Open Access

Development of an in vitro model to study clot lysis activity of thrombolytic drugs

  • Sweta Prasad1,
  • Rajpal S Kashyap1,
  • Jayant Y Deopujari1,
  • Hemant J Purohit2,
  • Girdhar M Taori1 and
  • Hatim F Daginawala1Email author
Thrombosis Journal20064:14

https://doi.org/10.1186/1477-9560-4-14

Received: 17 June 2006

Accepted: 12 September 2006

Published: 12 September 2006

Abstract

Background

Thrombolytic drugs are widely used for the management of cerebral venous sinus thrombosis patients. Several in vitro models have been developed to study clot lytic activity of thrombolytic drugs, but all of these have certain limitations. There is need of an appropriate model to check the clot lytic efficacy of thrombolytic drugs. In the present study, an attempt has been made to design and develop a new model system to study clot lysis in a simplified and easy way using a thrombolytic drug, streptokinase.

Methods

Whole blood from healthy individuals (n = 20) was allowed to form clots in a pre-weighed sterile microcentrifuge tubes; serum was removed and clot was weighed. After lysis by streptokinase fluid was removed and remnants of clot were again weighed along with the tube. Percentage of Clot lysis was calculated on the basis of the weight difference of microcentrifuge tubes obtained before and after clot lysis.

Results

There was a significant percentage of clot lysis observed when streptokinase was used. On the other hand with water (negative control), minimal (2.5%) clot lysis was observed. There was a significant difference between clot lysis done by streptokinase and water.

Conclusion

Our study could be a rapid and effective methodology to study clot-lytic effect of newly developed drugs as well as known drugs.

Background

Cerebral venous sinus thrombosis (CVST) is a common disorder that is often accompanied by significant morbidity and mortality [1, 2]. In anticoagulation therapy the intravenous heparin [2, 3] is the first line of treatment for CVST, because of its efficacy, safety and feasibility. However, thrombolytic therapy, with its ability to produce rapid clot lysis, has long been considered an attractive alternative [4]. Thrombolytic drugs like tissue plasminogen activator (t-PA), urokinase, streptokinase etc. play a crucial role in the management of patients with CVST. In India though streptokinase and urokinase are widely used due to its lower cost, as compared to other thrombolytic drugs, these are dangerous [59] because they might cause serious bleeding complications along with reocclusion and reinfarction.

Various methods were developed to measure the clot lysis activity of thrombolytic drugs. The best way to study thrombolytic drugs is through in vitro clot lysis model [1019]. Earlier Basta et al [10] worked on artificial clots and used ultrasound methods to measure the thrombolytic activity of streptokinase. Several other models have been reported which either uses complicated mathematical [11] or computing [12] skills, but all these methods are very costly and not affordable in developing countries. The above-mentioned problems demand a need of simple and cost effective clot lytic model for measurement of clot lysis activity of thrombolytic drugs. In the present study an attempt has been made to develop an in-vitro clot lytic model using a known thrombolytic drug, streptokinase.

Materials and methods

Specimen

Venous blood was drawn from healthy human volunteers (n = 20) without a history of oral contraceptive or anticoagulant therapy (using a protocol approved by our Institutional Ethics Committee). 500 μl of blood was transferred to each of the previously weighed microcentrifuge tubes to form clots.

Streptokinase

To the commercially available lyophilized streptokinase vial (15, 00,000 I.U.) 5 ml phosphate buffered saline (PBS) was added and mixed properly. This suspension was used as a stock from which appropriate dilutions were made to observe the thrombolytic activity using the in vitro model developed in our laboratory.

Study design

Venous blood drawn from healthy volunteers (n = 20) was transferred in different pre weighed sterile microcentrifuge tube (500 μl/tube) and incubated at 37°C for 45 minutes. After clot formation, serum was completely removed (aspirated out without disturbing the clot formed) and each tube having clot was again weighed to determine the clot weight (clot weight = weight of clot containing tube – weight of tube alone). Each microcentrifuge tube containing clot was properly labeled and 100 μl of streptokinase along with various dilutions in sterile distilled water (undiluted, 3:4, 1:2 and 1:3) was added to the tubes. Water was also added to one of the tubes containing clot and this serves as a negative thrombolytic control. All the tubes were then incubated at 37°C for 90 minutes and observed for clot lysis. After incubation, fluid obtained was removed and tubes were again weighed to observe the difference in weight after clot disruption. Difference obtained in weight taken before and after clot lysis was expressed as percentage of clot lysis. The test was repeated ten times with all the four dilutions of the thrombolytic drugs (Streptokinase) in blood samples of twenty different healthy volunteers.

Statistical analysis

The mean clot lysis percent of streptokinase with different concentrations was compared with water using the repeated measures ANOVA with Tukey post test. Data are expressed as mean ± standard deviation. A p value ≤ 0.05 was considered to be statistically significant.

Results

A clear, visual representation of clot lysis is shown in figure no. 1. When 100 μl water was added to the control clot negligible clot lysis was observed. Whereas, tubes to which different dilutions of streptokinase was added, significant clot lysis could be visually seen. Percent clot lysis obtained after treating clots with streptokinase and control group is shown in figure 2. Maximum clot lysis was observed when undiluted streptokinase (100 μl) was added to the clots. With water 2.55% weight difference was seen. Though undiluted streptokinase (30,000 I.U.) showed maximum clot lysis (p = 0.004) but another three dilutions (22,500 I.U., 15,000 I.U., 7,500 I.U.) of streptokinase also showed approximately same percent of clot lysis (p < 0.05). The mean clot lysis % of streptokinase with all different concentration was found to differ significantly when compared with water (i.e, p < 0.05 for all the concentrations of streptokinase).
Figure 1

Clot-lysis of blood samples of normal subjects (positive and negative control). Tube no. 1 is a control clot (negative control) to which water was added. No clot lysis was observed in tube no.1; a black arrow indicates the intact clot. Tube no. 2–5 (positive control) was lysed by four different concentrations of streptokinase with decreasing order. After dissolution of the clots, tubes were inverted and fluid (blue arrow) along with the remnants of clots (red arrow) could be clearly seen

Figure 2

Clot lysis of blood samples of normal subjects by different concentrations of Streptokinase. Effect of streptokinase on the dissolution of the clots prepared with blood of normal subjects. The clots were treated by four different dilutions of streptokinase i.e., 30,000 I.U, 22,500 I.U., 15,000 I.U., and 7,500 I.U.) and clot lysis % was 70.80, 69.68, 68.93 and 62.16 respectively. Clot lysis % by each dilution of streptokinase differs significantly with water (control) (p < 0.05). The percentage of clot lysis by different concentration of streptokinase ranges from 62–71%

Discussion

This study shows in vitro dissolution of clots by four different dilutions of streptokinase, assayed by an in vitro clot lysis model designed and developed in our laboratory. Most of the in vitro methods that were conventionally or currently applied to study thrombolysis have certain limitations. Some involve complex computation and mathematical skills that too give only theoretical prediction of the outcome and most are expensive to be performed in a laboratory. In context with the current scenario we thought of developing a clot lysis model that would be easy to perform & that should be cost effective too.

Keeping this idea in the prime focus, weight of the clot before lysis and after lysis was considered as appropriate determinant of calculating clot lysis percentage. Blood collected by the standard protocol of venipuncture was allowed to form clots naturally and the weight difference obtained before and after lysis was noted and clot lysis percentage was calculated. In other methods, there are different parameters to analyze the extent of clot lysis. For example, radiolabelling of factors that are actively involved in clot lysis mechanism [13], MRI [15], ultrasound frequency [10], turbidity determination using microtiter plate reader (euglobulin lysis test) [16, 17], study of fibrinolytic activity by circulating fibrinolytic enzymes and monitoring the effect by calculating shear rate [18, 19]. All these methods are sophisticated whereas, our model is simple and easy to perform and one can even visually observe the lysis of clots (as shown in figure 1).

Conclusion

To check the efficacy of thrombolytic drugs or herbs one can compare the data with positive and negative control. In our study we took a known thrombolytic drug; streptokinase as a positive control and water as a negative control. Test drug can also be analyzed in the same way. Thus, our study could be an instant and effective methodology to study clot lytic effect of newly developed drugs as well as known drugs.

Declarations

Acknowledgements

We thank Rashmi. N. Mishra for assistance in statistical analysis.

Authors’ Affiliations

(1)
Biochemistry Research Laboratory, Central India Institute of Medical Sciences
(2)
Environmental Genomics Unit, NEERI, Nehru Marg

References

  1. Allroggen H, Abbott RJ: Cerebral venous sinus thrombosis. Postgrad Med J 2000, 76: 12-15. 10.1136/pmj.76.891.12PubMed CentralView ArticlePubMedGoogle Scholar
  2. Watson RD, Chin BS, Lip GY: Antithrombotic therapy in acute coronary syndromes. BMJ 2002, 325: 1348-1351. 10.1136/bmj.325.7376.1348PubMed CentralView ArticlePubMedGoogle Scholar
  3. Biousse V, Newman NJ: Venous disease of the central nervous system. Seminars in Cardiovascular Diseases and Stroke 2004, 4: 2-17. 10.1053/j.scds.2004.07.001View ArticleGoogle Scholar
  4. Baruah DB, Dash RN, Chaudhari MR, Kadam SS: Plasminogen activators: A comparison. Vascular pharmacology 2006, 44: 1-9. 10.1016/j.vph.2005.09.003View ArticlePubMedGoogle Scholar
  5. Anticoagulant, Antiplatelet, and Fibrinolytic (Thrombolytic) Drugs[http://connection.lww.com/products/craig/documents/Ch22.pdf]
  6. Patel SC, Mody A: Cerebral hemorrhagic complications of thrombolytic therapy. Progress in Cardiovascular Diseases 1999, 42: 217-233. 10.1016/S0033-0620(99)70004-6View ArticlePubMedGoogle Scholar
  7. Haines ST, Bussey HI: Thrombosis and the pharmacology of antithrombotic agents. Ann Pharmacother 1995, 29: 892-904.PubMedGoogle Scholar
  8. Arcasoy SM, Kreit JW: Thrombolytic Therapy of Pulmonary Embolism. Chest 1999, 115: 1695-1707. 10.1378/chest.115.6.1695View ArticlePubMedGoogle Scholar
  9. Khalid Almoosa : Is Thrombolytic Therapy Effective for Pulmonary Embolism? American Family Physician 2002, 65: 1097-1102.PubMedGoogle Scholar
  10. Giuseppina Basta , Cristiana Lupi , Guido Lazzerini , Piero Chiarelli , Antonio L'Abbate , Daniele Rovai : Therapeutic effect of diagnostic ultrasound on enzymatic thrombolysis. An in vitro study on blood of normal subjects and patients with coronary artery disease. Thromb Haemost 2004, 91: 1078-1083.PubMedGoogle Scholar
  11. Beltrami E, Jesty J: Mathematical analysis of activation thresholds in enzyme-catalyzed positive feedbacks: Application to the feedbacks of blood coagulation. Proc Natl Acad Sci USA 1995, 92: 8744-8748. 10.1073/pnas.92.19.8744PubMed CentralView ArticlePubMedGoogle Scholar
  12. Anand S, Diamond SL: Computer Simulation of Systemic Circulation and Clot Lysis Dynamics During Thrombolytic Therapy That Accounts for Inner Clot Transport and Reaction. Circulation 1996, 94: 763-774.View ArticlePubMedGoogle Scholar
  13. Tsuchida-Straeten N, Ensslen S, Schafer C, Woltje M, Denecke B, Moser M, Graber S, Wakabayashi S, Koide T, Jahnen-Dechent W: Enhanced blood coagulation and fibrinolysis in mice lacking histidine-rich glycoprotein (HRG). J Thromb Haemost 2005, 3(5):865-872. 10.1111/j.1538-7836.2005.01238.xView ArticlePubMedGoogle Scholar
  14. Tratar G, Blinc A, Strukelj M, Mikac U, Sersa I: Turbulent axially directed flow of plasma containing rt-PA promotes thrombolysis on non-occlusive whole blood clots in vitro. Thromb Haemost 2004, 91: 487-496.PubMedGoogle Scholar
  15. Strief TW: In vitro simulation of therapeutic thrombolysis with microtiter plate clot-lysis assay. Clin Appl Thrombosis/Hemostatis 2006, 12: 21-32. 10.1177/107602960601200105View ArticleGoogle Scholar
  16. Couto LT, Donato JL, de Nucci G: Analysis of five streptokinase formulations using the euglobulin lysis test and the plasminogen activation assay. Braz J Med Biol Res 2004, 37: 1889-1894. 10.1590/S0100-879X2004001200015View ArticlePubMedGoogle Scholar
  17. Boudjeltia KZ, Cauchie P, Remacle C, Guillaume M, Brohee D, Hubert JL, Vanhaeverbeek M: A new device for measurement of fibrin clot lysis: application to the Euglobulin Clot Lysis Time. BMC Biotechnology 2002, 2: 8. 10.1186/1472-6750-2-8PubMed CentralView ArticlePubMedGoogle Scholar
  18. Greenberg RK, Ouriel K, Srivastava S, Shortell C, Ivancev K, Waldman D, Illig K, Green R: Mechanical versus Chemical Thrombolysis: An In Vitro Differentiation of Thrombolytic Mechanisms. Journal of Vascular and Interventional Radiology 2000, 11: 199-205.View ArticlePubMedGoogle Scholar
  19. Erzsebet Komorowicz , Krasimir Kolev , Istvan Lerant , Raymund Machovich : Flow Rate-Modulated Dissolution of Fibrin With Clot-Embedded and Circulating Proteases. Circulation Research 1998, 82: 1102-1108.View ArticleGoogle Scholar

Copyright

© Prasad et al; licensee BioMed Central Ltd. 2006

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.