Designing a multifunctional staphylokinase variant (SAK-2RGD-TTI) with appropriate thrombolytic activity in vitro

  • Habibollah Faraji
  • Fatemeh Soltani
  • Mohammad Ramezani
  • Hamid Reza Sadeghnia
  • Reza Nedaeinia
  • Hamid Moghimi Benhangi
  • Baratali MashkaniEmail author
Original Research Paper



Thrombin, platelets, and plasmin are three key factors involved in hemostasis and thrombolysis. Thrombolytic therapy with clinically approved drugs is often followed by recurrent thrombosis caused by thrombin-induced platelet aggregation from the clot debris. In order to minimize these problems, new constructs were designed for the expression of recombinant staphylokinase (rSAK) and also a fusion protein composed of staphylokinase, 20 amino acids containing 2 RGD followed by tsetse thrombin Inhibitor (SAK-2RGD-TTI) in Pichia pastoris.


Modeling the tertiary structure of SAK-2RGD-TTI showed that the linker containing RGD and TTI did not interfere with proper folding of SAK. In laboratory testing, the purified SAK-2RGD-TTI (420 μg/mL) dissolved an average of 45% of the blood clot. The activity of the SAK-2RGD-TTI was also confirmed in various tests including human plasminogen activation assay, fibrin clot lysis assay, well diffusion method, activated partial thromboplastin time and platelet rich clot lysis assay.


Our findings suggest that SAK-2RGD-TTI has improved therapeutic properties preventing reocclussion. It further confirms that it is practicable to assemble and produce a hybrid multifunctional protein that targets hemostatic process at various stages.


Process engineering Staphylokinase Tsetse thrombin inhibitor peptide Yeast 



We appreciated Dr. Mohammad Soukhtanloo, Dr. Mostafa Khedri and Dr. Manouchehr Teymouri for their valuable comments.

Supporting information

Supplementary Figure 1—The secondary structure of the SAK-2RGD-TTI protein predicted by SOPMA online software. Amino acid residues of N-terminal, which is indeed a catalytic site for fibrinolytic activity, has more the random coil structure.

Supplementary Figure 2—Ramachandran plot analysis of the SAK-2RGD-TTI using the RAMPAGE online software. It is based on torsional angles (Phi or ф and Psi or ψ) of amino acid residues (A) led to their falling in energetically favoured, allowed or outlier regions. More amino acids in favoured and allowed regions, the model more valid. The conformations and location of amino acids is shown in four individual plots having heading as general (all amino acids except Glycine, Proline and Pre-Pro (amino acid before proline), Glycine, Pre-Pro and Proline. Amino acids marked with red rectangleplace in outlier region.


This study was supported by the Pharmaceutical Research Center, Buali (Avicenna) Research Institute, Mashhad University of Medical Sciences with Grant No. 921731.

Compliance with ethical standards

Conflict of interest

The authors have declared no conflict of interest with the current work or its publication.

Ethical approval

All procedures were performed in accordance with the ethical standards of the local ethics committee of Mashhad University of Medical Sciences (Iran) and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards (Grant No: 921731).

Informed consent

All authors are aware of and agree to the content of the manuscript.

Research involving human participants and/or animals


Supplementary material

10529_2019_2748_MOESM1_ESM.docx (721 kb)
Supplementary material 1 (DOCX 721 kb)


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Copyright information

© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Department of Laboratory Sciences, Faculty of Para-medicineHormozgan University of Medical SciencesBandar AbbasIran
  2. 2.Pharmaceutical Research Center, Pharmaceutical Technology InstituteMashhad University of Medical SciencesMashhadIran
  3. 3.Pharmacological Research Center of Medicinal PlantsMashhad University of Medical SciencesMashhadIran
  4. 4.Department of Pharmacology, Faculty of MedicineMashhad University of Medical SciencesMashhadIran
  5. 5.Isfahan Cardiovascular Research Center, Cardiovascular Research InstituteIsfahan University of Medical SciencesIsfahanIran
  6. 6.Department of ToxicologyIslamic Azad UniversityShahrezaIran
  7. 7.Bioinformatics Research GroupMashhad University of Medical SciencesMashhadIran

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