PLATELET AMYLOID PRECURSOR PROTEIN INFLUENCES HEMOSTASIS

形成这一提议基础的假设是，蛋白酶 连接蛋白-2/淀粉样β蛋白前体（PN-2/APP）在 止血因子XIa和IXa的生理调节。 人类 血小板是PN-2/APP的主要血管内储存库， 这种蛋白质的生理性血管内递送系统。 人类的大脑， 富含PN-2/APP，利用这种蛋白质作为重要的 抗凝剂 PN-2/APP的异常加工和/或表达， 血小板和脑血管有助于发病机制 阿尔茨海默病与遗传性脑出血 淀粉样变性-荷兰型（HCHWA-D）。 本提案的目标是 研究PN-2/APP对止血蛋白的影响， 人血小板和脑血管中的表达/加工 组织. 具体目标如下：（1）研究将 确定PN-2/APP与因子Ixa和Xia的相互作用。 的 将表征PN-2/APP抑制因子Ixa的动力学。 Pn- 2/因子Xia和Ixa的APP失活率将在 不存在或存在肝素和血小板或培养的内皮细胞。 研究将确定当过量时，因子Ixa和因子Xia 蛋白水解PN-2/APP。（2）将进行研究， 人血小板PN-2/APP的加工和表达。 以确定在人血小板溶解后，内源性钙蛋白酶 在正常和AD血小板中产生淀粉样蛋白片段。 研究将确定凝血酶或血小板活化因子是否能 在不存在细胞裂解的情况下诱导PN-2/APP的钙蛋白酶加工。 将进行研究以表征膜APP的结构 在PN-2/APP分泌后保留血小板。 PN-2/APP将 在完整裂解物中定量，并从正常和AD血小板中释放。 (3)研究将确定PN-2/APP的表达和加工 在脑血管中。 免疫组织化学和原位杂交 对来自HCHWA-D患者、AD患者和 正常人脑组织中PN-2/APP的合成和形成 血管壁 Pn-2/APP失活速率的影响 确定培养的平滑肌细胞的存在。 培养 将研究平滑肌细胞以确定它们如何表达和 过程PN-2/APP.更好地了解生物化学和生理学 的PN-2/APP将提供深入了解病理生物化学和病理- AD和HCHWA-D的生理学，两种与PN密切相关的疾病， 2/APP。

The hypotheses that form the basis of this proposal are that protease nexin-2/amyloid beta-protein precursor (PN-2/APP) has a critical role in the physiologic regulation of hemostatic factors XIa and IXa. Human platelets, the major intravascular repository for PN-2/APP, function as the physiologic, intravascular delivery system for this protein. Human brain, enriched with PN-2/APP, utilizes this protein as an important anticoagulant. Abnormal processing an/or expression of PN-2/APP by platelets and cerebral blood vessels contribute to the pathogenesis of Alzheimer's Disease (AD) and Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch Type (HCHWA-D). The objectives of this proposal are to study the effect of PN-2/APP on hemostatic proteins and its expression/processing by human platelets and in cerebral blood vessel tissue. The specific aims are as follows: (1) Studies will be performed to ascertain the interaction of PN-2/APP with factors Ixa and Xia. The kinetics of factor Ixa inhibition by PN-2/APP will be characterized. Pn- 2/APP inactivation rates of factors Xia and Ixa will be determined in the absence or presence of heparin and platelets or cultured endothelial cells. Investigations will determine if, when in excess, factor Ixa and factor Xia proteolyze PN-2/APP. (2) Investigations will be performed to study the processing and expression of human platelet PN-2/APP. Studies will be performed to determine if upon lysis of human platelets, endogenous calpain produces an amyloidogenic fragment in normal and AD platelets. Investigations will determine if thrombin or platelet activating factor can induce calpain processing of PN-2/APP in the absence of cell lysis. Studies will be conducted to characterize the structure of membrane APP remaining with platelets after PN-2/APP secretion. PN-2/APP will be quantitated in whole lysates and releasates from normal and AD platelets. (3) Investigations will determine the expression and processing of PN-2/APP in cerebral blood vessels. Immunohistochemical and in situ hybridization studies on human brain tissue from HCHWA-D patients, AD patients, and normals will determine the synthesis and form of PN-2/APP in cerebral vessel walls. Pn-2/APP inactivation rates of factors Xia and Ixa in the presence of cultured smooth muscle cells will be determined. Cultured smooth muscle cells will be studied to determine how they express and process PN-2/APP. Better understanding of the biochemistry and physiology of PN-2/APP will provide insight into the patho-biochemistry and patho- physiology of AD and HCHWA-D, two disorders intimately involved with PN- 2/APP.