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MATERIALS AND METHODS ...330 RESULTS ...331 DISCUSSION ...333 REFERENCES ...335 SEcnON VI: THE MARATEA CONFERENCE ...337 PHOTO LAYOUT ...339 PARTICIPANTS ...341 INDEX ...*. **. ****************************** 345 xxv SECTION I Biochemistry and cell biology of serine proteases and serpins THROMBIN STRUCIURAL REGIONS IN DETERMINING BIOREGULATORY FUNCTIONS JOHN W. FENTON n Wadsworth Center for Laboratories & Research, New York State Department of Health, Empire State Plaza, Box 509, Albany, NY 12201, and Department of Physiology, The Albany Medical College of Union University, Albany, NY 12208, USA INTRODUCTION Thrombin (EC 3. 4. 21. 5) is the activation product of its blood-circulating or cellular-sequestered zymogen, prothrombin. Unlike the majority of enzymes and activated proteins of the blood-coagulation, fibrinolytic, and complement systems, thrombin has multiple bioregulatory functions, which are central in hemostasis, wound 1 6 healing, and perhaps developmental, as well as certain disease processes. - Hemostasis occurs at three levels, which are: i) plasma proteins (e. g. , coagulation factors V, VIII, and IX; proteins C and S, complement components), ii) blood cells (e. g.
, platelets, monocytes, neutrophils), and iii) blood vessels (e. g. , endothelium, smooth muscles). Moreover, thrombin functions at all three levels of hemostasis, while the majority of cell types known are responsive to greater or lesser extents to thrombin. Thrombin furthermore functions both as a proteolytic enzyme with arginine- or lysine-directed specificities or as a nonenzymic activated protein 1 6 (hormone-like) involving receptor occupancy on certain cells.

Contents:

Section I: Biochemistry and Cell Biology of Serine Proteases and Serpins.- 1: Thrombin Structural Regions in Determining Bioregutatory Functions.- Acknowledgments.- References.- 2: Regulation and Control of the Fibrinolytic System.- Main Components of the Fibrinolytic System.- Plasminogen.- Physiological Plasminogen Activators.- Tissue-type plasminogen activator (tPA).- Urokinase-type plasminogen activator (uPA).- Inhibitors of the I1brinolytic System.- Alpha2-Antiplasmin.- Plasminogen Activator Inhibitor-1 (PAL-1).- Regulation and Control of Physiological Fibrinolysis.- Molecular Interactions Between the Components of the Fibrinolytic System.- Mechanism of Action of tPA.- Mechanism of Action of uPA.- Pathophysiological Aspects of Fibrinolysis.- Excessive Fibrinolysis.- Alpha2-Antiplasmin Deficiency and Bleeding.- Excess tPA Levels and Bleeding.- Impairment of Fibrinolysis.- PAI Activity and Thrombosis.- Plasminogen Deficiency and Thrombosis.- Plasminogen Activator Deficiency and Thrombosis.- Conclusion.- References.- 3: A Key Molecule Dictating and Regulating Surface Plasmin Formation: The Receptor for Urokinase Plasminogen Activator.- Physiologically Invasive Processes.- Plasmin Formation.- The uPA Receptor.- Receptor-binding region in uPA.- Structure of the uPA receptor.- uPA Receptors and the Focal Adhesion Sites.- Plasminogen Activation with Surface-Bound Reactants.- Regulation of the uPA-uPA Receptor Interaction.- Conclusions.- References.- 4: Regulation of Tissue Plasminogen Activator Secretion from Human Endothelial Cells.- Results.- Discussion.- References.- 5: Thrombin Disintegrates Cell Surface Urokinase Focal Adhesion Plaques and Decreases Cell Extension: Implications for Axonal Growth.- Materials and Methods.- Materials.- Methods.- Cell culture.- Interference reflection microscopy.- Immunofluorescence labeling of urokinase and vinculin.- Assay of the Growth Factors.- Results.- Discussion.- Acknowledgments.- References.- 6: Structure and Function of Tissue-Type Plasminogen Activator.- Biology of tPA.- tPA is a Mosaic Protein.- Interaction with plasminogen.- Interaction with PAI-1.- Interaction with fibrin.- Interaction with receptors in the liver.- Extravascular Cofactors.- Heparin.- Fibronectin.- Thrombospondin and Histidine-Rich Glycoprotein.- Miscellaneous.- References.- 7: The Heparin Binding Site and Activation of Protease Nexin I.- Protease Nexin I.- Antithrombin III.- Heparin Cofactor II.- Methods.- Results and Discussion.- Heparin Binding Serpins: Structure and Reactive Centre.- Protease Nexin I: Identification of the Heparin Binding Site.- Evidence From Antithrombin mutants....- Modelling of the Site.- Conclusion.- Acknowledgments.- References.- 8: Polypeptide Chain Structure of Inter-?-Trypsin Inhibitor and Pre-?-Trypsin Inhibttor: Evidence for Chain Assembly by Glycan and Comparison with Other "Kunin"-Containing Proteins.- Experimental Procedures.- Materials.- Polyacrylamide Gel Electrophoresis.- Trypsin Inhibitor Counterstained Gels.- Protein Purification.- Protein Fragmentation.- Amino Acid Sequence Analysis.- Chemical Deglycosylation.- Enzymatic Deglycosylation.- Zone Electrophoresis of I?I and P?I kDa.- Results.- Purification of SDS-Stable Trypsin Inhibitors from Human Plasma.- Identity of the Inhibitors.- Chain Composition.- Chain Stoichiometry.- Composition of the Crosslinks(s).- Location of the Crosslink.- Multiple Forms of Kunin-Containing Proteins.- Summary.- Acknowledgments.- References.- 9: Regulation of Protease Nexin I Activity and Target Protease Specificity by the Extracellular Matrix.- Results.- Fibroblasts Accelerate the Inactivation of Thrombin by PNI.- Fibroblasts Block the Ability of PNI to Inactivate Urokinase and Plasmin.- Discussion.- Summary.- Acknowledgments.- References.- Section II: Molecular Biology of Serine Proteases and Serpins.- 10: Induction of the Urokinase-Type Plasminogen Activator Gene by Cytoskeeton-Disrupting Agents.- Materials and Methods.- Materials.- Cell Culture.- cDNA Probes.- RNA Analysis.- Nuclear run on-transcription.- CAMP-dependent Protein Kinase Assay.- DNA Transfection and CAT Assay.- Transgenic Mice.- Results.- Colchicine Induces uPA mRNA and Gene Transcription.- Cytochalasin B Also Induces uPA Gene Expression.- Induction of uPA mRNA by Colchicine or Cytochalasin B Treatment Involves Protein kinase C.- Induction of uPA Gene Expression is Mediated by Cis-Acting Element.- Expression of uPA LacZ Chimeric Gene in Transgenic Mice.- Discussion.- References.- 11: Use of Protein Chemistry and Molecular Biology to Determine Interaction Areas Between Proteases and Their Inhibitors: The Thrombin-Hirudin Interaction as an Example.- Properties of Thrombin and Hirudin.- Thrombin.- Hirudin.- Kinetic Mechanism of the Inhibition of Thrombin by Hirudin.- Identification of Interaction Areas on Thrombin.- Derivatives of ?-thrombin With Modified Active Sites.- Proteolysed Forms of Thrombin and Peptide-Specific Antibodies.- Protection Against Chemical Modification and Proteolysis.- Identification of Interaction Areas on Hirudin.- Importance of Basic Amino Acid Residues.- Ionic Interactions Involving the C-terminal Region.- Interaction With the N-terminus.- Conclusions.- References.- 12: Signal Transduction Chains Involved in the Control of the Fibrinolytic Enzyme Cascade.- Hormonal Modulation of the Fibrinolytic System.- Protein Kinase C-dependent Pathway.- Tyrosine Specific Protein Kinase Dependent Pathway.- Regulation by Steroid Hormones.- Regulation by Cyclic AMP.- Regulation Via the Hypothalamic-Gonadal Axis.- Regulation by Cytokines.- Summary.- References.- 13: Rodent Serpins: Accelerated Evolution and Novel Specificities.- Materials and Methods.- Genetic Analysis.- Cosmid Clone Analysis.- Sequence Analysis of Exon 5.- Results and Discussion.- Serpin Genes and Genetics.- Structure of the Spi-2 Locus.- Evolution of the Reactive Centre Region of the Serpins.- Accelerated Evolution in the Reactive Centre Domain.- Reactive Centre Regions Within the Spi-2 Gene Cluster.- Expression of the Spi-2 Genes in Mouse.- References.- 14: Structure of the Human Protease Nexin Gene and Expression of Recombinant forms of PNI.- Materials and Methods.- Tissue Culture.- Preparation of mRNA and Northern Blot Analysis.- Construction of Human Genomic Library.- Hybridization Procedures.- DNA Manipulations.- DNA Sequencing and Analysis.- Expression of PNI in CHO Cells.- Expression of Recombinant PNI in Insect Cells.- Thrombin Inhibition Assay of PNI.- PNI/Protease Complex Formation.- Results.- Identification of Two Different Forms of PNI.- Analysis of PNI mRNA Expression.- Differential Expression of PNI and Plasminogen Activator.- Structure of the PNI Gene.- Comparison of the PNI Gene and the PAI-1 Gene.- Expression of Recombinant PNI in Mammalian and Insect Cells.- Characterization of CHO Recombinant Alpha PNI.- Discussion.- Summary.- References.- 15: Evolutionary Adaptations of Serpins and Their Use in Designing New Proteinase Inhibitors.- How Do Serpins Function as Inhibitors?.- Is Serpin Complex Formation Reversible?.- How are Serpins Regulated?.- Is Carbohydrate Required for Inhibitory Activity?.- Are all Elastase Specific Serpins Oxidative sensitive?.- What are the P1 Residues in Naturally Occurring Oxidation Resistant Elastase Inhibitors?.- How Have Elastase Inhibitors Evolved?.- Can We Change Inhibitor Specificity?.- Summary.- References.- Section III: Serine Proteases in the Nervous System.- 16: Plasminogen Activator in the Developing Nervous System.- Methods.- Cell Cultures.- Plasminogen Activator Assays.- 125I-tPA Binding Assay.- Proteolysis of Fibronectin.- tPA mRNA.- Results.- Discussion.- Acknowledgments.- References.- 17: Multiple Roles for Plasminogen Activator System in Nervous System Development.- Neuronal Migration.- Glial Proliferation.- Neuritic Fasciculation.- Neuritogenesis.- Conclusions.- References.- 18: Interaction of Plasminogen Activators with the Neuronal Surface.- Neuronal uPA.- Neuronal Inhibitor of uPA.- Neuronal tPA.- References.- 19: Glial Plasminogen Activators in Developing and Regenerating Neural Tissue.- Results and Discussion.- Modulation of Neural Cytoarchitecture by PA/Plasmin In Vitro.- PAs in Schwann Cells.- PAs in Differentiating Astroglia.- Role of Plasmin/PA Activities in PNS Regeneration.- Concluding Remarks.- Acknowledgment.- References.- 20: Plasminogen Activators in Development, Injury and Pathology of the Neuromuscular System.- Release of PAs from Muscle after Axotomy or Nerve Crush.- Muscle PAs Increase After Muscle Axotomy.- The Nerve Crush Paradigm.- Degradation of Muscle Adhesive Macromolecules by PAs.- Muscle PAs Decline During Synapse Elimination.- PAs in Lower Motor Neuron Diseases.- The Mutant Wobbler Mouse.- Study of PA and Inhibitors in ALS.- Conclusion.- Acknowledgments.- References.- 21: Relationship Between Plasminogen Activators and Regeneration Capacities of Rat Skeletal Muscle.- Materials and Methods.- Chemicals and Reagents.- Preparation of Muscle Extracts.- Cell Cultures.- Electron Microscopy.- Regeneration Studies.- Assay of PAs.- Miscellaneous.- Results.- In Vivo Activities.- In Vitro Activities.- Regeneration Studies.- tPA.- uPA.- Discussion.- Conclusion.- Acknowledgments.- References.- Section IV: Balance of Proteases and Serpins in the Nervous System.- 22: A Cascade Approach to Synapse Formation Based on Thrombogenic and Fibrinolytic Models.- Proteases at the Neuromuscular Junction.- Inhibitors of Serine Proteases.- Kunitz-Type Inhibitors (Kunins).- Serpins.- Protease nexin I.- Plasminogen activator inhibitors 1,2 and 3.- Protease nexin II (PNII).- Serpins in yhe Nervous System.- Serpins in the Central Nervous System.- Neurite Outgrowth Promotion.- Neuronal Survival.- Distribution of Serpins in the CNS.- Serpins in the Peripheral Nervous System.- Thrombogenic and Fibrinolytic Cascade Models of Synapse Formation and Plasticity.- Summary.- References.- 23: Localized Extracellular Proteolysis May Convey Inductive Signals Between Nerve and Muscle Cells During Synaptogenesis.- Short-Range Cellular Interactions.- Neural Regulation of Muscle Basal Lamina Proteoglycan.- Neural Remodeling of the Muscle Basal Lamina.- Desorptive Actions of Inductive Polymer Microbeads.- Alternative Modes of Inductive Cell Communication.- Possible Transduction of Pericellular Proteolysis by Ecm Receptors.- Indirect Proteolytic Regulation of Growth Factor Receptors.- Regulation of Cellular Adhesion, Proliferation and Differentiation.- Summary.- References.- 24: Steps in Establishing a Biological Relevance for Gliaderived Nexin.- Neurite Outgrowth Due to Serine Protease Inhibition.- Characterization of The Primary Sequence.- GDN Biological Effects in Vitro.- Localization of GDN.- Up-Regulation of GDN Following Rat Sciatic Nerve Lesion.- Conclusion.- References.- Section V: Serpins in Degenerative and Malignant Neurologic Diseases.- 25: Protease Inhibitors in Neurologic Diseases.- Protease Inhibitors in Amyotrophic Lateral Sclerosis.- Protease Inhibitors in Familial Amyloidotic Polyneuropathy.- Direct measurement of inhibitors in sera.- Indirect assumption of the state of protease and protease inhibitors from the clinical symptoms of FAP.- Possible Application of Proteases to the Management of FAP.- Protease Inhibitors and Other Neurologic Diseases.- Concluding Comments.- Acknowledgments.- References.- 26: Presence and Significance of ?1-Antitrypsin in Human Brain Tumors.- Material and Methods.- Biochemical Studies.- Immunohistochemical Methods.- Results.- Biochemical Results.- Immunohistochemical Results.- Discussion.- Summary.- Acknowledgment.- References.- 27: Serpins and Brain Tumors: Roles in Pathogenesis.- Experimental Procedures.- Materials.- Methods.- Cell culture.- Preparation of conditioned media from 9L cells.- Tissue processing.- Concanavalin-A chromatography.- Inhibition or release of labeled extracellular matrix.- Fibrin zymography.- Reverse fibrin zymography.- Radioiodination of proteins.- Complex formation studies.- Extraction and purification of RNA.- Northern hybridization.- Results.- Inhibition of Matrix Degradation by 9L Conditioned Medium.- Fibrin Zymography.- Reverse Fibrin Zymography.- Complex Formation Studies with Labeled Urokinase or Thrombin.- Northern Hybridization with Protease Nexin I cDNA.- Discussion.- Summary.- Acknowledgments.- References.- 28: A Serine Protease Inhibitor Domain Encoded Within the Alzheimer Disease Associated Amyloid ?-Protein Precursor Gene.- The Relationship Between the APP Gene and Familial Alzheimer's Disease.- A Kunitz-Type Serine Protease Inhibitor Domain within APP.- Expression of Alternate Transcipts for the APP Gene.- Final Considerations.- References.- 29: The Serpin, al-Antichymotrypsin, in Brain Aging and Diseases of The Nervous System.- ?1-Antichymotrypsin in Alzheimer Amyloid Deposits 321.- Amyloid Deposition During Normal Aging.- The Origin of Amyloid Proteins in Alzheimer's Disease.- Role of Proteases and Inhibitors in Amyloid Formation.- Special Association Between ?1-Antichymotrypsin and ?-Protein in Amyloid.- Is ?1-Antichymotrypsin Part of a Brain 'Acute Phase Response'?.- Summary.- References.- 30: Protease Nexin I Immunostaining in Alzheimer's Disease.- Materials and Methods.- Results.- Discussion.- References.- Section VI: The Maratea Conference.- Photo Layout.- Participants.