Kedees Mamdouh H, Babinska Anna, Swiatkowska Maria, Deitch Jonathan, Hussain M Mahmood, Ehrlich Yigal H, Kornecki Elizabeth
Department of Anatomy and Cell Biology, State University of New York, Downstate Medical Center at Brooklyn, Brooklyn, NY 11203, USA.
Platelets. 2005 Mar;16(2):99-109. doi: 10.1080/09537100400010329.
The F11 receptor (F11R/JAM) is a member of the immunoglobulin superfamily localized on the membrane surface of human platelets and a component of tight junctions of endothelial and epithelial cells. F11R was demonstrated to participate in the adhesion of human platelets to cytokine-inflamed endothelial cells (EC), indicating an important role for F11R in inflammatory thrombosis and atherosclerosis. Domains responsible for the formation of tight junctions, the adhesion of platelets to EC, activation of platelets resulting in granule release, the activation of IIb/3 integrin and platelet aggregation, were identified in the external portion of F11R. To further examine critical sites of F11R, we utilized the baculovirus system to generate the F11R recombinant protein with the sequence of the extracellular domain, in two types of insect cells, Sf9 and H5. The F11R recombinant protein was detected in the cytoplasm of both infected Sf9 and H5 insect cells, but only infected H5 cells secreted a soluble F11R protein. The purified recombinant F11R proteins, obtained from both types of insect cells, were recognizeable by a conformation-dependent monoclonal antibody, M.Ab.F11, directed against domains within the N-terminus and the first Ig-like fold of F11R. Assessment of the phosphorylation state in the recombinant F11R protein revealed phosphorylation of serine, threonine and tyrosine amino acid residues within the external domain. Real-time biomolecular interaction analysis, performed to assess kinetic constants associated with the binding of active molecules to the purified recombinant F11R protein revealed high affinity binding of the phosphorylated recombinant protein by M.Ab.F11 with K(a) of 5.47 x 10(6) and K(d) of 1.83 x 10(-7), comparable to values measured with intact human platelets. The findings reported here provide new information on specific domains of F11R that can lead to the generation of therapeutic agents expected to be useful in the treatment of cardiovascular diseases.
F11受体（F11R/JAM）是免疫球蛋白超家族的成员，定位于人血小板的膜表面，也是内皮细胞和上皮细胞紧密连接的一个组成部分。F11R已被证明参与人血小板与细胞因子炎症内皮细胞（EC）的黏附，这表明F11R在炎症性血栓形成和动脉粥样硬化中起重要作用。在F11R的外部区域鉴定出了负责紧密连接形成、血小板与内皮细胞黏附、血小板激活导致颗粒释放、IIb/3整合素激活和血小板聚集的结构域。为了进一步研究F11R的关键位点，我们利用杆状病毒系统在两种昆虫细胞Sf9和H5中产生具有细胞外结构域序列的F11R重组蛋白。在感染的Sf9和H5昆虫细胞的细胞质中均检测到F11R重组蛋白，但只有感染的H5细胞分泌可溶性F11R蛋白。从两种昆虫细胞中获得的纯化重组F11R蛋白可被一种针对F11R N端和第一个免疫球蛋白样结构域内结构域的构象依赖性单克隆抗体M.Ab.F11识别。对重组F11R蛋白磷酸化状态的评估显示，其外部结构域内的丝氨酸、苏氨酸和酪氨酸氨基酸残基发生了磷酸化。进行实时生物分子相互作用分析以评估活性分子与纯化重组F11R蛋白结合的动力学常数，结果显示M.Ab.F11对磷酸化重组蛋白具有高亲和力结合，K(a)为5.47×10(6)，K(d)为1.83×10(-7)，与用人完整血小板测得的值相当。本文报道的研究结果为F11R的特定结构域提供了新信息，这些信息可能有助于开发有望用于治疗心血管疾病的治疗药物。
