Thrombospondin forms complexes with single-chain and two-chain forms of urokinase.
Thrombospondin (TSP), an adhesive glycoprotein found in platelets and extracellular matrix, has been shown previously to interact with plasminogen and tissue plasminogen activator, resulting in efficient plasmin generation. We now demonstrate specific complex formation of TSP with both the single-chain and two-chain forms of urokinase (scuPA and uPA). Binding of uPA and scuPA to immobilized TSP was detected and quantified using colorimetric immunoassays and a functional amidolytic assay. Binding was time and concentration dependent with apparent affinity constants of 40-50 nM. Binding was not affected by serine protease inhibitors, EDTA, or epsilon-aminocaproic acid. scUPA and uPA bound to TSP retained functional activity. Using a sensitive amidolytic assay we found that TSP. scuPA complexes were efficiently converted to TSP. uPA by catalytic plasmin concentrations. Additionally, TSP.uPA complexes were found to have plasminogen-activating activity equivalent to fluid-phase uPA and to be protected from inhibition by plasminogen activator inhibitor type 1, the major plasma and matrix plasminogen activator inhibitor. Using immunohistochemical techniques, we also demonstrated co-distribution of TSP and uPA in normal and malignant breast tissue. Complex formation of TSP with uPA may serve to localize, concentrate, and protect these enzymes on cell surfaces and within the extracellular matrix, thereby providing a reservoir of plasminogen activator activity.
Silverstein
R L
RL
Department of Medicine, Cornell University Medical College, New York, New York 10021.
Nachman
R L
RL
Pannell
R
R
Gurewich
V
V
Harpel
P C
PC
D000595:Amino Acid Sequence; D001792:Blood Platelets / Q000032:analysis*; D001940:Breast / Q000032:analysis; D001943:Breast Neoplasms / Q000032:analysis; D003124:Colorimetry; D004797:Enzyme-Linked Immunosorbent Assay; D005109:Extracellular Matrix / Q000032:analysis; D005341:Fibrinolysin / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D007700:Kinetics; D008562:Membrane Glycoproteins / Q000378:metabolism* / Q000494:pharmacology; D008969:Molecular Sequence Data; D010958:Plasminogen / Q000378:metabolism; D010960:Plasminogen Activators / Q000494:pharmacology; D019699:Thrombospondins; D014018:Tissue Distribution; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism* / Q000494:pharmacology
D008562:Membrane Glycoproteins; D019699:Thrombospondins; D010958:Plasminogen; D010960:Plasminogen Activators; D005341:Fibrinolysin; D014568:Urokinase-Type Plasminogen Activator