PLATELET AMYLOID PRECURSOR PROTEIN INFLUENCES HEMOSTASIS

形成这一提议基础的假设是，蛋白酶 Nexin-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，其功能是 这种蛋白质的生理、血管内递送系统。人脑， 富含Pn-2/APP，利用该蛋白作为重要的 抗凝剂。Pn-2/APP的异常处理和/或表达 血小板和脑血管参与了高血压的发病机制。 阿尔茨海默病(AD)和遗传性脑出血 荷兰型淀粉样变性(HCHWA-D)。这项建议的目标是 PN-2/APP对止血蛋白的影响及其机制的研究 人血小板和脑血管的表达/加工 组织。具体目标如下：(1)研究 确定Pn-2/APP与因子IXA和XIA的交互作用。这个 Pn-2/APP抑制因子IXA的动力学将被表征。PN- 2/APP因素XIA和IXA的灭活率将在 没有或存在肝素、血小板或培养的内皮细胞。 调查将确定，当超过时，因子IxA和因子Xia Pn-2/APP蛋白水解酶。(2)进行调查，以研究 人血小板PN-2/APP的加工和表达研究将会是 以确定在人的血小板溶解时，内源性钙蛋白酶 在正常和阿尔茨海默病患者的血小板中产生淀粉样蛋白。 研究将确定凝血酶或血小板激活因子是否可以 在没有细胞裂解的情况下，诱导pN-2/APP的钙蛋白酶加工。 将对膜APP的结构进行表征 PN-2/APP分泌后仍与血小板在一起。PN-2/APP将是 在正常和AD血小板的完整裂解和释放物中定量。 (3)调查将确定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 HCHWA-D患者、AD患者和 正常人将决定Pn-2/APP在大脑中的合成和形式 血管壁。Pn-2/APP中Xia和IXA因子的失活 培养的平滑肌细胞的存在将被确定。文化修养 将对平滑肌细胞进行研究，以确定它们如何表达和 工艺PN-2/APP。更好地理解生物化学和生理学 Pn-2/APP的研究将有助于深入了解Pn-2/APP的致病生化和致病机制。 与PN密切相关的两种疾病AD和HCHWA-D的生理学 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.