RESEARCH ARTICLE


Study of Optimum Condition for Rapid Preparation of Thrombin using Russell’s Viper Venom Factor X Activator



Narin Kijkriengkraikul1, *, Issarang Nuchprayoon2
1 Technopreneurship and Innovation Management Program, Graduate School, Chulalongkorn University, Bangkok 10330, Thailand
2 Department of Pediatrics, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand


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© 2018 Kijkriengkraikul and Nuchprayoon.

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution 4.0 International Public License (CC-BY 4.0), a copy of which is available at: (https://creativecommons.org/licenses/by/4.0/legalcode). This license permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to this author at the Technopreneurship and Innovation Management Program, Graduate School, Chulalongkorn University, 1405-1409 14th Floor, Chamchuri Square Building, Phayathai Road, Bangkok 10330, Thailand; Tel: +66851355353; E-mail: narin4k@yahoo.com


Abstract

Background:

The purpose of this study is to investigate a simple method with the optimum condition for rapid thrombin preparation from Cryoprecipitate-depleted Plasma (CDP) using RVV-X in the process.

Methods:

Thrombin preparation from human CDP was studied with the presence of different factors in batch condition including: 1) RVV-X; 2) volume of calcium chloride solution; 3) volume of sodium chloride solution for final extraction; and 4) incubation time. The properties of the prepared sample were analyzed for fibrin clot formation, total protein by Kjeldahl method, thrombin time, molecular weight and protein patterns by SDS-PAGE, and thrombin concentration by coagulation analyzer. The method and process of preparing thrombin and the study of optimum condition for rapidly preparing the highest yield of thrombin from starting CDP 100 ml were introduced.

Results:

The best four conditions were concluded: 1) RVV-X 50 mcg should be present in the process; 2) volume of 0.25 M calcium chloride should be 3 ml; 3) volume of 0.85% sodium chloride for the final protein precipitate extraction should be 10 ml and; 4) no incubation time needed for prothrombin activation process. A solution prepared from the optimum condition showed an obvious band on SDS-PAGE at a molecular weight about 36,000 Da which is our target protein thrombin. The prepared solution had a total protein content of 0.065 g/dl and gave satisfactory results of thrombin time (9 seconds) and fibrin clot formation. The test results of thrombin concentration between the method with and without incubation time were 269.4 and 295.2 IU/ml, respectively.

Conclusion:

This result showed that the method with RVV-X but without incubation time for prothrombin activation (optimum condition) gave the highest yield of thrombin.

Keywords: Preparation of Thrombin, Russell’s Viper Venom Factor X Activator, Fibrinogen, Blood coagulation, Fibrin clot, Plasma.