Role Of Tissue-nonspecific Alkaline Phosphatase Research Articles

Abstract 19: Loss Of Endothelial Tissue-nonspecific Alkaline Phosphatase Modifies Sensorimotor Deficits In Chronic Ischemic Stroke

Tissue nonspecific alkaline phosphatase (TNAP) is an ectoenzyme that is known for its anti-inflammatory properties in the immune system. Although TNAP is present in nearly every cell, it is highly abundant in brain endothelial cells (BEC). However, the role of TNAP in the BECs and other cells in the neurovascular unit is poorly understood. Our laboratory has previously identified a role for BEC TNAP in vascular integrity. While the loss of cortical TNAP activity has been observed in ischemic stroke, the functional outcomes that result from the loss of the enzyme are less clear. The objective of this study is to determine how TNAP affects post-stroke functional outcomes. We hypothesized that the loss of endothelial TNAP worsens post-stroke sensorimotor deficits. To test this, we generated mice with a conditional deletion of Alpl , the gene that codes for TNAP, in endothelial cells (VE-cKO mice) and compared the results to their littermate controls ( Alpl fl/fl ). Male and female six month-old mice (n=40) received a photothrombotic ischemic stroke (PTS) or sham surgery followed by daily neurological assessment (Clark’s test) and comprehensive sensorimotor testing, i.e. corner test, rotarod, open field, and gait analysis (DigiGait), over a seven-day period. Laser speckle flowmetry confirmed that PTS mice exhibited a sustained loss of cerebral blood flow compared to sham mice (p<0.0001). Daily neurological scoring showed higher (worse) scores in VEcKO PTS-mice compared to littermate controls (p=0.004). Quantification of total spontaneous movement via open field testing showed a significant interaction among stroke, genotype, and time (p=0.014). Gait analysis showed that the post-stroke stance/swing ratio, a measure of stability, differed between the left and right hind paws in VEcKO mice compared to controls: right hind paw- genotype x stroke interaction (p=0.041), and left hind paw- genotype x surgery x sex interaction (p=0.005). Significant interactions between surgery and sex were also observed in the right fore paw for stride (p=0.02), stride frequency (p=0.00026), and stride length (p=0.02). These results demonstrate a novel role for endothelial TNAP in functional recovery post-stroke.

Anticalcification effects of DS-1211 in pseudoxanthoma elasticum mouse models and the role of tissue-nonspecific alkaline phosphatase in ABCC6-deficient ectopic calcification

Pseudoxanthoma elasticum (PXE) is a multisystem, genetic, ectopic mineralization disorder with no effective treatment. Inhibition of tissue-nonspecific alkaline phosphatase (TNAP) may prevent ectopic soft tissue calcification by increasing endogenous pyrophosphate (PPi). This study evaluated the anticalcification effects of DS-1211, an orally administered, potent, and highly selective small molecule TNAP inhibitor, in mouse models of PXE. Calcium content in vibrissae was measured in KK/HlJ and ABCC6-/- mice after DS-1211 administration for 13–14 weeks. Pharmacokinetic and pharmacodynamic effects of DS-1211 were evaluated, including plasma alkaline phosphatase (ALP) activity and biomarker changes in PPi and pyridoxal-phosphate (PLP). Anticalcification effects of DS-1211 through TNAP inhibition were further evaluated in ABCC6-/- mice with genetically reduced TNAP activity, ABCC6-/-/TNAP+/+ and ABCC6-/-/TNAP+/-. In KK/HlJ and ABCC6-/- mouse models, DS-1211 inhibited plasma ALP activity in a dose-dependent manner and prevented progression of ectopic calcification compared with vehicle-treated mice. Plasma PPi and PLP increased dose-dependently with DS-1211 in ABCC6-/- mice. Mice with ABCC6-/-/TNAP+/- phenotype had significantly less calcification and higher plasma PPi and PLP than ABCC6-/-/TNAP+/+ mice. TNAP plays an active role in pathomechanistic pathways of dysregulated calcification, demonstrated by reduced ectopic calcification in mice with lower TNAP activity. DS-1211 may be a potential therapeutic drug for PXE.

The Role of Tissue-Nonspecific Alkaline Phosphatase in Dental Tissue: A Review of Recent Literature

AbstractThis brief review describes the role of tissue-nonspecific alkaline phosphatase (TNAP) during dental development. Mutations within the ALPL gene, which is encoding the human ectoenzyme TNAP, lead to the rare disease Hypophosphatasia (HPP). Apart from the most prominent bone hypomineralization, the characteristic symptoms of HPP often include a dental phenotype. To investigate the molecular causes of distinct HPP symptoms in closer detail, a number of transgenic models in different animals are established, like mice (Mus musculus) and zebrafish (Danio rerio). We therefore also compare the processes of dental development in humans, mice, and zebrafish.

TNAP is a novel regulator of cardiac fibrosis after myocardial infarction by mediating TGF-β/Smads and ERK1/2 signaling pathways.

Cardiac fibrosis is the most important pathogenesis leading to cardiac remodeling and heart failure after myocardial infarction (MI). Tissue nonspecific alkaline phosphatase (TNAP) has recently been recognized as a potential prognostic factor for MI. Nevertheless, the role of TNAP in cardiac fibrosis after MI has not been explicitly delineated. A systematic review and meta-analysis was conducted to assess the effect of serum TNAP levels on mortality in patients with ischemic heart disease (IHD). A correlation analysis was performed to investigate the relationship between serum levels of TNAP and biomarkers of fibrosis. Heart biopsies from patients with MI and a mouse model of MI were used to detect the expression and distribution of TNAP. Furthermore, we established adenovirus-mediated knockdown and overexpression of TNAP, using a combination of in vivo and in vitro studies in mice, to determine the role and mechanism of TNAP in cardiac fibrosis after MI. In the in vitro studies, cardiac fibroblasts were cultured on soft plates. After searching the main databases and performing a detailed assessment of the full-text articles, eight studies with 14,816 individuals were included in the quantitative analysis. We found that a high serum TNAP level was associated with an increased risk of mortality in patients with IHD and MI. The correlation analysis revealed a positive correlation between serum TNAP levels and the concentration of fibrosis biomarkers (PICP/PIIINP). The expression of TNAP was upregulated in the myocardium of patients with MI and in a mouse model of MI, accompanied by fibroblast activation and the deposition of collagen fibers. In the in vivo study, TNAP knockdown ameliorated cardiac fibrosis and improved cardiac function in mice. TNAP overexpression aggravated cardiac fibrosis and worsened cardiac function. In the in vitro study, TNAP promoted cardiac fibroblast differentiation, migration and proliferation. Mechanistically, the pro-fibrotic effect of TNAP on cardiac fibroblasts was at least partially achieved by activating the TGF-β1/Smads and ERK1/2 signaling pathways. Based on these findings, TNAP plays an important pro-fibrotic role in cardiac fibrosis after MI by activating TGF-β/Smads and ERK1/2 signaling, indicating that it functions as a potential regulator of and therapeutic target in cardiac fibrosis. This work was supported by the National Natural Science Foundation of China.

A novel role for tissue-nonspecific alkaline phosphatase at the blood-brain barrier during sepsis.

A novel role for tissue-nonspecific alkaline phosphatase at the blood-brain barrier during sepsis.

Systemic inhibition of tissue-nonspecific alkaline phosphatase alters the brain-immune axis in experimental sepsis

Tissue-nonspecific alkaline phosphatase (TNAP) is a ubiquitous enzyme present in many cells and tissues, including the central nervous system. Yet its functions at the brain-immune axis remain unclear. The goal of this study was to use a novel small molecular inhibitor of TNAP, SBI-425, to interrogate the function of TNAP in neuroimmune disorders. Following intraperitoneal (IP) administration of SBI-425, mass spectrometry analysis revealed that the SBI-425 does not cross the blood-brain barrier (BBB) in healthy mice. To elucidate the role of TNAP at the brain-immune axis, mice were subjected to experimental sepsis and received either vehicle or SBI-425 (25 mg/kg, IP) daily for 7 days. While SBI-425 administration did not affect clinical severity outcomes, we found that SBI-425 administration suppressed CD4 + Foxp3+ CD25− and CD8 + Foxp3+ CD25− splenocyte T-cell populations compared to controls. Further evaluation of SBI-425’s effects in the brain revealed that TNAP activity was suppressed in the brain parenchyma of SBI-425-treated mice compared to controls. When primary brain endothelial cells were treated with a proinflammatory stimulus the addition of SBI-425 treatment potentiated the loss of barrier function in BBB endothelial cells. To further demonstrate a protective role for TNAP at endothelial barriers within this axis, transgenic mice with a conditional overexpression of TNAP were subjected to experimental sepsis and found to have increased survival and decreased clinical severity scores compared to controls. Taken together, these results demonstrate a novel role for TNAP activity in shaping the dynamic interactions within the brain-immune axis.

Abstract TMP104: Upregulation of Tissue-Nonspecific Alkaline Phosphatase in Endothelial Cells Leads to Intracranial Vascular Calcification: A Useful Model of Primary Familial Brain Calcification

Introduction: Vascular calcification is an important pathophysiological factor contributing to neurodegenerative diseases such as primary familial brain calcification (PFBC) and is a potential therapeutic target. Hypothesis: Given the essential role of tissue-nonspecific alkaline phosphatase (TNAP) in biomineralization, we tested the hypothesis that upregulation of TNAP activity can lead to intracranial calcification. Methods: We previously reported that overexpression of TNAP in endothelial cells (eTNAP) leads to arterial calcification. Here we analyzed intracranial calcification in eTNAP on the original B6;129 and on the C57BL/6 (B6) genetic backgrounds. Histology was performed on formalin-fixed cryo-preserved tissues. Locomotion testing was performed at 23 weeks and analyzed in Matlab. Gene expression was analyzed by qPCR. Results: On the B6;129 background, eTNAP mice developed progressive intracranial calcification (0% were affected at 8 weeks, 71% at 13 weeks, and 100% at 23 weeks, n=7 per group). At 23 weeks, calcification was undetectable in the middle cerebral arteries but was associated with microvasculature in the basal ganglia, thalamus, hindbrain, and cerebellum. Calcified lesions were accompanied by astro- and micro-gliosis. Extravasation of IgG into the brain parenchyma was evident in eTNAP; blood-brain barrier was intact in controls (n=3 per group; 23 weeks). There were no significant differences in the locomotion or behavior (open field exploration) between the eTNAP and controls on the B6;129 background (n=6 per group). On B6 background, eTNAP mice displayed significant motor deficits - reduced ambulation (p<0.01), rearing (p< 0.01), speed (p<0.05), and acceleration (p<0.05; n=7-9 per group). There were no changes in osteogenic gene expression or phosphate transporters in eTNAP brains compared with controls. However, we detected significant regional differences between the thalamus and cortex in the levels of RumX2 , Spp1 , SLC17a7 , SLC17a6 of both control and eTNAP mice. Conclusions: Upregulation of TNAP activity can lead to intracranial vascular calcification. Given the similarities in presentation between eTNAP mice and PFBC patients, this model can advance the understanding of PFBC disease progression.

Tissue nonspecific alkaline phosphatase promotes calvarial progenitor cell cycle progression and cytokinesis via Erk1,2.

Bone growth is dependent upon the presence of self-renewing progenitor cell populations. While the contribution of Tissue Nonspecific Alkaline Phosphatase (TNAP) enzyme activity in promoting bone mineralization when expressed in differentiated bone forming cells is well understood, little is known regarding the role of TNAP in bone progenitor cells. We previously found diminished proliferation in the calvarial MC3T3E1 cell line upon suppression of TNAP by shRNA, and in calvarial cells and tissues of TNAP-/- mice. These findings indicate that TNAP promotes cell proliferation. Here we investigate how TNAP mediates this effect. Results show that TNAP is essential for calvarial progenitor cell cycle progression and cytokinesis, and that these effects are mediated by inorganic phosphate and Erk1/2. Levels of active Erk1/2 are significantly diminished in TNAP deficient cranial cells and tissues even in the presence of inorganic phosphate. Moreover, in the absence of TNAP, FGFR2 expression levels are high and FGF2 rescues phospho-Erk1/2 levels and cell cycle abnormalities to a significantly greater extent than inorganic phosphate. Based upon the data we propose a model in which TNAP stimulates Erk1/2 activity via both phosphate dependent and independent mechanisms to promote cell cycle progression and cytokinesis in calvarial bone progenitor cells. Concomitantly, TNAP feeds back to inhibit FGFR2 expression. These results identify a novel mechanism by which TNAP promotes calvarial progenitor cell renewal and indicate that converging pathways exist downstream of FGF signaling and TNAP activity to control craniofacial skeletal development.

Alkaline Phosphatase Controls Lineage Switching of Mesenchymal Stem Cells by Regulating the LRP6/GSK3β Complex in Hypophosphatasia.

Lineage differentiation of bone marrow mesenchymal stem cells (BMMSCs) is the key to bone-fat reciprocity in bone marrow. To date, the regulators of BMMSC lineage switching have all been identified to be transcription factors, and researchers have not determined whether other genes control this process. This study aims to reveal a previously unknown role of tissue-nonspecific alkaline phosphatase (TNSALP) in controlling BMMSC lineage selection.Methods: We compared the characteristics of cultured BMMSCs from patients with hypophosphatasia (HPP), which is caused by mutations in the liver/bone/kidney alkaline phosphatase (ALPL) gene, and an ALPL knockout (ko) mouse model. We performed ALPL downregulation and overexpression experiments to investigate the regulatory role of ALPL in BMMSC lineage switching. Using the PathScan array, coimmunoprecipitation experiments and pathway-guided small molecule treatments, we explored the possible mechanism underlying the regulatory effects of ALPL on cell differentiation and evaluated its therapeutic effect on ALPL ko mice.Results: BMMSCs from both patients with HPP and ALPL ko mice exhibited defective lineage differentiation, including a decrease in osteogenic differentiation and a parallel increase in adipogenic differentiation. Mechanistically, TNSALP directly interacted with LRP6 and regulated the phosphorylation of GSK3β, subsequently resulting in lineage switching of BMMSCs. Re-phosphorylation of GSK3β induced by LiCl treatment restored differentiation of BMMSCs and attenuated skeletal deformities in Alpl+/- mice.Conclusion: Based on our findings, TNSALP acts as a signal regulator to control lineage switching of BMMSCs by regulating the LRP6/GSK3β cascade.

Overexpression of tissue-nonspecific alkaline phosphatase increases the expression of neurogenic differentiation markers in the human SH-SY5Y neuroblastoma cell line

Patients suffering from the rare hereditary disease hypophosphatasia (HPP), which is based on mutations in the ALPL gene, tend to develop central nervous system (CNS) related issues like epileptic seizures and neuropsychiatric illnesses such as anxiety and depression, in addition to well-known problems with the mineralization of bones and teeth. Analyses of the molecular role of tissue-nonspecific alkaline phosphatase (TNAP) in transgenic SH-SY5YTNAPhigh neuroblastoma cells compared to SH-SY5YTNAPlow cells indicate that the enzyme influences the expression levels of neuronal marker genes like RNA-binding protein, fox-1 homolog 3 (NEUN) and enolase 2, gamma neuronal (NSE) as well as microtubule-binding proteins like microtubule-associated protein 2 (MAP2) and microtubule-associated protein tau (TAU) during neurogenic differentiation. Fluorescence staining of SH-SY5YTNAPhigh cells reveals TNAP localization throughout the whole length of the developed projection network and even synapsin Ι co-localization with strong TNAP signals at some spots at least at the early time points of differentiation. Additional immunocytochemical staining shows higher MAP2 expression in SH-SY5YTNAPhigh cells and further a distinct up-regulation of tau and MAP2 in the course of neurogenic differentiation. Interestingly, transgenic SH-SY5YTNAPhigh cells are able to develop longer cellular processes compared to control cells after stimulation with all-trans retinoic acid (RA). Current therapies for HPP prioritize improvement of the bone phenotype. Unraveling the molecular role of TNAP in extraosseous tissues, like in the CNS, will help to improve treatment strategies for HPP patients. Taking this rare disease as a model may also help to dissect TNAP's role in neurodegenerative diseases and even improve future treatment of common pathologies.

Signal Transduction Pathways of TNAP: Molecular Network Analyses.

Despite the growing body of evidence pointing on the involvement of tissue non-specific alkaline phosphatase (TNAP) in brain function and diseases like epilepsy and Alzheimer's disease, our understanding about the role of TNAP in the regulation of neurotransmission is severely limited. The aim of our study was to integrate the fragmented knowledge into a comprehensive view regarding neuronal functions of TNAP using objective tools. As a model we used the signal transduction molecular network of a pyramidal neuron after complementing with TNAP related data and performed the analysis using graph theoretic tools. The analyses show that TNAP is in the crossroad of numerous pathways and therefore is one of the key players of the neuronal signal transduction network. Through many of its connections, most notably with molecules of the purinergic system, TNAP serves as a controller by funnelling signal flow towards a subset of molecules. TNAP also appears as the source of signal to be spread via interactions with molecules involved among others in neurodegeneration. Cluster analyses identified TNAP as part of the second messenger signalling cascade. However, TNAP also forms connections with other functional groups involved in neuronal signal transduction. The results indicate the distinct ways of involvement of TNAP in multiple neuronal functions and diseases.

What Can We Learn About the Neural Functions of TNAP from Studies on Other Organs and Tissues?

To-date, the function of tissue-nonspecific alkaline phosphatase (TNAP) has largely been defined through studies in patients and mice affected by hypophosphatasia (HPP), a rare inborn-error-of-metabolism caused by mutation(s) in the TNAP gene (ALPL). The skeletal disease in HPP can be explained by alterations in the Pi/PPi ratio, with accumulation in the concentration of the mineralization inhibitor PPi as the culprit in preventing propagation of mineralization onto the collagenous extracellular matrix in bones and teeth. Accumulation of phosphorylated osteopontin increases the severity of HPP, at least in mice. Disruption in the metabolism of vitamin B6 leads to intracellular deficiency of pyridoxal, and this causes vitamin B6-responsive seizures in patients with the severe forms of the disease. Recent findings also implicate TNAP in the metabolism of ATP, in the production of adenosine and in the dephosphorylation of the bacterial toxin lipopolysaccharide, all molecules known to be involved in inflammation. The role of TNAP in establishing the ATP/adenosine ratio is important for purinergic signaling, and these mechanisms could be significant in determining axonal growth in the brain. Finally, the potential involvement of TNAP in dephosphorylating tau protein and its role in the pathogenesis of Alzheimer's disease is intriguing.

PHOSPHO1 is essential for mechanically competent mineralization and the avoidance of spontaneous fractures

Patients suffering from the rare hereditary disease hypophosphatasia (HPP), which is based on mutations in the ALPL gene, tend to develop central nervous system (CNS) related issues like epileptic seizures and neuropsychiatric illnesses such as anxiety and depression, in addition to well-known problems with the mineralization of bones and teeth. Analyses of the molecular role of tissue-nonspecific alkaline phosphatase (TNAP) in transgenic SH-SY5YTNAPhigh neuroblastoma cells compared to SH-SY5YTNAPlow cells indicate that the enzyme influences the expression levels of neuronal marker genes like RNA-binding protein, fox-1 homolog 3 (NEUN) and enolase 2, gamma neuronal (NSE) as well as microtubule-binding proteins like microtubule-associated protein 2 (MAP2) and microtubule-associated protein tau (TAU) during neurogenic differentiation. Fluorescence staining of SH-SY5YTNAPhigh cells reveals TNAP localization throughout the whole length of the developed projection network and even synapsin Ι co-localization with strong TNAP signals at some spots at least at the early time points of differentiation. Additional immunocytochemical staining shows higher MAP2 expression in SH-SY5YTNAPhigh cells and further a distinct up-regulation of tau and MAP2 in the course of neurogenic differentiation. Interestingly, transgenic SH-SY5YTNAPhigh cells are able to develop longer cellular processes compared to control cells after stimulation with all-trans retinoic acid (RA). Current therapies for HPP prioritize improvement of the bone phenotype. Unraveling the molecular role of TNAP in extraosseous tissues, like in the CNS, will help to improve treatment strategies for HPP patients. Taking this rare disease as a model may also help to dissect TNAP's role in neurodegenerative diseases and even improve future treatment of common pathologies.

Tissue-nonspecific Alkaline Phosphatase Is Required for the Calcification of Collagen in Serum: A POSSIBLE MECHANISM FOR BIOMINERALIZATION

Previous studies have shown that the type I collagen of tendon and demineralized bone both calcify rapidly in serum. The speed, collagen matrix-type specificity, and extent of the re-calcification of demineralized bone in serum suggest that the serum calcification activity identified in these studies may participate in normal biomineralization. Because of its presence in serum and its long history of association with the normal mineralization of the collagen matrix of bone, tissue-nonspecific alkaline phosphatase (TNAP) is an obvious candidate for a protein that could be a component of serum calcification activity, and experiments were therefore carried out to test this possibility. These experiments show that the inactivation of TNAP in serum prevents collagen calcification, and that the addition of physiological levels of purified TNAP restores the ability of TNAP-deficient serum to calcify collagen. Additional experiments show that the role of TNAP in collagen calcification is to activate a serum nucleator of apatite crystal formation. Based on these and earlier studies, the mechanism of collagen calcification in serum requires at least four elements as follows. 1) A matrix (collagen fibrils) that is accessible to small apatite crystals but not large molecules ( Toroian, D., Lim, J. E., and Price, P. A. (2007) J. Biol. Chem. 282, 22437-22447 ). 2) A large serum nucleator that generates small crystals, some of which diffuse into the fibrils. 3) A source of TNAP to activate the serum nucleator. 4) A large protein (fetuin) that selectively inhibits growth of crystals remaining in solution, thereby ensuring that only crystals within fibrils grow ( Toroian, D. T., and Price, P. A. (2008) Calcif. Tissue Int. 82, 116-126 ).

The role of tissue-nonspecific alkaline phosphatase in the phosphate-induced activation of alkaline phosphatase and mineralization in SaOS-2 human osteoblast-like cells

Tissue-nonspecific alkaline phosphatase (TNAP) plays a key role in mineralization by degrading inorganic pyrophosphate and providing free inorganic phosphate. We have previously reported that TNAP is induced by beta-glycerophosphate and NaH(2)PO(4) in short-term cultures of SaOS-2 human osteoblast-like cells and that PHEX (phosphate-regulating gene with homologies to endopeptidase on the X chromosome) mRNA is also induced after TNAP induction. In the present study, we have investigated the effects of levamisole, a TNAP inhibitor, and phosphonoformic acid (PFA), a type III sodium-phosphate cotransporter inhibitor, on the phosphate-induced expression of TNAP and mineralization. Levamisole inhibited beta-glycerophosphate-induced mineralization, TNAP and PHEX expression, and the increase in enzymatic activity of NPP1 (5'-nucleotide pyrophosphatase phosphodiesterase 1), but did not inhibit NaH(2)PO(4)-induced mineralization. PFA completely inhibited NaH(2)PO(4)-induced mineralization and NPP1 enzymatic activation, and partly inhibited beta-glycerophosphate-induced mineralization, but did not affect the increase in TNAP activity. These results suggest that phosphate derived from TNAP-induced hydrolysis of beta-glycerophosphate yields signals that induce TNAP expression and mineralization, and that PHEX expression may be linked to TNAP expression. However, luciferase assays failed to detect any transcriptional activation of the promoter region of the human TNAP gene by beta-glycerophosphate or NaH(2)PO(4), suggesting that the effects of these phosphates may be indirect.

Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 +/- 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.