysmal wall. Immediately after sampling the thrombus was washed in saline and the
homogenate was prepared. The tissue factor (TF), antiheparin (AH) and antithrombin (AT) activities, plasminogen (PLG) and plasminogen activators levels (PA) and
content of D-Dimers (D-D) in the samples were measured.
The TF and AH activities were expressed by shortening of
the coagulation time compared to the NaCl control. The
AT activity was expressed by prolongation of the coagulation time as compared to the saline. PLG and PA levels
were measured by the chromogenic and Elisa methods,
respectively. D-D content were measured by the immunoturbidimetric assay.
Results: The activities of TF and AH in the abluminal
layer of the ILT were statistically significantly higher in
the thin thrombi than in the thick ones (p < 0.001). Moreover, thin thrombi revealed significantly higher PLG and
D-D levels when compared to thick thrombi (p < 0.001),
while the activity of PA was higher in the thick thrombi
(p < 0.05).
Conclusions: The abluminal layer of the thin thrombus
(up to 10 mm) of the AAA shows higher activities of coagulative processes when compared to thicker thrombi
(over 25 mm). Moreover, this site reveals strong secondary activation of fibrinolytic system. Further investigation
of association between coagulation/fibrinolytic activity
and proteolysis occurring within the AAA wall requires
evaluation of such processes with regard to differences in
the thickness of the thrombus.
42nd Meeting of the Polish Biochemical Society
Vol. 54 O1.7
O1.8
Fibroblast growth factor in varicose veins and
varicose veins complicated by thrombophlebitis
A new thrombolytic, antiplatelet agent with
higher fibrin affinity – a staphylokinase variant
Radosław Kowalewski2, Andrzej Małkowski1,
Krzysztof Sobolewski1*, Marek Gacko2
Katarzyna Oszajca*
1Department
of Medical Biochemistry, 2Department of
Vascular Surgery and Transplantology, Medical Academy of
Bialystok, Białystok, Poland
*e-mail: Krzysztof Sobolewski < [email protected]>
Mechanical properties of the vein wall depend to a great
extent on connective tissue extracellular components, as
well as on their quantitative interrelations. The varicose
vein wall is an example of extracellular matrix remodeling, which has been demonstrated in our numerous previously published reports. Peptide growth factors play
a key role in tissue remodeling. One to the best-known
growth factors is fibroblast growth factor, whose reservoir is extracellular matrix. The aim of the study was to
evaluate expression and contents of fibroblast growth
factors (aFGF and bFGF) in the wall of varicose veins.
Segments of varicose saphenous veins and thrombophlebitic varicose saphenous veins were the studied material,
whereas normal saphenous veins served as the control.
FGFs were evaluated with Western blot and ELISA methods. Growth factors were extracted from investigated tissues in the form of macromolecular complexes. Basic FGF
(bFGF) and acidic FGF (aFGF) were present in varicose,
as well as in varicose veins complicated by thrombophlebitis. Significant increase in aFGF content was demonstrated in studied material with varicose veins complicated by thrombophlebitis in particular, when compared
to normal veins. Whereas, bFGF content in varicose veins
and varicose veins complicated by thrombophlebitis was
comparable to that one in normal veins. The results may
indicate aFGF as a factor responsible for extracellular matrix remodeling in varicose veins and varicose veins complicated by thrombophlebitis.
Medical University of Łódź, Łódź, Poland
*e-mail: Katarzyna Oszajca < [email protected]>
Background: The incidence of thromboembolic disorders
has been increasing for several years now. Effective treatment in myocardial infarction, pulmonary embolism or
deep vein thrombosis is associated with early thrombolytic therapy. The most common strategy of the thrombolytic therapy involves the activation of fibrinolytic system
with intravenous plasminogen activators. Aggregation of
blood platelets constitutes an important part of the thrombus, and therefore antiplatelet agents have been used in
the treatment and prevention of arterial thrombosis. The
RGD peptide is a well known component of ligands recognizing platelet integrins. A chimeric proteins, consisting of the staphylokinase and the Kringle 2 domain (K2)
of t-PA for activation of fibrinolysis, the Arg-Gly-Asp
sequence for the prevention of platelet aggregation were
constructed.
Methods: The current study was aimed to assess the
thrombolytic activity of recombinant staphylokinase
(rSAK) variants with antithrombotic and antiplatelet
properties in a rat model of arterial and venous thrombolysis.
Results: We have shown that the addition of the RGD sequence to r-SAK resulted in acquisition of the ability to
prevent platelet aggregation. The insignificant inhibitory
influence of r-SAK on the aggregation of washed platelets in vitro was also demonstrated. It may result from
accidental plasmin formation (lack of a2 anti-plasmin)
causing degradation of fibrinogen and other proteins involved in the aggregation process. This phenomenon has
not been observed in platelet-rich plasma. The efficiency
of platelet–platelet interaction blocking by SAK-RGD-K2
was comparable with that of the RGD sequence alone.
These results showed that the recombinant protein SAKRGD-K2 containing the RGD sequence possesses the
ability to block platelet aggregation and hence should be
more effective in clot lysis than r-SAK. In the arterial and
venous thrombolysis animal model we have observed
reperfusion, without reocclusion, in all animals treated
with SAK-RGD-K2 - protein containing antiplatelet RGD
sequence and specific to fibrin kringle 2 domain (K2) of
t-PA
Conclusion: In conclusion, we have shown that recombinant SAK-RGD-K2, SAK-RGD proteins are strong
thrombolytic agents in rat model of arterial and venous
thrombolysis.
Acknowledgements:
This work was supported by the research project 6PO 5A 11121
of the Polish Committee for Scientific Research and research
project of the Fundation for Development of Polish Pharmacy
and Medicine — Polpharma.
Abstracts
O1.9
The influence of anthocyanins from Aronia
melanocarpa on platelet aggregation in
patients with metabolic syndrome
Joanna M. Sikora1*, Barbara Kostka2, Marzena
Koziróg-Kołacińska3, Julita Chojnowska-Jezierska3,
Elżbieta Mikiciuk-Olasik1, Marlena Broncel3
1Department
of Pharmaceutical Chemistry and Drug
Analysis, 2Department of Pharmaceutical Biochemistry,
3Department of Internal Diseases with Clinical Pharmacology
and Therapy Monitoring Unit, Medical University of Łódź,
Łódź, Poland
*e-mail: Joanna M. Sikora < [email protected]>
Background: Anthocyanins are part of a large and widespread group of plant constituents known collectively
as flavonoids. In recent years, a growing interest in their
biological activities and possible health benefits in protecting against some chronic diseases, including cancer,
cardio- and celebrovascular, atherosclerosis and diabetes
is observed. One of the richest sources of anthocyanins
are fruits of Aronia melanocarpa.
The aim of this study was to evaluate the effects of anthocyanins from Aronia melanocarpa on the parameters of
ADP-induced platelet aggregation.
Material and methods: The study group included 25 subjects with metabolic syndrome. Including criteria were:
visceral obesity (waist circumference for men > 94cm, for
women > 80 cm), HDL < 50 mg/dL (women), HDL < 40
mg/dL (men) and TG >150 mg/dL. In all patients extract
from Aronia melanocarpa (Aronox, Agropharm) was administrated 3 × 100 mg daily for 4 week. Before and after
the period of anthocyanins administration a blood sample was taken and lipidogram and platelet aggregation
were estimated.
Aggregation of platelets was measured in PRP by the turbidimetric method. Curves triggered by addition of 5 μl
of ADP (10 mmol/L) were recorded and evaluated by using our own computer program. This program estimated
5 parameters of platelet aggregation: maximal aggregation (Amax), initial velocity (v0), the time needed to reach
maximal aggregation (Tmax), the aggregation level after
5 min (A5min) from Amax (which enables to estimate the
disaggregation), and platelet shape change (PSC) (which
is only a rough estimation of this process).
Results: Four-week intake of extract from chokeberry
fruits reduced significantly the level of total cholesterol
(242.8 ± 34.5 vs. 229.2 ± 33.1 mg/dL, p = 0.002), LDL-cholesterol (158.7 ± 35.8 vs. 150.0 ± 34.6 mg/dL, p = 0.032) and
triglycerides (215.9 ± 63.6 vs. 184.6 ± 79.3 mg/dL, p = 0.017).
No significant influence on HDL-cholesterol (42.9 ± 5.0 vs.
44.3 ± 6.1 mg/L, p = 0.112) was observed.
Anthocyanins also exchanged significantly the kinetic parameters of aggregation: Amax (26.8 ± 13.1 vs. 18.9 ± 9.7 %T,
p = 0.017), v0 (18.3 ± 14.2 vs. 11.5 ± 8.3 %T/min, p = 0.022)
and Tmax (375.2 ± 174.0 vs. 431.8 ± 119.4 s, p = 0.04), no
influence on PSC (4.2 ± 2.4 vs. 3.4 ± 4.6 %T, p = 0.497) and
disaggregation was observed.
Conclusion: Anthocyanins from chokeberry (Aronia
melanocarpa) after four-week intake beneficially modified
2007
platelet aggregation and cholesterol level and therefore
may be potentially useful for atherosclerosis prevention.
Acknowledgements:
This study was supported by grants No 502-13-542 of Medical
University of Lodz, Poland.
42nd Meeting of the Polish Biochemical Society
Vol. 54 O1.10
Optimization of refolding and
purification of recombinant NTPDase2
from Arabidopsis thaliana
Dorota Ściesińska*, Mariusz Banach, Joanna
Czarnecka, Michał A. Komoszyński
Institute of Biochemistry, University of Mikołaj Kopernik,
Toruń, Poland
*e-mail: Dorota Ściesińska < [email protected]>
NTPDases — apyrases (ATP diphosphohydrolases; EC
3.6.1.5.) are enzymes capable of hydrolyzing 5’-, di- and
triphosphate nucleotides to di- and monophosphate nucleotides and an inorganic phosphate. These enzymes
have been detected in plants, vertebrates, insects, parasites, protozoa and yeast, in almost all kinds of tissues
and cells. The animals have eight different enzymes encoded by eight different genes. Despite the high similarity of nucleotide and amino acid sequences, NTPDases reveal different substrate specificity. Basic role of
NTPDases is regulation of levels of intra- and extracellular nucleotides and nucleosides. They participate in
the metabolism of ecto-nucleotides (ATP, ADP), which
play a significant role in regulation of blood pressure
and platelet aggregation. Disorders in these processes
might cause atherosclerosis and myocardial infarction.
Aim of the research is to develop a new anticoagulant
enzymatic drug based on the nucleotidase activity of the
plant NTPDases — apyrases.
The research was conducted with cDNA of A2F2 NTPDase from A. thaliana and E. coli BL21(DE3)Codon+
bacteria. The apyrase gene was cloned into pET-28a
plasmide. That vector was used to transform bacteria.
The overexpressed protein accumulated in inclusion
bodies.
The refolding process by dilution was performed to restore the biological activity. Different contents of the
inclusion body proteins were tested to optimize the
conditions of refolding with the selected method. The
maximum efficiency was achieved with the largest (36
mg/ml) initial concentration of the protein. However,
only 7% of the used protein was reactivated. The largest
specific activity was obtained for refolding performed
with the 1.98 mg/ml protein of inclusion bodies. The
active protein was purified with the ion exchange
chromatography. Separation with the MonoQ column
gave two catalytically active fractions (A and B) differing by the substrate specificity against adenine nucleotides (ATP and ADP). Fraction A was eluted from the
column without using KCl. That enzyme hydrolyzed
both ATP and ADP. Second fraction, eluted with 0.7 M
KCl, degraded only ADP. Activity of both enzymes was
stimulated by the presence of Mg2+ ions. Electroforesis
of both fractions under denaturing conditions revealed
the presence of bands with a molecular mass corresponding to the product of the apyrase gene. Separation of these proteins under native conditions revealed,
that they have significantly different mobility. Western
Blotting confirmed that the refolded and purified proteins are NTPDases.
Summarizing, two enzymes degrading adenine nucleotides were reactivated during the refolding procedure.
They seem to be products of the same gene, and the observed differences in the kinetic properties might result
from their different molecular forms.