Establishment of a cardiac biobank in a Department of Pathology and Laboratory Medicine.

Biobanks are considered to be important resources of Departments of Pathology and Laboratory Medicine allowing the clarification of relevant disease mechanisms and the improvement of the diagnosis, prognosis, and treatment of both pediatric and adult cardiovascular diseases. To successfully establish a cardiovascular biobank, it is important to consider the public opinion and views on it and the factors involved in the willingness of the public to participate in the donation of genetic material. The literature was systematically reviewed to identify the attitude and willingness of patients affected by congenital and acquired heart disease to participate in biobanking research. Six relevant studies were identified in which it was indicated that psychosocial and demographic characteristics, as well as the patient's medical condition, could influence patient and family members' attitudes and willingness to participate in research. In both congenital and acquired heart diseases, participation in biobank research activities was higher if patients and their families were approached when hospitalized, but not during the acute moment of their illness. Other quantitative and qualitative studies are required to improve patient and family participation in these research initiatives.

The criterion of proportionality in the activation of Left Ventricular Assist Device implants: the method of "four boxes" to analyze the pre-implant phase.

BACKGROUND: Life-saving technologies have completely changed the normal conception of medical treatments. Left Ventricular Assist Devices (LVAD) can prolong survival for patients who are not candidates for heart transplantation. In order to analyze the pre-implantation phase, which involves a shared-decision making process before activation of the device, attention should be paid to the criterion of "proportionality" in order to properly assess the risks and benefits of implantation. AIM: The aim of our analysis is to provide an useful tool for the assessment of LVAD proportionality during the physicians' decision making. METHODS: The method of the "four boxes", developed by Jonsen et al, was chosen to analyze the notion of proportionality and the other main ethical issues regarding LVAD activation in adult patients. RESULTS: Medical issues are not the sole factors, which influence the choice of implantation by patients. Indeed, patient preferences, his/her quality of life, and contextual features should be taken into consideration when proposing LVADs: these factors are as important as clinical issues where outcomes are concerned. CONCLUSIONS: In order to assess the proportionality of such a device, we present, discuss and examine, in the framework of the pre-implant phase, the content of each topic treated by the "four boxes method", that is, an essential tool for the assessment of the proportionality of the treatment for LVAD candidates.

Donation After Circulatory Death: When Withdrawing Life-Sustaining Treatments Is Ethically Acceptable.

The possibility to determine death based on cardiocirculatory criteria in controlled cases, namely when there is a request to withhold treatment-or, more frequently, withdraw it-specifically recalls the recent Italian law on advance treatment directives and leaves the following question unanswered: Under what conditions is the patient's request legally and ethically acceptable? We present three ethical proportionality criteria for supporting physicians' decision-making facing patients' requests of treatment withdrawal, namely: 1. irreversible pathology with an ominous and worsening prognosis; 2. within an evaluation considering both clinical data and the patient's history; and 3. facing burdens that are no longer bearable. We finally argue that reflection over controlled donor may be a model for giving medicine the chance to responsibly deal with broader end-of-life issues.

A rapid response magnitude scale for timely assessment of the high frequency seismic radiation.

In this work the scaling of seismic moment (M0) and radiated energy (Er) is investigated for almost 800 earthquakes of the 2016-17 Amatrice-Norcia sequences in Italy, ranging in moment magnitude (Mw) from 2.5 to 6.5. The analysis of the M0-to-Er scaling highlights a breaking of the source self-similarity, with higher stress drops for larger events. Our results show the limitation of using M0, and in turn Mw, to capture the variability of the high frequency ground motion. Since the observed seismicity does not agree with the assumptions on stress drop in the definition of Mw, we exploit the availability of both Er and M0 to modify the definition of Mw and introduce a rapid response magnitude (Mr), which accounts for the dynamic properties of rupture. The new Mr scale allows us to improve the prediction of the earthquake shaking potential, as shown by the reduction of the between-event residuals computed for the peak ground velocity. The procedure we propose is therefore a significant step towards a quick assessment of earthquakes damage potential and timely implementation of emergency plans.

Munchausen Syndrome by Proxy: balancing ethical and clinical challenges for healthcare professionals Ethical consideration in factitious disorders.

Munchausen syndrome by proxy is a relatively rare behavioral disorder affecting a child's primary caregiver, typically the mother. Ethical dilemmas that physicians may face in such situations mainly concern the medical options for best protecting the child's welfare, that are important, in clinical pediatric practice, because critical conflicts might arise between health professionals and parents. In such cases, the physician's primary obligation is to protect the children involved, whose family environment may be essential to their wellbeing. Ev- ery ethical choice should be tailored to a given child's story, which should be viewed as a whole, considering the possible consequences for the family balance, and taking the complexity of the emotional and psychological dimensions of children's relationship with their parents into account. Specific protocols, discussion of clinical cases, open communi- cation of feelings will help doctors to deal more effectively with the families involved and ensure that treatment decisions are made in the child's best interest.

Evidence for a difference in rupture initiation between small and large earthquakes.

The process of earthquake rupture nucleation and propagation has been investigated through laboratory experiments and theoretical modelling, but a limited number of observations exist at the scale of earthquake fault zones. Distinct models have been proposed, and whether the magnitude can be predicted while the rupture is ongoing represents an unsolved question. Here we show that the evolution of P-wave peak displacement with time is informative regarding the early stage of the rupture process and can be used as a proxy for the final size of the rupture. For the analysed earthquake set, we found a rapid initial increase of the peak displacement for small events and a slower growth for large earthquakes. Our results indicate that earthquakes occurring in a region with a large critical slip distance have a greater likelihood of growing into a large rupture than those originating in a region with a smaller slip-weakening distance.

Thrombin interaction with platelet GpIB: role of the heparin binding domain.

The platelet membrane glycoprotein Ib (GpIb) has a high affinity binding site for alpha-thrombin whose occupancy is thought to positively modulate the thrombin-induced platelet activation. In this study, aimed at further characterizing the thrombin-GpIb interaction, two thrombin anion exosites referred to as "heparin binding site" (HBS) and "fibrinogen recognition site" (FRS) were investigated as the possible domains involved in GpIb binding. The role of thrombin HBS was explored by performing binding measurements of 125I-alpha-thrombin to purified glycocalicin (GC), the extracytoplasmic portion of GpIb, in the presence of heparin as well as after chemical modifications of the thrombin heparin binding site (thrombin-HBS phosphopyridoxylation). These studies showed that a) thrombin binding to GC could be competitively inhibited by heparin and b) the equilibrium association constant for thrombin-GC interaction was reduced up to ten-fold by chemical modifications at the HBS. On the other hand, the role of FRS in the thrombin-GC interaction could be excluded by other experiments showing that GC in solution could not influence the interaction of alpha-thrombin with two substrates which bind to both the catalytic site and the fibrinogen recognition site: 1) the thrombin receptor peptide 38-60 (TR, L38-E60) and 2) the A alpha-chain of fibrinogen. Altogether these results demonstrated that GC interaction with thrombin involves the enzyme heparin binding site, whereas the fibrinogen recognition site does not play a significant role.

Effect of sodium on the energetics of thrombin-thrombomodulin interaction and its relevance for protein C hydrolysis.

Measurements of the apparent affinity constant for thrombomodulin (TM) binding to human alpha-thrombin as a function of both NA+ and temperature at constant ionic strength (0.15 M) showed that TM affinity increases in the presence of Na+ and vice versa. Moreover, this experimental strategy allowed us to accurately split the free energy of sodium binding into its entropic and enthalpic components for both the TM-free and TM-bound enzyme. Namely, at 25 degrees C, the value of delta G of sodium binding was found equal to -2.4 kcal/mol in the absence of TM and -3.6 kcal/mol for the thrombin-TM complex. The enthalpic contribution to the free energy of sodium binding is equal to -27 kcal/mol and -21 kcal/mol in the TM-free and TM-bound thrombin forms, respectively. Finally, the entropy change for sodium binding was also affected by TM, being equal to -83 cal/(mol deg) and -58 cal/(mol deg) in TM-free and TM-bound thrombin species, respectively. Moreover, the thermodynamic parameters for TM binding to Na+-free thrombin species were solved. TM binding is characterized by an enthalpy and entropy change equal to -10 kcal/mol and 2 cal/(mol deg), respectively, for Na+-free thrombin. It is well known that Na+ binding to thrombin causes conformational transitions and functional activation of the enzyme molecule. The finding that binding of thrombomodulin enhances thrombin affinity for sodium and vice versa raises the question as to whether the change of Na+ ligation induced by TM binding could contribute to the change in thrombin specificity for the hydrolysis of Protein C. Therefore, the effect of sodium binding to thrombin on the hydrolysis of human Protein C was extensively investigated. At both 25 and 37 degrees C the value of kcat/Km for Protein C hydrolysis by thrombin in the absence of TM was found to be enhanced by Na+ over a concentration ranging from 0 to 150 mM. Application of thermodynamic principles demonstrated that the Na+-thrombomodulin linkage contributes, under physiological conditions of sodium activity and temperature, to reduce significantly the transition-state stabilization free energy for Protein C hydrolysis.

Thrombin-thrombomodulin interaction: energetics and potential role of water as an allosteric effector.

The interaction of rabbit lung thrombomodulin (TM) and C-terminal hirudin 54-65 fragment (Hir54-65) with human alpha-thrombin were investigated by exploiting their competitive inhibition of thrombin-fibrinogen interaction. Measurements of Ki values for TM and Hir54-65 interactions with human alpha-thrombin performed over a temperature range spanning from 10 to 40 degrees C showed a constant enthalpy for both ligands. The enthalpic and entropic contributions to the free energy of binding, however, are different for TM and the hirudin peptide. The calculated values of delta H and delta S, in fact, were -47.3 +/- 2.51 kJ (-11.3 +/- 0.6 kcal)/mol and -42.7 +/- 7.9 J (-10.2 +/- 1.9 cal)/mol.K for the hirudin peptide, while being -22.9 +/- 2.09 kJ (-5.47 +/- 0.5 kcal)/mol and 102.50 +/- 6.69 J (24.5 +/- 1.6 cal)/mol.K respectively for TM binding. These findings indicate that the interaction between thrombin and Hir54-65 is largely driven by the enthalpic contribution, whereas the positive entropy change is the driving force for the formation of the thrombin-TM complex. In other experiments performed in the presence of various concentrations of either sorbitol or sucrose it could be demonstrated that the value of the equilibrium association constant for thrombin-TM interaction increases as a function of the osmotic pressure, while the thrombin-Hir54-65 interaction was not affected by the same conditions. Moreover, control experiments showed that no major conformational changes are produced on TM by osmotic pressures used in the present study. From these experiments it was calculated that roughly 35 water molecules are released into the bulk water upon TM binding. Such a phenomenon, which is likely to be responsible for the entropic change described above, indicates the relevance of hydration processes for the formation of the thrombin-TM adduct.

Conformational transitions linked to active site ligation in human thrombin: effect on the interaction with fibrinogen and the cleavable platelet receptor.

An experimental strategy based on solution viscosity perturbation allowed us to study the energetics of amide-substrates, p-aminobenzamidine (p-ABZ) and proflavin binding to the catalytic site of two proteolyzed forms of alpha-thrombin, i.e. zeta- and gamma T-thrombin. These thrombin derivatives are cleaved at the Leu144-Gly150 loop and at the fibrinogen recognition exosite (FRS), respectively. A phenomenological analysis of thermodynamic data showed that the amide substrates and p-ABZ interactions with zeta-thrombin were respectively, associated with a chemical compensation (i.e. the linear relationship between entropy and enthalpy of binding) and a hydrophobic phenomenon (i.e. a change in the standard heat capacity). The latter was slightly lower than that previously observed for a alpha-thrombin (0.78 +/- 0.25 versus 1.01 +/- 0.17 kcal/mol K). Both phenomenon were absent in gamma T-thrombin. The interaction of a alpha-, zeta- and gamma T-thrombin with macromolecular substrates that "bridge-bind" to both the catalytic site (CS) and fibrinogen recognition exosite (FRS), such as fibrinogen and the cleavable platelet receptor (CPR), was also evaluated. These interactions were studied by following fibrinopeptide A (FpA) release and by measuring intraplatelet Ca2+ changes induced by thrombin-CPR interaction. It was found that the free energy of activation (RT ln Kcat/Km) for both fibrinogen and CPR hydrolysis followed the same hierarchy, i.e. alpha > zeta > gamma. Moreover, the values of delta Cp for alpha-, zeta- and gamma T-thrombin interaction with p-ABZ were found to be linearly correlated to the free energy of activation for both fibrinogen and CPR cleavage. In conclusion, these data demonstrate that: (1) the Leu144-Gly150 loop and the FRS are both involved in the conformational transition linked to the binding of p-aminobenzamidine to the thrombin active site; (2) the extent of thrombin's capacity to undergo conformational transitions in alpha-, zeta- and gamma T forms is positively correlated to the free energy of activation for hydrolysis of macromolecular substrates interacting with both the catalytic domain and the FRS.

Effects of protons on the thrombin-fibrinogen interaction.

Amidase activity of human alpha-thrombin toward the synthetic substrate Tosyl-Gly-Pro-Arg-NH-Ph and fibrinogen has been studied as a function of pH at t = 25 degrees C, under steady-state conditions. A viscosity-perturbation method allowed us to compute the equilibrium binding constant along with the rate constants for the acylation and deacylation reactions. The ionization constants for the groups affecting binding and hydrolysis of the synthetic substrate were measured by application of linkage thermodynamics principles. The binding of the synthetic substrate is controlled by two ionizable groups having pKa values of 7.5 and 8.7 in the free enzyme and 6.3 and 9.8 in the Michaelis adduct. These two groups were found to control the acylation process as well. Thrombin-fibrinogen interaction has been studied by measurements of steady-state hydrolysis of the synthetic substrate Phe-pipecolyl-Arg-NH-Ph in the presence of fibrinogen, used as a competitive inhibitor. This method allowed us to measure the Km of thrombin-fibrinogen interaction. The values of Km computed at different solution viscosities were used in order to calculate the equilibrium dissociation constant and both k2/k3 and k2/k-1 ratios. The same residues that were found to control binding of Tosyl-Gly-Pro-Arg-NH-Ph to alpha-thrombin, do modulate binding of fibrinogen as well. These residues shift their pKa values upon the formation of the Michaelis adduct from 7.5 to 5.7 and from 8.7 to 9.7, respectively. Furthermore the ratio kcat/Km as a function of pH has been obtained by HPLC measurements of fibrinopeptides release. The kcat/Km values along with the ratio k2/k-1, derived from viscometric experiments, allowed us to calculate the forward-rate constant, k+1, for the thrombin-fibrinogen interaction. The association process was found to depend on pH, namely in the alkaline region. The results for Tosyl-Gly-Pro-Arg-NH-Ph and fibrinogen are compared and discussed on the basis of the structural elements which differentiate the interactions of these substrates with human alpha-thrombin.

Effect of temperature on the association step in thrombin-fibrinogen interaction.

Kinetics of fibrinopeptide A release by human alpha-thrombin at low fibrinogen concentration allowed us to measure the specificity constant, i.e. kcat/Km, for the interaction between the enzyme and human fibrinogen. A study of the dependence of the ratio kcat/Km upon the viscosity of the medium revealed that fibrinogen acts as a 'sticky' substrate, or, in other words, as a substrate that dissociates from the Michaelis complex with a rate comparable with that for acylation of the active site. These experiments allowed us also to compute for the first time the second-order rate constant for thrombin-fibrinogen association. A study of the temperature-dependence of the association rate, carried out over the temperature range spanning from 10 degrees C to 37 degrees C (pH 7.50; I0.15) permitted the estimation of the enthalpy and entropy of activation, delta H++ and delta S++, which were found to be equal to 5.69 +/- 0.77 kJ.mol-1 and -80.25 +/- 1.79 kJ.K-1.mol-1 respectively. In addition, the values of Km for thrombin-fibrinogen reaction were measured at different solution viscosities in order to derive the equilibrium dissociation constant, Ks, of this interaction. These experiments showed that the Ks values for thrombin-fibrinogen binding was equal to 1.8 microM at 25 degrees C. Altogether these results indicated that fibrinogen, though interacting with both the catalytic pocket and the fibrinogen recognition site on the thrombin molecule, dissociates from Michaelis complex with a rate comparable with that shown by amide substrates, which interact only with the catalytic site.