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THE INFLUENCE OF FIBRINOGENASE ISOLATED FROM THE ANTARCTIC SCALLOP ON BLOOD COAGULATION
Author(s):
Nataliia Raksha
,
Nataliia Raksha
Affiliations:
Biochemistry,Educational and Scientific Center “Institute of Biology and Medicine” Taras Shevchenko National University of Kyiv,Kyiv,Ukraine
Dmytro Gladun
,
Dmytro Gladun
Affiliations:
Biochemistry,Educational and Scientific Center “Institute of Biology and Medicine” Taras Shevchenko National University of Kyiv,Kyiv,Ukraine
Tetyana Ishchuk
Tetyana Ishchuk
Affiliations:
Biochemistry,Educational and Scientific Center “Institute of Biology and Medicine” Taras Shevchenko National University of Kyiv,Kyiv,Ukraine
(Abstract release date: 05/18/17) EHA Library. Raksha N. 05/18/17; 182921; PB2208
Nataliia Raksha
Nataliia Raksha
Contributions
PDF Abstract
Abstract

Abstract: PB2208

Type: Publication Only

Background

At the present time, cardiovascular diseases such as acute myocardial infarction, ischemic heart diseases, and stroke are the most important causes of the human mortality around the world. Thrombosis is probably the most common symptom among cardiovascular diseases. Thrombolytic agents have been extensively used in the therapeutic treatment of thrombosis. But most of them have some serious shortcomings, including limited efficacy, short plasma half-life, large therapeutic dose or allergic response. Considering the global burden, the search continues for a safe and cheap thrombolytic agent to treat cardiovascular diseases. To date, many investigators have been trying to improve the safety and efficacy of thrombolytic therapy. Fibrinogenolytic enzymes or fibrinogenases because of their role in dissolving of blood clots as well as prevention their formation have attracted special medical and scientific attention. Enzymes that affect hemostasis have been isolated from different sources. In recent years, special attention is paid to the hydrobionts from the Antarctic region which are poorly explored and potentially can be a valuable source of new bioactive compounds, in particular enzymes.

Aims

The main goal of current research was to test the effect of fibrinogenase from marine hydrobiont the Antarctic scallop Adamussium colbecki on platelet aggregation and blood coagulation.

Methods
Fibrinogenase from the crude tissue extract of A. colbecki was isolated by three-step procedure (affinity chromatography on SBTI-sepharose following affinity chromatography on Blue-Sepharose and size exclusion chromatography on Superdex 75-PG). Platelet aggregation was determined by AT-02 aggregometer (Medtech, RF). The platelet count was adapted to 2.5х105 platelets/μL with platelet-poor plasma. Then, fibrinogenase (12.5 μg/mL or 6.25 μg/mL) was added 2 min before the addition of the platelet aggregation inducer (5х10-6 M ADP). The changes in light transmittance were continuously monitored during 8 min, and the percentage of aggregation, in the presence of the samples, was calculated comparing the transmittance against the controls. Activated partial thromboplastin time (APTT) and prothrombin time (PT) were measured in CLOTimer analyzer.

Results
According to our result investigated enzyme inhibited ADP-induced platelet aggregation and the inhibition increased with increasing concentration of the enzyme. Data analysis revealed a significant (p<0.05) inhibition of ADP-induced platelet aggregation by 80.5 % for 12.5 μg/mL of fibrinogenase and by 31.8 % for 6.25 μg/mL of fibrinogenase. Isolated fibrinogenase may inhibit platelet aggregation by hydrolyzing the Aα-chain of fibrinogen to prevent fibrinogen from combining with fibrinogen receptor on platelet membrane. In addition, fibrinogen degradation products may act as competitive inhibitors of platelet–fibrinogen bridging formation. We also investigated the effect of fibrinogenase on blood coagulation by determination of APTT and PT. According to our result APTT increased in 2.25 and 1.25 times at the concentration of fibrinogenase of 12.5 μg/mL and 6.25 μg/mL, respectively. The fibrinogenase also prolonged PT from 18 s to 68 s when the concentration of enzyme was 6.25 μg/mL.

Conclusion
Further investigations of fibrinogenase from A. colbecki are interesting and would probably help to develop new therapeutic agents to treat thrombotic disorders.

Session topic: 34. Thrombosis and vascular biology

Keyword(s): Platelet aggregation, Blood coagulation

Abstract: PB2208

Type: Publication Only

Background

At the present time, cardiovascular diseases such as acute myocardial infarction, ischemic heart diseases, and stroke are the most important causes of the human mortality around the world. Thrombosis is probably the most common symptom among cardiovascular diseases. Thrombolytic agents have been extensively used in the therapeutic treatment of thrombosis. But most of them have some serious shortcomings, including limited efficacy, short plasma half-life, large therapeutic dose or allergic response. Considering the global burden, the search continues for a safe and cheap thrombolytic agent to treat cardiovascular diseases. To date, many investigators have been trying to improve the safety and efficacy of thrombolytic therapy. Fibrinogenolytic enzymes or fibrinogenases because of their role in dissolving of blood clots as well as prevention their formation have attracted special medical and scientific attention. Enzymes that affect hemostasis have been isolated from different sources. In recent years, special attention is paid to the hydrobionts from the Antarctic region which are poorly explored and potentially can be a valuable source of new bioactive compounds, in particular enzymes.

Aims

The main goal of current research was to test the effect of fibrinogenase from marine hydrobiont the Antarctic scallop Adamussium colbecki on platelet aggregation and blood coagulation.

Methods
Fibrinogenase from the crude tissue extract of A. colbecki was isolated by three-step procedure (affinity chromatography on SBTI-sepharose following affinity chromatography on Blue-Sepharose and size exclusion chromatography on Superdex 75-PG). Platelet aggregation was determined by AT-02 aggregometer (Medtech, RF). The platelet count was adapted to 2.5х105 platelets/μL with platelet-poor plasma. Then, fibrinogenase (12.5 μg/mL or 6.25 μg/mL) was added 2 min before the addition of the platelet aggregation inducer (5х10-6 M ADP). The changes in light transmittance were continuously monitored during 8 min, and the percentage of aggregation, in the presence of the samples, was calculated comparing the transmittance against the controls. Activated partial thromboplastin time (APTT) and prothrombin time (PT) were measured in CLOTimer analyzer.

Results
According to our result investigated enzyme inhibited ADP-induced platelet aggregation and the inhibition increased with increasing concentration of the enzyme. Data analysis revealed a significant (p<0.05) inhibition of ADP-induced platelet aggregation by 80.5 % for 12.5 μg/mL of fibrinogenase and by 31.8 % for 6.25 μg/mL of fibrinogenase. Isolated fibrinogenase may inhibit platelet aggregation by hydrolyzing the Aα-chain of fibrinogen to prevent fibrinogen from combining with fibrinogen receptor on platelet membrane. In addition, fibrinogen degradation products may act as competitive inhibitors of platelet–fibrinogen bridging formation. We also investigated the effect of fibrinogenase on blood coagulation by determination of APTT and PT. According to our result APTT increased in 2.25 and 1.25 times at the concentration of fibrinogenase of 12.5 μg/mL and 6.25 μg/mL, respectively. The fibrinogenase also prolonged PT from 18 s to 68 s when the concentration of enzyme was 6.25 μg/mL.

Conclusion
Further investigations of fibrinogenase from A. colbecki are interesting and would probably help to develop new therapeutic agents to treat thrombotic disorders.

Session topic: 34. Thrombosis and vascular biology

Keyword(s): Platelet aggregation, Blood coagulation

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