Importance of individual activated protein C cleavage site regions in coagulation Factor V for Factor Va inactivation and for Factor Xa activation

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/66371/1/j.1432-1327.1999.00137.x.pd

Antimicrobial resistance (AMR) nanomachines: mechanisms for fluoroquinolone and glycopeptide recognition, efflux and/or deactivation

In this review, we discuss mechanisms of resistance identified in bacterial agents Staphylococcus aureus and the enterococci towards two priority classes of antibiotics—the fluoroquinolones and the glycopeptides. Members of both classes interact with a number of components in the cells of these bacteria, so the cellular targets are also considered. Fluoroquinolone resistance mechanisms include efflux pumps (MepA, NorA, NorB, NorC, MdeA, LmrS or SdrM in S. aureus and EfmA or EfrAB in the enterococci) for removal of fluoroquinolone from the intracellular environment of bacterial cells and/or protection of the gyrase and topoisomerase IV target sites in Enterococcus faecalis by Qnr-like proteins. Expression of efflux systems is regulated by GntR-like (S. aureus NorG), MarR-like (MgrA, MepR) regulators or a two-component signal transduction system (TCS) (S. aureus ArlSR). Resistance to the glycopeptide antibiotic teicoplanin occurs via efflux regulated by the TcaR regulator in S. aureus. Resistance to vancomycin occurs through modification of the D-Ala-D-Ala target in the cell wall peptidoglycan and removal of high affinity precursors, or by target protection via cell wall thickening. Of the six Van resistance types (VanA-E, VanG), the VanA resistance type is considered in this review, including its regulation by the VanSR TCS. We describe the recent application of biophysical approaches such as the hydrodynamic technique of analytical ultracentrifugation and circular dichroism spectroscopy to identify the possible molecular effector of the VanS receptor that activates expression of the Van resistance genes; both approaches demonstrated that vancomycin interacts with VanS, suggesting that vancomycin itself (or vancomycin with an accessory factor) may be an effector of vancomycin resistance. With 16 and 19 proteins or protein complexes involved in fluoroquinolone and glycopeptide resistances, respectively, and the complexities of bacterial sensing mechanisms that trigger and regulate a wide variety of possible resistance mechanisms, we propose that these antimicrobial resistance mechanisms might be considered complex ‘nanomachines’ that drive survival of bacterial cells in antibiotic environments

Species-specific anticoagulant and mitogenic activities of murine protein S

Mice and other animals have proved extremely useful testing grounds for hypotheses and for potential therapies. However, significant interspecies differences can confound correct interpretation and attempts to extrapolate results to man. In this study Fernandez and co-workers demonstrate that the human and murine protein C-protein S systems function in a similar way but do not interact efficiently with one another

Plasma protein S residues 37-50 mediate its binding to factor Va and inhibition of blood coagulation

Protein S (PS) is an anticoagulant plasma protein whose deficiency is associated with increased risk of venous thrombosis. PS directly inhibits thrombin generation by the blood coagulation pathways by several mechanisms, including by binding coagulation factors (F) Va and Xa. To identify PS sequences that mediate inhibition of FVa activity, antibodies and synthetic peptides based on PS sequence were prepared and employed in plasma coagulation assays, purified component prothrombinase assays, binding assays, and immunoblots. In the absence of activated protein C, monoclonal antibody (Mab) S4 shortened FXa-induced clotting in normal plasma but not in PS-depleted plasma. Mab S4 also blocked PS inhibition of FVa-dependent prothrombinase activity in purified component assays in the absence or presence of phospholipids and inhibited binding of PS to immobilized FVa. Epitope mapping identified N-terminal region residues 37–67 of PS as this antibody's epitope. A peptide representing PS residues 37–50 inhibited FVa-dependent prothrombinase activity in a noncompetitive manner, with 50% inhibition observed at 11 μM peptide, whereas a peptide with a D-amino acid sequence of 37–50 was ineffective. FVa, but not FXa, bound specifically to the immobilized peptide representing residues 37–50, and the peptide inhibited binding of FVa to immobilized PS. These data implicate PS residues 37–50 as a binding site for FVa that mediates, at least in part, the direct inhibition of FVa-dependent procoagulant activity by PS