Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

There is frequent use of human and animal proteins as stabilizers during lyophilization of a variety of biological substances with a view to long term stable storage. This report describes the comparative excellent stabilizing effect of a porcine collagen peptide fraction (CPF) during the lyophilization and subsequent storage of three commonly used biological substances, alkaline phosphatase, tissue plasminogen activator and thrombin. The CPF was heated to 150 degrees C for one hour before use. The CPF was shown to have some advantage during lyophilized storage over human serum albumin. Solutions of thrombin stored in CPF at room temperature and at 37 degrees C for one week retained nearly all activity, while storage of thrombin in human serum albumin solution at 37 degrees C lost nearly all biological activity. These preliminary data suggest that porcine CPF is a safe and advantageous stabilizer for addition to biological products with a view to long-term lyophilized storage and short-term liquid storage.
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PMID:A pig collagen peptide fraction. A unique material for maintaining biological activity during lyophilization and during storage in the liquid state. 891 Aug 48

By means of CM-Sephadex C-25, DEAE-Sephadex A-50, Sephadex G-200, and Sephadex G-75 chromatographies, a lupus anticoagulant like protein (LALP) from Agkistrodon halys brevicaudus was purified. On SDS-PAGE, the purified LALP had a molecular weight of 25,500 daltons under non-reducing condition and 15,000 daltons under reducing condition. The isoelectric point was pH 5.6. Its N terminal amino acid sequencing revealed a mixture of 2 sequences: DCP(P/S)(D/G)WSSYEGH(C/R)(Q/K). It was devoid of phospholipase A, fibrino(geno)lytic, 5'-nucleotidase, L-amino acid oxidase, phosphomonoesterase, phosphodiesterase and thrombin-like activities, which were found in crude venom. In the presence of LALP, PT, aPTT, and dRVVT of human plasma were markedly prolonged and its effects were concentration-dependent but time-independent. The inhibitory effect of LALP on the plasma clotting time was enhanced by decreasing phospholipid concentration in TTI test. The individual clotting factor activity was not affected by LALP when higher dilutions of LALP-plasma mixture were used for assay. Russell's viper venom time was shortened when high phospholipid confirmatory reagent was used. Therefore, the protein has lupus anticoagulant property.
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PMID:Purification and characterization of lupus anticoagulant like protein from Agkistrodon halys brevicaudus venom. 897 23

Adhesion molecules such as P-selectin are potential markers for evaluating platelet activation and studying the role of cell-cell interactions in numerous biological processes related to hemostasis and inflammation. The expression of P-selectin and related molecules has previously been quantified with different techniques. As an alternative to the most common method. flow cytometry, we have developed a useful ELISA method to simultaneously analyse 96 samples for platelet expression of P-selectin. Samples may be stored for at least 7 days at 4 degrees C prior to analysis. The method is simple, reproducible, flexible and requires only standard equipment. Washed platelets (WP) from healthy male volunteers, at a concentration of 1 x 10(7)/microtiter plate well, were stimulated with various known platelet activators and fixed with 0.1% formaldehyde for 10 min. The fixed WP were centrifuged to form a confluent layer in the wells and then incubated with optimal dilutions of primary antibodies (1/2000) directed against P-selectin, CD41, CD9 and secondary antibodies conjugated with alkaline phosphatase. Our results show that P-selectin expression on WP increases significantly upon stimulation with thrombin (0.1-1.0 U/ml), ADP (10 microM) and epinephrine (100 microM). The induction of P-selectin expression by thrombin is fast and has different kinetics depending on the concentration of the agonist. Prior incubation with the nitric oxide donor SNAP (10 microM) inhibits the up-regulation of P-selectin induced by sub-maximal concentrations of thrombin (p < 0.05). This ELISA is suitable for studying the expression and regulation of P-selectin and other surface molecules on human platelets in various pathological states.
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PMID:Modulation of P-selectin expression on isolated human platelets by an NO donor assessed by a novel ELISA application. 900 52

Annexins are a family of calcium-binding proteins that have been implicated in a wide range of intracellular processes. We have previously reported that stimulation of platelets with agents that increase intracellular [Ca2+] induces the relocation of annexin V to membranes, and that this annexin V may be binding to a 50 kDa protein located within platelet membranes. We report here, using an in vitro reconstitution system, that the relocation of annexin V to membranes is enhanced by ATP. We also demonstrate that when adenosine 5'-[gamma-thio]-triphosphate, which can replace ATP in phosphorylation reactions, is substituted for ATP, the amount of annexin V that binds to membranes is further increased. In separate experiments using intact cells, we show that the protein phosphatase inhibitor okadaic acid mimics the action of the physiological agonist thrombin, in that it induces annexin V to bind to membranes and that the addition of the protein kinase inhibitor staurosporine inhibits A23187-induced relocation of annexin V. In addition, alkaline phosphatase, when added to isolated membranes, was found to remove endogenous annexin V from the membranes. Furthermore, immunoprecipitation of 33P-labelled proteins indicated that annexin V may form a multi-protein complex including phosphoproteins of 25, 50 and 83 kDa. Taken together these observations suggest that, following physiological activation, the phosphorylation of one or more proteins is responsible for the tight association of annexin V with platelet membranes and the subsequent regulation of membrane localized processes.
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PMID:Relocation of annexin V to platelet membranes is a phosphorylation-dependent process. 937

PtdIns(4,5)P2 production by the enzyme PtdIns4P 5-kinase C (PIPkin C) was examined in thrombin-stimulated human platelets. Thrombin caused a rapid, transient 2-3-fold increase in PIPkin activity and a transient net dephosphorylation of the enzyme. PIPkin C was phosphorylated on serine and threonine residues in unstimulated platelets; no evidence for tyrosine phosphorylation was found. The phosphatase inhibitor okadaic acid promoted PIPkin C hyperphosphorylation and a concomitant marked inhibition of its activity in immunoprecipitates. Activity was restored by treatment with alkaline phosphatase, suggesting the existence of an inhibitory phosphorylation site. In support of this idea, alkaline phosphatase treatment of PIPkin C immunoprecipitated from unstimulated platelets caused a modest (1.6-fold) but significant activation of the enzyme. However, alkaline phosphatase treatment of PIPkin C immunoprecipitated from thrombin-stimulated platelets caused a decrease in activity to approximately the same levels, suggesting that the phosphorylation of PIPkin C also contributes to the observed stimulation. Two-dimensional phosphopeptide mapping of immunoprecipitated PIPkin C revealed that the enzyme is multiply phosphorylated and that, whereas some phosphopeptides are indeed lost on stimulation, consistent with the net dephosphorylation of the enzyme, at least two novel sites become phosphorylated. This suggests that thrombin causes complex changes in the phosphorylation state of PIPkin C, one consequence of which is its activation.
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PMID:Regulation of PtdIns4P 5-kinase C by thrombin-stimulated changes in its phosphorylation state in human platelets. 940 83

In the present study we have investigated whether platelet derived microvesicles can bind soluble fibrinogen, bind to immobilized fibrinogen, and coaggregate with platelets. Flow cytometry was used for studies on binding of soluble fibrinogen and coaggregation, whereas ELISA wells were used to study binding of microvesicles to immobilized fibrinogen. Biotinylated microvesicles produced by stimulation with A23187, thrombin or SFLLRN of platelets which had been surface-labelled with biotin, were used both for the coaggregation experiments and for the binding studies with immobilized fibrinogen. Unlabelled microvesicles and biotinylated fibrinogen were employed when studying binding of soluble fibrinogen to the microvesicles. For the flow cytometry, the biotinylated proteins were reacted with avidin or streptavidin which was PE-conjugated, whereas the same substances were conjugated with alkaline phosphatase for the ELISA studies. The microvesicles formed after stimulation of platelets by SFLLRN or A23187 clearly bound the soluble, biotinylated fibrinogen. Moreover, isolated biotinylated microvesicles added to washed platelets prior to activation, were associated to the microaggregates that formed after stimulation. A significant binding of biotinylated microvesicles to immobilized fibrinogen could also be detected. The binding of microvesicles to soluble and immobilized fibrinogen and association to platelets was clearly specific and at least partly dependent on the GPIIb-IIIa complex, as all of these phenomena could be prevented or reduced by addition of the c7E3 Fab which blocks the activated form of this receptor complex. From these in vitro results it is clear that microvesicles can bind to immobilized fibrinogen, bind soluble fibrinogen and are able to coaggregate with platelets. It may be speculated that these results also reflect a haemostatic role of microvesicles in vivo.
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PMID:Microvesicles bind soluble fibrinogen, adhere to immobilized fibrinogen and coaggregate with platelets. 949 96

Twenty-two cats with liver disease were evaluated for coagulation abnormalities including alterations in prothrombin time, activated partial thromboplastin time, thrombin time, factor VII activity, and platelet count. The purpose of the study was to determine the prevalence of coagulation abnormalities in this population of cats, classify abnormalities according to underlying pathogenesis, and determine if serum biochemical parameters typically used as indicators of liver disease showed any correlation with the coagulation abnormalities present. Study results indicated that at least 1 coagulation abnormality was present in 82% of the cats. Prolongation of prothrombin time was most common (16/22 cats) and factor VII activity was below reference range (< 60%) in 15 cats. When classified according to underlying pathogenesis, vitamin K deficiency was the most common abnormality found (11/22). Other abnormalities were less common and included hepatic synthetic failure (3/22), indeterminate (3/22), and disseminated intravascular coagulation (1/22). Increase in alkaline phosphatase (ALP) activity was the only biochemical abnormality that showed statistically significant correlation with coagulation abnormalities (P = .023). Cats with marked increases in ALP activity were more likely to have coagulation abnormalities than those with only mild increases in ALP activity.
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PMID:Coagulation abnormalities in 22 cats with naturally occurring liver disease. 956 Jul 61

Previous studies have indicated that thrombin can activate pulp cells, including fibroblasts. Because pulp cells and periodontal ligament (PDL) fibroblasts can express thrombin receptor mRNA, the specific aim of this study was to determine whether thrombin can activate the growth, attachment, protein synthesis, alkaline phosphatase activities and cellular clustering of cultured human PDL fibroblasts. Thrombin can stimulate the growth of PDL fibroblasts in a dose dependent manner (as analyzed by MTT assay). At concentrations of 5 and 10 U/ml, thrombin increased the cell numbers to 141% and 153% greater than that of the control after 5 days of incubation, respectively. Thrombin (5-20 U/ml) also stimulated the protein synthesis rate (assayed by [3H]proline incorporation) to 1.88-2.13 fold that of the control. However, pretreatment of PDL fibroblasts with thrombin (1-20 U/ml) could not promote the attachment of PDL fibroblasts to type I collagen and fibronectin. Moreover, thrombin could induce clustering of PDL fibroblasts within a concentration range of 5-20 U/ml. However, thrombin (1-20 U/ml) exerted neither stimulatory nor inhibitory effect on cellular alkaline phosphatase activities. In conclusion, it appears that the presence of thrombin seems to have effects on PDL fibroblasts in terms of cell growth, protein synthesis and cell clustering. This suggests that thrombin might be important in the early healing process of periodontium following periodontal surgery.
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PMID:Effects of thrombin on the growth, protein synthesis, attachment, clustering and alkaline phosphatase activity of cultured human periodontal ligament fibroblasts. 985 May 96

In addition to playing a central role in thrombosis and hemostasis, the serine protease thrombin is a specific agonist for a variety of functional responses in cells including osteoblast-like cells. Many of the cellular responses to thrombin are mediated by protease-activated receptor-1 (PAR-1). Since osteoblasts express PAR-1 in vivo during development, the effect of PAR-1 activation on proliferation and differentiation in primary rat osteoblast-like cells was investigated. Thrombin or the rat PAR-1-activating peptide SFFLRNPSENTFELVPL (SFFL) stimulated cell proliferation (as assessed by 3H- thymidine incorporation) of primary osteoblast-like cells derived from long bone or calvaria, and treatment with antibodies to PAR-1 abolished the proliferative response to thrombin. Activation of PAR-1 by thrombin or SFFL inhibited endogenous alkaline phosphatase (ALP) activity and caused a transient elevation of intracellular calcium in the osteoblast-like cells. Calcium mobilization was not, however, required for thrombin's effect on proliferation or ALP activity. The ability of a number of growth factors and hormones to regulate expression of PAR-1 in osteoblast-like cells was investigated. Expression of PAR-1 transcript and protein by osteoblast-like cells in vitro was markedly increased by treatment with transforming growth factor-beta (TGF-beta), and the proliferative response to thrombin was enhanced by TGF-beta pretreatment. Platelet-derived growth factor-BB caused a slight but significant down-regulation of PAR-1 mRNA expression. Thrombin caused a transient increase in PAR-1 expression, whereas neither parathyroid hormone-related peptide nor 1, 25-dihydroxyvitamin D3 had any effect. The observations described here suggest that PAR-1 mediates thrombin-induced osteoblast proliferation, which in turn may contribute to responses of osteoblasts to osteogenic growth factors.
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PMID:Modulation of osteoblast-like cell behavior by activation of protease-activated receptor-1. 1045 64

Osteoblasts express protease-activated receptor-1 (PAR-1), which is activated by thrombin or by synthetic peptides corresponding to the new "tethered ligand" N-terminus of PAR-1 created by receptor cleavage. Both thrombin and human PAR-1-activating peptide stimulate an elevation of [Ca2+]i in the human SaOS-2 osteoblast-like cell line, but the peptide stimulates receptor-mediated Ca+ entry, whereas thrombin does not. Stimulation of proliferation in rat primary osteoblast-like cells is greater in response to rat PAR-1-activating peptide than to thrombin. Because the PAR-1-activating peptides are now known to activate PAR-2, the current study was undertaken to investigate whether osteoblasts express this receptor and, if so, whether this could account for the observed discrepancies between responses of osteoblasts to thrombin and to PAR-1-activating peptides. Reverse transcriptase-polymerase chain reaction (RT-PCR) and immunocytochemical studies demonstrated expression of PAR-2 by primary cultures of rat calvarial osteoblast-like cells. In immunohistochemical studies of embryonic mouse bones, osteoblasts showed positive staining for the presence of PAR-2. Activators of PAR-2 include trypsin, mast cell tryptase, gingipain-R, and synthetic peptides corresponding to the PAR-2 tethered ligand sequence. Treatment of primary rat osteoblast-like cells with rat PAR-2-activating peptide (SLIGRL), or SaOS-2 cells with human PAR-2-activating peptide (SLIGKV), caused a dose-dependent increase in [Ca2+]i. Trypsin or gingipain-R also induced an increase in intracellular calcium concentration, and caused reciprocal cross desensitization. Activators of PAR-2 caused a sharp peak in [Ca2+]i followed by a sustained plateau; [Ca2+]i returned to baseline levels upon treatment with ethylene-glycol tetraacetic acid (EGTA). Treatment of rat osteoblast-like cells in vitro with SLIGRL did not affect thymidine incorporation or endogenous alkaline phosphatase activity. The results presented here demonstrate that osteoblasts express PAR-2, and that such expression is able to account for the observed discrepancies between thrombin and PAR-1-activating peptides in their ability to evoke calcium entry, but not proliferative responses.
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PMID:Expression of protease-activated receptor-2 by osteoblasts. 1061 51


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