Chavakis Triantafyllos, Santoso Sentot, Clemetson Kenneth J, Sachs Ulrich J H, Isordia-Salas Irma, Pixley Robin A, Nawroth Peter P, Colman Robert W, Preissner Klaus T
Department of Internal Medicine I, University Heidelberg, D-69115 Heidelberg, Germany.
J Biol Chem. 2003 Nov 14;278(46):45375-81. doi: 10.1074/jbc.M304344200. Epub 2003 Sep 2.
Leukocyte-platelet interaction is important in mediating leukocyte adhesion to a thrombus and leukocyte recruitment to a site of vascular injury. This interaction is mediated at least in part by the beta2-integrin Mac-1 (CD11b/CD18) and its counter-receptor on platelets, glycoprotein Ibalpha (GPIbalpha). High molecular weight kininogen (HK) was previously shown to interact with both GPIbalpha and Mac-1 through its domains 3 and 5, respectively. In this study we investigated the ability of HK to interfere with the leukocyte-platelet interaction. In a purified system, HK binding to GPIbalpha was inhibited by HK domain 3 and the monoclonal antibody (mAb) SZ2, directed against the epitope 269-282 of GPIbalpha, whereas mAb AP1, directed to the region 201-268 of GPIbalpha had no effect. In contrast, mAb AP1 inhibited the Mac-1-GPIbalpha interaction. Binding of GPIbalpha to Mac-1 was enhanced 2-fold by HK. This effect of HK was abrogated in the presence of HK domains 3 or 5 or peptides from the 475-497 region of the carboxyl terminus of domain 5 as well as in the presence of mAb SZ2 but not mAb AP1. Whereas no difference in the affinity of the Mac-1-GPIbalpha interaction was observed in the absence or presence of HK, maximal binding of GPIbalpha to Mac-1 doubled in the presence of HK. Moreover, HK/HKa increased the Mac-1-dependent adhesion of myelomonocytic U937 cells and K562 cells transfected with Mac-1 to immobilized GPIbalpha or to GPIbalpha-transfected Chinese hamster ovary cells. Finally, Mac-1-dependent adhesion of neutrophils to surface-adherent platelets was enhanced by HK. Thus, HK can bridge leukocytes with platelets by interacting via its domain 3 with GPIbalpha and via its domain 5 with Mac-1 thereby augmenting the Mac-1-GPIbalpha interaction. These distinct molecular interactions of HK with leukocytes and platelets contribute to the regulation of the adhesive behavior of vascular cells and provide novel molecular targets for reducing atherothrombotic pathologies.
白细胞与血小板的相互作用在介导白细胞黏附于血栓以及白细胞募集至血管损伤部位的过程中起着重要作用。这种相互作用至少部分是由β2整合素Mac-1（CD11b/CD18）及其在血小板上的反受体糖蛋白Ibalpha（GPIbalpha）介导的。先前研究表明，高分子量激肽原（HK）分别通过其结构域3和5与GPIbalpha和Mac-1相互作用。在本研究中，我们调查了HK干扰白细胞与血小板相互作用的能力。在纯化系统中，HK结构域3和针对GPIbalpha表位269 - 282的单克隆抗体（mAb）SZ2可抑制HK与GPIbalpha的结合，而针对GPIbalpha区域201 - 268的mAb AP1则无此作用。相反，mAb AP1抑制Mac-1与GPIbalpha的相互作用。HK使GPIbalpha与Mac-1的结合增强了2倍。在存在HK结构域3或5、结构域5羧基末端475 - 497区域的肽以及mAb SZ2但不存在mAb AP1的情况下，HK的这种作用被消除。尽管在不存在或存在HK的情况下，未观察到Mac-1与GPIbalpha相互作用亲和力的差异，但在存在HK的情况下，GPIbalpha与Mac-1的最大结合量增加了一倍。此外，HK/HKa增加了髓单核细胞U937细胞和转染了Mac-1的K562细胞对固定化GPIbalpha或对转染了GPIbalpha的中国仓鼠卵巢细胞的Mac-1依赖性黏附。最后，HK增强了中性粒细胞对表面黏附血小板的Mac-1依赖性黏附。因此，HK可通过其结构域3与GPIbalpha相互作用以及通过其结构域5与Mac-1相互作用，将白细胞与血小板连接起来，从而增强Mac-1与GPIbalpha的相互作用。HK与白细胞和血小板的这些独特分子相互作用有助于调节血管细胞的黏附行为，并为减少动脉粥样硬化血栓形成性疾病提供了新的分子靶点。
