Thrombin Poster Slidesx

Download Report

Transcript Thrombin Poster Slidesx

Introduction
Thrombin and Blood Clotting
Thrombin is the final enzyme in the coagulation cascade (Figure 1) at sites of
vascular injury and serves as the link between vessel injury, coagulation, and
platelet response.
When leeches draw blood, the blood does not clot until after the leech releases
its grip. This is due to a protein that the leech secretes called hirudin. Hirudin is
an anticoagulant protein and stops the function of the thrombin.1 Our body also
has naturally occurring anticoagulants such as heparin cofactor II and antithrombin in order to keep thrombin activity in check.1
α-Thrombin is a trypsin-like serine protease, a protein-cutting enzyme that uses
serine to perform the cleavage.4 Other examples of serine proteases are trypsin
and chymotrypsin, enzymes involved in digestion.
Coagulation Cascade
Figure 2: In the coagulation cascade, thrombin catalytically cleaves the bonds of
fibrinogen into networks of fibrin to form the solid fibrin gel of a hemostatic plug or a
pathologic thrombus. It activates platelets as well as the blood coagulation factors V,
VIII, and XIII that are essential for formation of the blood clot.
Crystal Structure
Human α-Thrombin
First crystallized by Bode et al (Human Thrombin).
Heterodimer; disulfide linked; hydrophilic (Hydropathy Plot).
Light (L)
- 36 residues, 1 alpha helix
Heavy (H)
- 289 residues, 2 beta barrels,
several alpha helices
The active site is located in between the 2 beta barrels (Active Site).
A conserved feature of trypsin-like protease is the presence of His406, Asp462, and
Ser568 in the active site (Sequence Homology).
Human α-Thrombin
Hydropathy Plot
Figure 3: A hydropathy plot for human thrombin, a plasma soluble blood clotting protein
Active Site
Sequence Homology
Catalytic Mechanism
Figure 4: Diagram showing interaction between fibrinogen A-strand and thrombin at
thrombin's active site, including Ser568 and His406.5
Medical Relevance
Venous and arterial thrombosis (blood clots) are one of the
most common causes of death.
Thrombin inhibitors stop the function of thrombin and
prevent the formation of blood clots.
Blood clots can travel through the body and cause several
medical conditions ultimately leading to a heart attack.2
Hirudin – Inhibits thrombin by binding to exosite I and the
active site, the area where fibrinogen binds.7
Hirudin
Hirudin is the most potent and specific natural
thrombin inhibitor.
Hirudin consists of a polypeptide chain of 65
residues with three disulfide bridges.
The three N-terminal amino acids of hirudin bind in
the active site of thrombin to induce inhibition.
Hirudin-Thrombin
The C-terminal fragment of hirudin binds to exosite1 of thrombin making both the electrostatic and
hydrophobic interactions essential for tight binding.
Key electrostatic interactions: hAsp-55 with Arg-73
and hGlu-57 with Arg-75.
A large number of hydrophobic interactions between
thrombin and hPhe-56, hIle-59, hPro-60, hTyr-63, and
hLeu-64 of the C-terminal fragment of hirudin are
made upon binding as well.
Mutagenesis Study
Numerically, hPhe-56 makes the most contacts with
thrombin, and its loss through replacement with
alanine causes a decrease in the free energy of
binding (Betz et al.,1991b).
It was shown that hPhe-56 can be replaced with
tyrosine without loss, but tryptophan seems to be
too large.
Replacement with branched amino acids (Leu, Val,
Ile, or Thr) causes an even larger loss of energy.
Thrombin-Hirudin
References
1.
2.
3.
4.
5.
6.
7.
8.
9.
Davie, E. W., Kulman, J. D. (2006) An overview of the structure and function of Thrombin.
Semin Thromb Hemost. 32, 3-15. PMID: 16673262.
Tanaka-Azevedo A.M, Morais-Zani K, Torquato R.J.S, Tanaka A.S. (2010) Journal of
Biomedicine and Biotechnology.
Di Cera, E. (2008) Mol Aspects Med. 29, 203–254.
Kraut, J. Serine Proteases: Structure and Mechanism of Catalysis (1977). Ann. Rev.
Biochem., 46, 331-358.
Martin, P. D., Robertson, W. Turk, D.,Huber, R., Bode, W. Edwards, B.F.P. (1992) The
Structure of Residues 7-16 of the Aa-Chain of Human Fibrinogen Bound to Bovine
Thrombin at 2.3-A Resolution. The Journal of Biological Chemistry. 267, 7911-7920.
Baum, B.; Muley, L.; Heine, A.; Smolinski, M.; Hangauer, D.; Klebe, G. (2009) J Mol Biol.
391, 552-64.
Grutter MG, Priestle JP, Rahuel J, Grossenbacher H, Bode W, Hofsteenge J, Stone SR. 1990
Crystal structure of the thrombin-hirudin complex: a novel mode of serine protease
inhibition. EMBO J. 9, 2361–2365.
Pineda, A.; Carrell, C.; Bush, L.; Prasad, S.; Caccia, S.; Chen, Z.; Mathews, F.; Di Cera, E.
(2004) J Biol Chem. 279, 31842-53.
Rydel TJ, Tulinsky A, Bode W, Huber R. 1991 Refined structure of the hirudin-thrombin
complex. J Mol Biol. 221, 583–601.