Composition exhibiting a von willebrand factor (vWF) protease activity comprising a polypeptide chain with the amino acid sequence AAGGILHLELLV
The invention relates to vWF cleaving entities having a molecular weight of 180 kD, 170 kD, 160 kD, 120 kD or 110 kD and an N-terminal amino acid sequence of AAGGILHLELLV (SEQ ID NO: 1), vWF cleaving complexes and methods for their production.
1. A composition exhibiting vWF protease activity comprising at least one single isolated peptide chain having a molecular weight from about 180 kD to about 120 kD as determined by SDS-PAGE under reducing conditions and comprising the amino acid sequence AAGGILHLELLV (SEQ ID NO: 1), wherein the peptide chain is obtained from human plasma.
2. A composition according to claim 1 wherein said sequence is located at the N-terminus of the peptide chain.
3. A composition according to claim 1 wherein said peptide chain has a molecular weight of about 180 kD.
4. A composition according to claim 1 wherein said peptide chain has a molecular weight of about 170 kD.
5. A composition according to claim 1 wherein said peptide chain has a molecular weight of about 160 kD.
6. A composition according to claim 1 wherein said peptide chain has a molecular weight of about 120 kD.
7. A composition according to claim 1 wherein said composition cleaves vWF at the peptide bond 842Tyr-843Met.
8. A composition according to claim 1 wherein said composition retains activity in the presence of the serine protease inhibitor diisopropyl fluorophosphate or a calpain protease inhibitor having the structure Z-Leu-Leu-Tyr-CHN 2 , where Z is carbobenzyloxy.
9. A composition according to claim 8 , wherein said protease inhibitor is diisopropyl fluorophosphate.
10. A composition according to claim 8 , wherein said calpain protease inhibitor is Z-Leu-Leu-Tyr-CHN 2 .
11. A composition according to claim 1 wherein said peptide chain further comprises the amino acid sequence AVGPDVFQAHQEDTERYVLTNLNI GAELLRDPSLGAQPRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPG HADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI (SEQ ID NO:15) following the sequence AAGGILHLELLV (SEQ ID NO:1).
12. A composition according to claim 1 , further comprising Ca 2+ , Sr 2+ or Ba 2+ ions.
13. A composition according to claim 1 , further comprising Ca 2+ ions in a concentration of about 1 to 10 6 per selected polypeptide molecule.
14. A composition according to claim 1 , wherein said composition is essentially free of vWF or vWF fragments.
15. A composition according to claim 1 , further comprising clusterin or an analog or derivative thereof.
16. An isolated polypeptide exhibiting vWF activity, having a molecular weight between 180 kD and 120 kD as determined by SDS-PAGE under reducing conditions and comprising the amino acid sequence AAGGILHLELLV (SEQ ID NO:1), wherein the polypeptide is obtained from human plasma.
17. An isolated polypeptide according to claim 16 , wherein said polypeptide comprises the amino acid sequence AVGPDVFQAHQEDTE RYVLTNLNIGAELLRDPSLGAQFRVHLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTI NPEDDTDPGHADLVLYITRFDLELPDGNRQVRGVTQLGGACSPTWSCLITEDTGFDLGV TI (SEQ ID NO:15) directly following the sequence AAGGILHLELLV (SEQ ID NO:1).
18. An isolated polypeptide according to claim 17 having a molecular weight of about 170 kD.
19. An isolated polypeptide according to claim 17 having a molecular weight of about 160 kD.
20. An isolated polypeptide according to claim 17 having a molecular weight of about 120 kD.
21. A vWF cleaving complex comprising a polypeptide according to claim 17 and a divalent ion selected from the group consisting of Ca ++ , Sr ++ and Ba ++ .
22. A vWF cleaving complex according to claim 21 wherein the divalent cation is Ca ++ .
23. A vWF cleaving complex according to claim 21 , further containing vWF.
24. A composition comprising a polypeptide according to claim 16 .
25. An isolated polypeptide according to claim 17 , wherein the amino acid sequence is encoded by the polynucleotide set forth in SEQ ID NO: 3.
26. An isolated polypeptide having vWF protease activity wherein said polypeptide is obtained from human plasma and comprises the amino acid sequence AAGGILHLELLVAVGDVFQAHQEDTERYVLTNLNIGAELLRDPSLGAQFRV HLVKMVILTEPEGAPNITANLTSSLLSVCGWSQTINPEDDTDPGHADLVLYITRFDLELPD GNRQVRGVTQLGGACSPTWSCLITEDTGFDLGVTI (SEQ ID NO:4).
27. An isolated polypeptide according to claim 26 wherein said polypeptide is encoded by a polynucleotide sequence, which comprises SEQ ID NO: 3.