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((R)) (BCS (R)) and fluorogenic assays via Calibrated Automated Thrombography((R)) (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-factorVIII antibody BO2CII revealed that the BCS detected extrinsic coagulation exclusively, at all TF concentrations tested. In contrast, at lowTF concentrations (I 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 ETPCAT versus ETPBCS 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

Thrombospondin-1 deficiency accelerates 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.status: publishe

Functional polymorphisms in the paternally expressed XLαs and its cofactor ALEX decrease their mutual interaction and enhance receptor-mediated cAMP formation resulting in platelet Gs hyperfunction

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Dextran sulfate triggers platelet aggregation via direct activation of PEAR1

Dextran sulfate (DxS; Mr 500 kD) induces fibrinogen receptor (αIIbβ3) 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.peerreview_statement: The publishing and review policy for this title is described in its Aims & Scope. aims_and_scope_url: http://www.tandfonline.com/action/journalInformation?show=aimsScope&journalCode=iplt20status: publishe

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

BackgroundPulmonary 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.MethodsWe 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 ¿g MWCNTs or 32 or 64 ¿g ZnO NPs.ResultsMWCNTs 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.ConclusionsThe 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.status: publishe

Functional Significance of Calcium Binding to Tissue-Nonspecific Alkaline Phosphatase

<div><p>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 <i>ALPL</i> 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.</p></div

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.status: publishe

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

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 (αIIbβ3) 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.publisher: Elsevier articletitle: Absence of Pear1 does not affect murine platelet function in vivo journaltitle: Thrombosis Research articlelink: http://dx.doi.org/10.1016/j.thromres.2016.08.026 content_type: article copyright: © 2016 Elsevier Ltd. All rights reserved.status: publishe