Protein Families sequence homology ― gene & protein Swiss Prot blastp similar protein structure – includes S-S, 2ndary structure patterns, 3D conformation. chymotrypsin trypsin 3. related function

Reasons to Cleave Proteins Destroy a) Digest food protein b) Eliminate function after usefulness Activate ― Proproteins  Active Protein Digestive Serine Proteases Trypsin – cleaves after Lys/Arg Chymotrypsin – cleaves after aromatic made in pancreas & secreted to intestines activated by enterokinase

Complement C1 +  activates C2 & C4 Degrading Serine Proteases Plasmin (digestion of fibrin) kidney  plasma Elastase (digestion of elastin, etc.) varied sources Myeloblastin (digests laminin, fibronectin, elastase, vitronectin, collagens) Granzymes (target cell lysis in immune response) Proteinase C (deactivates some Clotting factors) Activating Serine Proteases enteropeptidase & Trypsin +... Blood Clotting Factors ― thrombin, VII, IX, X, XI, XII, Kallikreins Tissue Plasminogen Activator Complement C1 +  activates C2 & C4 HGF Activator

Serine proteases form a covalent intermediate 1. E + S  ES 2. ES  E-P2 + P1 fast 3. E-P2  E + P2 slow Chymotrypsin cleavage of N-acetyl-phenylalanine p-nitrophenyl ester

.. .. R Serine Proteases O Gly - N - CH - C N - CH - C - H O - CH2 H specificity pocket Serine Proteases H - N - CH - C N - CH - C - R O Gly O - CH2 H .. Ser N HN .. His Asp O C

.. R O Gly - N - CH - C N - CH - C - H O - CH2 H Ser Asp N O C His HN Serine Protease - N - CH - C N - CH - C - R O Gly covalent (acyl) intermediate O - CH2 Ser N HN .. His H Asp O C

Lys ― X Arg ― X Ala ― X small Large aromatic basic Trp ― X Tyr ― X Phe ― X Lys ― X Arg ― X Ala ― X small Large aromatic basic

Blood Clotting Cascade Intrinsic Cascade Kallikrein released Extrinsic + Intrinsic Cascade Kallikrein + TF released

Intrinsic (Kininogen & Kallikrein) Extrinsic XII XIIa Trauma XI XIa IX IXa VIIa VII VIIIat TF & Ca X Xa X Vat (Vit. K) Prothrombin Thrombin Fibrinogen Fibrin XIIIat Clot The extrinsic (external laceration) & intrinsic (internal wall trauma, bruising, atherosclerotic plaque etc.) pathways to clot formation. Blood Clotting Cascade

Fibrinogen: Soluble monomer unit 3 globular segments A A B B 2 rod segments (AB Each monomer is a dimer of 3 subunits containing a central core with all the amino terminal ends, two, 3-strand helical rod segments and a peripheral globular units. Disulfide bonds stabilize the dimer.

Fibrinogen Thrombin Fibrin II A A B B Thrombin A A B B Thrombin is a serine protease that cuts a specific Arg-Gly link between B &  in the  subunit and between A &  in the  subunit. Fibrin II

This exposes specific binding sites for the peripheral ends.

46 nm 23 nm A B .... that leads to a “soft” clot; a 2D polymer of fibrin units. Covalent cross links between Gln & Lys residues from different clot sheets (catalyzed by factor VIII*) forms a 3D “hard” clot.

N-(CH2)3-CH2 H O CH2-CH2-C-NH2 GLN H2N-(CH2)3-CH2 LYS Factor XIII Transglutaminase

Fibrin Stabilizing Factor (XIII) is a transglutaminase - forms GLN-LYS, covalent cross-links. A B .... that leads to a “soft” clot; a 2D polymer of fibrin units. Covalent cross links between Gln & Lys residues from different clot sheets (catalyzed by factor VIII*) forms a 3D “hard” clot. HARD CLOT

Prothrombin Sequence g-carboxy Glu chelates Ca++ Binds membrane His Asp Ser g-carboxy Glu chelates Ca++ Disulfide bond 336-482

Serine Protease Domain 328-362 C Prothrombin 10 Glu residues, 49-75 converted to g-carboxy Glu by Vit K dependent carboxylase N Activation Peptide #1 44-198 cleaved by Thrombin (in vitro only?) Activation Peptide #2 199-327 cleaved by Xa 363-364 cleaved by Xa - still held together by disulfide bond (336-482). Serine Protease Domain 328-362 C Prothrombin

CH2-CH2-COO- COO- + CH2-CH Ca++ Vit K dependent Carboxylase Modified Glu residues anchor Prothrombin to platelets (Ca2+) While Kringle domains bind damaged tissue + CH2-CH2-COO- Vit K dependent Carboxylase CH2-CH COO- Ca++

Heparin - released by damaged mast cells Control of Clotting Antithrombin III (inhibits soluble thrombin, XII, XI, IX, & X) Heparin - released by damaged mast cells (enhances Antithrombin III) Protein C - activated by thrombin (destroys factor VIII & V)

Heparin: sulfated polysaccharide binds to AntiThrombin III and helps it bind thrombin etc. OSO3- O O hyaluronic acid OH O O OH NH Cellulose, also a pure polymer of glucose, differs from amylose only in the beta connection. However, this radically effects its function (and its digestability). COO- O SO3- OSO3- etc. glucosamine

Heparin a natural polysaccharide released by mast cells an anticoagulent in human blood

Blood Clot time after trauma Kallikrein & TF released Anti-Thrombin III + Heparin prevents off-site clotting Kallikrein & TF released Protein C: inhibits additional clot Blood Clot TPA & Plasmin: dissolves clot time after trauma

Reversal of Clotting TPA (72K) Plasminogen (bound)  Plasmin Clot  Dissolved clot A B

Plasmin TPA Thrombin Serine Protease Kringle : binds tissue Fibrin binding Growth Factor Domain Thrombin Kringle : binds tissue Serine Protease

Medical Reasons to prevent Clotting (heparin) Angioplasty (restenosis & rethrombosis 6-8%) Deep-Vein Thrombosis Unstable Angina Medical Reasons to dissolve Clots (cloned TPA) Myocardial infarction (MI) or stroke Hemophilia & Factor VIII 1. X linked - 1 in 10,000 males 2. 50% severe : < 1% F8 activity 3. 10% moderate : 2-5% F8 4. 40% mild : 5-30% F8

NSAIDs Glucose Acetyl CoA cholesterol cortisone NSAIDs (aspirin) Membrane Lipids arachadonic acid prostaglandins (Cox-2) platelet activating factor (Cox-1) (+ gastric protection) leukotrienes NSAIDs (aspirin)

Aspirin binds more tightly to COX-1 than COX-2 Aspirin binds more tightly to COX-1 than COX-2. This means it will block platelet aggregation at much lower [ ] than is required to block inflammation. NSAIDS can be harmful to individuals at risk of internal bleeding. One ‘holy grail’ of pharmacology was to find a selective COX-2 inhibitor.

Aspirin S – CH3 | O Vioxx S – NH2 | O F3C Celebrex COOH O N COX-2 has an extension of the hydrophobic pocket binding site for aspirin relative to Cox-1.

designed to show gastric protection S – CH3 | O Vioxx recall FDA approved May 1999 Vioxx VIGOR submitted Feb 2001 designed to show gastric protection also illustrated ↑cardiovascular risk Withdrawn Sept 2004 Vioxx effects ― desired effect - ↓prostacyclin - ↓inflammation response Additional effect - ↑thromboxanes - ↑bp and ↑clotting