Abstract: Objectives The purpose of this study was to show the contrast-enhanced sonographic features of various levels of renal artery rupture and to validate the therapeutic effects of percutaneous 915-MHz microwave ablation compared to hemostatic drug injection (batroxobin) using an in vivo canine renal artery injury model Methods Three renal artery hemorrhage models (A, diameter <1 mm, subcapsular artery; B, diameter 1–2 mm, interlobar artery; and C, diameter 2–3 mm, segmental artery) were created in 24 canines for this study Contrast-enhanced sonography was used to show the bleeding features and guide hemostatic therapies using 915-MHz microwave ablation and local batroxobin injection Success rates were assessed according to amounts of bleeding, times required for hemostatic action, and volumes of fluid infusion required using pathologic examination as a reference standard Results Contrast-enhanced sonography clearly showed renal artery ruptures with active bleeding at various levels and degrees and was very useful to make diagnoses and guide therapies The success rate in the microwave treatment group was higher than that in the drug injection group (except group A; P< 05) The time required for hemostasis and the volume of fluid infusion required in the microwave group were notably less than those in the drug injection group (P < 05) Conclusions Contrast-enhanced sonography is a useful imaging method for assessing renal vessel injury and guide interventional therapies Contrast-enhanced sonographically guided percutaneous 915-MHz microwave ablation is a preferred hemostatic technique for treatment of renal artery injury, with greater effectiveness and less tissue damage compared to local drug injection

Abstract: Objective To investigate the interactive effects between batroxobin and low molecular weight heparin（LMWH）in reducing peri-operative blood loss and coagulation function in patients who undergone the total hip replacement surgery. Methods 240 ASA Ⅰ-Ⅲ patients received 4 000 IU LMWH 12 hours preoperatively before undergoing the total hip replacement operation,were randomly divided into two groups：testing group （Group A,n=120） and control group（Group B,n=120）receiving 2 U batroxobin or 50 mg mannitol 10 minutes before incision respectively. Perioperative blood loss,postoperative 24 hours drainage and blood routine test, prothrombin time （PT）,activated partial thromboplastin time（APTT） and fibrinogen（FIB） were measured respectively. Deep vein thrombosis （DVT） were measured through color Doppler B-ultrasound 3 days after the operation. Results The perioperative blood loss in Group A （422.64 ml）was less than that in Group B（667.67 ml）（P0.05）. There were no drug-related adverse effects found in the two groups,neither the difference in hospitalization between the two groups（P〉0.05）. Conclusion Batroxobin （2 U） could reduce the perioperative blood loss in patients with LMWH who had undergone the total hip replacement operation but did not show adverse effect on DVT. Key words: Low molecular weight heparin; Batroxobin; Total hip replacement; Blood loss

Abstract: ........................................................................... iii ACKNOWLEDGEMENTS......................................................... vii LIST OF FIGURES.................................................................. xv LIST OF TABLES.................................................................... xvii LIST OF ABBREVIATIONS...................................................... xviii 1.0 CHAPTER 1: GENERAL INTRODUCTION.................... 1 1.1 OVERVIEW OF HEMOSTASIS...................................... 1 1.2 OVERVIEW OF THROMBOSIS..................................... 2 1.3 OVERVIEW OF THE COAGULATION SYSTEM................ 3 1.4 EXTRINSIC (OR TISSUE FACTOR) PATHWAY OF COAGULATION......................................................... 7 1.5 INTRINSIC (OR CONTACT) PATHWAY OF COAGULATION......................................................... 8 1.6 COMMON PATHWAY OF COAGULATION...................... 10 1.7 THROMBIN............................................................... 10 1.8 FIBRIN(OGEN).......................................................... 11 1.8.1 γ'-Fibrinogen........................................................... 14 1.8.2 The γ'-chain modulates activity of fibrin-bound thrombin....... 14 1.8.3 The γ'-chain modulates fibrin clot formation and structure...... 16 1.8.4 Impact of γ'-chain on fibrin(ogen) binding to platelets.......... 17 1.8.5 Role of γA/γ'-fibrin(ogen) in pathophysiology.................... 17 1.9 BATROXOBIN.......................................................... 19 1.10 ROLE OF THE EXTRINSIC AND INTRINSIC PATHWAYS IN HEMOSTASIS & THROMBOSIS................................ 20 1.10.1.11.10.1.2 Evidence for the extrinsic pathway in hemostasis & thrombosis............................................................. 20 1.10.2.11.10.2.2 Evidence for the intrinsic pathway in hemostasis & thrombosis............................................................. 22 1.11 FXII STRUCTURE, ACTIVATION & REGULATION........... 24 1.11.1 FXII structure......................................................... 24 1.11.2 FXII activation........................................................ 27 1.11.3 Inhibitors of FXIIa................................................... 28 1.11.4 Novel inhibitors of FXIIa........................................... 29 1.11.5 Mechanisms of HRG inhibition of FXIIa......................... 29 1.11.6 Epidemiological studies of FXII and the risk for thrombosis... 30 1.12 POLYANIONIC ACTIVATORS OF THE INTRINSIC PATHWAY............................................................... 31 1.12.1 Polyphosphate.......................................................... 31 1.12.2 DNA and RNA........................................................ 35 1.12.3 Role of DNA in thrombosis.......................................... 35 1.12.4 Role of RNA in thrombosis.......................................... 36 x 1.12.5 Circulating DNA and RNA as prognostic markers of thrombosis............................................................ 37 1.12.6 Mechanisms of nucleic acid-mediated activation of the intrinsic pathway...................................................... 38 1.12.7 Role of nucleases in regulating nucleic acid-mediated activation of coagulation............................................. 39 1.13 HISTIDINE-RICH GLCYOPROTEIN................................ 40 1.13.1 Protein synthesis and plasma levels................................ 40 1.13.2 Domain structure and proteolytic cleavage by plasmin and kallikrein............................................................... 41 1.13.3 HRG ligands in coagulation and fibrinolysis..................... 43 1.13.4 HRG modulation of hemostasis and thrombosis.................. 44 2.0 CHAPTER 2: OVERVIEW, HYPOTHESIS & OBJECTIVES........................................................... 48 2.1 Thesis overview.......................................................... 48 2.2 Rationale.................................................................. 48 2.3 Hypothesis................................................................ 49 2.4 Overall objectives........................................................ 49 2.4 Objective 1: Characterization of the HRG interaction with γA/γAand γA/γ' fibrin(ogen)..................................................... 49 2.4.1 Objective 2: Characterization of the mechanisms of batroxobin binding to γA/γAand γA/γ'-fibrin(ogen)................................. 49 2.4.2 Objective 3: Characterization of the mechanisms of HRG attenuation of DNAand RNA-mediated activation of the contact pathway................................................................... 50 2.4.3 Objective 4: Characterization of the role of HRG in hemostasis and thrombosis using a mouse model ................................ 51 3.0 CHAPTER 3: Histidine-rich glycoprotein binds fibrin(ogen) with high affinity and competes with thrombin for binding to the γ'-chain............................................................... 52 3.1 Forward.................................................................... 52 3.2 Abstract............ ....................................................... 53 3.3 Introduction............................................................... 54 3.4 Experimental Procedures................................................ 56 3.4.1 Materials................................................................... 56 3.4.1.1 Reagents............................................................... 56 3.4.1.2 Preparation of fibrinogen fragments............................... 57 3.4.1.3 Preparation of γ'-directed IgG Fab fragments..................... 58 3.4.1.4 Labeling of proteins.................................................. 58 3.4.2 Methods................................................................... 59 3.4.2.1 Surface plasmon resonance (SPR)................................. 59 3.4.2.2 SPR data analysis.................................................... 61

Abstract: Many types of hemostatic agents have been studied for the effective control of bleeding In this study, a powdery medical adhesive composed of aldehyded dextran and e-poly (L-lysine) was used with the recombinant batroxobin Batroxobin is a venomous component from the snake Bothrops atrox moojeni and catalyzes fibrinogen conversion to form soluble fibrin clots This research aims to examine the performance of the batroxobin-containing adhesive for hemostasis, and evaluate its potential as a novel hemostatic adhesive The fibrinogen conversion ability of batroxobin was evaluated by a fibrinogen clotting assay and a whole blood clotting assay Both experiments demonstrated the effectiveness of the batroxobin-containing adhesive for blood clot formation Animal experiments were also conducted After a pricking wound was made in an ICR (imprinting control region) mouse liver, the adhesive and various concentrations of batroxobin were applied The total amount of blood loss was reduced with increasing concentrations of batroxobin For excessive bleeding conditions, the femoral artery wound model of SD (Sprague-Dawley) rats was adopted With higher concentrations of batroxobin, hemostasis was more rapidly achieved Histological analysis of the liver model also supports the hemostatic effects through fibrin clot formation In conclusion, batroxobin and medical adhesive effectively facilitate blood coagulation, and could be developed for clinical use