Novel recombinant anticoagulant proteins

Abstract

Novel recombinant anticoagulation proteins, methods of their use and methods of their production are described. In particular, recombinant fusions of annexin V (ANV) and Kunitz protease inhibitors (KPI) that possess potent anticoagulant activity are provided. The fusions, abbreviated ANV:KPI, utilize ANV having high affinity for phosphatidyl-L-serine with various KPI's to target serine proteases in membrane-associated coagulation complexes in the blood coagulation cascade. ANV:KPIs are potentially useful antithrombotic drugs permitting localized passivation of thrombogenic vessel walls and associated thrombi.

1 . A method of producing a recombinant anticoagulant protein comprising linking annexin V (ANV) (SEQ ID NO: 10) and a Kunitz protease inhibitor (KPI). 
     
     
         2 . A method according to  claim 1  comprising generating a recombinant DNA molecule comprising a first DNA sequence encoding annexin V (ANV) (SEQ ID NO: 10) and a second DNA sequence encoding a Kunitz protease inhibitor (KPI). 
     
     
         3 . A method according to  claim 2  wherein the second DNA sequence is selected from the group consisting of Tick Anticoagulant Peptide (TAP) (SEQ ID NO: 1), a variant of aprotinin (6L15) (SEQ ID NO: 2), Kunitz-inhibitory domain of amyloid β-protein precursor (K APP ) (SEQ ID NO: 3), and KK TFPI 22-160  (SEQ ID NO: 4). 
     
     
         4 . A recombinant DNA molecule comprising a first DNA sequence encoding annexin V (ANV) (SEQ ID NO: 10) and second DNA sequence encoding a Kunitz protease inhibitor (KPI). 
     
     
         5 . A recombinant DNA molecule according to  claim 5  wherein said first DNA sequence comprises SEQ. ID. NO. 9, cDNA mutation of Cys 316 -to-Ala of SEQ ID NO: 9, or conservatively substituted variants thereof. 
     
     
         6 . A recombinant DNA molecule according to  claim 5  comprising a DNA sequence selected from the group consisting of TAP-ANV (SEQ ID NO: 5), ANV-6L15 (SEQ ID NO: 6), ANV-K APP  (SEQ ID NO: 7), and ANV-KK TFPI  (SEQ ID NO: 8), or conservatively substituted variants thereof. 
     
     
         7 . A host cell comprising a recombinant DNA molecule according to  claim 5 . 
     
     
         8 . A stably co-transfected cell line expressing a recombinant anticoagulant protein according to  claim 1 . 
     
     
         9 . A prokaryotic cell line according to  claim 8 . 
     
     
         10 . A eukaryotic cell line according to  claim 8 . 
     
     
         11 . A process for the preparation of a cell line expressing a recombinant anticoagulant protein comprising a fusion of annexin V (ANV) (SEQ ID NO: 10) and a Kunitz protease inhibitor (KPI), said process comprising stably co-transfecting a host cell with at least one recombinant expression vector comprising a cDNA sequence encoding ANV or conservatively substituted variants thereof, and at least one recombinant expression vector comprising a cDNA sequence encoding a KPI. 
     
     
         12 . A recombinant expression vector comprising a first nucleotide sequence encoding annexin V (ANV) (SEQ ID NO: 9), cDNA mutation of Cys 316 -to-Ala of SEQ ID NO: 9, or conservatively substituted variants thereof, and a second nucleotide sequence of a Kunitz protease inhibitor (KPI) together with additional sequences capable of directing the synthesis of a recombinant anticoagulant protein comprising a fusion of ANV and a KPI, in a culture of stably co-tranfected cells. 
     
     
         13 . A recombinant expression vector according to  claim 12  in a culture of stably co-transfected prokaryotic cells. 
     
     
         14 . A recombinant expression vector according to  claim 12  in a culture of stably co-transfected eukaryotic cells. 
     
     
         15 . A recombinant expression vector according to  claim 12  wherein said first nucleotide sequence comprises SEQ ID NO: 9, cDNA mutation of Cys 316 -to-Ala of SEQ ID NO: 9, or conservatively substituted variants thereovariants thereof. 
     
     
         16 . A recombinant expression vector according to  claim 12  comprising a nucleotide sequence selected from the group of TAP-ANV (SEQ ID NO: 5), ANV-6L15 (SEQ ID NO: 6), ANV-K APP  (SEQ ID NO: 7), and ANV-KK TFPI  (SEQ ID NO: 8), or conservatively substituted variants thereof.