Properties of Some Variants of Human β2-Microglobulin and Amyloidogenesis

Three variants of human beta(2)-microglobulin (beta(2)-m) were compared with wild-type protein. For two variants, namely the mutant R3Abeta(2)-m and the form devoid of the N-terminal tripeptide (DeltaN3beta(2)-m), a reduced unfolding free energy was measured compared with wild-type beta(2)-m, whereas an increased stability was observed for the mutant H31Ybeta(2)-m. The solution structure could be determined by (1)H NMR spectroscopy and restrained modeling only for R3Abeta(2)-m that showed the same conformation as the parent species, except for deviations at the interstrand loops. Analogous conclusions were reached for H31Ybeta(2)-m and DeltaN3beta(2)-m. Precipitation and unfolding were observed over time periods shorter than 4-6 weeks with all the variants and, sometimes, with wild-type protein. The rate of structured protein loss from solution as a result of precipitation and unfolding always showed pseudo-zeroth order kinetics. This and the failure to observe an unfolded species without precipitation suggest that a nucleated conformational conversion scheme should apply for beta(2)-m fibrillogenesis. The mechanism is consistent with the previous and present results on beta(2)-m amyloid transition, provided a nucleated oligomeric species be considered the stable intermediate of fibrillogenesis, the monomeric intermediate being the necessary transition step along the pathway from the native protein to the nucleated oligomer

Circadian variations in platelet aggregability in non insulin dependent diabetes patients (NIDDM).

Circadian rhythms of platelet activity have been extensively investigated in recent years in view of a possible correlation with the increased morning frequency of thrombotic events. Since a role of enhanced platelet activity has been suggested in diabetic macro- and microvascular disease, the aim of the present study was to analyze the circadian variations of in vitro platelet aggregability in patients with non insulin-dependent diabetes mellitus. Eighteen in-patients, ranging in age from 49 to 73 years, all in good metabolic control, were included in the study. Aggregometric patterns were analyzed in blood samples collected at 0700, 0900, 1100 and 1700. A significant increase of the threshold concentrations for ADP-collagen- and arachidonic acid-induced platelet aggregation has been observed in the interval 0700-1100 or 0700-1700, suggesting, also in diabetic patients as in normal subjects, a maximum of platelet activity in the morning, regardless of postural changes and timing of meals

IN-VIVO THROMBIN GENERATION AND PLATELET HYPERACTIVITY IN PATIENTS WITH CHRONIC OBSTRUCTIVE PULMONARY-DISEASE

[No abstract available

Chronic obstructive pulmonary disease. A possible correlation between platelet aggregation tests and respiratory indexes. ECOB Group.

It has been suggested that thrombotic events occurring in patients with chronic obstructive pulmonary disease could be partially due to abnormalities in blood coagulation and fibrinolysis, as well as to changes in platelet function. In the present study in vitro platelet aggregation, in addition to clotting activity, has been studied in 20 patients with chronic obstructive pulmonary disease and different degrees of alteration of blood gas analysis. The results obtained suggest that the presence of a condition of platelet hypoaggregability characterizes patients with marked hypercapnia and/or hypoxemia, whereas the improvement of respiratory indexes is followed by a trend to normalization, or, more often, to an increase of platelet activity

Potential usefulness of antiplatelet agents in patients with chronic obstructive pulmonary disease

Thrombotic complications of pulmonary circulation might occur in patients with chronic obstructive pulmonary disease (COPD) (1-3). It has been suggested that these thrombotic events could be partly tied to the abnormalities found in platelet function. In fact, in vitro and in vivo platelet activation could actually occur in systemic and pulmonary circulation as a result of hypoxemia, acidosis or hyperviscosity, all characteristic findings of COPD. A shortened platelet half-life, as well as increased plasma levels of beta-thromboglobulin (beta-TG) and enhanced platelet aggregation have been reported in patients with COPD (4-7). Moreover, a decrease in platelet survival time has been correlated with chronic hypoxemia, thus suggesting an increased platelet consumption due to in vivo platelet activation (6, 8-10). In 1991, Segall and Goetzman (11), in a study carried out on newborn lambs, demonstrated the occurrence of platelet content release in the lung during hypoxia-induced pulmonary hypertension. This observation is in good agreement with the above reported findings of short platelet half-life and in vivo platelet activation in patients with chronic hypoxemia. Moreover, the possibility of platelet content release in the lung during hypoxic conditions may account for impaired respiratory parameters, such as forced expiratory volume in one second (FEV1)

Solution structure of beta(2)-microglobulin and insights into fibrillogenesis

The solution structure of human b2-microglobulin (b2-m) was determined by 1H NMR spectroscopy and restrained modeling calculations. Compared to the crystal structure of type I major histocompatibility complex (MHC-I), where the protein is associated to the heavy-chain component, several differences are observed, i.e., increased separation between strands A and B, displacements of strand CV and loop DE, shortening of strands D and E. These modifications can be considered as the prodromes of the amyloid transition. Even minor charge changes in response to pH, as is the case with H31 imidazole protonation, trigger the transition that starts with unpairing of strand A. The same mechanism accounts for the partial unfolding and fiber formation subsequent to Cu2+ binding which is shown to occur primarily at H31. Solvation of the protected regions in MHC-I decreases the tertiary packing by breaking the contiguity of the surface hydrophobic patches via surface charge cluster. Mutants or truncated forms of h2-m can be designed to remove the instability from H31 titration or to enhance the instability through surface charge suppression. By monitoring the conformational evolution of wild-type protein and variants thereof, either in response or absence of external perturbation, valuable insights into intermediate structure and fibrillogenesis mechanisms are gained

Monitoring the interaction between β2-microglobulin and the molecular chaperone αB-crystallin by NMR and mass spectrometry

The interaction at neutral pH between wild-type and a variant form (R3A) of the amyloid fibril-forming protein β2-microglobulin (β2m) and the molecular chaperone αB-crystallin was investigated by thioflavin T fluorescence, NMR spectroscopy, and mass s