Protease nexin-2 (PN-2), a soluble form of amyloid beta-protein precursor (APP) containing a Kunin protease inhibitor domain, has been shown to be a potent, reversible and competitive inhibitor of blood coagulation factor XIa (FXIa). We have analyzed progress curves of the hydrolysis of a sensitive fluorogenic substrate by FXIa in the presence of PN-2 to ascertain the kinetic rate constants governing the inhibition of FXIa by PN-2. The mechanism of this inhibition is best described as a slow equilibration between the free enzyme and inhibitor directly, without prior formation of a loosely-associated complex. The association rate constant (kon) and the dissociation rate constant (koff) were found to be 2.1 +/- 0.2 x 10(6) M-1 s-1 and 8.5 +/- 0.8 x 10(-4) s-1, respectively (n = 23). The inhibition constant calculated from these parameters (Ki) is 400 pM, in good agreement with previous reports. High molecular weight kininogen (HK) and Zn2+ ions exert opposite effects on the inhibition of FXIa by PN-2. HK protects FXIa from inactivation in a dose dependent and saturable manner (EC50 = 61 nM) whereas Zn2+ augments the ability of PN-2 to inhibit FXIa. When both Zn2+ ions and HK are present, only the accessory effect of Zn2+ is observed. PN-2 is known to be an abundant platelet alpha-granule protein (Van Nostrand et al., 1990a; Smith & Broze, 1992). We conducted sensitive measurements of FXIa activity in the presence of human platelets before and after their being activated with the thrombin receptor agonist peptide, SFLLRN-amide. We found that platelet activation, and ostensibly the release of PN-2, limits the lifetime of FXIa activity within the locus of activated platelets. As in the purified system, HK protects FXIa from inactivation and Zn2+ increases the inactivation of FXIa. However, when HK and Zn2+ are both present, it is the protective effect of HK which predominates and prolongs the lifetime of FXIa after platelet activation.

Background NIBP/TRAPPC9 is expressed in brain neurons, and human NIBP mutations are associated with neurodevelopmental disorders. The cellular distribution and function of NIBP in the enteric nervous system (ENS) remain unknown. Methods Western blot and RT-PCR analysis were used respectively to identify the protein and mRNA expression of NIBP and other neuronal markers. Multilabeled immunofluorescent microscopy and confocal image analysis were used to examine the cellular distribution of NIBP-like immunoreactivity (IR) in whole mount intestine. Enteric neuronal cell line (ENC) was infected with lentivirus carrying NIBP or its shRNA expression vectors and treated with vehicle or TNFα. Key Results NIBP is expressed at both mRNA and protein levels in different regions and layers of the mouse intestine. NIBP-like-IR was co-localized with various neuronal markers, but not with glial, smooth muscular, or ICC markers. A small population of NIBP-expressing cells and fibers in extra-ganglionic and intra-ganglionic area were negative for pan-neuronal markers HuD or Peripherin. Relatively high NIBP-like-IR was found in 35-44% of myenteric neurons and 9-10% of submucosal neurons. Approximately 98%, 87% and 43% of these relatively high NIBP-expressing neurons were positive for ChAT, nNOS and Calretinin, respectively. NIBP shRNA knockdown in ENC inhibited TNFα-induced NFkB activation and neuronal differentiation, whereas NIBP overexpression promoted it. Conclusions & Inferences NIBP is extensively expressed in the ENS with relatively high level in a subpopulation of enteric neurons. Various NIBP expression levels in different neurons may represent dynamic trafficking or posttranslational modification of NIBP in some functionally-active neurons and ultimately regulate ENS plasticity.

A prototype weakly interacting massive particle (WIMP) detector system is described. The detection efficiency is much larger when the incident neutral particle flux is perpendicular to the detector plane than when it is parallel to the plane. A greatly scaled-up system based on this device would therefore be sensitive to the diurnal rotation of the "WIMP wind" produced by the solar system's rapid motion through the galactic halo. Experimental results are reported which exhibit the detection efficiency anisotropy when neutrons were used to simulate the interactions of WIMPS.

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