The kinetic mechanism of Na ؉ binding to thrombin was resolved by stopped-flow measurements of intrinsic fluorescence. Na ؉ binds to thrombin in a two-step mechanism with a rapid phase occurring within the dead time of the spectrometer (<0.5 ms) followed by a single-exponential slow phase whose k obs decreases hyperbolically with increasing [Na ؉ ]. The rapid phase is due to Na ؉ binding to the enzyme E to generate the E:Na ؉ form. The slow phase is due to the interconversion between E* and E, where E* is a form that cannot bind NaTemperature studies in the range from 5 to 35°C show significant enthalpy, entropy, and heat capacity changes associated with both Na ؉ binding and the E to E* transition. As a result, under conditions of physiologic temperature and salt concentrations, the E* form is negligibly populated (<1%) and thrombin is almost equally partitioned between the E (40%) and E:Na ؉ (60%) forms. Single-site Phe mutations of all nine Trp residues of thrombin enabled assignment of the fluorescence changes induced by Na ؉ binding mainly to Trp-141 and Trp-215, and to a lesser extent to Trp-148, Trp-207, and Trp-237. However, the fast phase of fluorescence increase is influenced to different extents by all Trp residues. The distribution of these residues over the entire thrombin surface demonstrates that Na ؉ binding induces long-range effects on the structure of the enzyme as a whole, contrary to the conclusions drawn from recent structural studies. These findings elucidate the mechanism of Na ؉ binding to thrombin and are relevant to other clotting factors and enzymes allosterically activated by monovalent cations.

The activating effect of Na ؉ on thrombin is allosteric and depends on the conformational transition from a low activity Na ؉ -free (slow) form to a high activity Na ؉ -bound (fast) form. The structures of these active forms have been solved. Recent structures of thrombin obtained in the absence of Na ؉ have also documented inactive conformations that presumably exist in equilibrium with the active slow form. The validity of these inactive slow form structures, however, is called into question by the presence of packing interactions involving the Na ؉ site and the active site regions. Here, we report a 1.87 Å resolution structure of thrombin in the absence of inhibitors and salts with a single molecule in the asymmetric unit and devoid of significant packing interactions in regions involved in the allosteric slow 3 fast transition. The structure shows an unprecedented self-inhibited conformation where Trp-215 and Arg-221a relocate >10 Å to occlude the active site and the primary specificity pocket, and the guanidinium group of Arg-187 penetrates the protein core to fill the empty Na ؉ -binding site. The extreme mobility of Trp-215 was investigated further with the W215P mutation. Remarkably, the mutation significantly compromises cleavage of the anticoagulant protein C but has no effect on the hydrolysis of fibrinogen and PAR1. These findings demonstrate that thrombin may assume an inactive conformation in the absence of Na ؉ and that its procoagulant and anticoagulant activities are closely linked to the mobility of residue 215.

Thrombin is a Na(+)-activated, allosteric serine protease that plays multiple functional roles in blood pathophysiology. Binding of Na(+) is the major driving force behind the procoagulant, prothrombotic and signaling functions of the enzyme. This review summarizes our current understanding of the molecular basis of thrombin allostery with special emphasis on the kinetic aspects of Na(+) activation. The molecular mechanism of thrombin allostery is a remarkable example of long-range communication that offers a paradigm for many other biological systems.

Abstract.Photoacoustic endoscopy offers in vivo examination of the visceral tissue using endogenous contrast, but its typical B-scan rate is ∼10  Hz, restricted by the speed of the scanning unit and the laser pulse repetition rate. Here, we present a transvaginal fast-scanning optical-resolution photoacoustic endoscope with a 250-Hz B-scan rate over a 3-mm scanning range. Using this modality, we not only illustrated the morphological differences of vasculatures among the human ectocervix, uterine body, and sublingual mucosa but also showed the longitudinal and cross-sectional differences of cervical vasculatures in pregnant women. This technology is promising for screening the visceral pathological changes associated with angiogenesis.

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In this large cohort of patients who were followed prospectively for carotid stenosis, pulse pressure and HDL were found to be the key risk factors for carotid stenosis progression. The fact that pulse pressure superseded systolic pressure in multivariate modeling may shed light on the biology of carotid plaque progression. Further, our identification of these modifiable risk factors may help in the design of therapeutic trials for the prevention of progression of carotid atherosclerosis.

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Despite a higher endoleak rate, the nonsupported Dacron Ancure endografts were associated with greater sac shrinkage at up to 24 months following implantation.