Blood Coagulation Cascade and Fibrinolysis

Blood coagulation is an enzyme-driven bodily function that results in the formation of insoluble threadlike protein called fibrin. It is a physiological homeostatic process which, while is often associated with thrombotic or hyperfibrinolytic conditions that eventually cause cardiac diseases, is essential in the body’s natural response to wounds. Coagulation prevents excessive bleeding, as fibrin, together with platelets enable repair of vascular injuries.

Coagulation

1. When damage to blood vessel is detected, proteins called clotting factors.

• Trigger platelet activation => platelets attach to walls of the damaged area and to each other forming a haemostatic plug

• Initiate vasoconstriction (=narrowing of blood vessels), locally

• Mediates conversion of prothrombin (inactive precursor) into thrombin

2. Soluble strands of fibrinogen = thrombin => insoluble strands of fibrin. Fibrin cumulatively forms a mesh-like structure that can trap more red blood cells and platelets, while stabilizing the haemostatic plug, leading to the formation of a temporary clot that limits blood loss as repair continues.

Thrombo/Fibrino-lysis

1. Endothelial cells secrete tissue plasminogen activator protein.

2. Plasminogen (inactive precursor) = tPA => plasmin (active postcursor, serine protease, responsible for dissolution of clot).

Decreased plasminogen levels can be problematic, compromising the body’s ability to dissolve fibrin, hence predisposing to thrombosis.

Acute myocardial infarction is a form of coronary thrombosis that occurs when a blood clot forms within the coronary arteries, the vessels that supply the heart with oxygenated blood.

1. Cholesterol plaques rupture: LDL accumulates in arterial walls (atherosclerosis) => triggers clotting factors to begin coagulation cascade.

2. Cholesterol narrows coronary arteries: restricts blood flow,  increases blood pressure => stress => arterial damage => repair by cascade

Failed haemostasis may also result in pulmonary embolisms, ischemic strokes.

Lumbrokinase and Existing Fibrinolytic Agents

Existing Fibrinolytic Agents and their Limitations

Existing Medication: t-PA, streptokinase, urokinase.

Despite their wide use, these thrombolytic agents are:

1. Inaccessible due to excessive cost.

2. Inefficient due to low specificity, accused of its undesirable side effects one of which is gastrointestinal bleeding.

Lumbokinase and its Mechanism

Earthworms have long been recognized by East Asian countries, (China and Japan) and incorporated into their traditional medicine.  

• Evidenced to secrete a group of 6 novel proteolytic enzymes, derived from the earthworm species, Lumbricus rubellus.

• Unlike the options currently available, LK demonstrates high specificity to fibrin (no effect on other proteins in blood plasma) => no side effects: excessive bleeding, haemorrhage due to excessive fibrinolysis.

Lumbrokinase Mechanism

Its mechanism is two-fold

1. Dissolve/proteolyze the fibrin itself

2. Convert plasminogen into its post-cursor serine-protease plasmin by activating local, endogenous t-PA, inducing it to act on the fibrin via body’s own cascade.

Molecular Docking

The computer simulation procedure enables scientists to understand protein-protein and protein-ligand interactions: binding modes and affinity for each other.

• Often hampered by the practical difficulty and costs of experimental methods

• Attempts to mimic the natural course of interaction between a ligand and a receptor via the lowest energy pathway

• Computations involve consideration of intermolecular forces such as hydrophobic, van der Waals, hydrogen bonding, electrostatic forces between molecules

• Data driven approach: provide information the algorithm can use

Protein Modelling from its Gene Sequence

The gene sequence of the LK, derived from organism Lumbricus rubellus, was retrieved from UniProtKB in the FASTA format, ready for the next step. 

The 3D structure of the enzyme constructed using the SWISS-MODEL Workspace

The SWISS-MODEL program is able to generate predicted protein structures through homology modelling

• Utilizing templates of existing proteins whose sequence is known and is similar to the protein of interest.

Protein-Protein Docking

Consensus Prediction Of interface Residues in Transient complexes (CPORT) web server

• Predicts the active and passive residues forming the interaction sites of LK and fibrin.

• Physical and knowledge-based approach to predict the interactive/binding sites.

High Ambiguity Driven protein-protein DOCKing (HADDOCK) webserver

• Protein-protein docking setting.

• Docking repeated 6 times for the 6 chains/domains of the fibrin.

PRODIGY (Protein Binding Energy Prediction) webserver

• Produces predictive values for binding affinity (ΔG) and dissociation constant (Kd) of protein-protein complex.

LIGPLOT+ Webserver

• Generates schematic diagrams of LK and fibrin interaction.

Protein-Ligand Docking

PyMol software

• Visualization of the protein model

• Renders animation of 3D structure of protein

• Enables identification of amino acid residues comprising the catalytic triad within pockets

IBM RPG

• Programming language

• Create fragments of the protein to act as ligands

• Fragmentation follows cleavage rules derived from substrate specificity patterns (LK’s preference for location of digestion of peptide bond)

• RPG then hydrolyses fibrin at these specific cleavage sites

• Fragments undergo same process as in protein-protein docking: CPORT, then docked with HADDOCK etc..

Result and Conclusion

The binding affinity (ΔG) represents whether the interaction/complex formation between a receptor (protein) with its ligand/binding partner (another protein) takes place favourably

• A more negative ΔG value is indicative of a stronger affinity for each.

• Determined by noncovalent intermolecular. interactions (e.g. hydrogen bonding, electrostatic interactions, hydrophobic interactions).

Bolstered by equilibrium dissociation constant (Kd)

• Higher the Kd value, the lower the binding affinity of the ligand to its target site.

The most negative ΔG with the lowest Kd value was found in the β chain (domains B and E), specifically chain/domain E of the protein-protein complex generated between fibrin and LK enzyme.

These results are then further reinforced by the LIGPLOT+ diagrams showing mostly hydrogen bonding (hydrophilic interactions, stronger than hydrophobic, amongst all intermolecular forces, H-bonds are strongest) between the fibrin and the LK’s catalytic triad composed of amino acids: Ser195, His50, Asp91.

Protein-ligand docking further supplemented these findings, demonstrating that in the β chain the following interactions occurred.

• Hydrophobic interactions between Asp189 of the LK and fibrin

• Hydrogen bonds between Ser195 of the LK and fibrin

• Hydrogen bonds between HIs50 of the LK and fibrin

This docking analysis, hence, provides insight on the molecular mechanism by which the LK protein acts on the human fibrin, thus evidences the potential of LK as an effective novel anti-coagulant.