PLATELET AMYLOID PRECURSOR PROTEIN INFLUENCES HEMOSTASIS

构成这一提议基础的假设是蛋白酶

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.