Inhibitors for use in hemostasis and immune function

Abstract

The present invention relates to polynucleotide and polypeptide molecules for use as inhibitors in hemostasis and immune function. Such inhibitors are members of the family of proteins bearing a collagen-like domain and a globular domain. The inhibitors are useful for promoting blood flow in the vasculature by reducing thrombogenic and complement activity. The inhibitors are also useful for pacify collagenous surfaces and modulating wound healing.

What is claimed is:  
     
         1 . A method of promoting blood flow within the vasculature of a mammal comprising administering to said mammal a therapeutically effective amount of an adipocyte complement related protein; in a pharmaceutically acceptable vehicle; whereby said adipocyte complement related protein reduces thrombogenic and complement activity within said vasculature.  
     
     
         2 . A method according to  claim 1  wherein said adipocyte complement related protein comprises a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 26-281of SEQ ID NO: 2, wherein said sequence comprises: 
 Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid, and  
 a carboxy-terminal globular portion.  
 
     
     
         3 . A method according to  claim 2 , wherein said polypeptide comprises a sequence of amino acid residues that is at least 90% identical in amino acid sequence to residues 22-281 of SEQ ID NO: 2.  
     
     
         4 . A method according to  claim 2 , wherein said polypeptide comprises an amino acid sequence that is at least 90% identical in amino acid sequence to residues 26-281 of SEQ ID NO: 2.  
     
     
         5 . A method according to  claim 3 , wherein any differences between said polypeptide and SEQ ID NO: 2 are due to conservative amino acid substitutions.  
     
     
         6 . A method according to  claim 3 , wherein said collagen domain consists of 13 Gly-Xaa-Xaa repeats and 1 Gly-Xaa-Pro repeat.  
     
     
         7 . A method according to  claim 3 , wherein said globular domain consists of ten beta sheets.  
     
     
         8 . A method according to  claim 7 , wherein said beta sheets are associated with amino acid residues corresponding to 147-151, 170-172, 178-181, 191-203, 207-214, 219-225, 227-239, 244-250, and 269-274 of SEQ ID NO: 2.  
     
     
         9 . A method according to  claim 2 , wherein said polypeptide comprises residues 1-281 of SEQ ID NO: 2 or residues 1-281 of SEQ ID NO: 44.  
     
     
         10 . A method according to  claim 2 , wherein said polypeptide is complexed to a second polypeptide to form a oligomer.  
     
     
         11 . A method according to  claim 10 , wherein said polypeptides are complexed by intermolecular disulfide bonds.  
     
     
         12 . A method according to  claim 11 , wherein said oligomer is a trimer.  
     
     
         13 . A method according to  claim 11  wherein said oligomer is a hexamer.  
     
     
         14 . A method according to  claim 11  wherein said oligomer is an 18mer.  
     
     
         15 . A method according to  claim 1 , wherein said polypeptide reduces thrombogenic and complement activity by inhibition of the complement pathway and inhibition collagen-mediated platelet adhesion, activation or aggregation.  
     
     
         16 . A method according to  claim 1 , wherein said polypeptide reduces thrombogenic and complement activity by inhibition of the complement pathway.  
     
     
         17 . A method according to  claim 1 , wherein said polypeptide reduces thrombogenic and complement activity by inhibition of collagen-mediated platelet adhesion, activation or accretion.  
     
     
         18 . A method according to  claim 1 , wherein said polypeptide reduces thrombogenic and complement activity by inhibition of collagen-mediated platelet aggregation.  
     
     
         16 . A method according to  claim 1 , wherein said polypeptide is administered prior to, during or following an acute vascular injury in said mammal.  
     
     
         17 . A method according to  claim 16 , wherein said injury is due to vascular reconstruction.  
     
     
         18 . A method according to  claim 17 , wherein said vascular reconstruction comprises angioplasty, coronary artery bypass graft, endarterectomy, microvascular repair or anastomosis of a vascular graft.  
     
     
         19 . A method according to  claim 18 , wherein said injury is due to trauma, stroke or aneurysm.  
     
     
         20 . A method of pacifying damaged collagenous tissues within a mammal comprising administering to said mammal a therapeutically effective amount of an adipocyte complement related protein; whereby said protein renders the damaged collagenous tissue inert towards complement activation, thrombotic activity or immune activation  
     
     
         21 . A method according to  claim 20 , wherein said adipocyte complement related protein comprises a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 26-281 of SEQ ID NO: 2, wherein said sequence comprises: 
 Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid, and    a carboxy-terminal globular portion.    
     
     
         22 . A method according to  claim 21 , wherein said polypeptide comprises a sequence of amino acid residues that is at least 90% identical in amino acid sequence to residues 22-281 of SEQ ID NO: 2.  
     
     
         23 . A method according to  claim 21 , wherein said polypeptide comprises an amino acid sequence that is at least 90% identical in amino acid sequence to residues 26-281 of SEQ ID NO: 2.  
     
     
         24 . A method according to  claim 22 , wherein any differences between said polypeptide and SEQ ID NO: 2 are due to conservative amino acid substitutions.  
     
     
         25 . A method according to  claim 22 , wherein said collagen domain consists of 13 Gly-Xaa-Xaa repeats and 1 Gly-Xaa-Pro repeat.  
     
     
         26 . A method according to  claim 22 , wherein said globular domain consists of ten beta sheets.  
     
     
         27 . A method according to  claim 26 , wherein said beta sheets are associated with amino acid residues corresponding to 147-151, 170-172, 178-181, 191-203, 207-214, 219-225, 227-239, 244-250, and 269-274 of SEQ ID NO: 2.  
     
     
         28 . A method according to  claim 22 , wherein said polypeptide comprises residues 1-281 of SEQ ID NO: 2 or residues 1-281 of SEQ ID NO: 44.  
     
     
         29 . A method according to  claim 22 , wherein said polypeptide is complexed to a second polypeptide to form a oligomer.  
     
     
         30 . A method according to  claim 29 , wherein said polypeptides are complexed by intermolecular disulfide bonds.  
     
     
         31 . A method according to  claim 29 , wherein said oligomer is a trimer.  
     
     
         32 . A method according to  claim 29  wherein said oligomer is a hexamer.  
     
     
         33 . A method according to  claim 29  wherein said oligomer is an 18mer.  
     
     
         34 . A method according to  claim 20 , wherein said damaged collagenous tissues are due to injury associated with ischemia and reperfusion.  
     
     
         35 . A method according the  claim 34 , wherein said injury comprises trauma injury ischemia, intestinal strangulation, or injury associated with pre- and post-establishment of blood flow.  
     
     
         36 . A method according to  claim 20 , wherein said polypeptide is administered to a mammal suffering from cardiopulmonary bypass ischemia and recesitation, myocardial infarction, or post-trauma vasospasm.  
     
     
         37 . A method according to  claim 36 , wherein said post-trauma vasospasm comprises stroke, percutanious transluminal angioplasty, endarterectomy, accidental vascular trauma or surgical-induced vascular trauma.  
     
     
         38 . A method of pacifying the surface of a prostatic biomaterial for use in association with a mammal comprising administering to said mammal a therapeutically effective amount of an adipocyte complement related protein; 
 whereby said polypeptide renders the surface of said prosthetic biomaterial inert towards complement activation, thrombotic activity or immune activation.    
     
     
         39 . A method according to  claim 38 , wherein said adipocyte complement related protein comprises a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 26-281 of SEQ ID NO: 2, wherein said sequence comprises: 
 Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid, and    a carboxy-terminal globular portion.    
     
     
         40 . A method according to  claim 39 , wherein said polypeptide comprises a sequence of amino acid residues that is at least 90% identical in amino acid sequence to residues 22-281 of SEQ ID NO: 2.  
     
     
         41 . A method according to  claim 39 , wherein said polypeptide comprises an amino acid sequence that is at least 90% identical in amino acid sequence to residues 26-281 of SEQ ID NO: 2.  
     
     
         42 . A method according to  claim 40 , wherein any differences between said polypeptide and SEQ ID NO: 2 are due to conservative amino acid substitutions.  
     
     
         43 . A method according to  claim 40 , wherein said collagen domain consists of 13 Gly-Xaa-Xaa repeats and 1 Gly-Xaa-Pro repeat.  
     
     
         44 . A method according to  claim 40 , wherein said globular domain consists of ten beta sheets.  
     
     
         45 . A method according to  claim 44 , wherein said beta sheets are associated with amino acid residues corresponding to 147-151, 170-172, 178-181, 191-203, 207-214, 219-225, 227-239, 244-250, and 269-274 of SEQ ID NO: 2.  
     
     
         46 . A method according to  claim 40 , wherein said polypeptide comprises residues 1-281 of SEQ ID NO: 2 or residues 1-281 of SEQ ID NO: 44.  
     
     
         47 . A method according to  claim 39 , wherein said polypeptide is complexed to a second polypeptide to form a oligomer.  
     
     
         48 . A method according to,  claim 47 , wherein said polypeptides are complexed by intermolecular disulfide bonds.  
     
     
         49 . A method according to  claim 47 , wherein said oligomer is a trimer.  
     
     
         50 . A method according to  claim 47  wherein said oligomer is a hexamer.  
     
     
         51 . A method according to  claim 47  wherein said oligomer is an 18mer.  
     
     
         52 . A method of pacifying the surface of a prostatic biomaterial according to  claim 38 , wherein the surface of said prostatic biomaterial is coated with collagen or collagen fragments, gelatin, fibrin or fibronectin.  
     
     
         53 . A method of mediating wound repair within a mammal comprising administering to said mammal a therapeutically effective amount of an adipocyte complement related protein; whereby said polypeptide enhances progression in wound healing.  
     
     
         54 . A method according to  claim 53 , wherein said adipocyte complement related protein comprises a polypeptide comprising a sequence of amino acid residues that is at least 75% identical in amino acid sequence to residues 26-281 of SEQ ID NO: 2, wherein said sequence comprises: 
 Gly-Xaa-Xaa or Gly-Xaa-Pro repeats forming a collagen domain, wherein Xaa is any amino acid, and    a carboxy-terminal globular portion.    
     
     
         55 . A method according to  claim 54 , wherein said polypeptide comprises a sequence of amino acid residues that is at least 90% identical in amino acid sequence to residues 22-281 of SEQ ID NO: 2.  
     
     
         56 . A method according to  claim 54 , wherein said polypeptide comprises an amino acid sequence that is at least 90% identical in amino acid sequence to residues 26-281 of SEQ ID NO: 2.  
     
     
         57 . A method according to  claim 55 , wherein any differences between said polypeptide and SEQ ID NO: 2 are due to conservative amino acid substitutions.  
     
     
         58 . A method according to  claim 55 , wherein said collagen domain consists of 13 Gly-Xaa-Xaa repeats and 1 Gly-Xaa-Pro repeat.  
     
     
         59 . A method according to  claim 55 , wherein said globular domain consists of ten beta sheets.  
     
     
         60 . A method according to  claim 59 , wherein said beta sheets are associated with amino acid residues corresponding to 147-151, 170-172, 178-181, 191-203, 207-214, 219-225, 227-239, 244-250, and 269-274 of SEQ ID NO: 2.  
     
     
         61 . A method according to  claim 55 , wherein said polypeptide comprises residues 1-281 of SEQ ID NO: 2 or residues 1-281 of SEQ ID NO: 44.  
     
     
         62 . A method according to  claim 55 , wherein said polypeptide is complexed to a second polypeptide to form a oligomer.  
     
     
         63 . A method according to  claim 62 , wherein said polypeptides are complexed by intermolecular disulfide bonds.  
     
     
         64 . A method according to  claim 62 , wherein said oligomer is a trimer.  
     
     
         65 . A method according to  claim 62 , wherein said oligomer is a hexamer.  
     
     
         66 . A method according to  claim 62 , wherein said multimer is an 18mer.  
     
     
         67 . A method according to  claim 62 , wherein progression in wound healing comprises reduction in inflammation, reduction in fibroblast recruitment, wound retraction, or reduction in infection.  
     
     
         68 . A method for inhibiting platelet adhesion comprising administering to a patient in need of such inhibition a therapeutically effective amount of an adipocyte complement related protein in a pharmaceutical acceptable vehicle.  
     
     
         69 . A method for inhibiting platelet activation comprising administering to a patient in need of such inhibition a therapeutically effective amount of an adipocyte complement related protein in a pharmaceutical acceptable vehicle.  
     
     
         70 . A method for inhibiting platelet accretion comprising administering to a patient in need of such inhibition a therapeutically effective amount of an adipocyte complement related protein in a pharmaceutical acceptable vehicle.  
     
     
         71 . A method for minimizing vascular occlusion by increasing patency time in a patient in need of such treatment comprising administering an adipocyte complement related protein in a pharmaceutically acceptable vehicle, wherein said adipocyte complement related protein promotes blood flow.  
     
     
         72 . A method according to  claim 71 , wherein said adipocyte complement related protein is administered concurrently with, or within an effective time period before, an acute vascular injury.  
     
     
         73 . A method according to  claim 71  wherein said adipocyte complement related protein is administered following an acute vascular injury.  
     
     
         74 . A method according to  claim 71 , wherein blood flow is increased by inhibition of platelet adhesion, activation or accretion.  
     
     
         75 . A method for inducing vasodilation within the vasculature of a mammal comprising administrating to said mammal a therapeutically effective amount of an adipocyte complement related protein; in a pharmaceutically acceptable vehicle; whereby said adipocyte complement related protein elicits a vasorelaxant response.  
     
     
         76 . A method for protecting ischemic myocardium from reperfusion injury comprising administering an adipocyte complement related protein to a patient in need of such protection, wherein said adipocyte complement related protein inhibits complement.  
     
     
         77 . A method for mediating tumor metastasis comprising administering an adipocyte complement related protein to a patient in need of such inhibition, wherein said adipocyte complement related protein mediates tumor cell binding to collagen.  
     
     
         78 . A method for inhibiting platelet adhesion comprising administering to a patient a therapeutically effective amount of a formulation comprising: 
 a polypeptide comprising amino acid residues 26 to 281 of SEQ ID NO: 2 and;    a pharmaceutical acceptable vehicle.    
     
     
         79 . The method of  claim 78  wherein the polypeptide is SEQ ID NO: 2.  
     
     
         80 . The method of  claim 78  wherein the formulation is administered prior to, during, or following an acute vascular injury.  
     
     
         81 . The method of  claim 80  wherein the formulation is administered at or near the vascular injury site.  
     
     
         82 . The method of  claim 80  wherein the vascular injury is due to vascular reconstruction.  
     
     
         83 . The method of  claim 82  wherein the vascular reconstruction comprises angioplasty, coronary artery bypass graft, endarterectomy, microvascular repair or anastomosis of a vascular graft.  
     
     
         84 . The method of  claim 80  wherein the vascular injury is due to trauma, stroke or aneurysm.  
     
     
         85 . The method of  claim 80  wherein the vascular injury is due to plaque rupture or degradation of the vasculature.  
     
     
         86 . The method of  claim 80  wherein the vascular injury comprises trauma injury ischemia, intestinal strangulation or injury associated with pre- and post-establishment of blood flow.  
     
     
         87 . The method of  claim 78  wherein the patient suffers from cardiopulmonary bypass ischemia and recesitation, diabetes, atherosclerosis, disseminated intravascular coagulation, myocardial infarction, or post-trauma vasospasm.  
     
     
         88 . The method of  claim 87  wherein the post-trauma vasospasm comprises stroke, percutanious transluminal angioplasty, accidental vascular trauma or surgical-induced vascular trauma.  
     
     
         89 . The method of  claim 78  wherein the formulation is administered parenterally, orally, nasally, rectally, topically or transdermally.  
     
     
         90 . The method of  claim 78  wherein the polypeptide is complexed to a second polypeptide to form an oligomer.  
     
     
         91 . The method of  claim 90  wherein the-polypeptides are complexed by intermolecular disulfide bonds.  
     
     
         92 . The method of  claim 91  wherein the oligomer is a trimer.  
     
     
         93 . The method of  claim 91  wherein the oligomer is a hexamer.  
     
     
         94 . The method of  claim 91  wherein the oligomer is an 18mer.  
     
     
         95 . A method for inhibiting platelet activation comprising administering to a patient a therapeutically effective amount of a formulation comprising: 
 a polypeptide comprising amino acid residues 26 to 281 of SEQ ID NO: 2 and;    a pharmaceutical acceptable vehicle.    
     
     
         96 . The method of  claim 95  wherein the polypeptide is SEQ ID NO: 2.  
     
     
         97 . The method of  claim 95  wherein the formulation is administered prior to, during, or following an acute vascular injury.  
     
     
         98 . The method of  claim 97  wherein the formulation is administered at or near the vascular injury site.  
     
     
         99 . The method of  claim 97  wherein the vascular injury is due to vascular reconstruction.  
     
     
         100 . The method of  claim 99  wherein the vascular reconstruction comprises angioplasty, coronary artery bypass graft, endarterectomy, microvascular repair or anastomosis of a vascular graft.  
     
     
         101 . The method of  claim 97  wherein the vascular injury is due to trauma, stroke or aneurysm.  
     
     
         102 . The method of  claim 97  wherein the vascular injury is due to plaque rupture or degradation of the vasculature.  
     
     
         103 . The method of  claim 97  wherein the vascular injury comprises trauma injury ischemia, intestinal strangulation or injury associated with pre- and post-establishment of blood flow.  
     
     
         104 . The method of  claim 95  wherein the patient suffers from cardiopulmonary bypass ischemia and recesitation, diabetes, atherosclerosis, disseminated intravascular coagulation, myocardial infarction, or post-trauma vasospasm.  
     
     
         105 . The method of  claim 104  wherein the post-trauma vasospasm comprises stroke, percutanious transluminal angioplasty, accidental vascular trauma or surgical-induced vascular trauma.  
     
     
         106 . The method of  claim 95  wherein the formulation is administered parenterally, orally, nasally, rectally, topically or transdermally.  
     
     
         107 . The method of  claim 95  wherein the polypeptide is complexed to a second polypeptide to form an oligomer.  
     
     
         108 . The method of  claim 107  wherein the polypeptides are complexed by intermolecular disulfide bonds.  
     
     
         109 . The method of  claim 108  wherein the oligomer is a trimer.  
     
     
         110 . The method of  claim 108  wherein the oligomer is a hexamer.  
     
     
         111 . The method of  claim 108  wherein the oligomer is an 18mer.  
     
     
         112 . A method for inhibiting platelet accretion comprising administering to a patient a therapeutically effective amount of a formulation comprising: 
 a polypeptide comprising amino acid residues 26 to 281 of SEQ ID NO: 2 and;    a pharmaceutical acceptable vehicle.    
     
     
         113 . The method of  claim 112  wherein the polypeptide is SEQ ID NO: 2.  
     
     
         114 . The method of  claim 112  wherein the formulation is administered prior to, during, or following an acute vascular injury.  
     
     
         115 . The method of  claim 114  wherein the formulation is administered at or near the vascular injury site.  
     
     
         116 . The method of  claim 114  wherein the vascular injury is due to vascular reconstruction.  
     
     
         117 . The method of  claim 116  wherein the vascular reconstruction comprises angioplasty, coronary artery bypass graft, endarterectomy, microvascular repair or anastomosis of a vascular graft.  
     
     
         118 . The method of  claim 114  wherein the vascular injury is due to trauma, stroke or aneurysm.  
     
     
         119 . The method of  claim 114  wherein the vascular injury is due to plaque rupture or degradation of the vasculature.  
     
     
         120 . The method of  claim 114  wherein the vascular injury comprises trauma injury ischemia, intestinal strangulation or injury associated with pre- and post-establishment of blood flow.  
     
     
         121 . The method of  claim 112  wherein the patient suffers from cardiopulmonary bypass ischemia and recesitation, diabetes, atherosclerosis, disseminated intravascular coagulation, myocardial infarction, or post-trauma vasospasm.  
     
     
         122 . The method of  claim 121  wherein the post-trauma vasospasm comprises stroke, percutanious transluminal angioplasty, accidental vascular trauma or surgical-induced vascular trauma.  
     
     
         123 . The method of  claim 112  wherein the formulation is administered parenterally, orally, nasally, rectally, topically or transdermally.  
     
     
         124 . The method of  claim 112  wherein the polypeptide is complexed to a second polypeptide to form an oligomer.  
     
     
         125 . The method of  claim 124  wherein the polypeptides are complexed by intermolecular disulfide bonds.  
     
     
         126 . The method of  claim 125  wherein the oligomer is a trimer.  
     
     
         127 . The method of  claim 125  wherein the oligomer is a hexamer.  
     
     
         128 . The method of  claim 125  wherein the oligomer is an 18mer.