Polysaccharide-peptide conjugates for use as thrombin substrates
View Patent ↗The present invention relates to macromolecular polysaccharide-peptide conjugates which contain a peptide portion which C-terminally contains the amino acid sequence Ala-Gly-Arg, which is cleaved by thrombin.
1. A polysaccharide-peptide conjugate having a molecular weight of more than 10 kDa, wherein the peptide portion comprises the sequence Ala-Gly-Arg-R on its C-terminus, wherein R is a signal group cleavable by thrombin, and wherein at least five peptide molecules are bonded per polysaccharide molecule.
2. The polysaccharide-peptide conjugate as claimed in claim 1 , wherein the peptide portion comprises an amino acid sequence of three to five amino acid residues, wherein the peptide is not more than eight amino acid residues long.
3. The polysaccharide-peptide conjugate as claimed in claim 1 , wherein the polysaccharide is constructed from monosaccharide units which contain vicinal diols or hydroxyl/amino groups or hydroxyl/carbonyl groups or carbonyl/carbonyl groups.
4. The polysaccharide-peptide conjugate as claimed in claim 1 , wherein the polysaccharide portion comprises dextran.
5. The polysaccharide-peptide conjugate as claimed in claim 1 , wherein the polysaccharide portion has a molar mass of approximately 10,000 g/mol to approximately 40,000 g/mol.
6. The polysaccharide-peptide conjugate as claimed in claim 1 , wherein the cleavable signal group R is a chromogenic group.
7. The polysaccharide-peptide conjugate as claimed in claim 6 , wherein the chromogenic group is para-nitroaniline (pNA).
8. The polysaccharide-peptide conjugate as claimed in claim 1 , wherein the cleavable signal group R is a fluorogenic group.
9. The polysaccharide-peptide conjugate as claimed in claim 1 , wherein the peptide portion is bonded to the polysaccharide via a secondary amine bond.
10. A method for the determination of thrombin generation in a sample, the method comprising
(a) obtaining a blood or plasma sample of a patient,
(b) mixing the sample with a polysaccharide-peptide conjugate and a suitable activator, and
(c) measuring the amount of signal group released over time,
wherein the polysaccharide-peptide conjugate has a molecular weight of more than 10 kDa, wherein the peptide portion comprises the sequence Ala-Gly-Arg-R on its C-terminus, wherein R is a signal group cleavable by thrombin, and wherein at least five peptide molecules are bonded per polysaccharide molecule.
11. The polysaccharide-peptide conjugate as claimed in claim 5 , wherein the polysaccharide portion has a molar mass of approximately 12,000 g/mol to approximately 20,000 g/mol.
12. The polysaccharide-peptide conjugate as claimed in claim 11 , wherein the polysaccharide portion has a molar mass of approximately 15,000 g/mol.
13. The polysaccharide-peptide conjugate as claimed in claim 1 , wherein at least ten peptide molecules are bonded per polysaccharide molecule.
14. The conjugate of claim 4 , wherein the conjugate has a molecular weight of approximately 20 kDa.
15. The conjugate of claim 4 , comprising seven to ten peptide molecules.
16. The conjugate of claim 8 , wherein the fluorogenic group is 7-amino-4-methoxycoumarin (AMC).
17. The method of claim 10 , wherein the activator is recombinant human tissue factor.