Matrix vesicle biomimetics harboring Annexin A5 and alkaline phosphatase bind to the native collagen matrix produced by mineralizing vascular smooth muscle cells.

BACKGOUND: Vascular smooth muscle cells (VSMCs) transdifferentiated ectopically trigger vascular calcifications, contributing to clinical cardiovascular disease in the aging population. AnxA5 and TNAP play a crucial role in (patho)physiological mineralization. METHODS: We performed affinity studies between DPPC and 9:1 DPPC:DPPS-proteoliposomes carrying AnxA5 and/or TNAP and different types of collagen matrix: type I, II, I + III and native collagenous extracellular matrix (ECM) produced from VSMCs with or without differentiation, to simulate ectopic calcification conditions. RESULTS: AnxA5-proteoliposomes had the highest affinity for collagens, specially for type II. TNAP-proteoliposomes bound poorly and the simultaneous presence of TNAP in the AnxA5-proteoliposomes disturbed interactions between AnxA5 and collagen. DPPC AnxA5-proteoliposomes affinities for ECM from transdifferentiating cells went up 2-fold compared to that from native VSMCs. The affinities of DPPC:DPPS-proteoliposomes were high for ECM from VSMCs with or without differentiation, underscoring a synergistic effect between AnxA5 and DPPS. Co-localization studies uncovered binding of proteoliposomes harboring AnxA5 or TNAP+AnxA5 to various regions of the ECM, not limited to type II collagen. CONCLUSION: AnxA5-proteoliposomes showed the highest affinities for type II collagen, deposited during chondrocyte mineralization in joint cartilage. TNAP in the lipid/protein microenvironment disturbs interactions between AnxA5 and collagen. These findings support the hypothesis that TNAP is cleaved from the MVs membrane just before ECM binding, such facilitating MV anchoring to ECM via AnxA5 interaction. GENERAL SIGNIFICANCE: Proteoliposomes as MV biomimetics are useful in the understanding of mechanisms that regulate the mineralization process and may be essential for the development of novel therapeutic strategies to prevent or inhibit ectopic mineralization.

Bacterial adherence to graft tissues in static and flow conditions.

BACKGROUND: Various conduits and stent-mounted valves are used as pulmonary valve graft tissues for right ventricular outflow tract reconstruction with good hemodynamic results. Valve replacement carries an increased risk of infective endocarditis (IE). Recent observations have increased awareness of the risk of IE after transcatheter implantation of a stent-mounted bovine jugular vein valve. This study focused on the susceptibility of graft tissue surfaces to bacterial adherence as a potential risk factor for subsequent IE. METHODS: Adhesion of Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus sanguinis to bovine pericardium (BP) patch, bovine jugular vein (BJV), and cryopreserved homograft (CH) tissues was quantified under static and shear stress conditions. Microscopic analysis and histology were performed to evaluate bacterial adhesion to matrix components. RESULTS: In general, similar bacteria numbers were recovered from CH and BJV tissue surfaces for all strains, especially in flow conditions. Static bacterial adhesion to the CH wall was lower for S sanguinis adhesion (P < .05 vs BP patch). Adhesion to the BJV wall, CH wall, and leaflet was decreased for S epidermidis in static conditions (P < .05 vs BP patch). Bacterial adhesion under shear stress indicated similar bacterial adhesion to all tissues, except for lower adhesion to the BJV wall after S sanguinis incubation. Microscopic analysis showed the importance of matrix component exposure for bacterial adherence to CH. CONCLUSIONS: Our data provide evidence that the surface composition of BJV and CH tissues themselves, bacterial surface proteins, and shear forces per se are not the prime determinants of bacterial adherence.

Absence of Pear1 does not affect murine platelet function in vivo.

BACKGROUND: Platelet Endothelial Aggregation Receptor-1 (PEAR1) is a transmembrane platelet receptor that amplifies the activation of the platelet fibrinogen receptor (αIIbß3) during platelet aggregation. In man, Pear1 polymorphisms are associated with changes in platelet aggregability. In this report, we characterized Pear1 expression and function in murine platelets. METHODS: Pear1 phosphorylation and signaling, platelet aggregation, α-degranulation and clot retraction were studied in WT and Pear1-/- platelets. The function of Pear1 in haemostasis and thrombosis was studied in a mouse tail vein bleeding and ferric chloride-induced mesenteric thrombosis model. RESULTS: Mature murine platelets express Pear1 on their membrane and clustering of Pear1 by anti-Pear1 antibodies triggered platelet aggregation. Pear1 was weakly phosphorylated during collagen-induced murine platelet aggregation and was translocated to the cytoskeleton. Absence of murine Pear1 impaired dextran sulfate-induced platelet aggregation, but did not impact collagen-, AYPGK and ADP-induced platelet aggregation, coupled to a lower Pear1 expression in murine than in human platelets and to weaker Pear1-mediated downstream signaling. Neither clot retraction nor α-degranulation was affected in Pear1-/- mice. Likewise, in vivo tests like the tail vein bleeding time and thrombus formation in mesenteric veins were similar in WT and Pear1-/- mice. CONCLUSION: Murine platelet Pear1 shares a number of characteristics with human platelet PEAR1. Nevertheless, murine Pear1 contributes less to platelet function as does human PEAR1 and does not overtly impact haemostasis and thrombosis in mice.

Dextran sulfate triggers platelet aggregation via direct activation of PEAR1.

UNLABELLED: Dextran sulfate (DxS; Mr 500 kD) induces fibrinogen receptor (αIIbß3) activation via CLEC-2/Syk signaling and via a Syk-independent SFK/PI3K/Akt-dependent tyrosine kinase pathway in human and murine platelets. The platelet surface receptor, responsible for the DxS-induced Syk-independent Akt-activation, has hitherto not been identified. We found that DxS elicited a concentration-dependent aggregation of human platelets resulting from direct PEAR1 activation by DxS. Blocking the PEAR1 receptor, in combination with a selective Syk-inhibitor, completely abrogated the DxS-driven platelet aggregation. The DxS-induced Syk-phosphorylation was not affected in Pear1(-/-) platelets, but Akt-phosphorylation was largely abolished. As a result, the aggregation of Pear1(-/-) platelets was reduced and reversible, i.e. aggregates were less stable compared to wild-type platelet aggregates. Moreover, DxS-induced Pear1(-/-) platelet aggregation was fully abrogated by Syk inhibition, indicating that the remaining platelet aggregation of Pear1(-/-) platelets was Syk dependent. Hence, the Pear1/c-Src/PI3K/Akt- and CLEC-2/Syk-signaling pathways are independently and additively activated during platelet aggregation by DxS. CONCLUSION: The DxS-induced aggregation of human and murine platelets is the result of activation of PI3K/Akt through direct PEAR1 phosphorylation and parallel Syk-signaling through CLEC-2.

Functional significance of calcium binding to tissue-nonspecific alkaline phosphatase.

The conserved active site of alkaline phosphatases (AP) contains catalytically important Zn2+ (M1 and M2) and Mg2+-sites (M3) and a fourth peripheral Ca2+ site (M4) of unknown significance. We have studied Ca2+ binding to M1-4 of tissue-nonspecific AP (TNAP), an enzyme crucial for skeletal mineralization, using recombinant TNAP and a series of M4 mutants. Ca2+ could substitute for Mg2+ at M3, with maximal activity for Ca2+/Zn2+-TNAP around 40% that of Mg2+/Zn2+-TNAP at pH 9.8 and 7.4. At pH 7.4, allosteric TNAP-activation at M3 by Ca2+ occurred faster than by Mg2+. Several TNAP M4 mutations eradicated TNAP activity, while others mildly influenced the affinity of Ca2+ and Mg2+ for M3 similarly, excluding a catalytic role for Ca2+ in the TNAP M4 site. At pH 9.8, Ca2+ competed with soluble Zn2+ for binding to M1 and M2 up to 1 mM and at higher concentrations, it even displaced M1- and M2-bound Zn2+, forming Ca2+/Ca2+-TNAP with a catalytic activity only 4-6% that of Mg2+/Zn2+-TNAP. At pH 7.4, competition with Zn2+ and its displacement from M1 and M2 required >10-fold higher Ca2+ concentrations, to generate weakly active Ca2+/Ca2+-TNAP. Thus, in a Ca2+-rich environment, such as during skeletal mineralization at pH 7.4, Ca2+ adequately activates Zn2+-TNAP at M3, but very high Ca2+ concentrations compete with available Zn2+ for binding to M1 and M2 and ultimately displace Zn2+ from the active site, virtually inactivating TNAP. Those ALPL mutations that substitute critical TNAP amino acids involved in coordinating Ca2+ to M4 cause hypophosphatasia because of their 3D-structural impact, but M4-bound Ca2+ is catalytically inactive. In conclusion, during skeletal mineralization, the building Ca2+ gradient first activates TNAP, but gradually inactivates it at high Ca2+ concentrations, toward completion of mineralization.

Pulmonary and hemostatic toxicity of multi-walled carbon nanotubes and zinc oxide nanoparticles after pulmonary exposure in Bmal1 knockout mice.

BACKGROUND: Pulmonary exposure to nanoparticles (NPs) may affect, in addition to pulmonary toxicity, the cardiovascular system such as procoagulant effects, vascular dysfunction and progression of atherosclerosis. However, only few studies have investigated hemostatic effects after pulmonary exposure. METHODS: We used Bmal1 (brain and muscle ARNT-like protein-1) knockout (Bmal1(-/-)) mice which have a disturbed circadian rhythm and procoagulant phenotype, to study the pulmonary and hemostatic toxicity of multi-walled carbon nanotubes (MWCNTs) and zinc oxide (ZnO) NPs after subacute pulmonary exposure. Bmal1(-/-) and wild-type (Bmal1(+/+)) mice were exposed via oropharyngeal aspiration, once a week, during 5 consecutive weeks, to a cumulative dose of 32 or 128 µg MWCNTs or 32 or 64 µg ZnO NPs. RESULTS: MWCNTs caused a pronounced inflammatory response in the lung with increased cell counts in the broncho-alveolar lavage and increased secretion of interleukin-1ß and cytokine-induced neutrophil chemo-attractant (KC), oxidative stress (increased ratio of oxidized versus reduced glutathione and decreased total glutathione) as well as anemic and procoagulant effects as evidenced by a decreased prothrombin time with increased fibrinogen concentrations and coagulation factor (F)VII. In contrast, the ZnO NPs seemed to suppress the inflammatory (decreased neutrophils in Bmal1(-/-) mice) and oxidative response (increased total glutathione in Bmal1(-/-) mice), but were also procoagulant with a significant increase of FVIII. The procoagulant effects, as well as the significant correlations between the pulmonary endpoints (inflammation and oxidative stress) and hemostasis parameters were more pronounced in Bmal1(-/-) mice than in Bmal1(+/+) mice. CONCLUSIONS: The Bmal1(-/-) mouse is a sensitive animal model to study the procoagulant effects of engineered NPs. The MWCNTs and ZnO NPs showed different pulmonary toxicity but both NPs induced procoagulant effects, suggesting different mechanisms of affecting hemostasis. However, the correlation analysis suggests a causal association between the observed pulmonary and procoagulant effects.

Thrombogenic changes in young and old mice upon subchronic exposure to air pollution in an urban roadside tunnel.

Epidemiological studies indicate that elderly persons are particularly susceptible to the cardiovascular health complications of air pollution, but pathophysiological mechanisms behind the increased susceptibility remain unclear. Therefore, we investigated how continuous traffic-related air pollution exposure affects haemostasis parameters in young and old mice. Young (10 weeks) and old (20 months) mice were placed in an urban roadside tunnel or in a clean environment for 25 or 26 days and markers of inflammation and endothelial cells or blood platelet activation were measured, respectively. Plasma microvesicles and pro/anticoagulant factors were analysed, and thrombin generation analysis was performed. Despite elevated macrophage carbon load, tunnel mice showed no overt pulmonary or systemic inflammation, yet manifested reduced pulmonary thrombomudulin expression and elevated endothelial von Willebrand factor (VWF) expression in lung capillaries. In young mice, soluble P-selectin (sP-sel) increased with exposure and correlated with soluble E-selectin and VWF. Baseline plasma factor VIII (FVIII), sP-sel and VWF were higher in old mice, but did not pronouncedly increase further with exposure. Traffic-related air pollution markedly raised red blood cell and blood platelet numbers in young and old mice and procoagulant blood platelet-derived microvesicle numbers in old animals. Changes in coagulation factors and thrombin generation were mild or absent. Hence, continuous traffic-related air pollution did not trigger overt lung inflammation, yet modified pulmonary endothelial cell function and enhanced platelet activity. In old mice, subchronic exposure to polluted air raised platelet numbers, VWF, sP-sel and microvesicles to the highest values presently recorded, collectively substantiating a further elevation of thrombogenicity, already high at old age.

Thrombopoietic effect of VPAC1 inhibition during megakaryopoiesis.

Megakaryocytes and platelets express the stimulatory G protein (Gs)-coupled VPAC1 receptor, for which the pituitary adenylyl cyclase-activating peptide (PACAP) and vasoactive intestinal peptide (VIP) are agonists. The neuropeptide PACAP and VPAC1 were previously found to negatively regulate megakaryopoiesis, and inhibition of their physiological pathway was found to have a thrombopoietic effect in conditions where megakaryopoiesis and thrombopoiesis were impaired, such as chemotherapy-induced thrombocytopenia and congenital thrombocytopenia. The present study explored the thrombopoietic effect of VPAC1 inhibition in a murine model of syngeneic bone marrow transplantation (BMT) and in passive immune thrombocytopenia. Treatment of donor mice with a neutralizing anti-VPAC1 antibody stimulated the initial, most critical recovery of the platelets in irradiated mice. In the passive immune thrombocytopenia model, we observed a thrombopoietic effect, resulting in a less severe platelet drop after induction of their removal in the spleen by an anti-platelet antibody. We concluded that inhibition of the physiological PACAP/VPAC1 pathway could stimulate in vivo megakaryopoiesis. This inhibition can be applied to attenuate thrombocytopenia in conditions where platelets are destroyed as the major pathogenetic mechanism, e.g. immune thrombocytopenia purpura, or need to be produced de novo, e.g. after irradiation and BMT.

The uremic retention solute p-cresyl sulfate and markers of endothelial damage.

BACKGROUND: Cardiovascular disease is highly prevalent in patients with chronic kidney disease. In hemodialysis patients, the protein-bound uremic retention solute p-cresol is independently associated with cardiovascular disease. The underlying mechanisms have not been elucidated. STUDY DESIGN: (1) Prospective observational study of humans and (2) in vitro study in human umbilical vein endothelial cells. SETTING: Hemodialysis patients. FACTOR: p-Cresol and its main derivative p-cresyl sulfate. OUTCOMES: Endothelial dysfunction. MEASUREMENTS: We studied: (1) the relation between p-cresol and blood markers of endothelial dysfunction, including soluble P-selectin and endothelial microparticles; and (2) direct effects of p-cresol and p-cresyl sulfate on endothelial cell cultures. RESULTS: (1) In a cohort of 100 maintenance hemodialysis patients, free serum p-cresol concentrations (median, 11.7 micromol/L; interquartile range, 15.2) were directly associated with circulating endothelial microparticles (P = 0.007), but not with soluble P-selectin (mean, 37.7 +/- 14.4 [SD] pg/mL). Other independent determinants of the degree of circulating microparticles were greater serum phosphorus (mean, 4.8 +/- 1.5 mg/dL; P = 0.008) and serum calcium concentrations (mean, 9.3 +/- 0.8 mg/dL; P = 0.03), whereas treatment with active vitamin D (P = 0.008) and vintage (median, 25 months; P = 0.04) were inversely associated. (2) In vitro, p-cresyl sulfate induced a dose-dependent increase in the shedding of endothelial microparticles (P < 0.001) by human umbilical vein endothelial cells. Shedding was reduced, but not completely aborted, in the presence of albumin, whereas the selective Rho kinase inhibitor Y-27632 abrogated the p-cresyl sulfate-induced generation of endothelial microparticles. LIMITATIONS: The relationship between p-cresyl sulfate and shedding of endothelial microparticles in vivo was not mechanistically explored. CONCLUSION: p-Cresyl sulfate induces shedding of endothelial microparticles in the absence of overt endothelial damage in vitro and is independently associated with the number of endothelial microparticles in hemodialysis patients. These findings suggest that p-cresyl sulfate alters endothelial function in hemodialysis patients.

Thrombospondin-1 deficiency accelerates atherosclerotic plaque maturation in ApoE-/- mice.

Thrombospondin (TSP)1 is implicated in various inflammatory processes, but its role in atherosclerotic plaque formation and progression is unclear. Therefore, the development of atherosclerosis was compared in ApoE(-/-) and Tsp1(-/-)ApoE(-/-) mice kept on a normocholesterolemic diet. At 6 months, morphometric analysis of the aortic root of both mouse genotypes showed comparable lesion areas. Even when plaque burden increased approximately 5-fold in ApoE(-/-) and 10-fold in Tsp1(-/-)ApoE(-/-) mice, during the subsequent 3 months, total plaque areas were comparable at 9 months. In contrast, plaque composition differed substantially between genotypes: smooth muscle cell areas, mostly located in the fibrous cap of ApoE(-/-) plaques, both at 6 and 9 months, were 3-fold smaller in Tsp1(-/-)ApoE(-/-) plaques, which, in addition, were also more fibrotic. Moreover, inflammation by macrophages was twice as high in Tsp1(-/-)ApoE(-/-) plaques. This correlated with a 30-fold elevated incidence of elastic lamina degradation, with matrix metalloproteinase-9 accumulation, underneath plaques and manifestation of ectasia, exclusively in Tsp1(-/-)ApoE(-/-) mice. At 9 months, the necrotic core was 1.4-fold larger and 4-fold higher numbers of undigested disintegrated apoptotic cells were found in Tsp1(-/-)ApoE(-/-) plaques. Phagocytosis of platelets by cultured Tsp1(-/-) macrophages revealed the instrumental role of TSP1 in phagocytosis, corroborating the defective intraplaque phagocytosis of apoptotic cells. Hence, the altered smooth muscle cell phenotype in Tsp1(-/-)ApoE(-/-) mice has limited quantitative impact on atherosclerosis, but defective TSP1-mediated phagocytosis enhanced plaque necrotic core formation, accelerating inflammation and macrophage-induced elastin degradation by metalloproteinases, speeding up plaque maturation and vessel wall degeneration.

Thrombin generation in plasma of healthy adults and children: chromogenic versus fluorogenic thrombogram analysis.

Coagulation tests and coagulation factor assays have been complemented recently with experimental tests to measure the total amount of thrombin formed. We have presently analyzed thrombin generation of healthy adult and paediatric plasma samples via a fluorogenic and a chromogenic method. The chromogenic method was performed on the fully automated Behring Coagulation System (BCS) and fluorogenic assays via Calibrated Automated Thrombography (CAT), after coagulation induction by various tissue factor (TF) concentrations. Sample distribution and variability were analyzed for the four main coagulation parameters, derived via computerized curve analysis in each method. Results for both methods were correlated. At the recommended TF concentration (300 pM), thrombin generation via BCS was less variable than via CAT (1-6 pM), but at comparable TF concentrations (1-6 pM), the CAT sensitivity was higher than that of BCS. Inhibition of intrinsic coagulation with the anti-factor VIII antibody BO2C11 revealed that the BCS detected extrinsic coagulation exclusively, at all TF concentrations tested. In contrast, at low TF concentrations (1 and 2.5 pM), via CAT, intrinsic coagulation pathway amplification was measured. At standardized TF concentrations (300 pM in BCS vs. 2.5 pM in CAT), different reference values between adults and children were found, for all parameters, except Tmax. In adult samples, the best correlation between both methods was observed for ETP(CAT) versus ETP(BCS) and for Peak height(CAT) versus Cmax(BCS), when thrombin generation was exclusively extrinsic (300 pM in BCS vs. 6 pM in CAT). In conclusion, differential thrombin generation characteristics in BCS and CAT are relevant for their clinical applicability.

Mechanical stress activates platelets at a subhemolysis level: an in vitro study.

A feasibility study is performed to quantify sheep platelets (PLTs) and to identify the relationship between PLT count and hemolysis as a consequence of mechanical stress. Six adult, healthy Dorset sheep have been used for in vitro blood sampling test procedures in a hemoresistometer device (HRM). In each experiment, blood of the same animal was exposed to six different shear rates. Free hemoglobin levels and PLT count for each shear rate were detected. In all animals (A-F), hemolysis increased significantly between the shear rates of 2325 and 3100/s (P < 0.05) and the mean PLT count dropped immediately (contact, low shear) 40% in the beginning, between the shear rates of 0 and 775/s (P < 0.05). PLT count increased slightly as soon as hemolysis started. At higher shear rates, hemolysis increased and PLTs reduced further. Precise counting of PLTs indicates that PLTs are consumed dramatically at very low shear (by contact) and further by applied mechanical stress when hemolysis is obvious. A repetition of these tests with human blood could indicate species differences.

Mammalian alkaline phosphatase catalysis requires active site structure stabilization via the N-terminal amino acid microenvironment.

In mammalian alkaline phosphatase (AP) dimers, the N-terminus of one monomer embraces the other, stretching toward its active site. We have analyzed the role of the N-terminus and its microenvironment in determining the enzyme stability and catalysis using human placental (PLAP) and tissue-nonspecific AP (TNAP) as paradigms. Deletion of nine amino acid (aa) residues in PLAP reduced its AP activity and heat stability, while deletion of 25 aa resulted in an inactive enzyme. In turn, deletion of five and nine N-terminal aa in TNAP reduced and abolished AP activity, respectively. The N-terminal aa deletions in both isozymes affected the rate of substrate catalysis (k(cat)), with an only minor effect on the Michaelis constant (K(m)) explained by decelerated intramolecular transition rates in the active site. Arg370 in PLAP, and the corresponding Arg374 in TNAP, critically control the structure and function of the enzymes, but the Glu6-Arg370 bond predicted by the PLAP crystal structure appeared to be irrelevant with respect to PLAP stability or catalysis. Structural disruption was also noted in [R374A]TNAP, [Delta5]TNAP, [Delta9]TNAP, and [Delta25]TNAP using a panel of 19 anti-TNAP antibodies illustrating the structural role of the N-terminus. Our data reveal that the N-terminal alpha-helical folding is more crucial for the structural stability of the second monomer in TNAP than in PLAP. The correct folding of the N-terminus and of interacting loops in its immediate environment is essential for overall structural integrity and for execution of intramolecular transitions during enzyme catalysis. These findings provide a mechanistic interpretation for loss-of-function mutations of N-terminal TNAP residues in cases of hypophosphatasia.