Platelets are responsible for the accumulation of ß-amyloid in blood clots inside and around blood vessels in mouse brain after thrombosis.

INTRODUCTION: Platelets contain beta-amyloid precursor protein (APP) as well as Aß peptide (Aß) that can be released upon activation. During thrombosis, platelets are concentrated in clots and activated. METHODS: We used in vivo fluorescent analysis and electron microscopy in mice to determine to what degree platelets are concentrated in clots. We used immunostaining to visualize Aß after photothrombosis in mouse brains. RESULTS: Both in vivo results and electron microscopy revealed that platelets were 300-500 times more concentrated in clots than in non-clotted blood. After thrombosis in control mice, but not in thrombocytopenic animals, Aß immunofluorescence was present inside blood vessels in the visual cortex and around capillaries in the entorhinal cortex. CONCLUSION: The increased concentration of platelets allows enhanced release of Aß during thrombosis, suggesting an additional source of Aß in the brains of Alzheimer's patients that may arise if frequent micro-thrombosis events occur in their brains.

Soluble TLT-1 modulates platelet-endothelial cell interactions and actin polymerization.

Triggering receptor expressed on myeloid cells (TREM) like transcript-1 (TLT-1) is a membrane protein receptor found in alpha-granules of platelets and megakaryocytes. Upon platelet activation TLT-1 is rapidly brought to the surface of platelets. Recently, we demonstrated that activated platelets release a soluble form of TLT-1 (sTLT-1) that is found in serum but not in the plasma of healthy individuals and can enhance platelet aggregation in vitro. Furthermore, evaluation of patients diagnosed with inflammatory diseases, such as sepsis, show that these patients have significantly elevated levels of sTLT-1 in their blood. Accordingly, mice deficient in TLT-1 are predisposed to bleeding in response to an inflammatory challenge; however, the mechanism of TLT-1 function remains unknown. In this investigation, we demonstrate an increase in the amount of platelets that adhere to endothelial cell monolayers in the presence of recombinant sTLT-1 (rsTLT-1). Additionally, we present evidence that rsTLT-1 increases platelet adherence to glass slides by stimulating actin polymerization in platelets, as determined by increased staining of rodamine phalloidin. These results suggest that during inflammation, sTLT-1 may mediate hemostasis by enhancing actin polymerization, resulting in increased platelet aggregation and adherence to the endothelium.

Membrane potential and pH-dependent accumulation of decynium-22 (1,1'-diethyl-2,2'-cyanine iodide) flourencence through OCT transporters in astrocytes.

1,1 '-Diethyl-2,2'-cyanine iodide (decynium22; D22) is a potent blocker of the organic cation family of transporters (EMT/OCT) known to move endogenous monoamines like dopamine and norepinephrine across cell membranes. Decynium22 is a cation with a relatively high affinity for all members of the OCT family in both human and rat cells. The mechanism through which decynium22 blocks OCT transporters are poorly understood. We tested the hypothesis that denynium22 may compete with monoamines utilizing OCT to permeate the cells. Using the ability of D22 to aggregate and produce fluorescence at 570 nm, we measured D22 uptake in cultured astrocytes. The rate of D22 uptake was strongly depressed by acid pH and by elevated external K+. The rate of uptake was similar to that displayed by 4-(4-(dimethylamino)-styryl)-N-methylpyridinium (ASP+), a well established substrate for OCT and high-affinity Na+-dependent monoamine transporters. These data were supported by measurement of electrogenic uptake using whole cell voltage clamp recording. Decynium22 depressed norepinephrine, but not glutamate uptake. These data are also consistent with the described OCT transporter characteristics. Taken together, our results suggest that decynium22 accumulation might be a useful instrument to study monoamine transport in the brain, and particularly in astrocytes, where they may play a prominent role in monoamine uptake during brain dysfunction related to monoamines (like Parkinson disease) and drug addiction.