Plasmin Variants Resistant to Autocatalytic Degradation
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Solution Overview
Problem
Current methods fail to provide a plasmin variant or plasminogen variant that is intrinsically resistant to autocatalytic degradation, which is essential for efficient and safe long-term storage and therapeutic use in thrombolytic therapy or other applications.
Innovation Solution
Plasminogen or plasmin variants with mutations in the catalytic domain, specifically at positions 138, 137, and 158, where lysine or arginine residues are replaced with non-lysine, non-arginine amino acids, reducing autoproteolytic degradation and maintaining proteolytic activity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If plasmin is used as a direct fibrinolytic agent, then thrombolytic activity is achieved, but autocatalytic degradation reduces stability and shelf life
Solution Approach 1:
The patent applies parameter changes by mutating specific amino acid residues (Lys137, Arg158, Lys147) in the catalytic domain of plasmin to non-lysine, non-arginine residues. This changes the chemical parameters of the enzyme to eliminate autocatalytic degradation while preserving fibrinolytic activity, thereby improving both stability and shelf life
Solution Approach 2:
The patent creates modified copies of plasmin with altered amino acid sequences at specific positions. These copied variants retain the essential catalytic function but have eliminated vulnerability to autocatalytic degradation, solving the stability-shelf life contradiction
2Reliability
If plasmin is stored at acidic pH to prevent autocatalytic degradation, then stability is improved, but acid hydrolysis becomes a destabilizing factor
Solution Approach 1:
The patent converts the harmful effect of autocatalytic degradation into a benefit by using site-directed mutagenesis to eliminate the autocatalytic sites. The mutated plasmin variants can then be stored at physiological pH without suffering from autocatalytic degradation, while avoiding the harmful acid hydrolysis that occurs at low pH
Solution Approach 2:
The patent changes the amino acid composition parameters of plasmin at specific positions (137, 158, 147) to eliminate autocatalytic activity. This allows the enzyme to maintain stability at physiological pH, avoiding the need for acidic storage conditions that cause acid hydrolysis
3Reliability
If plasmin is stored at neutral pH with stabilizers, then some stability is maintained, but autocatalytic cleavage still occurs
Solution Approach 1:
The patent creates modified copies of plasmin with specific amino acid substitutions that eliminate autocatalytic cleavage sites. These copied variants can be stored at neutral pH without suffering from autoproteolytic degradation, solving the stability-degradation contradiction
Solution Approach 2:
The patent changes the chemical parameters of plasmin by substituting lysine and arginine residues with non-lysine, non-arginine residues at critical positions. This parameter change eliminates the substrate recognition sites for autocatalytic cleavage while preserving fibrinolytic function, allowing stable storage at neutral pH
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
These variants exhibit significantly reduced autoproteolytic degradation while retaining proteolytic activity, enhancing stability and efficacy in therapeutic applications such as thrombolytic therapy and vitreous liquefaction.
Implementation Method 1
Activation of plasminogen to plasmin, triggered by the cleavage of the R561-V562 peptide bond in human plasminogen, induces a large conformational change in the light chain, said change resulting in the priming, or activation, of the catalytic triad within said light chain
Implementation Method 2
plasmin is, like many proteases, subject to autocatalytic proteolytic degradation which follows second order kinetics subject to product inhibition
Implementation Method 3
After activation, an autolytic cleavage removes an N-terminal segment from the heavy chain (78 amino acids of human plasmin; 77 amino acids of bovine plasmin)
Implementation Method 4
Bacterial plasminogen activators such as streptokinase and staphylokinase form a complex with plasminogen and, without cleavage of the R561-V562 peptide bond of plasminogen, the catalytic site of plasminogen is activated due to conformational changes upon activator-plasminogen complex formation
Implementation Method 5
Both chains are held together via 2 disulfide bonds
Data Source
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AI summary
The invention relates to variants of plasminogen and plasmin comprising one or more point mutations in the catalytic domain which reduce or prevent autocatylic destruction of the protease activity of plasmin. Compositions, uses and methods of using said variants of plasminogen and plasmin are also disclosed.