Effects of Ambient Air Pollution on Hemostasis and Inflammation

BACKGROUND: Air pollution has consistently been associated with increased morbidity and mortality due to respiratory and cardiovascular disease. Underlying biological mechanisms are not entirely clear, and hemostasis and inflammation are suggested to be involved. OBJECTIVES: Our aim was to study the association of the variation in local concentrations of airborne particulate matter (PM) with aerodynamic diameter < 10 mu m, carbon monoxide, nitrogen monoxide, nitrogen dioxide, and ozone with platelet aggregation, thrombin generation, fibrinogen, and C-reactive protein (CRP) levels in healthy individuals. METHODS: From 40 healthy volunteers, we collected 13 consecutive blood samples within a 1-year period and measured light-transmittance platelet aggregometry, thrombin generation, fibrinogen, and CRP. We performed regression analysis using generalized additive models to study the association between the hemostatic and inflammatory variables, and local environmental concentrations 0 air pollutants for time lags within 24 hr before blood sampling or 24-96 hr before blood sampling. RESULTS: In general, air pollutants were associated with platelet aggregation [average, +8% per interquartile range (IQR), p < 0.01] and thrombin generation (average, +1% per IQR, p < 0.015). Platelet aggregation was not affected by in vitro incubation of plasma with PM. We observed no relationship between any of the air pollutants and fibrinogen or CRP levels. CONCLUSIONS:. Air pollution increased platelet aggregation as well as coagulation activity but had no clear effect on systemic inflammation. These prothrombotic effects may partly explain the relationship between air pollution and the risk of ischemic cardiovascular disease

Total chemical synthesis of human matrix Gla protein

Human matrix Gla protein (MGP) is a vitamin K–dependent extracellular matrix protein that binds Ca2+ ions and that is involved in the prevention of vascular calcification. MGP is a 10.6-kD protein (84 amino acids) containing five γ-carboxyglutamic acid (Gla) residues and one disulfide bond. Studies of the mechanism by which MGP prevents calcification of the arterial media are hampered by the low solubility of the protein (<10 μg/mL). Because of solubility problems, processing of a recombinantly expressed MGP-fusion protein chimera to obtain MGP was unsuccessful. Here we describe the total chemical synthesis of MGP by tBoc solid-phase peptide synthesis (SPPS) and native chemical ligation. Peptide Tyr1-Ala53 was synthesized on a derivatized resin yielding a C-terminal thioester group. Peptide Cys54-Lys84 was synthesized on Lys-PAM resin yielding a C-terminal carboxylic acid. Subsequent native chemical ligation of the two peptides resulted in the formation of a native peptide bond between Ala53 and Cys54. Folding of the 1–84-polypeptide chain in 3 M guanidine (pH 8) resulted in a decrease of molecular mass from 10,605 to 10,603 (ESI-MS), representing the loss of two protons because of the formation of the Cys54-Cys60 internal disulfide bond. Like native MGP, synthetic MGP had the same low solubility when brought into aqueous buffer solutions with physiological salt concentrations, confirming its native like structure. However, the solubility of MGP markedly increased in borate buffer at pH 7.4 in the absence of sodium chloride. Ca2+-binding to MGP was confirmed by analytical HPLC, on which the retention time of MGP was reduced in the presence of CaCl2. Circular dichroism studies revealed a sharp increase in α-helicity at 0.2 mM CaCl2 that may explain the Ca2+-dependent shift in high-pressure liquid chromatography (HPLC)-retention time of MGP. In conclusion, facile and efficient chemical synthesis in combination with native chemical ligation yielded MGP preparations that can aid in unraveling the mechanism by which MGP prevents vascular calcification

Platelet adhesion receptors do not modulate infarct volume after a photochemically induced stroke in mice.

Photochemically induced cerebral infarction has been considered a clinically relevant model for ischemic stroke. We evaluated various transgenic mice to study the role of platelet adhesion molecules in this model. Infarction to the sensorimotoric cortex was induced by erythrosin B and laser light. Infarct volumes were calculated from triphenyltetrazolium chloride stained brain slices. Thrombus formation and vessel leakage were observed in vivo by multiphoton microscopy. Mice mutant in VWF, GPIbalpha, beta3 integrin, and P-selectin did not show any significant differences in infarct volume compared to wild type (WT). This is in contrast to the intraluminal middle cerebral artery occlusion model in which alphaIIbbeta3 integrin, GPIbalpha, and P-selectin are known to modulate infarct size. Multiphoton microscopy showed that small, non-occlusive embolizing platelet thrombi formed in the photochemically injured brains. Massive vessel leakage was observed within 25 min of laser injury. Interestingly, we observed a significant increase in infarct size with aging, accordant with heightened fragility of the blood brain barrier (BBB) in older mice. This model of photochemically induced stroke is closer to a BBB injury model than a thrombotic stroke model in which platelets and their adhesion molecules are crucial. This model will be useful to study mechanisms regulating BBB permeability

Impaired glucose metabolism and type 2 diabetes are associated with hypercoagulability: potential role of central adiposity and low-grade inflammation - The Hoorn Study

AbstractIntroductionType 2 diabetes (DM2) is associated with greater risk for cardiovascular disease (CVD), which may, at least partially, be explained by prothrombotic alterations. We therefore investigated; first, the extent to which individuals with impaired glucose metabolism (IGM) and/or DM2 had greater levels of thrombin generation than those with normal glucose metabolism (NGM); and second, whether any differences were independent of other cardiovascular risk factors, such as smoking, hypertension, dyslipidaemia, (micro)albuminuria, glycemic control and (central) adiposity, and/or were potentially ‘mediated’ by low-grade inflammation (high-sensitivity C-reactive protein (hsCRP)).Materials and methodsWe studied 744 individuals from the Hoorn Study (275 NGM, 176 IGM and 293 DM2, mean age 68.6±7.1years). Thrombin generation in platelet-poor plasma was measured using the Calibrated Automated Thrombogram and three parameters were derived: lag time, peak height and endogenous thrombin potential (ETP). Data were analyzed with multiple linear regression analyses.ResultsAfter adjustment for age, sex, prior CVD and smoking status, individuals with IGM or DM2 had a longer lag time [ß=0.14min (95% CI: 0.02; 0.26)], higher peak height [ß=7.29 nM (−1.33; 15.91)] and ETP [ß=35.65nM*min (0.97; 70.34)] than those with NGM. These differences were attenuated to ß=0.06min (−0.07; 0.19), 3.82nM (−5.46; 13.10) and 16.34nM*min (−20.92; 53.59), respectively, when further adjusted for waist circumference and hsCRP.ConclusionIndividuals with IGM or DM2 had up to 4% higher thrombin generation compared with NGM, which may be explained, to a great extent, by the greater levels of central adiposity and related low-grade inflammation characterizing these individuals