Platelet Thrombospondin Forms

Thrombospondin (TSP), a multifunctional a-granule glycoprotein of human platelets binds fibrinogen, fibronectin, heparin, histidine-rich glycoprotein (HRGP), and plasminogen (Pig), and thus, may play an important role in regulating thrombotic influences at vessel surfaces. In this study we have demonstrated that purified human platelet TSP formed a trimolecular complex with human Pig and HRGP. Complex formation was detected by a specific binding enzyme-linked immunosorbent assay (ELISA) which demonstrated simultaneous binding of fluidphase Pig and HRGP to TSP adsorbed to microtitration wells. While neither ligand inhibited complex formation of the other with TSP, 10 mM E-amino-n-caproic acid selectively blocked incorporation of Pig into the complex, suggesting that TSP contains independent binding sites for Pig and HRGP. Comparable extent of trimolecular complex formation was also detected when TSP monomer was substituted for whole TSP in the ELISA. HRGP covalently cross-linked to Sepharose 4B simultaneously bound both 12SI-TSP and "'I-P, confirming trmolecular complex formation. Rocket immunoelectrophoresis of mixtures of the purified radiolabeled proteins into anti-Pig containing agarose also confirmed trimolecular complex formation. The TSP-HRGP-PIg complex bound a similar amount of heparin as the TSP-HRGP complex, demonstrating that the HRGP within the trimolecular complex maintained functional capability. Similarly, using a fluorometric plasmin substrate, the trmolecular complex was shown to be an effective substrate for tissue plasminogen activator. Significant amounts of plasmin were generated from the TSP-HRGP-Plg complex (equivalent to that from the TSP-Pig complex), but the rate of plasmin generation from the trimolecular complex was greater than from the bimolecular complex, suggesting an important interaction of HRGP with Pig when both are complexed to TSP. The macromolecular assembly of these three proteins on cellular surfaces, such as the platelet, may serve important regulatory functions, both prothrombotic at sites of active fibrin deposition and proteolytic in nonfibrin-containing microenvironments.