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BLOOD COAGULATION: AT THE INTERFACE BETWEEN PHYSICAL AND CHEMICAL KINETICS Slide 2 2006 UpToDate 2006 UpToDate www.uptodate.com Contact Uswww.uptodate.comContact Us e-mail this to a colleague Slide 3 2006 UpToDate 2006 UpToDate www.uptodate.com Contact Uswww.uptodate.comContact Us e-mail this to a colleague Slide 4 FUSTER et al, 2005 Slide 5 Slide 6 FUSTER et al., 2005 Slide 7 Local homeostasis maintained by continuous adjustments to changes threshold Tissue PERTURBATION RESPONSE COAGULATION Hemostasis thrombophilia hemophilia Disseminated coagulation INFLAMMATION Immunity hypersensitivity autoimmunity Immunodeficiency REACTION RATE CHANGE Slide 8 PLASMA COAGULATION / FIBRINOLYSIS PROCOAGULANT SURFACES FLOW Slide 9 Slide 10 COAGULATION CONTROL INHIBITORS HEPARIN (Glycosaminoglycans) Warfarin (Vit.-K antagonist) Anti-platelets PROCOAGULANTS COAGULATION FACTORS BLOOD TRANSFUSIONS STATINS ? Inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A Reduce cholesterol levels and cardiovascular disease but Under the best circumstances 2/3 cardiovascular events remain. Slide 11 Contact activator autoactivation of fXII Pre to kallikrein H-kininogen to bradikinin B1 and B2 receptors Of endothelial cells PLA 2 ->PG->cAMP Ca 2 +->eNOS->NO-> cGMP RELAXATION Bradikinin - tPA Plasminogen Fibrin Plasmin FIBRINOLYSIS Hereditary angioedema Slide 12 Spaan J. A. E. et al., 2003 Slide 13 Slide 14 Slide 15 Slide 16 Slide 17 Slide 18 A + B AB PRODUCT (1nmol ~ 10 14 molecules) FLUX : convective pressure gradient diffusive concentration gradients FLOW-DEPENDENT TRANSFER? SURFACE DIFFUSION ON CELLS? Slide 19 A + B K A K d [AB] K f >> PRODUCTS PROTOTYPE REACTION SCHEME K eff = K A.K f / K d + K f K d K eff = K A K f Slide 20 Gla Domain Gamma-carboxylated Glutamic acid residues Serine proteinase Domain Aspartic acid Histidine SERINE Growth factor-like domain MODULAR STRUCTURE OF COAGULATION ZYMOGENS Slide 21 Slide 22 Slide 23 Slide 24 Slide 25 DIFFUSION-CONTROLLED REACTIONS IN BIOLOGICAL SYSTEMS Soluble enzyme kinetics Steady-state diffusion-controlled reaction (Smoluchowski) Influence of intermolecular forces (Kramers, Debye) Hydrodynamics effects (Friedman, Deutch) (osmotic stress techniques?) Convective flow (Levich, Delichatsios) Diffusion toward an array of reactive traps. Spherical Influence of chemical reaction on diffusion in the reactive system Mean first passage times. Slide 26 Glycosaminoglycans role in the local regulation of interstitial fluid volume Local hydration = competitive affinity of macromolecules for water Glycosaminoglycans form hydrophilic gels that store dehydration energy In vivo manifested as a dehydrating potential. In vitro detected as swelling pressure CARTILAGE VASCULATURE Slide 27 Polymer rubber elasticity H20H20 Polymer-polymer affinity H + Pressure OSMOTIC FORCES Slide 28 GALACTOSAMINOGLYCANS Slide 29 Slide 30 Slide 31 AT + GAGATGAG+fXaATGAG-fXa AT-fXa GAG K 1/2 V sat Slide 32 G/ = V K Antithrombin surface1.4 under 4.0 Water activity Slide 33 Factor Xa nM Slide 34 Osmotic Pressure (atm) G ( kcal/mol) Slide 35 TABLE 1 Probe radius, () (kcal/mol/atm)(water mol/mol) PEG: 300 4 -0.072 0.011 165 25 600 7 -0.221 0.060 508 138 1500 9 -0.336 0.031 772 71 3400 17 -1.16 0.11 2667 252 8000 26 -1.083 0.2072489 475 Dextran T10 24 -1.287 0.113 2958 260 PVP 40 NA - 1.314 0.176 3028 404 Water Transfer by Osmotic Stress Technique with Polymers of Different Size and Chemical Structure Slide 36 ABC PI RCL C-sheet hH B-sheet hD hB hE A-sheet hC hF hl hA hG Slide 37 Dermatan Sulfate, M RATE s -1 X10 3 1 1 1 Slide 38 Dermatan Disulfate, M RATE s- 1 X 10 3 Slide 39 GAG solutionStandardOsmotic Stress (0.5 atm) CSE in: K 1/2, ( V sat (s -1 x 10 3 ) K 1/2, ( V sat (s -1 x 10 3 ) 0.15 N, NaCl3.3 1.95.3 1.34.3 1.616 3 0.15 N, NaCl, 2mM CaCl 2 2.7 0.85.7 0.72.9 0.420 1 0.075 N NaCl4.3 2.86.1 1.85.2 2.315 3 0.075 N NaCl,2mM CaCl 2 1.3 0.413.8 0.10.34 .0544 1 0.15 N NaCl, 5mM CaCl 2 4.8 0.813.2 0.12.7 0.434 2 HS in: 0.15 N, NaCl4.0 0.648 45.5 0.9104 10 15 N, NaCl, 2mM CaCl 2 6.9 0.997 56.3 0.4189 5.075 N NaCl6.6 0.878 44.8 0.3119 3.075 N NaCl, 2mM CaCl 2 9.2 1.8204 19.45.1 0.8283 17 Slide 40 GAG solution Standard Osmotic Stress (0.5 atm) DS in: K 1/2, ( V sat (s -1 x 10 3 ) K 1/2, ( V sat (s -1 x 10 3 ) 0.15 N, NaCl 74 784 5 37 6 120 8 0.15 N, NaCl, 2mM CaCl 2 17 397 6 16 1 136 5 0.075 N NaCl 62 4125 5 26 2 136 5 0.075 N NaCl,2mM CaCl 2 19 6163 21 3 1 116 8 DDS in: 0.15 N, NaCl 17 356 5 9.4 2 104 10 15 N, NaCl, 2mM CaCl 2 5.1 0.473 2 1.7 0.3 106 5.075 N NaCl 7.5 0.656 2 3.8 0.2 97 2.075 N NaCl, 2mM CaCl 2 2.1 0.273 2.5 0.4 0.8 107 1 Slide 41 TABLE 3 Disaccharide Structure and Anticoagulant Function. Increased Efficiency with Osmotic Stress and Calcium Hexuronic Acid Hexosamine sulfation pattern Efficiency M -1 s -1 Dermatan Sulfate Iduronic C4 4 X 10 4 (34) Dermatan Disulfate Iduronic C4, C6 2 X 10 5 (81) Chondroitin Sulfate E Glucuronic C4, C6 1 X 10 5 (81) Heparan SulfateIduronic C2, N (variable) 5 X 10 4 (5) Efficiency was estimated as the ratio between V sat and K 1/2 determined in titrations of antithrombin activity, under an osmotic stress of 0.5 atm, with the indicated glycosaminoglycans in TRIS buffer pH 7.4, 0.075 N NaCl and 2mM CaCl 2 The increases in efficiency relative to reactions in standard TRIS buffer pH 7.4, 0.15N NaCl are indicated in parentheses. Slide 42 GLYCOSAMINOGLYCANS REGULATE REACTIONS IN BIOGELS Slide 43 Qui alium sequitur, nihil invenit, immo nee quaerit. SENECA (He who follows another not only discover nothing, but is not even investigating. Translation by RG Gummere)