PLATELET-FIBRINOGEN INTERACTIONS

Vitronectin，也被称为血清扩散因子，是一种主要的细胞黏附因子 存在于血浆和内皮下基质中的分子。至少 三种截然不同的细胞表面玻璃连结蛋白受体(αVb3， 属于整合素超家族的αIIbbeta3和alphaVbeta5)具有 已被确认身份。RGD介导的配体-受体相互作用 玻璃凝集素测序。尽管所有的配体都有 RGD序列，受体能够区分各种含有RGD的 配基。因此，纤维连接蛋白受体不与 Vitronectin。在三种玻璃连结蛋白受体中，只有αVbeta5具有这种作用 不与纤维蛋白原和von Willebrand蛋白相互作用，并且只有 除了RGD之外，AlphaIIbbeta3还识别HLGGAKQAGDV序列 纤维蛋白原。受体特异性的作用机制尚不清楚。一 假说认为RGD位点附近的氨基酸序列 确定其构象，从而赋予其特异性，而其他 假说提出了额外的受体特异性结合位点(S)。我们有 重组人野生型Vitronectin在金黄地鼠肾脏中的表达 细胞。分离的野生型Vitronectin促进细胞黏附的作用类似 到血浆中提取的维他命联素。3个突变体，VN(Arg45~Leu 45)， VN(Gly46~Ala46)和VN(Asp47~Glu47)也得到了表达和分离 来自BHK细胞。含αVbeta5的PANC-1细胞明显 减少对这些突变体的粘附性(超过50-100倍)。相比之下， AlphaIIbbeta3和AlphaVbeta3的相互作用受到的抑制较少 戏剧性的(2-10倍)。因此，这些受体表现出不同程度的 RGD位点的特异性，aVb5的特异性最强，耐受性最差 当alphaIIbbeta3和alphaVb3显示时，RGD位点上的替换 松弛的特异性，即使在RGD位点也能容忍替换。这个 这项建议的主要目的是进一步定义氨基酸残基， 除了RGD，这是玻璃体凝集素识别所必需的 受体aVb3、aVb5和alphaIIbbeta3，使用定点和缺失 Vitronectin的突变。Vitronectin还与 血浆和血浆中的纤溶酶原激活物抑制物-1 内皮下，使其稳定，使其免于快速失活。这种互动 可能调节纤溶酶介导的内皮下层的降解，这是一个关键的 在新血管生成、伤口愈合和转移中的事件。的其他目标 这项建议是为了定义参与PAI-I结合的氨基酸和 为了描述糖胺多糖结合部位在粘连中的作用， 内皮细胞的铺展和应力纤维的形成。

Vitronectin, also known as serum spreading factor, is a major cell adhesion molecule present in the plasma and in the subendothelial matrix. At least three distinct cell surface receptors for vitronectin (alphaVb3, alphaIIbbeta3 and alphaVbeta5) belonging to the integrin superfamily, have been identified. The ligand-receptor interaction is mediated by the RGD sequence in vitronectin. Despite the fact that all the ligands have the RGD sequence, the receptors are able to distinguish various RGD-containing ligands. Thus, the fibronectin receptor does not interact with vitronectin. Among the three vitronectin receptors, only alphaVbeta5 does not interact with fibrinogen and von Willebrand protein, and only alphaIIbbeta3 recognize, in addition the RGD, the sequence HLGGAKQAGDV in fibrinogen. The mechanism of receptor specificity is not clear. One hypothesis suggests that amino acid sequences adjacent to the RGD site determine its conformation thereby imparting specificity while other hypothesis suggest additional receptor-specific binding site(s). We have expressed human recombinant wild-type vitronectin in Baby Hamster Kidney cells. The isolated wild-type vitronectin promotes cell adhesion similar to plasma derived vitronectin. Three mutants, VN(Arg45 to Leu 45), VN(Gly46 to Ala46) and VN(Asp47 to Glu47) were also expressed and isolated from BHK cells. Panc-1 cells containing alphaVbeta5, showed markedly decreased adhesion (more than 50-100-fold) to these mutants. In contrast, the interactions of alphaIIbbeta3 and alphaVbeta3 were inhibited less dramatically (2-10-fold). Thus, these receptors display varying degrees of specificity to the RGD site, aVb5 being most specific and least tolerating substitutions at the RGD site while alphaIIbbeta3 and alphaVb3 display relaxed specificity tolerating substitutions even at the RGD site. The primary aim of this proposal is to further define the amino acid residues, in addition to RGD, that are necessary for recognition by the vitronectin receptors aVb3, aVb5 and alphaIIbbeta3, using site-directed and deletion mutations of vitronectin. Vitronectin also forms a complex with plasminogen activator inhibitor-1 both in the plasma and in the subendothelium, stabilizing it from rapid inactivation. This interaction may modulate plasmin-mediated degradation of subendothelium, a critical event in neoangiogenesis, wound healing and metastasis. Additional aims of this proposal are to define the amino acids involved in PAI-I binding and to delineate the role of glycosaminoglycan binding site in the adhesion, spreading and stress fiber formation by the endothelial cells.