Heidi J. Long

p amyloid fragments derived from activated platelets deposit in cerebrovascular endothelium: usage of a novel blood brain barrier endothelial cell model system

Amyloid precursor protein (AβPP) processing results in generation of amyloid β peptide (Aβ) which deposits in the brain parenchyma and cerebrovasculature of patients with Alzheimers disease (AD). Evidence that the vascular deposits derive in part from AβPP fragments originating from activated platelets includes findings that individuals who have had multiple small strokes have a higher prevalence of AD compared to individuals who have taken antiplatelet drugs. Thus, determination ofwhether platelet AβPP fragments are capable of traversing the blood-brain barrier (BBB) is critical. We have established that activated platelets from patients with AD retain more surface trans-membrane-bound AβPP (mAβPP) than control platelets. We report here that this mAβPP can be cleaved to Aβ-containing fragments which pass through a novel BBB model system. This model utilizes human BBB endothelial cells (BEC) isolated from brains of patients with AD. These BEC, after exposure to activated platelets which have been surface-labeled with fluorescein and express surface-retained mAβPP, cleave fluorescein-tagged surface proteins, including mAβPP, resulting in passage to the BEC layer. The data confirm that BEC contribute to processing of platelet-derived mAβPP and show that the processing yields Aβ con-tainingfragments which could potentially contribute to cerebrovascular Aβ deposition.

Blood brain barrier endothelial cells express candidate amyloid precursor protein-cleaving secretases

Proteolytic cleavage of the amyloid precursor protein (A beta PP) results in the generation of the amyloidogenic fragment known as amyloid beta peptide (A beta). Deposition of A beta in the brain parenchyma and cerebrovasculature is a feature of Alzheimers disease (AD). To date, the process whereby A beta is generated and deposited remains unclear. We have previously established that activated platelets from AD patients retain more A beta PP on their surface than control platelets. We report here that an endothelial cell-derived enzyme can cleave this surface platelet A beta PP. Human blood brain barrier endothelial cells from brains of AD patients were assayed for potential A beta PP-cleaving enzymes using synthetic peptide substrates encompassing the A beta N-terminus cleavage site. A protease activity capable of cleaving A beta PP on the surface of AD platelets was noted. The A beta PP cleavage is partially inhibited by EDTA, by ZincOV, as well as by a specific inhibitor of the Zn metalloprotease E.C.3.4.24.15. Furthermore, the protease is recognized by an antibody directed against it, using immunohistochemistry, Western blot analysis and flow cytometry. The protease is not secreted, but rather resides intracellularly as well as on the surface of the endothelial cells. The data suggest that E.C.3.4.24.15 synthesized by brain endothelial cells may process the platelet-derived A beta PP, yielding fragments which could contribute to cerebrovascular A beta deposits.

Initial cytoplasmic and phagosomal consequences of human neutrophil exposure to Staphylococcus epidermidis.

Microorganisms are recognized by specific phagocyte surface receptors. Liganded receptors then signal a series of events leading to phagocytosis and destruction of the organism by oxidative, lytic, and associated processes. Some organisms, such as Mycobacterium tuberculosis (Mtb), Cryptococcus neoformans (Cf), and others, evade such destruction, surviving and sometimes multiplying within the phagosome to later cause disease. To study such evasion, we developed protocols which permit simultaneous kinetic measurement of early cytoplasmic signaling and of phagosomal pH (pHp) and oxidative burst, on a cell‐by‐cell basis, of polymorphonuclear (PMN) leukocytes exposed to fluorescently labeled, nonpathogenic Staphylococcus epidermidis (Se). The availability of a new, highly sensitive pH probe, pHrodo™, permits observation of increasing pHp. Simultaneous labeling of the organism, applicable to any phagocyte target, with a probe insensitive to pH and oxidative species, such as AlexaFluor350™, permits distinction between binding and functional responses to it by ratioing fluorescences. Addition of an extracellular‐specific quencher (Trypan blue) permits distinction between bound and phagosome‐enclosed targets, so that conditions within the closed phagosome can be studied. We found that opsonization is required for functional activation of PMN by Se, that the organism causes early alkalinization of the phagosome (in contrast to Cf which hyperacidifies it), and that extracellular Ca2+ is not required for cytoplasmic Ca2+ signaling but contributes markedly to binding of Se to PMN and to ensuant bactericidal functions. These findings lead to a new approach to the study of select organisms, like Cf and Mtb, which evade killing by manipulating the phagosomal environment.

Platelets and DAMI megakaryocytes possess β-secretase-like activity

Abstract We report here the discovery of two novel human platelet and megakaryocytic DAMI cell enzymes that have β-secretase-like activity. These activities could potentially effect cleavage of the amyloid precursor protein (APP) at the β-amyloid peptide N-terminus, by an EC 3.4.24.15-like metalloprotease, and the N terminus-1 position, by a serine protease. Thus both enzymes may generate the amyloidogenic β-peptide. Studies of intact and Triton X-100-lysed DAMI cells, as well as intact versus subcellular fractions of platelets, demonstrate the presence of these proteolytic activities. The resting platelet has (1) a surface serine protease, demonstrated by its ability to cleave a β-secretase substrate and by its inhibitor sensitivity; and (2) a metalloprotease, recognized by an antibody to EC 3.4.24.15, which resides intracellularly in the α-granule membrane, is translocated to the surface on activation, and shows β-secretase-like activity by cleaving the same substrate. This metalloprotease can also cleave recombinant APP to a potentially amyloidogenic fragment. Surface metalloprotease was identified in DAMI cells by flow cytometry and Western blotting with a specific anti-EC 3.4.24.15 monoclonal antibody, while activity was identified by using two β-secretase substrates. This article is the first to document two previously unknown endoproteinases with β-secretase-like activity in platelets and DAMI cells. These proteases are capable of effecting cleavage of APP and could therefore contribute to Aβ deposition in the cerebrovasculature. (J Lab Clin Med 1999;133:507-15)

Brain endothelial cell enzymes cleave platelet-retained amyloid precursor protein

We have previously demonstrated that thrombin-activated platelets from patients with advanced Alzheimers disease (AD) retain significantly more surface membrane-bound amyloid precursor protein (mAPP) than platelets from non-demented age-matched individuals (AM). We have studied interactions between these platelets and the cerebrovascular endothelium to which activated platelets adhere in a model system, investigating their involvement in the formation of amyloid beta peptide (Abeta) deposits in AD patients. We report here that there appear to be alpha and beta secretase-like activities in primary human blood brain barrier endothelial cell (BEC) cultures from both AD patients and AM control subjects (AD-BEC and AM-BEC, respectively) as well as a gamma secretase-like activity that appears only in AD-BEC. No such activities were observed in human umbilical vein endothelial cells (HUVECs). Furthermore, there is more penetration of the platelet-released products platelet factor 4 and soluble APP through the BEC layer grown from AD patients than that grown from AM individuals, whereas none penetrate through a HUVEC layer. Thus the interaction between platelets, the APP they have retained or released, and cerebral vascular endothelial cells may be at least partially responsible for amyloidogenic deposits around the cerebral vasculature of AD patients.

Platelets from patients with Alzheimer's disease or other dementias exhibit disease-specific and apolipoprotein E correlatable defects

Platelets carry over 95% of the circulating Alzheimers β-amyloid precursor protein (AβPP), and release soluble and hydrophobic proteolytic fragments of AβPP upon activation. These cells may be the source of cerebrovascular amyloid peptides, a part of Alzheimers disease (AD) pathology. Our previous studies showed that platelets from patients with advanced AD exhibit both signal transduction (hyperacidification) and AβPP processing defects. Here, we show further that a similar hyperacidification also exists in patients with Picks disease (a dementia with AD-like symptoms but a different amyloid pathology) or Down syndrome (trisomy and hence overproduction of AβPP), while the AβPP processing defect and consequent AβPP retention on the membrane is absent and is thus likely to be AD-specific. The hyperacidiftcation defect correlates with all three dementias and with the presence of apolipoprotein E4 which has been implicated as a risk factorial-AD.

Sequential Chemotactic and Phagocytic Activation of Human Polymorphonuclear Neutrophils

ABSTRACT Human polymorphonuclear neutrophils (PMN) chemotax to a foreign entity. When the chemoattractants’ origins are reached, specific receptors bind to the invaders surface, initiating phagocytosis, phagosome formation, and fusion with granule membranes, generating the bactericidal oxidative burst, and releasing lytic enzymes, specific peptides, and proteins. We explored the initial signaling involved in these functions by observing naïve, unprimed PMN in suspension using fluorescent indicators of cytoplasmic signals (Δ[Ca2+]i and ΔpHi) and of bactericidal entities (oxidative species and elastase) exposed to N-formyl-methionyl-leucyl-phenylalanine (fMLP) and/or multivalent immune complexes (IC). fMLP and IC each initiate a rapid transient rise in [Ca2+]i, mostly from intracellular stores, simultaneously with a drop in pHi; these are followed by a drop in [Ca2+]i and a rise in pHi, with the latter being due to a Na+/H+ antiport. The impact of a second stimulation depends on the order in which stimuli are applied, on their dose, and on their nature. Provided that [Ca2+]i is restored, 10−7 M fMLP, previously shown to elicit maximal Δ[Ca2+]i but no bactericidal functions, did not prevent the cells’ responses with Δ[Ca2+]i to a subsequent high dose of fMLP or IC; conversely, cells first exposed to 120 μg/ml IC, previously shown to elicit maximal Δ[Ca2+]i and bactericidal functions, exhibited no subsequent Δ[Ca2+]i or ΔpHi to either stimulus. While exposure to 10−7 M fMLP, which saturates the PMN high-affinity receptor, did not elicit bactericidal release from these naïve unprimed PMN in suspension, 10−5 M fMLP did, presumably via the low-affinity receptor, using a different Ca2+ source.

β amyloid fragments derived from activated platelets deposit in cerebrovascular endothelium : Usage of a novel blood brain barrier endothelial cell model system

Amyloid precursor protein (A betaPP) processing results in generation of amyloid beta peptide (A beta) which deposits in the brain parenchyma and cerebrovasculature of patients with Alzheimers disease (AD). Evidence that the vascular deposits derive in part from A betaPP fragments originating from activated platelets includes findings that individuals who have had multiple small strokes have a higher prevalence of AD compared to individuals who have taken anti-platelet drugs. Thus, determination of whether platelet A betaPP fragments are capable of traversing the blood-brain barrier (BBB) is critical. We have established that activated platelets from patients with AD retain more surface transmembrane-bound A betaPP (mA betaPP) than control platelets. We report here that this mA betaPP can be cleaved to A beta-containing fragments which pass through a novel BBB model system. This model utilizes human BBB endothelial cells (BEC) isolated from brains of patients with AD. These BEC, after exposure to activated platelets which have been surface-labeled with fluorescein and express surface-retained mA betaPP, cleave fluorescein-tagged surface proteins, including mA betaPP, resulting in passage to the BEC layer The data confirm that BEC contribute to processing of platelet-derived mA betaPP and show that the processing yields A beta containing fragments which could potentially contribute to cerebrovascular A beta deposition.