The exposure of phosphatidylserine (PS) and other aminophospholipids (aminoPL) on the surface of activated or injured blood cells and endothelium is thought to play a key role in the initiation and regulation of blood coagulation. De novo surface exposure of aminophospholipids has also been implicated in the activation of both complement and coagulation systems after tissue injury, and in removal of injured or apoptotic cells by the reticuloendothelial system. Although migration of these phospholipids (PL) from inner-to-outer plasma membrane leaflets is known to be triggered by elevated intracellular [Ca2+] ([Ca2+]) and to be associated with vesicular blebbing of the cell surface, little is known about the cellular constituents that participate in this process.
As described in Ser. No. 08/790,186, cell surface PS has a role in coagulation, programmed cell death and clearance by the reticuloendothelial system. Ser. No. 08/790,186 also describes regulation of the transmembrane distribution of PS, the role of calcium in the collapse of phospholipid asymmetry, and the role PL translocation in Scott Syndrome.
Bassxc3xa9, et al. and Stout, et al. recently reported the purification and preliminary characterization of an integral RBC membrane protein that, when reconstituted in liposomes, mediates a Ca2+-dependent transbilayer movement of PL mimicking plasma membrane PL reorganization evoked upon elevation of [Ca2+]c (F. Bassxc3xa9, et al., J. Biol. Chem. 271:17205-17210, 1996; J. G. Stout, et al., J. Clin. Invest. 99:2232-2238, 1997). Evidence that a protein of similar function must also be present in platelets was recently reported by Comfurius, et al. (P. Comfurius, et al., Biochemistry 35:7631-7634, 1996).
Needed in the art is a method for modulating the activity of phospholipid scramblase within a cell, organ or tissue in which one wishes either to reduce the potential for thrombosis, clot formation, or cell clearance (by decreasing cellular PL scramblase expression or activity) or to promote hemostasis or cell clearance (by increasing cellular PL scramblase expression or activity).
The present invention relates to the creation and use of antithrombotic and thrombostatic reagents that rely on the properties of a protein preparation that mediates Ca2+-dependent transbilayer movement of membrane phospholipids.
In one embodiment, the present invention is a preparation of a plasma membrane phospholipid scramblase (xe2x80x9cPL scramblasexe2x80x9d). Preferably, the protein is approximately 35-37 kD as measured on a 12.5% SDS-polyacrylamide gel under reducing conditions. In a most preferred form of this invention, the preparation is a human or a mouse PL scramblase.
In one preferred embodiment of the present invention, the PL scramblase comprises SEQ ID NO:2, representing human PL scramblase, possibly with conservative or functionally equivalent substitutions.
In the most preferred embodiment of the present invention, the PL scramblase, preferably comprising SEQ ID NO:2, has been modified by the action of mammalian cellular enzymes to covalently incorporate phosphorous at one or more Thr, Ser, or Tyr residues or a fatty acid, preferably palmitate, at a cysteine residue.
The present invention is also a preparation of a murine cell protein, wherein the protein is a plasma membrane phospholipid scramblase, preferably wherein the protein is approximately 35 kD as measured on a 12.5% SDS-polyacrylamide gel under reducing conditions.
In one preferred embodiment of the present invention, the murine PL scramblase comprises SEQ ID NO:4, possibly with conservative or functionally equivalent substitutions.
In the most preferred embodiment of the present invention, the murine PL scramblase comprising SEQ ID NO:4 has been modified by the action of mammalian cellular enzymes to covalently incorporate phosphorous one or more Thr, Ser, or Tyr residues, and a fatty acid, preferably palmitate, at a cysteine residue.
The present invention is also a DNA sequence encoding the PL scramblase. Preferably, this DNA sequence comprises SEQ ID NO:1. Most preferably, this DNA sequence comprises residues 211-1164 of SEQ ID NO:1.
The present invention is also a DNA sequence encoding the murine PL scramblase. Preferably, this DNA sequence comprises SEQ ID NO:3. Most preferably, the DNA sequence comprises residues 192-1112 of SEQ ID NO:3.
In another embodiment, the present invention is a method of preventing the surface exposure of plasma membrane phospholipids and reducing the procoagulant properties of a cell by delivering to the cell a mutant phospholipid scramblase. This scramblase is preferably mutated at a site of post-translational modification, most preferably the site is selected from the group consisting of Asp273-Asp284, Thr161 and Cys297 of human PL scramblase SEQ ID NO:2 or the corresponding conserved residues in mouse or other mammalian PL scramblase.
In one embodiment, a gene construct encoding a mutant phospholipid scramblase is delivered to the cell. In an alternative embodiment, the mutant protein itself is delivered.
The present invention is also an inhibitor of the PL scramblase activity of PL scramblase. This inhibitor may be an antisense nucleotide derived from the DNA sequence of PL scramblase. In another embodiment, the inhibitor is a peptide sequence that is a competitive inhibitor of PL scramblase activity. In another embodiment, the inhibitor is an antibody, preferably a monoclonal antibody, raised against PL scramblase.
In another embodiment, the inhibitor works by modifying or inhibiting the post-translational modifications of the PL scramblase that are disclosed below in the Examples. For example, analysis of the primary PL scramblase sequence reveals a potential site of phosphorylation by protein kinase C or other cellular kinase (Thr161), a potential site for acylation by fatty acid (Cys297), and a potential binding site for Ca2+ ion provided by an EF-hand-like loop spanning residues Asp273-Asp284. These residues and motifs are conserved 25 in the mouse PL scramblase.
In one embodiment, the inhibitor is a compound that prevents thioacylation of the protein.
In another embodiment, a mutant phospholipid scramblase is provided in which cysteine residues, preferably Cys297 of SEQ ID NO:2 (or the equivalent residue in the conserved region of another PL scramblase), have been replaced by alanine or other non-conservative substitution.
The present invention is also a method for preventing the surface exposure of plasma membrane phosphatidylserine, phosphatidylethanolamine and cardiolipin on the surface of in vitro stored leukocytes, lymphocytes, platelets or red blood cells. This method comprises the steps of adding an inhibitor of PL scramblase activity to the stored blood cells.
The present invention is also a method for prolonging survival of transplanted organs comprising the step of adding an inhibitor of PL scramblase activity to an organ perfusate during in vitro organ storage. The present invention is also a method for prolonging the survival of transplanted cells, tissues, and organs by genetically engineering the cells to be transplanted so as to alter their expression of plasma membrane PL scramblase in order to reduce exposure of PS and other thrombogenic phospholipids at the plasma membrane surface, thereby reducing the risk of infarction due to fibrin clot formation.
The present invention is also a method for prolonging the in vivo survival of circulating blood cells (erythrocyte, platelets, lymphocyte, PMN""s, and monocytes) comprising the step of preventing surface exposure of plasma membrane phosphatidylserine on the surface of the cells by exposing the blood cells to an inhibitor of PL scramblase activity.
The present invention is also a method for preventing the procoagulant activities of erythrocytes in sickle cell disease comprising the step of inhibiting erythrocyte PL scramblase in a sickle cell patient.
The present invention is also a method for treating autoimmune and inflammatory diseases comprising the step of treating a patient with an inhibitor of the PL scramblase activity of PL scramblase.
The present invention is also a method for diagnosing individuals with reduced or elevated capacity for platelet-promoted or erythrocyte-promoted fibrin clot activity comprising the step of quantifying the cellular expression of PL scramblase. This quantitation may take the form of immunoblotting using an antibody to PL scramblase, an ELISA assay using an antibody to PL scramblase, flow cytometric analysis of the binding of monoclonal antibody reactive against the predicted extracellular domain of PL scramblase (residues Ser310-Tryp318 of sequence disclosed in SEQ ID NO:2 or the equivalent residue in the conserved region of another PL scramblase) or using oligonucleotides derived from PL scramblase cDNA and the polymerase chain reaction. In one method of the present invention, the quantitation is performed by isolating PL scramblase from a patient blood sample, measuring the amount of PL scramblase isolated and comparing the measurement with a control sample. The measurement may be by isolating PL scramblase from a patient blood sample and measuring via densitometry the amount of PL scramblase protein electrophoresed in a stained electrophoretic gel.
It is an object of the present invention to provide a preparation of a PL scramblase.
It is another object of the present invention to genetically alter the level of expression of PL scramblase by delivery of cDNA representing sense or antisense nucleotide sequence ligated into a suitable mammalian expression vector.
It is another object of the present invention to provide an inhibitor of PL scramblase PL scramblase activity.
It is another object of the present invention to provide an antithrombotic agent.
It is another object of the present invention to create cells, tissue, and organs for transplantation that have increased potential for survival and reduced potential for causing fibrin clot formation and vascular thrombosis when grafted into a recipient host.
It is another object of the present invention to create an animal, preferably a mouse or pig, that has been genetically engineered so that the PL scramblase gene is not expressed.
Other objects, advantages and features of the present invention will become apparent after one of skill in the art reviews the specification, claims and drawings herein.