Role of amyloid peptides in vascular dysfunction and platelet dysregulation in Alzheimer’s disease

Alzheimer’s disease (AD) is the most common neurodegenerative cause of dementia in the elderly. AD is accompanied by the accumulation of amyloid peptides in the brain parenchyma and in the cerebral vessels. The sporadic form of the AD accounts for about 95% of all cases. It is characterized by a late onset, typically after the age of 65, with a complex and still poorly understood aetiology. Several observations point towards a central role of cerebrovascular dysfunction in the onset of sporadic AD. According to the vascular hypothesis, AD may be initiated by vascular dysfunctions that precede and promote the neurodegenerative process. In accordance to this, AD patients show increased hemorragic or ischemic stroke risks. It is now clear that multiple bidirectional connections exist between AD and cerebrovascular disease, and in this new scenario, the effect of amyloid peptides on vascular cells and blood platelets appear to be central to AD. In this review we analyse the effect of amyloid peptides on vascular function and platelet activation and its contribution to the cerebrovascular pathology associated with AD and the progression of this disease

Increased platelet adhesion and thrombus formation in a mouse model of Alzheimer's disease

Vascular dysfunctions and Alzheimer's disease show significant similarities and overlaps. Cardiovascular risk factors (hypercholesterolemia, hypertension, obesity, atherosclerosis and diabetes) increase the risk of vascular dementia and Alzheimer's disease. Conversely, Alzheimer's patients have considerably increased predisposition of ischemic and hemorrhagic strokes. Platelets are major players in haemostasis and thrombosis and are involved in inflammation. We have investigated morphology and function of platelets in 3xTg-AD animals, a consolidate murine model for Alzheimer's disease. Platelets from aged 3xTg-AD mice are normal in number and glycoprotein expression, but adhere more avidly on matrices such as fibrillar collagen, von Willebrand factor, fibrinogen and amyloid peptides compared to platelets from age-matching wild type mice. 3xTg-AD washed platelets adherent to collagen also show increased phosphorylation of selected signaling proteins, including tyrosine kinase Pyk2, PI3 kinase effector Akt, p38MAP kinase and myosin light chain kinase, and increased ability to form thrombi under shear. In contrast, aggregation and integrin αIIbβ3 activation induced by several agonists in 3xTg-AD mice are similar to wild type platelets. These results demonstrated that Alzheimer's mutations result in a significant hyper-activated state of circulating platelets, evident with the progression of the disease

Extracellular Vesicles in Multiple Sclerosis: Role in the Pathogenesis and Potential Usefulness as Biomarkers and Therapeutic Tools

Although extracellular vesicles (EVs) were initially relegated to a waste disposal role, nowadays, they have gained multiple fundamental functions working as messengers in intercellular communication as well as exerting active roles in physiological and pathological processes. Accumulating evidence proves the involvement of EVs in many diseases, including those of the central nervous system (CNS), such as multiple sclerosis (MS). Indeed, these membrane-bound particles, produced in any type of cell, carry and release a vast range of bioactive molecules (nucleic acids, proteins, and lipids), conferring genotypic and phenotypic changes to the recipient cell. This means that not only EVs per se but their content, especially, could reveal new candidate disease biomarkers and/or therapeutic agents. This review is intended to provide an overview regarding current knowledge about EVs’ involvement in MS, analyzing the potential versatility of EVs as a new therapeutic tool and source of biomarkers

Novel pharmacological inhibitors demonstrate the role of the tyrosine kinase Pyk2 in adhesion and aggregation of human platelets

Pyk2 is a Ca(2+)-regulated kinase predominantly expressed in neuronal and in haematopoietic cells. Previous studies on Pyk2-null mice have demonstrated that Pyk2 plays a crucial role in platelet activation and thrombus formation, thus representing a possible target for antithrombotic therapy. Very limited information is available about the role of Pyk2 in human platelets, mainly because of the lack of specific pharmacological inhibitors. In this work, we have tested two novel Pyk2 inhibitors, PF-4594755 and PF-4520440, to validate their specificity and to investigate their ability to modulate platelet activation. Both molecules were able to efficiently block Pyk2 activity in human and mouse platelets stimulated with thrombin or with the Ca(2+)-ionophore. In wild-type murine platelets, PF-4594755 and PF-4520440 reduced thrombin-induced aggregation to the level observed in Pyk2 knockout platelets, but did not affect aggregation induced by GPVI stimulation. Importantly, neither compounds affected the residual thrombin-induced aggregation of Pyk2-null platelets, thus excluding possible off-target effects. In human platelets, PF-4594755 and PF-4520440 significantly reduced aggregation stimulated by thrombin, but not by the GPVI agonist convulxin. Both inhibitors reduced platelet adhesion on fibrinogen and prevented Akt phosphorylation in adherent cells, indicating that Pyk2 regulates PI3K and cell spreading downstream of integrins in human platelets. Finally, the Pyk2 inhibitors significantly inhibited thrombus formation upon blood perfusion on immobilized collagen under arterial flow rate. These results demonstrate that PF-4594755 and PF-4520440 are specific inhibitors of Pyk2 in intact platelets and allowed to reliably document that this kinase plays a relevant role in human platelet activation

Molecular mechanisms of platelet activation and aggregation induced by breast cancer cells

Tumor cell-induced platelet aggregation represents a critical process both for successful metastatic spread of the tumor and for the development of thrombotic complications in cancer patients. To get further insights into this process, we investigated and compared the molecular mechanisms of platelet aggregation induced by two different breast cancer cell lines (MDA-MB-231 and MCF7) and a colorectal cancer cell line (Caco-2). All the three types of cancer cells were able to induce comparable platelet aggregation, which, however, was observed exclusively in the presence of CaCl2 and autologous plasma. Aggregation was supported both by fibrinogen binding to integrin αIIbβ3 as well as by fibrin formation, and was completely prevented by the serine protease inhibitor PPACK. Platelet aggregation was preceded by generation of low amounts of thrombin, possibly through tumor cells-expressed tissue factor, and was supported by platelet activation, as revealed by stimulation of phospholipase C, intracellular Ca2+ increase and activation of Rap1b GTPase. Pharmacological inhibition of phospholipase C, but not of phosphatidylinositol 3-kinase or Src family kinases prevented tumor cell-induced platelet aggregation. Tumor cells also induced dense granule secretion and the stimulation of the P2Y12 receptor by released ADP was found to be necessary for complete platelet aggregation. By contrast, prevention of thromboxane A2 synthesis by aspirin did not alter the ability of all the cancer cell lines analyzed to induce platelet aggregation. These results indicate that tumor cell-induced platelet aggregation is not related to type of the cancer cells or to their metastatic potential, and is triggered by platelet activation and secretion driven by the generation of small amount of thrombin from plasma and supported by the positive feedback signaling through secreted ADP

Stimulation of mTORC2 by integrin αIIbβ3 is required for PI3Kβ-dependent activation of Akt but is dispensable for platelet spreading on fibrinogen

Phosphatidylinositol 3 kinase (PI3K) is a major player in platelet activation and regulates thrombus formation and stabilization. The β isoform of PI3K is implicated in integrin αIIbβ3 outside-in signaling, is required for the phosphorylation of Akt, and controls efficient platelet spreading upon adhesion to fibrinogen. In this study we found that during integrin αIIbβ3 outside-in signaling PI3Kβ-dependent phosphorylation of Akt on Serine473 is mediated by the mammalian target of rapamycin complex 2 (mTORC2). The activity of mTORC2 is stimulated upon platelet adhesion to fibrinogen, as documented by increased autophosphorylation. However, mTORC2 activation downstream of integrin αIIbβ3 is PI3Kβ-independent. Inhibition of mTORC2, but not mTORC1, also prevents Akt phosphorylation of Threonine308 and affects Akt activity, resulting in the inhibition of GSK3α/β phosphorylation. Nevertheless, mTORC2 or Akt inhibition does not alter PI3Kβ-dependent platelet spreading on fibrinogen. The activation of the small GTPase Rap1b downstream of integrin αIIbβ3 is regulated by PI3Kβ but is not affected upon inhibition of either mTORC2 or Akt. Altogether, these results demonstrate for the first time the activation of mTORC2 and its involvement in Akt phosphorylation and stimulation during integrin αIIbβ3 outside-in signaling. Moreover, the results demonstrate that the mTORC2/Akt pathway is dispensable for PI3Kβ-regulated platelet spreading on fibrinogen

Platelet amyloid precursor protein is a modulator of venous thromboembolism in mice

The amyloid precursor protein (APP), primarily known as the precursor of amyloid peptides that accumulate in the brain of patients with Alzheimer disease, is abundant in platelets, but its physiological function remains unknown. In this study, we investigated the role of APP in hemostasis and thrombosis, using APP knockout (KO) mice. Ex vivo aggregation, secretion, and integrin αIIbβ3 inside-out activation induced by several agonists were normal in APP-deficient platelets, but the number of circulating platelets was reduced by about 20%, and their size was slightly increased. Tail bleeding time was normal, and in vivo, the absence of APP did not alter thrombus formation in the femoral artery. In contrast, in a model of vein thrombosis induced by flow restriction in the inferior vena cava, APP-KO mice, as well as chimeric mice with selective deficiency of APP in blood cells, developed much larger thrombi than control animals, and were more sensitive to embolization. Consistent with this, in a pulmonary thromboembolism model, larger vessels were occluded. APP-KO mice displayed a shorter APTT, but not PT, when measured in the presence of platelets. Moreover, the activity of factor XIa (FXIa), but not FXIIa, was higher in APP-KO mice compared with controls. APP-KO mice presented a higher number of circulating platelet-leukocyte aggregates, and neutrophils displayed a greater tendency to protrude extracellular traps, which were more strongly incorporated into venous thrombi. These results indicate that platelet APP limits venous thromboembolism through a negative regulation of both fibrin formation and neutrophil function

Amyloid precursor protein is required for in vitro platelet adhesion to amyloid peptides and potentiation of thrombus formation

Amyloid precursor protein (APP) is the precursor of amyloid β (Aβ) peptides, whose accumulation in the brain is associated with Alzheimer's disease. APP is also expressed on the platelet surface and Aβ peptides are platelet agonists. The physiological role of APP is largely unknown. In neurons, APP acts as an adhesive receptor, facilitating integrin-mediated cell adhesion, while in platelets it regulates coagulation and venous thrombosis. In this work, we analyzed platelets from APP KO mice to investigate whether membrane APP supports platelet adhesion to physiological and pathological substrates. We found that APP-null platelets adhered and spread normally on collagen, von Willebrand Factor or fibrinogen. However, adhesion on immobilized Aβ peptides Aβ1-40, Aβ1-42 and Aβ25-35 was completely abolished in platelets lacking APP. By contrast, platelet activation and aggregation induced by Aβ peptides occurred normally in the absence of APP. Adhesion of APP-transfected HEK293 to Aβ peptides was significantly higher than that of control cells expressing low levels of APP. Co-coating of Aβ1-42 and Aβ25-35 with collagen strongly potentiated platelet adhesion when whole blood from wild type mice was perfused at arterial shear rate, but had no effects with blood from APP KO mice. These results demonstrate that APP selectively mediates platelet adhesion to Aβ under static condition but not platelet aggregation, and is responsible for Aβ-promoted potentiation of thrombus formation under flow. Therefore, APP may facilitate an early step in thrombus formation when Aβ peptides accumulate in cerebral vessel walls or atherosclerotic plaques