Grundy Jean E, Hancock Mark A, Meixner Scott C, MacKenzie Roger C, Koschinsky Marlys L, Pryzdial Edward L G
Canadian Blood Services, R&D Department, University of British Columbia/Centre for Blood Research, Department of Pathology and Laboratory Medicine, Vancouver.
Thromb Haemost. 2007 Jan;97(1):38-44.
Plasminogen binding to receptors involves both C-terminal lysine- dependent and -independent interactions. The latter are poorly understood. Our earlier work demonstrated a novel Ca (2+) -enhanced bivalent interaction between plasmin-cleaved FXa (FXa33/13) and plasminogen truncated at Lys78 (Lys-Pg). Here we hypothesized that the effects of Ca (2+) may enable dissection of the C-terminal lysine-dependent and -independent interactions. To evaluate the role of the Glu-plasminogen (Glu-Pg) amino acids 1 - 77, binding of FXa33/13 to immobilized Glu-Pg was compared to Lys-Pg by surface plasmon resonance. Under identical conditions, approximately half the amount of FXa33/13 bound to Glu-Pg. The simplest fit of data suggested a 2:1 plasminogen:FXa33/13 stoichiometry for both, which were proportionately enhanced by Ca (2+) . Only Lys-Pg demonstrated significant Ca (2+) -independent binding to FXa33/13. In the presence of Ca (2+) , weak C-terminal lysine-independent binding could be detected, but only for Glu-Pg. The elastase-generated plasminogen fragment encompassing the angiostatin-like kringle domains 1 to 3 (K1 - 3) inhibited binding of FXa33/13 to Lys-Pg, whereas fragments corresponding to kringle 4- and kringle 5-protease domain had no effect. Immobilized K1 - 3 binding to FXa33/13 had both Ca (2+) -dependent and -independent components. The principal K (d) for the interaction was 10-fold higher than Lys-Pg. In the presence of Ca (2+) , eACA inhibited FXa33/13 binding to K1 - 3 by 30%, but eliminated binding in the absence of Ca (2+) . These studies suggest that Ca (2+) -dependent and -independent binding of Lys-Pg to FXa33/13 are C-terminal lysine-dependent. The N-terminal 1 - 77 amino acids of Glu-Pg confer significant C-terminal lysine-independent binding, which may play a role during the initiating stages of plasminogen activation.
纤溶酶原与受体的结合涉及C端赖氨酸依赖性和非依赖性相互作用。后者目前了解较少。我们早期的研究表明，纤溶酶切割的FXa（FXa33/13）与在Lys78处截短的纤溶酶原（Lys-Pg）之间存在一种新型的Ca(2+)增强的二价相互作用。在此，我们假设Ca(2+)的作用可能有助于区分C端赖氨酸依赖性和非依赖性相互作用。为了评估谷氨酸纤溶酶原（Glu-Pg）1-77位氨基酸的作用，通过表面等离子体共振比较了FXa33/13与固定化Glu-Pg和Lys-Pg的结合情况。在相同条件下，与Glu-Pg结合的FXa33/13量约为与Lys-Pg结合量的一半。数据的最简单拟合表明，两者的纤溶酶原:FXa33/13化学计量比均为2:1，且均被Ca(2+)成比例增强。只有Lys-Pg表现出与FXa33/13显著的Ca(2+)非依赖性结合。在Ca(2+)存在的情况下，可以检测到较弱的C端赖氨酸非依赖性结合，但仅针对Glu-Pg。弹性蛋白酶产生的包含血管抑素样kringle结构域1至3（K1-3）的纤溶酶原片段抑制了FXa33/13与Lys-Pg的结合，而对应于kringle 4和kringle 5-蛋白酶结构域的片段则没有作用。固定化的K1-3与FXa33/13的结合既有Ca(2+)依赖性成分，也有非依赖性成分。相互作用的主要解离常数（Kd）比Lys-Pg高10倍。在Ca(2+)存在的情况下，eACA抑制FXa33/13与K1-3的结合达30%，但在无Ca(2+)时消除了结合。这些研究表明，Lys-Pg与FXa33/13的Ca(2+)依赖性和非依赖性结合是C端赖氨酸依赖性的。Glu-Pg的N端1-77位氨基酸赋予了显著的C端赖氨酸非依赖性结合，这可能在纤溶酶原激活的起始阶段发挥作用。
