- Email: [email protected]
Thrombospondin forms a surface for plasminogen activation by tissue activator: Formation of a ternary complex
FREE COMMUNICATIONS 5:
ADHESIVE PROTEINS WEDNESDAY, June 4, 1986 Abstract No. 104 - Abstract No. 109
104 ENDOTHELIAL CELL THROMBOSPONDIN: BINDING DOMAINS FOR FIBRONECTIN AND HEPARIN AND Ca DEPENDENT STRUCTURE. Rima Dardik, Olga Stein and Judith Lahav, polymer Research, Weizmann Institute of Science, Rehovot 76 100, Israel. The binding domains and proteolysis sensitive conformation of endothelial cell (EC) thrombospondin (TSP) were studied in the context of its role in the construction and cell adhesion properties of extracellular matrix. TSP was purified by a two step affinity chromatography procedure to higher than 95% purity. Limitted proteolytic digestion by thrcmbin trypsin and chymotrypsin in the presence of 2 mM Ca+2 or 2 mM EDTA showed a strict dependence of the conformation of EC TSP on Ca+2 ions. Removal of Ca+2 rendered the molecule generally more susceptible to proteolysis, though a 70 kd proteolysis resistant fragment persisted in the first two enzymes. The pattern of fragments obtained was similar but not identical to that of platelet TSP. Binding capacity to fibronectin and heparin was eliminated following enzymatic cleavage. However, digestion of TSP prebound to the ligand yielded fragments which retained their binding function. Monoclonal antibodies raised against human platelet TSP cross reacted with EC TSP. These immune precipitated the larger fragments of EC TSP but not the small ones, and their antigenicity appeared to be conformation sensitive. Thus EC TSP has a Ca dependent structure, its proteolysis resistant domains undergo conformational changes following cleavage of the intact molecule such that eliminate their binding function to fibronectin and heparin, but interaction of the intact molecule with these ligands protects from such structural changes upon subsequent fragmentation. 105 THROMBOSPONDIN FORMS A SURFACE FOR PLASMINOGEN ACTIVATION BY TISSUE ACTIVATOR: FORMATION OF A TERNARY COMPLEX. RL Nachman, Cornell University Medical College, N.Y., N.Y. Thrombospondin (TSP), a multifunctional a-granule platelet protein with a broad cellular distribution specifically interacts with plasminogen (Pig) and histidine-rich glycoprotein (HRGP) suggesting that TSP may modulate plasmin formation. The influence of immobilized TSP on Plg activation by TPA was studied. Plg was incubated in microtiter wells coated with TSP or control proteins and activated with TPA in the presence of a synthetic plasmin substrate. Kinetic analysis revealed an increase in affinity of TPA for Plg in the presence of TSP (KM decreased from 3uM to 0.08pM) resulting in a 35 fold increase in catalytic efficiency, Activation in wells coated with TSP-HRGP complexes resulted in a further 1.7 fold increase in rate. 81% of the plasmin activity generated remained bound to the TSP coated surfaces. This TSP-associated plasmin was protected from u -plasmin inhibitor, requiring 200 fold increase in inhibitor concen8ration to achieve a 50% inhibition. These kinetic data suggest that TPA may form a ternary complex with TSP and Plg (analagous to the fibrin-Plg-TPA complex generated on a clot) allowing efficient activation at plasma pig concentrations. Specific and saturable TPA binding to Plg-TSP complexes was directly demonstrated using both an ELISA binding assay and a rocket immunoelectrophoresis system. TSP may thus play an important role in effecting proteolytic events in a non-fibrin containing microenvironments. 54
ADHESIVE PROTEINS WEDNESDAY, June 4, 1986 Abstract No. 104 - Abstract No. 109
104 ENDOTHELIAL CELL THROMBOSPONDIN: BINDING DOMAINS FOR FIBRONECTIN AND HEPARIN AND Ca DEPENDENT STRUCTURE. Rima Dardik, Olga Stein and Judith Lahav, polymer Research, Weizmann Institute of Science, Rehovot 76 100, Israel. The binding domains and proteolysis sensitive conformation of endothelial cell (EC) thrombospondin (TSP) were studied in the context of its role in the construction and cell adhesion properties of extracellular matrix. TSP was purified by a two step affinity chromatography procedure to higher than 95% purity. Limitted proteolytic digestion by thrcmbin trypsin and chymotrypsin in the presence of 2 mM Ca+2 or 2 mM EDTA showed a strict dependence of the conformation of EC TSP on Ca+2 ions. Removal of Ca+2 rendered the molecule generally more susceptible to proteolysis, though a 70 kd proteolysis resistant fragment persisted in the first two enzymes. The pattern of fragments obtained was similar but not identical to that of platelet TSP. Binding capacity to fibronectin and heparin was eliminated following enzymatic cleavage. However, digestion of TSP prebound to the ligand yielded fragments which retained their binding function. Monoclonal antibodies raised against human platelet TSP cross reacted with EC TSP. These immune precipitated the larger fragments of EC TSP but not the small ones, and their antigenicity appeared to be conformation sensitive. Thus EC TSP has a Ca dependent structure, its proteolysis resistant domains undergo conformational changes following cleavage of the intact molecule such that eliminate their binding function to fibronectin and heparin, but interaction of the intact molecule with these ligands protects from such structural changes upon subsequent fragmentation. 105 THROMBOSPONDIN FORMS A SURFACE FOR PLASMINOGEN ACTIVATION BY TISSUE ACTIVATOR: FORMATION OF A TERNARY COMPLEX. RL Nachman, Cornell University Medical College, N.Y., N.Y. Thrombospondin (TSP), a multifunctional a-granule platelet protein with a broad cellular distribution specifically interacts with plasminogen (Pig) and histidine-rich glycoprotein (HRGP) suggesting that TSP may modulate plasmin formation. The influence of immobilized TSP on Plg activation by TPA was studied. Plg was incubated in microtiter wells coated with TSP or control proteins and activated with TPA in the presence of a synthetic plasmin substrate. Kinetic analysis revealed an increase in affinity of TPA for Plg in the presence of TSP (KM decreased from 3uM to 0.08pM) resulting in a 35 fold increase in catalytic efficiency, Activation in wells coated with TSP-HRGP complexes resulted in a further 1.7 fold increase in rate. 81% of the plasmin activity generated remained bound to the TSP coated surfaces. This TSP-associated plasmin was protected from u -plasmin inhibitor, requiring 200 fold increase in inhibitor concen8ration to achieve a 50% inhibition. These kinetic data suggest that TPA may form a ternary complex with TSP and Plg (analagous to the fibrin-Plg-TPA complex generated on a clot) allowing efficient activation at plasma pig concentrations. Specific and saturable TPA binding to Plg-TSP complexes was directly demonstrated using both an ELISA binding assay and a rocket immunoelectrophoresis system. TSP may thus play an important role in effecting proteolytic events in a non-fibrin containing microenvironments. 54