PLATELET THROMBOSPONDIN INTERACTIONS WITH HUMAN HIGH AND LOW-MOLECULAR-WEIGHT KININOGENS

Multifunctional proteins, e.g. high molecular weight kininogen (HK, 12 0 kDa) and the homotrimer, thrombospondin (TSP, 540 kDa), which have m ore than one domain on a single polypeptide chain, are particularly we ll-suited to be structural elements of extracellular matrices because of their ability to bind to several macromolecules. We now demonstrate that I-125-high molecular weight kininogen (HKa) cleaved by purified kallikrein forms a complex with purified intact platelet TSP (540 kDa) . HK also complexed with a proteolytic fragment (450 kDa) of TSP, lack ing its three identical heparin-binding domains (HBD, 30 kDa), but fai led to bind to a more extensively proteolysed molecule (210 kDa) lacki ng the C-terminal globular domain indicating that the binding on TSP-4 50 kDa is confined to the C-terminus. The binding of HK to intact TSP and to its 450 kDa fragment was of high affinity (K-d = 17-52 nM), spe cific, concentration dependent and saturable. Furthermore, we found bo th forms of the light chain (LC) of HK (56 and 46 kDa) resulting from cleavage by plasma kallikrein bound to both intact TSP and HBD indepen dent of the presence of calcium ions. However, neither the epitope rec ognized by monoclonal antibody (MAb) C11C1 on domain 5 nor the prekall ikrein binding site on domain 6 are involved, suggesting that the inte rvening proline-rich region may be the site of interaction. The heavy chain (HC) of HK required ionized calcium to bind to intact TSP or its 450 kDa homotrimer fragment. Purified immobilized low molecular weigh t kininogen (LK) which contains the identical HC as HK but a different LC was recognized by intact TSP but only in the presence of calcium i ons. Since MAb 2B5 inhibited the interaction, domain 2 and/or domain 3 of the HC contains the binding site for TSP. The TSP-HK interaction m ay play a role in the platelet function since both glycoproteins are p resent within the alpha-granules of platelets.