[Show abstract][Hide abstract] ABSTRACT: Calpain has been associated with the pathophysiology of Alzheimer's disease (AD) and with apoptotic neuronal cell death leading to microparticles (MPs) formation.
Alzheimer's & dementia: the journal of the Alzheimer's Association 09/2014; · 14.48 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Primary hemostasis is initiated by the adhesion of platelets to the exposed subendothelial matrix, a process that also initiates platelet activation and stimulates multiple platelet signaling pathways . One of the earliest events in platelet activation is the mobilization of stored Ca(2+) which is then able to form a complex with calmodulin (CaM) and subsequently activate a large number of CaM-binding proteins, including the myosin light chain kinase. The latter phosphorylates the myosin light chain and leads to the rapid rearrangement of the cytoskeleton that results in a shape change from discoid to fully spread cells. This article is protected by copyright. All rights reserved.
Journal of Thrombosis and Haemostasis 04/2014; · 6.08 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The Ca-sensing receptor (CaSR) is expressed in endothelial and smooth muscle cells but its role in regulating vascular reactivity is unclear, as are the effects of disease on CaSR function and expression.We studied vascular reactivity in aortic segments from healthy and diabetic mice, combined with in vitro proteolysis studies and Western blot analyses of CaSR expression in tissue samples.In endothelium-intact aortic rings, extracellular Ca elicited a nitric oxide-dependent relaxation that was attenuated by the CaSR antagonist, NPS2390. The calcimimetic, calindol, induced the endothelium-independent relaxation of aortic segments that was also sensitive to NPS2390. The antagonist failed to affect responses to acetylcholine or U46619 but attenuated contractions to phenylephrine and potassium. In mice fed a Western-type diet, phenylephrine-induced contractions and calindol-induced relaxations were markedly attenuated, and CaSR expression was decreased. The latter phenomenon could be attributed to the activation of the Ca-dependent protease, µ-calpain, and the subsequent proteolytic cleavage of the CaSR.CaSR activation in smooth muscle cells modulates vascular responsiveness to Ca-elevating agonists. These effects are blunted during metabolic stress because of the limited proteolysis of the CaSR by calpain. The loss of the CaSR function may predispose to the microvascular late complications associated with diabetes.
Journal of cardiovascular pharmacology 02/2013; · 2.83 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Diabetes is characterized by a dysregulation of glucose homeostasis and platelets from patients with diabetes are known to be hyper-reactive and contribute to the accelerated development of vascular diseases. Since many of the deleterious effects of glucose have been attributed to its metabolite methylgyloxal (MG) rather than to hyperglycemia itself, the aim of the present study was to characterize the effects of MG on platelet function. Washed human platelets were pre-incubated for 15 min with MG and platelet aggregation, adhesion on matrix-coated slides and signaling (Western blot) were assessed ex vivo. In vivo, the effect of MG on thrombus formation was determined using the FeCl3-induced carotid artery injury model. MG potentiated thrombin-induced platelet aggregation and dense granule release, but inhibited platelet spreading on fibronectin and collagen. In vivo, MG accelerated thrombus formation but decreased thrombus stability. At the molecular level, MG increased intracellular Ca(2+) and activated classical PKCs at the same time as inhibiting PI3K/Akt and the β3-integrin outside-in signaling. In conclusion, these findings indicate that the enhanced MG concentration measured in diabetic patients can directly contribute to the platelet dysfunction associated with diabetes characterized by hyperaggregability and reduced thrombus stability.
PLoS ONE 01/2013; 8(9):e74401. · 3.53 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Platelets from patients with diabetes are hyperreactive and demonstrate increased adhesiveness, aggregation, degranulation, and thrombus formation, processes that contribute to the accelerated development of vascular disease. Part of the problem seems to be dysregulated platelet Ca(2+) signaling and the activation of calpains, which are Ca(2+)-activated proteases that result in the limited proteolysis of substrate proteins and subsequent alterations in signaling. In the present study, we report that the activation of μ- and m-calpain in patients with type 2 diabetes has profound effects on the platelet proteome and have identified septin-5 and the integrin-linked kinase (ILK) as novel calpain substrates. The calpain-dependent cleavage of septin-5 disturbed its association with syntaxin-4 and promoted the secretion of α-granule contents, including TGF-β and CCL5. Calpain was also released by platelets and cleaved CCL5 to generate a variant with enhanced activity. Calpain activation also disrupted the ILK-PINCH-Parvin complex and altered platelet adhesion and spreading. In diabetic mice, calpain inhibition reversed the effects of diabetes on platelet protein cleavage, decreased circulating CCL5 levels, reduced platelet-leukocyte aggregate formation, and improved platelet function. The results of the present study indicate that diabetes-induced platelet dysfunction is mediated largely by calpain activation and suggest that calpain inhibition may be an effective way of preserving platelet function and eventually decelerating atherothrombosis development.
[Show abstract][Hide abstract] ABSTRACT: The Ca(2+)-activated proteases or calpains, play a crucial role in a spectrum of physiological processes such as cytoskeletal remodeling, cellular signaling, cell migration, apoptosis, cell survival and platelet activation, by the proteolytic cleavage of target proteins. Rather than eliciting protein degradation the calpains are responsible for their modification (e.g. activation, inhibition or altered sensitivity to intracellular signals) and therefore make a significant impact on intracellular signaling. Maintained calpain activation is known to be associated with disease development and in platelet calpains are involved in both physiological platelet activation as well as pathological platelet hyper-activation.
[Show abstract][Hide abstract] ABSTRACT: Platelets are involved in vascular homeostasis and inflammation through interaction with circulating blood cells and vascular wall. MiRNAs are small, conserved and non-coding RNA molecules, which interact directly with specific mRNAs regions regulating gene expression. The purpose of this review is to gather all known platelet miRNAs and summarize their role in platelet biogenesis and function. Increasing evidence supports the role of miR-34a and miR-150 in megakaryocytopoiesis and platelet production. Although 284 miRNAs are described to be present in platelets, their role is mostly unknown. The most abundant miRNA in platelets is miR-223 followed by miR-126. The miR-96, miR-200b, miR- 495, miR-107 and miR-223 are critically involved in platelet reactivity, aggregation, secretion and adhesion. The presence of miRNAs known to regulate angiogenesis in platelets is also discussed. Furthermore, platelet-derived microvesicles and microparticles contain several miRNAs, which may facilitate the communication between platelets with other vascular cells, a mechanism that may play an important role in vascular homeostasis and inflammation. Further studies are needed to elucidate the exact roles of platelet miRNAs in platelet function and vascular biology.
Current Vascular Pharmacology 02/2012; 10(5):524-31. · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Diabetes mellitus is associated with the accelerated development of vascular disease and there is evidence that platelets actively contribute to this process. Certainly, platelets are able to modulate the function of endothelial and vascular smooth muscle cells via the direct release of growth factors and pro-inflammatory chemokines but also via the production of microparticles which function as a transcellular delivery system for micro RNAs. This article reviews the intracellular signaling mechanisms underlying the increased activation of diabetic platelets and the involvement of platelets in atherothrombosis development.
Current Vascular Pharmacology 02/2012; 10(5):532-8. · 2.91 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The adenosine monophosphate (AMP)-activated protein kinase (AMPK) is a regulator of energy balance at the cellular and whole-body levels, but little is known about the role of AMPK in platelet activation. We report that both the α1 and α2 AMPK isoforms are expressed by human and murine platelets and that thrombin elicits the phosphorylation of AMPKα as well as the upstream kinase, liver kinase B1 (LKB1). In human platelets, the kinase inhibitors iodotubercidin and compound C significantly inhibited thrombin-induced platelet aggregation and clot retraction without affecting the initial increase in [Ca(2+)](i). Clot retraction was also impaired in platelets from AMPKα2(-/-) mice but not from wild-type littermates or AMPKα1(-/-) mice. Moreover, rebleeding was more frequent in AMPKα2(-/-) mice, and the FeCl(3)-induced thrombi formed in AMPKα2(-/-) mice were unstable. Mechanistically, AMPKα2 was found to phosphorylate in vitro the Src-family kinase, Fyn, and isoform deletion resulted in the attenuated threonine phosphorylation of Fyn as well as the subsequent tyrosine phosphorylation of its substrate, β3 integrin. These data indicate that AMPKα2-by affecting Fyn phosphorylation and activity-plays a key role in platelet αIIbβ3 integrin signaling, leading to clot retraction and thrombus stability.
[Show abstract][Hide abstract] ABSTRACT: Diabetes mellitus is a major risk factor for vascular diseases and is associated with accelerated atherosclerosis and a high rate of arterial thrombotic complications. A number of studies support the concept that platelets contribute to the pathogenesis and progression of the vascular complications of diabetes. µ-Calpain, a non-lysosomal, Ca(2+)-dependent cysteine protease, is expressed in platelets and is involved in physiological platelet activation. However, the inappropriate activation of calpain alters platelet function, partially degrades a spectrum of proteins and results in hyperaggregability. Changes in the activity of calpain in different cells involved in diabetes-related pathways, or the polymorphism of calpain genes have been associated with the development of type 2 diabetes but their relevance to the diabetes-related vascular complications is not really clear. This review will give an overview of the role of calpain in diabetes and analyze the role of calpain in platelet activation and the changes occurring during the onset of diabetes. Finally, we will discuss future therapeutic possibilities for the improvement of diabetes-associated vascular diseases.
Advances in pharmacology (San Diego, Calif.) 01/2010; 59:235-57.
[Show abstract][Hide abstract] ABSTRACT: Sphingosine-1-phosphate (S1P) is known to affect platelet responsiveness but the receptor mediating these effects and the mechanisms involved are poorly understood. This study was undertaken to examine S1P receptor expression in human platelets as well as potential changes associated with type 2 diabetes. S1P(2) receptor expression (Western blotting) was detected in washed human platelets from healthy volunteers. Stimulation of these platelets with exogenous S1P led to a concentration-dependent increase in intracellular Ca(2+) as well as to platelet aggregation. The S1P-induced increase in Ca(2+) was sensitive to the S1P(2) receptor antagonist JTE-013 but not the S1P(1/3) antagonist VPC23019. Both antagonists reduced the aggregation stimulated by S1P in a non-additive manner. S1P also elicited the translocation of RhoA to the membrane and RhoA activity was inhibited (by 50%) by the S1P receptor antagonists. Platelets from patients with type 2 diabetes demonstrated an attenuated aggregability to S1P as well as decreased levels of the full-length S1P(2) protein. The S1P(2) antibody used identified a 45 kDa receptor cleavage product in patients with diabetes that could also be generated from healthy human platelet lysates by the addition of the Ca(2+)-activated protease, mu-calpain. These results indicate that the S1P(2) receptor is involved in S1P-induced platelet aggregation and Rho kinase activation. Moreover, in platelets from patients with type 2 diabetes, responses to S1P are attenuated via a phenomenon attributed to the calpain-dependent cleavage of the S1P(2) receptor.
Archiv für Kreislaufforschung 02/2009; 104(3):333-40. · 7.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Recent studies indicate that platelets influence endothelial progenitor cell (EPC) recruitment to sites of vascular injury and promote their differentiation to an endothelial phenotype. Patients with cardiovascular risk factors (CVRF) demonstrate a reduced number and impaired function of EPC, as well as platelet hyper-reactivity. Therefore, we investigated the interaction of platelets and EPC from patients with CVRF.
Co-incubation of platelets and peripheral blood mononuclear cells, both from healthy volunteers, dose-dependently increased the number of adherent EPC. In contrast, patient-derived platelets failed to augment the number of adherent and migrating healthy and patient-derived EPC. However, co-incubation of platelets from healthy donors with mononuclear cells from patients with CVRF significantly enhanced the number of EPC, indicating that platelets from healthy volunteers are able to partially rescue the impairment of patient-derived EPC formation. Likewise, healthy donor-derived platelets augmented the impaired migration and clonal capacity of patient-derived EPC. Analysis of individual CVRF of platelet donors revealed that only diabetes mellitus inversely correlated with EPC number, colony formation and migration. The platelet supernatants from healthy volunteers that significantly increased EPC number contained IL-6, SDF-1, sCD40L and PDGF. While sCD40L and PDGF levels were comparable in platelet supernatants from healthy volunteers and patients with CVRF, the release of IL-6 and SDF-1 by patient-derived platelets was rather increased, thus, indicating that these soluble factors are not mediating the effect of platelet supernatants.
Healthy volunteer-derived platelets provide a source of soluble factors to improve the number and function of EPC from patients with cardiovascular risk factors, particularly diabetes mellitus.
Archiv für Kreislaufforschung 08/2008; 103(6):572-81. · 7.35 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Platelets from patients with type 2 diabetes mellitus display hyperaggregability and increased thrombogenic potential.
In platelets from patients with type 2 diabetes mellitus, we found enhanced tyrosine nitration and inactivation of the sarcoplasmic endoplasmic reticulum Ca2+-ATPase (SERCA-2), elevated platelet [Ca2+]i, and activation of mu-calpain. The tyrosine nitration of SERCA-2 and the activation of mu-calpain in vitro in platelets from healthy volunteers could be evoked in vitro by peroxynitrite. Platelet endothelial cell adhesion molecule-1 was identified as a mu-calpain substrate; its in vitro degradation was stimulated by peroxynitrite and prevented by calpain inhibitors. Calpain activation also was linked to hyperresponsiveness to thrombin and the loss of platelet sensitivity to nitric oxide synthase inhibitors. Platelets from patients with type 2 diabetes mellitus (hemoglobin A1c >6.6%) contained little or no intact platelet endothelial cell adhesion molecule-1, whereas degradation products were detectable. The peroxisome proliferator-activated receptor-gamma agonist rosiglitazone increased SERCA-2 expression in megakaryocytes, and treating patients with type 2 diabetes mellitus with rosiglitazone for 12 weeks increased platelet SERCA-2 expression and Ca2+-ATPase activity, decreased SERCA-2 tyrosine nitration, and normalized platelet [Ca2+]i. Rosiglitazone also reduced mu-calpain activity, normalized platelet endothelial cell adhesion molecule-1 levels, and partially restored platelet sensitivity to nitric oxide synthase inhibition.
These data identify megakaryocytes/platelets as additional cellular targets for peroxisome proliferator-activated receptor-gamma agonists and highlight potential benefits of rosiglitazone therapy in cardiovascular diseases.
[Show abstract][Hide abstract] ABSTRACT: Exogenous application of 20-hydroxyeicosatetraenoic acid (20-HETE) to small (300-500 microm) porcine coronary arteries elicits contraction by activating the Rho kinase and increasing the sensitivity of contractile proteins to Ca2+. Here, we determined whether 20-HETE is involved in the regulation of coronary artery tone as well as its role in the modulation of endothelium-derived hyperpolarizing factor (EDHF)-mediated responses.
Small porcine coronary arteries expressed cytochrome P450 (CYP) 4A, as demonstrated by Western blot analysis, and generated 20-HETE. Moreover, 20-HETE production was increased two- and threefold over basal levels in response to isometric stretch or the thromboxane analogue U46619, respectively, and was inhibited by the CYP 4A inhibitor N-methylsulfonyl-12,12-dibromododec-11-enamide (DDMS). In vascular reactivity studies, DDMS attenuated U46619-induced contractions and induced a concentration-dependent but endothelium-independent relaxation of precontracted arterial rings. Endogenously generated 20-HETE significantly inhibited the EDHF-mediated relaxation of coronary arteries, which was potentiated by the phospholipase A2 inhibitors AACOCF3 and ONO-RS-082, as well as by the omega-hydroxylase inhibitors 17-octadecynoic acid and DDMS. EDHF-mediated relaxation was not affected by either the nonselective epoxygenase inhibitors miconazole and clotrimazole or the CYP 2C inhibitor sulfaphenazole but was abolished by the Na-K-ATPase inhibitor, ouabain. Exogenous application of 20-HETE inhibited EDHF-mediated relaxations and caused a concomitant increase in the phosphorylation of protein kinase Calpha (PKCalpha). This effect was reversed by the PKC inhibitor Ro-318220 and mimicked by the PKC activator phorbol-12 myristate 13-acetate.
These results indicate that vascular tone in small porcine coronary arteries is partly determined by the endogenous production of 20-HETE. In addition, 20-HETE functionally antagonizes EDHF-mediated relaxation via a PKCalpha-dependent mechanism, probably involving the inhibition of the Na-K-ATPase.
Cardiovascular Research 03/2005; 65(2):487-94. · 5.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Platelets express the endothelial form of the nitric oxide synthase (eNOS) and generate NO. However, in contrast to eNOS in endothelial cells, eNOS in platelets is largely Ca(2+)-independent and the activity is regulated by phosphorylation. Platelet-derived NO plays an important role in the regulation of platelet aggregation and secretion. Changes in the activity of platelet eNOS are responsible for the abnormal platelet activation encountered in different pathological situations (e.g. hypertension and diabetes). In this review, we will summarize the current knowledge of the role of platelet eNOS and the regulation of its activity as well as the fate of platelet-derived NO in physiological and pathological situations.
[Show abstract][Hide abstract] ABSTRACT: Insulin-induced vasodilatation is sensitive to nitric oxide (NO) synthase (NOS) inhibitors. However, insulin is unable to relax isolated arteries or to activate endothelial NOS in endothelial cells. Since insulin can enhance platelet endothelial NOS activity, we determined whether insulin-induced vasodilatation can be attributed to a NO-dependent, platelet-mediated process. Insulin failed to relax endothelium-intact rings of porcine coronary artery. The supernatant from insulin-stimulated human platelets induced complete relaxation, which was prevented by preincubation of platelets with a NOS inhibitor, the soluble guanylyl cyclase inhibitor, NS 2028, or the G kinase inhibitor, KT 5823, and was abolished by an adenosine A2A receptor antagonist. Insulin induced the release of adenosine trisphosphate (ATP), adenosine, and serotonin from platelet-dense granules in a NO-dependent manner. This response was not detected using insulin-stimulated platelets from endothelial NOS-/- mice, although a NO donor elicited ATP release. Insulin-induced ATP release from human platelets correlated with the association of syntaxin 2 with the vesicle-associated membrane protein 3 but was not associated with the activation of alphaIIbbeta3 integrin. Thus, insulin elicits the release of vasoactive concentrations of ATP and adenosine from human platelets via a NO-G kinase-dependent signaling cascade. The mechanism of dense granule secretion involves the G kinase-dependent association of syntaxin 2 with vesicle-associated membrane protein 3.
Journal of Experimental Medicine 03/2004; 199(3):347-56. · 13.21 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: 20-HETE is a potent constrictor of small blood vessels and has been suggested to play a crucial role in the generation of myogenic tone and the development of hypertension. In the present study, we investigated the mechanisms by which exogenously applied 20-HETE modulates vascular tone in small porcine coronary arteries. In organ chamber experiments, 20-HETE elicited a concentration-dependent contraction of small porcine coronary artery rings that was partially inhibited by the cyclooxygenase inhibitor diclofenac, the thromboxane and endoperoxide receptor antagonist SQ29548, and the thromboxane A2 synthase inhibitor furegrelate. Removal of endothelium attenuated the response to 20-HETE, whereas preconstriction of endothelium-denuded vessels to 25% of the maximum response with KCl markedly enhanced the response to 20-HETE. This 20-HETE-induced contraction was not associated with a significant increase in the intracellular concentration of Ca2+. 20-HETE-induced contraction was also observed in beta-escin-permeabilized arteries precontracted with a submaximal concentration of Ca2+ and was abolished by the Rho-kinase inhibitor Y27632, but was insensitive to the PKC inhibitor RO 31-8220. 20-HETE elicited the phosphorylation of the myosin light chain (MLC20) in coronary artery rings, an effect that was sensitive to Y27632 and mimicked by the thromboxane analog U46619. These data suggest that in small porcine coronary arteries, 20-HETE can induce contraction by 2 mechanisms, one endothelium-dependent involving the cyclooxygenase-dependent generation of vasoconstrictor prostanoids, and the other endothelium-independent. The latter response is associated with the activation of Rho-kinase, phosphorylation of MLC20, and sensitization of the contractile apparatus to Ca2+.
[Show abstract][Hide abstract] ABSTRACT: Over the last 10 years it has become increasingly clear that cytochrome P450 (CYP) enzymes expressed within endothelial and vascular smooth muscle cells play a crucial role in the modulation of vascular homeostasis. There is strong evidence suggesting that the activation of a CYP 2C epoxygenase in endothelial cells is an essential step in nitric oxide (NO)- and prostacyclin (PGI(2))-independent vasodilatation of several vascular beds, particularly in the heart and kidney. Moreover, CYP epoxygenase products as well as CYP-derived reactive oxygen species are intracellular signal transduction molecules involved in several signaling cascades affecting numerous cellular processes, including vascular cell proliferation and angiogenesis. Various pharmacological compounds enhance vascular CYP 2C expression. One group of substances which highlight the possible effects of CYP induction in endothelial cells on vascular function are the HMG-CoA reductase inhibitors (statins). Cerivastatin and fluvastatin increase CYP 2C mRNA and protein in native and cultured endothelial cells, and enhance the bradykinin-induced NO/PGI(2)-independent relaxation of arterial segments as well as the generation of reactive oxygen species. However, statins also increase the expression of the endothelial NO synthase by approximately twofold. As a consequence, the probability that NO and reactive oxygen species react to generate peroxynitrite is increased and the treatment of vascular segments with statins resulted in enhanced protein tyrosine nitration. These data highlight the role played by CYP 2C in vascular homeostasis and its potential regulation by cardiovascular drugs.
Biochimica et Biophysica Acta 03/2003; 1619(3):332-9. · 4.66 Impact Factor