1. 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

2. 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

3. 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

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

5. .. .. 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

6. .. 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

7. 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

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

9. 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

10. 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.

11. 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

12. This exposes specific binding sites for the peripheral ends.

13. 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.

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

15. 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

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

17. 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 #
cleaved by Thrombin (in vitro only?)
Activation Peptide #
cleaved by Xa
cleaved by Xa - still held
together by disulfide bond ( ).
Serine Protease Domain C
Prothrombin

18. 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++

19. 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)

20. 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

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

22. 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

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

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

25. 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

26. 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)

27. 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.

28. 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.

29. 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