Secreted modular calcium-binding protein 1 binds and activates thrombin to account for platelet hyperreactivity in diabetes.

Secreted modular calcium-binding protein 1 (SMOC1) is an osteonectin/SPARC-related matricellular protein, whose expression is regulated by microRNA-223 (miR-223). Given that platelets are rich in miR-223, this study investigated the expression of SMOC1 and its contribution to platelet function. Human and murine platelets expressed SMOC1, whereas platelets from SMOC1+/- mice did not present detectable mature SMOC1 protein. Platelets from SMOC1+/- mice demonstrated attenuated responsiveness to thrombin (platelet neutrophil aggregate formation, aggregation, clot formation, Ca2+ increase, and ß3 integrin phosphorylation), whereas responses to other platelet agonists were unaffected. SMOC1 has been implicated in transforming growth factor-ß signaling, but no link to this pathway was detected in platelets. Rather, the SMOC1 Kazal domain directly bound thrombin to potentiate its activity in vitro, as well as its actions on isolated platelets. The latter effects were prevented by monoclonal antibodies against SMOC1. Platelets from miR-223-deficient mice expressed high levels of SMOC1 and exhibited hyperreactivity to thrombin that was also reversed by preincubation with monoclonal antibodies against SMOC1. Similarly, SMOC1 levels were markedly upregulated in platelets from individuals with type 2 diabetes, and the SMOC1 antibody abrogated platelet hyperresponsiveness to thrombin. Taken together, we have identified SMOC1 as a novel thrombin-activating protein that makes a significant contribution to the pathophysiological changes in platelet function associated with type 2 diabetes. Thus, strategies that target SMOC1 or its interaction with thrombin may be attractive therapeutic approaches to normalize platelet function in diabetes.

Structural, functional, and mechanistic insights uncover the fundamental role of orphan connexin-62 in platelets.

Connexins oligomerise to form hexameric hemichannels in the plasma membrane that can further dock together on adjacent cells to form gap junctions and facilitate intercellular trafficking of molecules. In this study, we report the expression and function of an orphan connexin, connexin-62 (Cx62), in human and mouse (Cx57, mouse homolog) platelets. A novel mimetic peptide (62Gap27) was developed to target the second extracellular loop of Cx62, and 3-dimensional structural models predicted its interference with gap junction and hemichannel function. The ability of 62Gap27 to regulate both gap junction and hemichannel-mediated intercellular communication was observed using fluorescence recovery after photobleaching analysis and flow cytometry. Cx62 inhibition by 62Gap27 suppressed a range of agonist-stimulated platelet functions and impaired thrombosis and hemostasis. This was associated with elevated protein kinase A-dependent signaling in a cyclic adenosine monophosphate-independent manner and was not observed in Cx57-deficient mouse platelets (in which the selectivity of 62Gap27 for this connexin was also confirmed). Notably, Cx62 hemichannels were observed to function independently of Cx37 and Cx40 hemichannels. Together, our data reveal a fundamental role for a hitherto uncharacterized connexin in regulating the function of circulating cells.

Platelet-derived calpain cleaves the endothelial protease-activated receptor 1 to induce vascular inflammation in diabetes.

Diabetes mellitus is a major risk factor for cardiovascular disease. Platelets from diabetic patients are hyperreactive and release microparticles that carry activated cysteine proteases or calpains. Whether platelet-derived calpains contribute to the development of vascular complications in diabetes is unknown. Here we report that platelet-derived calpain1 (CAPN1) cleaves the protease-activated receptor 1 (PAR-1) on the surface of endothelial cells, which then initiates a signaling cascade that includes the activation of the tumor necrosis factor (TNF)-α converting enzyme (TACE). The latter elicits the shedding of the endothelial protein C receptor and the generation of TNF-α, which in turn, induces intracellular adhesion molecule (ICAM)-1 expression to promote monocyte adhesion. All of the effects of CAPN1 were mimicked by platelet-derived microparticles from diabetic patients or from wild-type mice but not from CAPN1-/- mice, and were not observed in PAR-1-deficient endothelial cells. Importantly, aortae from diabetic mice expressed less PAR-1 but more ICAM-1 than non-diabetic mice, effects that were prevented by treating diabetic mice with a calpain inhibitor as well as by the platelet specific deletion of CAPN1. Thus, platelet-derived CAPN1 contributes to the initiation of the sterile vascular inflammation associated with diabetes via the cleavage of PAR-1 and the release of TNF-α from the endothelial cell surface.

Calpain 1 cleaves and inactivates prostacyclin synthase in mesenteric arteries from diabetic mice.

Diabetes is associated with a number of co-morbidities including an increased risk of developing cardiovascular diseases. The activation of Ca2+-activated proteases of the calpain family has been implicated in platelet activation associated with diabetes and this study aimed to determine the role of calpain activation in the development of endothelial dysfunction. Diabetes induction in mice attenuated acetylcholine-induced relaxation of mesenteric artery rings, an effect prevented in mice receiving a calpain inhibitor. A nitric oxide-independent but diclofenac-sensitive component of the relaxation-response was altered and correlated with a loss of prostacyclin (PGI2) generation and reduced vascular levels of PGI2 synthase. Calpain inhibition was also able to restore PGI2 synthase levels and PGI2 generation in arteries from diabetic animals. The effects of diabetes were reproduced in vitro by a combination of high glucose and palmitate, which elicited calpain activation, PGI2 synthase cleavage and inactivation as well as endothelial dysfunction in mesenteric arteries from wild-type mice. PGI2 cleavage was not observed in arteries from calpain 1-/- mice or mice overexpressing the endogenous calpain inhibitor calpastatin. Finally, proteomic analyses revealed that calpain 1 cleaved the C-terminal domain of PGI2 synthase close to the catalytic site of the enzyme. These data demonstrate that diabetes leads to the activation of calpain 1 in mesenteric arteries and can initiate endothelial dysfunction by cleaving and inactivating the PGI2 synthase. Given that calpain inhibition prevented diabetes-induced endothelial dysfunction in mesenteric arteries, calpains represent an interesting therapeutic target for the prevention of cardiovascular complication of diabetes.

Dicer cleavage by calpain determines platelet microRNA levels and function in diabetes.

RATIONALE: MicroRNAs (miRNAs) are short noncoding RNA species generated by the processing of longer precursors by the ribonucleases Drosha and Dicer. Platelets contain large amounts of miRNA that are altered by disease, in particular diabetes mellitus. OBJECTIVE: This study determined why platelet miRNA levels are attenuated in diabetic individuals and how decreased levels of the platelet-enriched miRNA, miR-223, affect platelet function. METHODS AND RESULTS: Dicer levels were altered in platelets from diabetic mice and patients, a change that could be attributed to the cleavage of the enzyme by calpain, resulting in loss of function. Diabetes mellitus in human subjects as well as in mice resulted in decreased levels of platelet miR-142, miR-143, miR-155, and miR-223. Focusing on only 1 of these miRNAs, miR-223 deletion in mice resulted in modestly enhanced platelet aggregation, the formation of large thrombi and delayed clot retraction compared with wild-type littermates. A similar dysregulation was detected in platelets from diabetic patients. Proteomic analysis of platelets from miR-223 knockout mice revealed increased levels of several proteins, including kindlin-3 and coagulation factor XIII-A. Whereas, kindlin-3 was indirectly regulated by miR-223, factor XIII was a direct target and both proteins were also altered in diabetic platelets. Treating diabetic mice with a calpain inhibitor prevented loss of platelet dicer as well as the diabetes mellitus-induced decrease in platelet miRNA levels and the upregulation of miR-223 target proteins. CONCLUSIONS: Thus, calpain inhibition may be one means of normalizing platelet miRNA processing as well as platelet function in diabetes mellitus.

MicroRNA-223 antagonizes angiogenesis by targeting ß1 integrin and preventing growth factor signaling in endothelial cells.

RATIONALE: Endothelial cells in situ are largely quiescent, and their isolation and culture are associated with the switch to a proliferative phenotype. OBJECTIVE: To identify antiangiogenic microRNAs expressed by native endothelial cells that are altered after isolation and culture, as well as the protein targets that regulate responses to growth factors. METHODS AND RESULTS: Profiling studies revealed that miR-223 was highly expressed in freshly isolated human, murine, and porcine endothelial cells, but those levels decreased in culture. In primary cultures of endothelial cells, vascular endothelial cell growth factor and basic fibroblast growth factor further decreased miR-223 expression. The overexpression of precursor-miR-223 did not affect basal endothelial cell proliferation but abrogated vascular endothelial cell growth factor-induced and basic fibroblast growth factor-induced proliferation, as well as migration and sprouting. Inhibition of miR-223 in vivo using specific antagomirs potentiated postnatal retinal angiogenesis in wild-type mice, whereas recovery of perfusion after femoral artery ligation and endothelial sprouting from aortic rings from adult miR-223(-/y) animals were enhanced. MiR-223 overexpression had no effect on the growth factor-induced activation of ERK1/2 but inhibited the vascular endothelial cell growth factor-induced and basic fibroblast growth factor-induced phosphorylation of their receptors and activation of Akt. ß1 integrin was identified as a target of miR-223 and its downregulation reproduced the defects in growth factor receptor phosphorylation and Akt signaling seen after miR-223 overexpression. Reintroduction of ß1 integrin into miR-223-ovexpressing cells was sufficient to rescue growth factor signaling and angiogenesis. CONCLUSIONS: These results indicate that miR-223 is an antiangiogenic microRNA that prevents endothelial cell proliferation at least partly by targeting ß1 integrin.

Calpain inhibition stabilizes the platelet proteome and reactivity in diabetes.

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-ß 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.

Ca2+-sensing receptor cleavage by calpain partially accounts for altered vascular reactivity in mice fed a high-fat diet.

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 macrovascular late complications associated with diabetes.

Loss of zinc transporters ZIP1 and ZIP3 augments platelet reactivity in response to thrombin and accelerates thrombus formation in vivo.

Zinc (Zn2+) is considered as important mediator of immune cell function, thrombosis and haemostasis. However, our understanding of the transport mechanisms that regulate Zn2+ homeostasis in platelets is limited. Zn2+ transporters, ZIPs and ZnTs, are widely expressed in eukaryotic cells. Using mice globally lacking ZIP1 and ZIP3 (ZIP1/3 DKO), our aim was to explore the potential role of these Zn2+ transporters in maintaining platelet Zn2+ homeostasis and in the regulation of platelet function. While ICP-MS measurements indicated unaltered overall Zn2+ concentrations in platelets of ZIP1/3 DKO mice, we observed a significantly increased content of FluoZin3-stainable free Zn2+, which, however, appears to be released less efficiently upon thrombin-stimulated platelet activation. On the functional level, ZIP1/3 DKO platelets exhibited a hyperactive response towards threshold concentrations of G protein-coupled receptor (GPCR) agonists, while immunoreceptor tyrosine-based activation motif (ITAM)-coupled receptor agonist signalling was unaffected. This resulted in enhanced platelet aggregation towards thrombin, bigger thrombus volume under flow ex vivo and faster in vivo thrombus formation in ZIP1/3 DKO mice. Molecularly, augmented GPCR responses were accompanied by enhanced Ca2+ and PKC, CamKII and ERK1/2 signalling. The current study thereby identifies ZIP1 and ZIP3 as important regulators for the maintenance of platelet Zn2+ homeostasis and function.

Platelet-Enriched MicroRNAs and Cardiovascular Homeostasis.

SIGNIFICANCE: Platelets are anucleate blood cells that are involved in hemostasis and thrombosis. Although no longer able to generate ribonucleic acid (RNA) de novo, platelets contain messenger RNA (mRNA), YRNA fragments, and premature microRNAs (miRNAs) that they inherit from megakaryocytes. Recent Advances: Novel sequencing techniques have helped identify the unexpectedly large number of RNA species present in platelets. Throughout their life time, platelets can process the pre-existing pool of premature miRNA to give the fully functional miRNA that can regulate platelet protein expression and function. CRITICAL ISSUES: Platelets make a major contribution to the circulating miRNA pool but platelet activation can have major consequences on Dicer levels and thus miRNA maturation, which has implications for studies that are focused on screening-stored platelets. FUTURE DIRECTIONS: It will be important to determine the importance of platelets as donors for miRNA-containing microvesicles that can be taken up and processed by other (particularly vascular) cells, thus contributing to homeostasis as well as disease progression. Antioxid. Redox Signal. 29, 902-921.

VASP regulates leukocyte infiltration, polarization, and vascular repair after ischemia.

In ischemic vascular diseases, leukocyte recruitment and polarization are crucial for revascularization and tissue repair. We investigated the role of vasodilator-stimulated phosphoprotein (VASP) in vascular repair. After hindlimb ischemia induction, blood flow recovery, angiogenesis, arteriogenesis, and leukocyte infiltration into ischemic muscles in VASP-/- mice were accelerated. VASP deficiency also elevated the polarization of the macrophages through increased signal transducer and activator of transcription (STAT) signaling, which augmented the release of chemokines, cytokines, and growth factors to promote leukocyte recruitment and vascular repair. Importantly, VASP deletion in bone marrow-derived cells was sufficient to mimic the increased blood flow recovery of global VASP-/- mice. In chemotaxis experiments, VASP-/- neutrophils/monocytes were significantly more responsive to M1-related chemokines than wild-type controls. Mechanistically, VASP formed complexes with the chemokine receptor CCR2 and ß-arrestin-2, and CCR2 receptor internalization was significantly reduced in VASP-/- leukocytes. Our data indicate that VASP is a major regulator of leukocyte recruitment and polarization in postischemic revascularization and support a novel role of VASP in chemokine receptor trafficking.

miR-223-IGF-IR signalling in hypoxia- and load-induced right-ventricular failure: a novel therapeutic approach.

AIMS: Pulmonary hypertension is a progressive disease with poor prognosis, characterized by pathological inward remodelling and loss of patency of the lung vasculature. The right ventricle is co-affected by pulmonary hypertension, which triggers events such as hypoxia and/or increased mechanical load. Initially the right ventricle responds with 'adaptive' hypertrophy, which is often rapidly followed by 'maladaptive' changes leading to right heart decompensation and failure, which is the ultimate cause of death. METHODS AND RESULTS: We report here that miR-223 is expressed in the murine lung and right ventricle at higher levels than in the left ventricle. Moreover, lung and right-ventricular miR-223 levels were markedly down-regulated by hypoxia. Correspondingly, increasing right-ventricular load by pulmonary artery banding, induced right-ventricular ischaemia, and the down-regulation of miR-223. Lung and right ventricle miR-223 down-regulation were linked with increased expression of the miR-223 target; insulin-like growth factor-I receptor (IGF-IR) and IGF-I downstream signalling. Similarly, miR-223 was decreased and IGF-IR increased in human pulmonary hypertension. Notably in young mice, miR-223 overexpression, the genetic inactivation or pharmacological inhibition of IGF-IR, all attenuated right-ventricular hypertrophy and improved right heart function under conditions of hypoxia or increased afterload. CONCLUSION: These findings highlight the early role of pulmonary and right-ventricular miR-223 and the IGF-IR in the right heart failure programme initiated by pulmonary hypoxia and increased mechanical load and may lead to the development of novel therapeutic strategies that target the development of PH and right heart failure.

Metformin reduces hyper-reactivity of platelets from patients with polycystic ovary syndrome by improving mitochondrial integrity.

Polycystic ovary syndrome (PCOS) is associated with decreased fertility, insulin resistance and an increased risk of developing cardiovascular disease. Treating PCOS patients with metformin improves fertility and decreases cardiovascular complications. Given that platelet activation contributes to both infertility and cardiovascular disease development, we assessed platelet reactivity in PCOS patients and the consequences of metformin treatment. Compared to washed platelets from healthy donors, platelets from PCOS patients demonstrated enhanced reactivity and impaired activation of the AMP-activated kinase (AMPK). PCOS platelets also demonstrated enhanced expression of mitochondrial proteins such as the cytochrome c reductase, ATP synthase and the voltage-dependent anion channel-1. However, mitochondrial function was impaired as demonstrated by a decreased respiration rate. In parallel, the phosphorylation of dynamin-related protein-1 (Drp-1) on Ser616 was increased while that on Ser637 decreased. The latter changes were accompanied by decreased mitochondrial size. In insulin-resistant PCOS patients (HOMA-IR> 2) metformin treatment (1.7 g per day for 4 weeks to 6 months) improved insulin sensitivity, restored mitochondrial integrity and function and normalised platelet aggregation. Treatment was without effect in PCOS patients with HOMA-IR< 2. Moreover, treatment of megakaryocytes with metformin enhanced mitochondrial content and in the same cells metformin enhanced the phosphorylation of the Drp-1 on Ser637 via an AMPKα1-dependent mechanism. In conclusion, the improvement of mitochondrial integrity and platelet reactivity may contribute to the beneficial effects of metformin on cardiovascular disease.

Methylglyoxal induces platelet hyperaggregation and reduces thrombus stability by activating PKC and inhibiting PI3K/Akt pathway.

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 ß3-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.