ISOLATION AND CHARACTERIZATION OF ANTICOAGULANT PROTEINS FROM THE VENOM OF HEMACHATUS HAEMACHATUS (AFRICAN RINGHALS COBRA) YAJNAVALKA BANERJEE (BSc., (Hons.)) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AT THE NATIONAL UNIVERSITY OF SINGAPORE DEPARTMENT OF BIOLOGICAL SCIENCES, FACULTY OF SCIENCE, NATIONAL UNIVERSITY OF SINGAPORE April, 2007 i Dedicated to the fond memories of late Swami Lokeshwarananda, Secretary, the Ramakrishna Mission Institute of Culture, Calcutta …………………… his life and works, have been a guiding beacon and inspiration for me, and could be best expressed in Paul Byrant’s famous quote “If you believe in yourself and have dedication and pride - and never quit, you'll be a winner. The price of victory is high but so are the rewards.”……….I miss him……… ii en ts em dg le kn ow Ac I am very appreciative of the good camaraderie and academic guidance that I have enjoyed the last few years. However, the initial transition into graduate school, including moving to Singapore, was a bit bumpy. In addition to missing friends and family, I remember feeling overwhelmed by the speed of research and the amount of knowledge that seemed required to design and carry out a thesis project. Having spent a few more years here, I have developed a deep appreciation for the NUS research community. First, I need to acknowledge those who paid the bills during my tenure as a graduate student in NUS. All the four years were supported by a Research Fellowship provided by the National University of Singapore, for which I am extremely grateful. It is probably one of the most generous fellowships available, providing not only tuition and stipend support for up to four years, but also funds for educational expenses and meeting travel. Additionally, I thank the Biomedical Research Council of Singapore, for providing a generous grant to Prof. Kini which funded my work described in this thesis. Next, I’d like to thank the faculty who helped me along the way. First, I will like to thank my supervisor and mentor Professor RM Kini (Prof Kini as I address him). He taught me not only most of what I know about protein chemistry and coagulation biochemistry, but also about how to think and work independently…and he somehow manage to stifle his laughter every time I had to tell him that I had forgotten to sequence a peptide, which I had freeze-dried a week earlier. One of my friends once showed me a quote on scientific research by Wernher Von Braun (1912-1977), “Research is what I'm doing when I don't know what I'm doing"; but working with Prof. Kini I always knew what I am doing and why I am doing a particular experiment; and was that experiment going to answer the question that we asked. In science it is common for other people to pursue what others have done earlier, but I think a good scientist should come up with innovative ideas of his own to tackle a problem. In this regard I remember the famous quote of Samuel Palmer “Wise men make proverbs, but fools repeat them”; Prof Kini always emphasized on designing on ones own experiment(s), which in turn has helped not only me but all others in the lab to develop an analytical frame of mind (a great asset to have when one is working as or trying to become a scientist). Thanks for that!!!!!!!!!!! In addition, I thank Prof Kini for imparting his logical and farsighted approach to scientific research, his attention to detail, and his ability to present complicated ideas with great clarity. I thank my co-supervisor Dr. Jayaraman Sivaraman (Prof. Shiva as everybody addresses him) for his help and helpful suggestions regarding X-ray crystallography. I have benefited greatly from his scientific expertise and career advice. iii I will like to thank my collaborators in Japan. Thank you Mizuguchi-san (not only for helping me in my work, but also for introducing me to the world of Japanese cuisine. I will never forget the taste of sashimi and tongkatsu, that I used to look forward to ones we were able to get the Ki for the inhibition). My hearty thanks to Professor Iwanagasensei at Kaketsuken, his insights into the structure-function relationship of factor VIIa were invaluable in my work. I also thank the factor VIIa group in Kaketsuken for their help and providing me with all the factor VIIa that I required to complete the studies depicted in this thesis. My thanks to Dr Egon Persson (Egon as he has asked me to address him) of Novonordisk. First, thank you for the light chain of FVIIa that you kindly provided for my studies. Second and most importantly thank you for the excellent suggestions that you kindly provided on the part describing the structure-function of TF-FVIIa complex in the introductory chapters of the thesis. If not for Egon it would have been impossible for me write that part. I thank Dr. Prakash Kumar for a large number of excellent suggestions during a meticulous and tireless shepherding process. Thanks to Dr Ganesh Anand, Dr Selvanayagam Nirthanan (Niru) and Dr. Sundarmurthy Kumar for useful discussions. I thank Professor Andre Ménez (Commissariat l’Energie Atomique, Saclay, France) for his time and helpful discussions concerning various aspects of science pertaining to my PhD project during his visits to our department in NUS. Thanks to everyone in Prof Kini’s lab with whom I had a chance to interact. The combination of graduate students and post-docs with diverse backgrounds has made it a tremendous place to learn. In particular, I would like to thank Tse Siang for teaching me the theoretical and practical basics of protein purification and chromatography, Vivek for helping me with nuclear magnetic resonance spectroscopic studies and Gayathri for teaching me how to use the DLS machine. Thanks to Lakshmi for teaching me the basics of circular dichroism. Thanks to Bee Ling for making the lab run so smoothly. Thanks to everyone else in Prof Kini’s lab and others in protein and proteomics centre including: Joanna, Rehana, Robin, Jegan, Ahsan, Arvind, Chow Yeow, Dileep, Shi Yang, Sin Min, Kathleen, Tram, Annabelle, Shifali, Say Tin and Shashikant Joshi. Finally and most importantly, I doubt anybody can make it through the frustrations of Ph.D. research without a social support system. I am fortunate to have two parents, who have not only supported me unconditionally in all my endeavors, but who also instilled in me the work ethic and values to be (more or less) successful at most of them. Not only that, but they never once uttered the parental phrase every grad student dreads: “So when are you gonna graduate and get a job?” I also need to specifically thank the friends who’ve listened to my bitching and moaning, chiefly Lakshmi (not only in Singapore but also after he went to US over the phone) who has endured more hours of complaints than anyone should have to; Of course, there’s no one better to commiserate with than a fellow iv grad student, especially one with whom you can tour the great breweries of the world, and for that purpose Kishore and Reza have always been available. Thanks to Srinivas Rao and Mandar for scientific discussions, beer drinking and excellent house warming parties. In this vein I also thank Naveen, Shilpa, Jaffar, Ali, Hari, Jaspreet, Akhilesh, Bobby, Deven, Vidya, Shalini, Divya and Anand. Thanks to all the people that make the Department run so smoothly. Thanks to Joanne, Reena, Mrs Chan and Annie. Thanks to Cynthia for providing me a separate cubicle for writing my thesis without getting disturbed. Thanks to Tammy for not letting me feel bored, while I was preparing my thesis. Thanks to my many inspirational teachers. In particular, thanks to Ajit Sengupta at Narendrapur Ramakrishna Mission for his excellent lectures at school on diversified topics in Biology and Dr. Biswanath Pyne at Presidency College Calcutta for his caring and clear instruction in biochemistry and human physiology. And there are plenty of other friends and colleagues too numerous to mention who’ve helped me, hopefully if you’re in this group, you know who you are and that I appreciate you! Yajnavalka Banerjee April, 2007 v TABLE OF CONTENTS Page ii Dedication iii Acknowledgement vi Table of contents ix Summary xii Research collaborations xiii Acknowledgement of copyright xix Abbreviation xv List of figures xviii List of tables Chapter One Introduction An overview of blood coagulation, including the anticoagulant pathways. Anticoagulants, targeting specific coagulation enzymes or steps in the coagulation pathway. with a focus on the ones targeting TF-FVIIa complex is given below. Snake venom anticoagulant proteins. Aim and scope of the thesis. 60 Chapter Two Purification of the Anticoagulant Protein Isolation and Purification of hemextin A and hemextin B. Assessment of homogeneity of hemextin A and hemextin B. Determination of complete amino acid sequence of hemextin A and hemextin B. Anticoagulant activity of hemextin A, B and formation of hemextin AB complex. Importance of proper folding of the proteins for mediating anticoagulant activity and complex formation. Preliminary characterization of the complex using gel-filtration chromatography. 87 Chapter Three Mechanism of Anticoagulant Activity vi Identification of the site of action of the anticoagulant protein and synergistic complex using “Dissection Approach”. Serine protease specificity. Kinetics of inhibition and determination of Ki Page 112 Chapter Four Biophysical Characterization of Hemextin AB Complex Conformational changes during complex formation. Changes in molecular diameters during the complex formation. Thermodynamics of hemextin AB complex formation. Effect of temperature on the complex formation. Effect of buffer ionization on the complex formation. Electrostatic interactions in hemextin AB complex formation. Hydrophobic interactions in the hemextin AB complex formation. Effect of buffer conditions on the conformation of hemextins. Model for hemextin AB complex assembly. 162 Chapter Five Molecular Interactions with FVIIa Binding of FVIIa to hemextin AB complex. The effect of temperature on hemextin AB-FVIIa complex formation. Conformational changes associated with hemextin ABFVIIa complex formation. Binding of FVIIa to hemextin A. Binding of hemextin AB complex dimer to FVIIa. Effect of soluble TF on the binding of anticoagulant proteins to FVIIa. Interaction of hemextin A and hemextin AB complex with individual chains of FVIIa. Interaction of hemextin A and hemextin AB complex with active site inhibited FVIIa. 195 Chapter Six Structural Characterization of Anticoagulant Protein Hemextin A Crystallization of hemextin A. Data Collection. Solution of structure and refinement. Analysis of the three-dimensional structure of hemextin A. 215 Chapter Seven Conclusion Conclusions. Future Prospects 224 Bibliography vii Journal, book and web-site references. 262 Publications Articles in internationally refereed journals. Patent. Conference abstracts. Appendix Classification of venomous snakes. Classification of snake venom anticoagulant proteins. Snake venom protein families. Interesting facts on spitting cobra. Publications. ------------------------------------------------------------------------------------------------------------ viii SUMMARY During vascular injury blood coagulation is initiated by the interaction of factor VIIa (FVIIa) present in blood with freshly exposed tissue factor (TF) forming TF-FVIIa complex. As, unwanted clot formation leads to death and debilitation due to vascular occlusion; hence anticoagulants are pivotal for treating thromboembolic disorders. Snake venoms are veritable gold mines of pharmacologically active polypeptide and proteins many of which exhibit anticoagulant activity. Two synergistically acting anticoagulant three-finger proteins, hemextin A and hemextin B were purified from the venom of the elapid Hemachatus haemachatus (African Ringhals cobra) using standard chromatographic procedures. Hemextin A, but not hemextin B has mild anticoagulant activity. However, hemextin B forms a complex (hemextin AB complex) with hemextin A and enhances its anticoagulant potency. Using biophysical techniques including circular dichroism, dynamic light scattering, isothermal titration calorimetry, mass spectrometry and nuclear magnetic resonance, the molecular interactions participating in complex formation were elucidated. Hemextin AB complex exists as a tetramer. Complex formation is enthalpically driven with a negative change in heat capacity, indicating the burial of hydrophobic surface area. The tetrameric complex behaves differently in buffers of higher ionic strength. It is also sensitive to the presence of glycerol in the buffer solution. Thus, a complex interplay of electrostatic and hydrophobic interactions drives the formation and stabilization of this novel anticoagulant protein complex. Based on the results of the above studies, a model was proposed for the assembly of this unique anticoagulant complex. ix Coagulation and kinetic assays showed that hemextin AB complex and hemextin A inhibit clot formation by inhibiting TF-FVIIa activity. Their specificity of inhibition was demonstrated by studying their effects on 12 serine proteases; hemextin AB complex potently inhibits the amidolytic activity of FVIIa either in the presence or in the absence of soluble tissue factor (sTF). This was further confirmed with biophysical experiments. The complex inhibits FVIIa-sTF non-competitively (Ki - 25 nM) and is the first natural inhibitor of FVIIa, which unlike tissue factor pathway and nematode anticoagulant peptide c2 does not use FXa as a scaffold for its inhibitory activity. Molecular interactions involved in the formation of hemextin AB-FVIIa complex and hemextin A-FVIIa complex were also investigated using size-exclusion chromatography and isothermal titration calorimetry. Hemextin A and hemextin AB complex bind to the heavy chain of FVIIa. Binding to FVIIa takes place with equal affinity irrespective of the presence or absence of co-factor. Binding also takes place even when the active site of FVIIa is blocked; this highlights the non-competitive nature of inhibition both for the anticoagulant protein and its complex, which is also supported by enzyme kinetic studies. Since, hemextin A is the only known protein belonging to the three-finger toxin family that exhibits FVIIa inhibitory activity, its three-dimensional strucuture was determined using X-ray crystallography. Hemextin A exhibits the characteristic three-finger fold consisting of six β-strands (β2↓β1↑β4↓β3↑β6↓β5↑) which forms two β-sheets. In conclusion, the present study provides a detailed characterization of an three-finger toxin, hemextin A and its synergistic complex with another three-finger toxin hemextin B. Hemextin AB complex is the only known heterotetrameric complex of three-finger x Submitted to Biochemistry 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 22 of 37 Tables Table 1. Thermodynamics of binding of hemextin AB complex/hemextin A to FVIIa in different buffer solutions KA × 105 (M-1) &H (kcal/mole) &S (cal/deg.mole) &G (kcal/mole) Number of Binding Sites (N) Tetramer in Tris-HCl Buffer 4.11 -7.931 -1.112 -7.586 1.01 Dimer in glycerol 1.77 -3.933 -1.1 -3.62 1.05 Dimer in salt 0.119 -2.994 -1.66 -2.54 0.89 In Tris-HCl buffer 0.125 -3.005 -1.7 -2.5 1.00 In glycerol 0.118 -2.821 -1.74 -2.3 0.97 In salt 0.121 -3.042 -1.56 -2.6 1.03 Binding to FVIIa Hemextin AB complex Hemextin A Note: All experiments were carried at 37 C 22 ACS Paragon Plus Environment Page 23 of 37 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Submitted to Biochemistry Table 2. Thermodynamic analysis of FVIIa binding to hemextin AB complex at different temperatures KA × 105 (M-1) &H (kcal/mole) &S (cal/deg.mole) IG (kcal/mole) Number of binding sites (N) 25 3.84 -6.56 3.322 -7.61 0.97 37 4.11 -7.931 -1.112 -7.586 1.01 45 1.55 -9.0010 -4.55 -7.548 1.08 Binding to FVIIa At different temperatures ( C) 23 ACS Paragon Plus Environment Submitted to Biochemistry 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 24 of 37 Table 3. Thermodynamic analysis of binding to FVIIa and its derivatives to hemextin AB complex/hemextin A KA × 105 (M-1) &H (kcal/mole) &S (cal/deg.mole) IG (kcal/mole) Number of binding site (N) FVIIa 4.11 -7.931 -1.112 -7.586 1.01 sTF-FVIIa 4.06 -7.84 -1.13 -7.49 1.16 FVIIa-Heavy chain 5.2 -7.82 -0.5 -7.67 0.921 FFRck-FVIIa 0.33 -5.01 -3.7 -4.74 1.08 FVIIa 0.125 -3.005 -1.7 -2.5 1.00 sTF-FVIIa 0.127 -3.1 -1.5 -2.69 1.07 FVIIa-Heavy chain 0.798 -4.1 -1.2 -3.7 0.96 FFRck-FVIIa 0.065 -2.3 -1.2 -1.3 0.94 Binding to FVIIa and its derivatives Hemextin AB complex Hemextin A Note : All experiments were carried at 37 C 24 ACS Paragon Plus Environment Page 25 of 37 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Submitted to Biochemistry Figures Figure 1. 25 ACS Paragon Plus Environment Submitted to Biochemistry Figure 2. 88 kDa 57 kDa (12.7 ml) (15 ml) Time (min) 50 100 µcal/s 50 kDa (16 ml) 28 kDa (19.7 ml) 150 B A Hemextin AB complex -16 kcal/mole of injectant 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 26 of 37 FVIIa 0.0 -3.5 -7.0 Hemextin AB complex with FVIIa Molar ratio Volume (ml) 26 ACS Paragon Plus Environment 30 Page 27 of 37 Figure 3. 2.5 A 0.5 T.S kcal/mole -1.5 -3.5 -5.5 .G -7.5 .H -9.5 295 300 305 310 315 320 Temperature (-K) -10 B -9 kcal/mole 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Submitted to Biochemistry .H -8 Ts .G -7 -6 -5 1.5 1.0 0.5 -0.5 -1 T.S (kcal/mole) 27 ACS Paragon Plus Environment -1.5 Submitted to Biochemistry Figure 4. 25 A 0.0 CD in millidegrees 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 28 of 37 225 260 225 260 -20.0 40 B -25 Wavelength in nm 28 ACS Paragon Plus Environment Page 29 of 37 Figure 5. 57 kDa (15 ml) Time (min) 25 50 A 50 kDa (16 ml) B µcal/s -5 kcal/mole of injectant 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Submitted to Biochemistry FVIIa -2 Hemextin A with FVIIa -4 Molar ratio Volume (ml) 29 ACS Paragon Plus Environment 30 Submitted to Biochemistry Figure 6. Time (min) µcal/s 50 100 88 kDa 64 kDa 57 kDa 28 kDa (12.7 ml) (14.6 ml) (15 ml) (19.7 ml) # C (24 ml) kcal/mole of injectant -1 Hemextin AB complex with FVIIa -2 A -4 µcal/s 50 100 Hemextin A with FVIIa 150 Hemextin AB complex with FVIIa in 250 mM glycerol -5 kcal/mole of injectant 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 30 of 37 Hemextin A with FVIIa in 250 mM glycerol -2 B -4 0.0 0.5 1.0 Molar ratio 1.5 Volume (ml) 30 30 ACS Paragon Plus Environment Page 31 of 37 Figure 7. Time (min) 100 200 300 µcal/s 0.0 kcal/mole of injectant # 88 kDa 57 kDa 28 kDa (12.7 ml) (15 ml) (19.7 ml) (24 ml) *(25 ml) C A -0.8 Hemextin AB complex with FVIIa -2 -4 100 200 300 Hemextin A with FVIIa µcal/s 0.0 Hemextin AB complex with FVIIa in 150 mM NaCl -0.5 0.0 kcal/mole of injectant 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Submitted to Biochemistry -1.5 Hemextin Awith FVIIa in 150 mM NaCl B -3.0 Molar ratio Volume (ml) 31 ACS Paragon Plus Environment 30 Submitted to Biochemistry Figure 8. Time (min) 50 kDa 77 kDa 84 kDa (16 ml) (13.84 ml) 27 kDa (13.26 ml) (17.2 ml) 28 kDa 116 kDa (19.7 ml) (11.2 ml) 60 120 µcal/s A Hemextin AB complex -1 -2 kcal/mole of injectant -3 FVIIa -2 B -4 sTF 0.0 0.5 1.0 1.5 2.0 10 20 30 40 µcal/s sTF-FVIIa Hemextin A with sTF-FVIIa Hemextin AB complex with sTFFVIIa Volume (ml) 30 kcal/mole of injectant 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 32 of 37 -5 -10 -15 -2 -4 -6 C -8 0.0 0.5 1.0 1.5 Molar ratio 32 ACS Paragon Plus Environment 2.0 Page 33 of 37 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Submitted to Biochemistry Figure 33 ACS Paragon Plus Environment Submitted to Biochemistry Figure 10 A Time (min) 0.25 50 100 Time (min) 150 200 40 80 120 0.0 ;cal/s 0.00 -0.2 -0.25 B kcal/mole if injectant 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 34 of 37 A C -0.4 -0.50 B -2 -2 -4 -6 -4 0.0 0.5 1.0 1.5 2.0 -8 0.0 Molar ratio 0.5 1.0 Molar ratio 34 ACS Paragon Plus Environment 1.5 2.0 Page 35 of 37 Figure 11 A AP B FVIIa FVIIa B TF 35 ACS Paragon Plus Environment EGF1 A TF EGF1 A B FVIIa TF A EGF1 C A A TF B A B TF FX EGF1 EGF1 TF FVIIa B FVIIa B EGF1 FVIIa EGF1 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Submitted to Biochemistry Submitted to Biochemistry 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 36 of 37 Figure legends Figure 1: Proposed model of hemextin AB complex. A. Schematic diagram depicting the formation of hemextin AB complex. Hemextins A and B, two structurally similar three-finger toxins, form a compact and rigid tetrameric complex with 1:1 stoichiometry. B. Schematic diagram showing the effect of salt and glycerol on the conformations of hemextins A and B. Hemextin A undergoes a conformational change in the presence of salt. C. Dissociation of the tetrameric hemextin AB complex in the presence of salt and glycerol. The dissociation probably occurs in two different planes. Thus the hemextin AB dimer in high salt is different from the dimer formed in the presence of glycerol. Two putative anticoagulant sites are shown with dotted semicircles (See text for details). Figure 2. Binding of hemextin AB complex to FVIIa. (A) ITC studies. Thermogram with the corresponding binding isotherm for the binding of hemextin AB complex to FVIIa in 50 mM Tris buffer (pH 7.4). (B) SEC studies. Elution profiles of the complex of FVIIa with hemextin AB complex in 50 mM Tris buffer (pH 7.4). Note: Upon complex formation there is a reduction in retention time. Figure 3. Thermodynamics of FVIIa-hemextin AB complex formation. (A) Effect of temperature on the energetics of FVIIa-hemextin AB complex interaction: enthalpy change (2H), change in entropy term (T2S) and free energy change (2G). (B) Enthalpy-entropy compensation in complex formation. (Note: Point of intersection of lines corresponding to 2H and 2G corresponds to Ts) Figure 4. Conformational changes associated with the formation of hemextin AB-FVIIa and hemextin A-FVIIa complexes. (A) Spectra for hemextin AB-FVIIa complex ( ), individual spectra of hemextin AB complex ( ) FVIIa ( ) are also shown for comparison. (B) Spectra for hemextin A-FVIIa complex ( ), individual spectra of hemextin A ( ) FVIIa ( ) are also shown for comparison. Figure 5. Binding of hemextin A to FVIIa. (A) ITC studies. Thermogram with the corresponding binding isotherm for the binding of hemextin A complex to FVIIa in 50 mM Tris buffer (pH 7.4).; Note the presence of only one kinetic phase, unlike two as observed in the case of hemextin AB-FVIIa formation (B) SEC studies. Elution profiles of the complex of FVIIa with hemextin AB complex in 50 mM Tris buffer (pH 7.4); Note on complex formation there is a reduction in retention time. Figure 6. Binding of hemextin AB complex or hemextin A to FVIIa in 50 mM Tris buffer (pH 7.4) containing 250 mM glycerol. Thermograms with the corresponding binding isotherms for the binding of (A) hemextin AB complex or (B) hemextin A to FVIIa in Tris buffer 36 ACS Paragon Plus Environment Page 37 of 37 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Submitted to Biochemistry containing 250 mM glycerol. (C) Elution profiles of hemextin AB complex or hemextin A with FVIIa in Tris-HCl buffer containing 250 mM glycerol. Figure 7. Binding of hemextin AB complex or hemextin A to FVIIa in 50 mM Tris buffer (pH 7.4) containing 150 mM salt. Thermograms with the corresponding binding isotherms for the binding of (A) hemextin AB complex or (B) hemextin A to FVIIa in Tris buffer containing 150 mM NaCl. (C) Elution profiles of hemextin AB complex or hemextin A with FVIIa in TrisHCl buffer containing 150 mM NaCl. Figure 8. Binding of hemextin A/hemextin AB complex to sTF-FVIIa. (A) Elution profiles of the complex of sTF-FVIIa with hemextin AB complex/hemextin A. Thermograms with the corresponding binding isotherms for the binding of (B) hemextin A and (C) hemextin AB complex to sTF-FVIIa. (Note: the presence of sTF does not affect the binding between FVIIa and the anticoagulant protein or its complex.). Figure 9. Binding of hemextin A/hemextin AB complex to active site blocked FVIIa (FFRck-FVIIa). (A) Elution profiles of the complex of FFRck-FVIIa with hemextin AB complex/hemextin A (inset, the pooled fractions corresponding to FFRck-FVIIa had [...]... representation of the approaches for TF-FVIIa inhibition Ribbon diagrams of the second and third kuniz domain of TFPI, Mechanism of action of TFPI, Ribbon diagram of the minimized mean structures of NAPc2, Mechanism of action of rNAPc2 The predicted anticoagulant region of anticoagulant PLA2 enzymes, Mechanism of anticoagulant activity of PLA2 Overall structure of FX-bp and FXGD1-44 complex, FIX binding protein, ... increases the rate of the reaction (Fujikawa et al., 1980) This result in the successive generation of two active forms of the factor: aFXIIa and bFXIIa The cleavage of the Arg353-Val354 bond of FXII results in the formation of aFXIIa form of the enzyme, which consists of a heavy and a light chain (353 and 243 amino acid residues, respectively) bound by a disulfide bond The bFXIIa form of the enzyme is... representation of the protein C anticoagulant system (Redrawn with kind permission from The Protein C Pathway” by Charles T Esmon, Chest:2003 Supplement; 26S-32S) In the presence of intact endothelium, thrombin binds to thrombomodulin and activates protein C Endothelial protein C receptor (EPCR) stimulates the activation of protein C Activated protein C counteracts coagulation by cleaving and inhibiting the cofactors... binding protein, Anticoagulant mechanism of factor IX/X-binding protein Anticoagulant mechanism of bothrojaracin Chapter Two 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10 2.11 2.12 2.13 2.14 Anticoagulant activity of the crude venom Size-exclusion chromatography (SEC) of Hemachatus haemachatus crude venom using a Superdex 30 column Cation exchange of peak 3 from SEC RP-HPLC profiles of hemextin A and hemextin... generated after the hydrolysis of two more peptide bonds Arg334-Asn335 and Arg343-Leu344 These cleavages result in the formation of a 30 kDa enzyme, consisting of the light chain of the enzyme supplemented with a small fragment of the heavy chain (Cochrane et al., 1973;Revak and Cochrane, 1976;Revak et al., 1977;Revak et al., 1978) Both the forms of FXIIa, activate FXI to FXIa by the hydrolysis of the Arg369-Ile370... addition, thrombin is the key activator of platelet aggregation at the site of injury (Davey and Luscher, 1967;Brass, 2003b) Platelets form a plug that stops the hemorrhage and prevents further blood loss Also, during the activation process a multitude of proteins is released at the site of injury that initiate the process of tissue repair These include plasma proteins such as von Willebrand factor (vWF),... converted to their serine proteases FIXa and FXa, which then form the intrinsic tenase and the prothrombinase complexes, respectively The combined actions of the intrinsic and extrinsic tenase and the prothrombinase complexes lead to an explosive burst of the enzyme thrombin (IIa) In addition to its multiple procoagulant roles, thrombin also acts in an anticoagulant capacity when combined with the cofactor... damage or activation of the endothelium This may occur due to the perforation of the vessel wall or activation of the endothelium (Geczy, 1994) Upon activation of endothelial cells (EC) by an injury, ECs immediately release vasoactive agents, such as endothelin (ET) (a potent vasoconstrictor) and nitric oxide (NO) that counteracts endothelin In states of EC dysfunction the concentrations of bioactive NO... Rechromatography of hemextin A and B ESI-MS of hemextin A and B Comparison of amino acid sequence of hemextin A and hemextin B with other sequences of the three-finger toxin family CD spectra Effects of hemextins A and B on prothrombin time Complex formation between hemextins A and B is illustrated by their effect on prothrombin time Gel filtration studies on the formation of hemextin AB complex Anticoagulant. .. popular under the name of the “waterfall” or the “cascade” hypothesis Over the years * “AFTER years of confusion, it seems that a relatively simple pattern is emerging from present theories of blood coagulation Its recognition is assisted by the Roman numeral terminology of the International Committee on Blood Clotting Factors, which, by displacing a profusion of synonyms, allows the basis of factual . i ISOLATION AND CHARACTERIZATION OF ANTICOAGULANT PROTEINS FROM THE VENOM OF HEMACHATUS HAEMACHATUS (AFRICAN RINGHALS COBRA) YAJNAVALKA BANERJEE (BSc., (Hons.)) A THESIS. synergistically acting anticoagulant three-finger proteins, hemextin A and hemextin B were purified from the venom of the elapid Hemachatus haemachatus (African Ringhals cobra) using standard chromatographic. Two Purification of the Anticoagulant Protein Isolation and Purification of hemextin A and hemextin B. Assessment of homogeneity of hemextin A and hemextin B. Determination of complete amino