DeLa Cadena R A, Wyshock E G, Kunapuli S P, Schultze R L, Miller M, Walz D A, Colman R W
Sol Sherry Thrombosis Research Center, Department of Pathology and Laboratory Medicine, Temple University School of Medicine, Philadelphia, PA 19140.
Thromb Haemost. 1994 Jul;72(1):125-31.
Multifunctional proteins, e.g. high molecular weight kininogen (HK, 120 kDa) and the homotrimer, thrombospondin (TSP, 540 kDa), which have more than one domain on a single polypeptide chain, are particularly well-suited to be structural elements of extracellular matrices because of their ability to bind to several macromolecules. We now demonstrate that 125I-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, lacking its three identical heparin-binding domains (HBD, 30 kDa), but failed to bind to a more extensively proteolysed molecule (210 kDa) lacking the C-terminal globular domain indicating that the binding on TSP-450 kDa is confined to the C-terminus. The binding of HK to intact TSP and to its 450 kDa fragment was of high affinity (Kd = 17-52 nM), specific, concentration dependent and saturable. Furthermore, we found both 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 independent of the presence of calcium ions. However, neither the epitope recognized by monoclonal antibody (MAb) C11C1 on domain 5 nor the prekallikrein binding site on domain 6 are involved, suggesting that the intervening 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.(ABSTRACT TRUNCATED AT 250 WORDS)
多功能蛋白，例如高分子量激肽原（HK，120 kDa）和同三聚体血小板反应蛋白（TSP，540 kDa），它们在单一多肽链上具有多个结构域，由于其能够结合多种大分子，因而特别适合作为细胞外基质的结构成分。我们现在证明，经纯化的激肽释放酶切割的125I-高分子量激肽原（HKa）与纯化的完整血小板TSP（540 kDa）形成复合物。HK还与TSP的一个蛋白水解片段（450 kDa）形成复合物，该片段缺乏其三个相同的肝素结合结构域（HBD，30 kDa），但未能与缺乏C末端球状结构域的更广泛蛋白水解的分子（210 kDa）结合，这表明与TSP-450 kDa的结合局限于C末端。HK与完整TSP及其450 kDa片段的结合具有高亲和力（Kd = 17 - 52 nM）、特异性、浓度依赖性且可饱和。此外，我们发现血浆激肽释放酶切割产生的HK轻链（LC）的两种形式（56和46 kDa）均与完整TSP和HBD结合，且与钙离子的存在无关。然而，结构域5上单克隆抗体（MAb）C11C1识别的表位和结构域6上的前激肽释放酶结合位点均未参与，这表明中间富含脯氨酸的区域可能是相互作用的位点。HK的重链（HC）需要离子化钙才能与完整TSP或其450 kDa同三聚体片段结合。（摘要截短于250字）
