PubMed · 12218558
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Evan G Pivalizza, R David Warters. 2002. Kaolin-activated thromboelastography.. https://doi.org/10.1097/00000542-200209000-00049
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Tryptase is a serine protease that is stored at low pH in the mast cell secretory granules in complex with heparin proteoglycan. When mast cells are activated, e.g. during allergic responses, the tryptase/heparin complexes are released together with a variety of other preformed inflammatory mediators. Previous crystallization of human beta-tryptase revealed a unique tetrameric structure with all of the active sites facing a central pore that has a limited accessibility both for potential substrates as well as for protease inhibitors. In this study we examined whether human beta-tryptase, in addition, could form active monomers. Incubation of recombinant tetrameric human beta-tryptase at neutral pH and 37 degrees C, followed by gel-filtration analysis using a running buffer containing pig mucosal heparin, led to the formation of enzymically active compounds that were of a size compatible with tryptase monomers in complex with heparin. The monomers were, in contrast to tryptase in the tetrameric form, inhibited by bovine pancreatic trypsin inhibitor. Further, the monomers, but not the tetramers, degraded fibronectin. Formation of active monomers was more pronounced at pH 7.5 than at pH 6.0 and was not detected at room temperature or at high heparin/tryptase ratios. The present findings thus introduce the possibility that human beta-tryptase, after mast cell degranulation and exposure to neutral pH in the tissue, may dissociate into active monomers with properties that are distinct from the tetrameric counterpart. Possibly, some of the biological activities of human tryptase may be attributable to active tryptase in its monomeric rather than tetrameric form.
NOESY and ROESY cross-peak intensities depend on internuclear distances and internal motion. Internal motion is usually ignored, and NOESY cross-peak intensities are interpreted in terms of internuclear distances only. Off-resonance ROESY experiments measure a weighted average of NOE and ROE. The weight can be described and experimentally set by an angle theta;. For large enough molecules, NOE and ROE have opposite signs. Therefore, each cross-peak intensity becomes zero for an angle theta;(0). For any sample, the maximum angle theta;(0) is determined by the overall motion of the molecule. Smaller theta;(0) values reflect the angular component of internal motions. Because individual cross-peaks are analyzed, the method evaluates internal motions of individual H,H vectors. The reduction of theta;(0) is largest for internal motions on a time scale of 100-300 ps. The sensitivity of theta;(0) for internal motions decreases with increasing molecular weight. We estimate that detecting internal motions will be practicable for molecules up to about 15 kDa. We describe a protocol to measure theta;(0) from a series of off-resonance ROESY spectra. For such a series, we describe the choice of experimental parameters, a procedure to extract theta;(0) from the raw data, and the interpretation of theta;(0) in terms of internal motions. In the small protein BPTI, we analyzed 75 cross-peaks. The precision of theta;(0) was 0.25 degrees, as compared to typical reductions of theta;(0) of 3 degrees. We found a well-defined maximum theta;(0) for cross-peaks in rigid parts of the molecule, which reflects the overall motion of the molecule. For BPTI, also many structurally important long-range cross-peaks appear rigid. The lower theta;(0) values of long-range contacts involving methyl groups are consistent with methyl rotation on the 25-ps time scale. The lower theta;(0) values of the flexible C-terminus and of flexible side chains translate into upper limits for the angular order parameter of 0.4 and 0.5-0.8, respectively. Off-resonance ROESY can monitor internal motions of H,H contacts that are used in a structure calculation. Because no isotope labeling is needed, off-resonance ROESY can be used to detect internal motions in a wide range of natural products.
Stroke is a major health problem worldwide, causing high morbidity and mortality. Intracerebral hemorrhage (ICH) accounts for 15% of stroke cases in the US and Europe and up to 30% in Asian populations. It is less treatable than other forms of stroke and causes higher morbidity and disability. Data suggest that early hematomy growth is the principal cause of early neurological deterioration after ICH. Prospective and retrospective studies indicate that early hematoma growth occurs in 18-38% of patients scanned within 3 h of ICH onset, and that hematoma volume is an important predictor of 30-day mortality. As hematoma growth in acute ICH is a dynamic process, intervention with ultra-early hemostatic therapy could lead to minimization and even prevention of early hematomy growth. Recombinant activated factor VII (rFVIIa, 'NovoSeven'), a powerful initiator of hemostasis, is approved for the treatment of bleeding in patients with hemophilia and inhibitors and may also promote hemostasis in patients with normal coagulation. rFVa acts locally at the bleeding site without activating systemic coagulation and may be a valuable therapy during the hyperacute stage of ICH. A randomized, double-blind, placebo-controlled, dose-ranging trial is currently in progress to investigate the potential of rFVIIa as an ultra-early hemostatic therapy to prevent or minimize hematoma growth in ICH patients without coagulopathy.