Where is prothrombin formed

Read More. Skip to content Main Navigation Search. Dictionary Articles Tutorials Biology Forum. Circulation The circulatory system is key to the transport of vital biomolecules and nutrients throughout the body. Macfarlane, R. Davie, E. Breckenridge, R. Jobin, F. Denson, K. Google Scholar. Esnouf, M. Williams, W. Article Google Scholar. Yin, E. Schwert, G. Acta , 16 , After a variable latent period, thrombin suddenly appears in large quantities, coincident with or immediately preceding the deposition of fibrin if fibrinogen is present.

The amount of thrombin formed in a mixture of prothrombin, Ca and platelets is independent of the platelet or Ca concentration, and depends primarily upon the amount of prothrombin used. Alternatively, stabilization of the open form via stalling of the prothrombinase complex after cleavage at Arg and generation of meizothrombin would afford an anticoagulant effect in vivo , as documented by the severe bleeding phenotype associated with the naturally occurring mutation prothrombin Padua, ArgHis, that abrogates cleavage site at Arg The mutation of the catalytic serine was necessary to avoid autoproteolytic degradation of meizothrombin Human recombinant prethrombin-2 was expressed and purified as described elsewhere All other chemicals were purchased from Sigma-Aldrich MO.

X-ray diffraction data were collected with a home source Rigaku 1. Crystals showed anisotropic diffraction with 3. Several dehydration methods were tried for improving crystal diffraction The structures of linker 2 and residues — were traced based on the extra electron density obtained from molecular replacement. For the structure at 4. Model building and analysis of the structures were conducted with COOT Statistics for data collection and refinement are summarized in Table 1S.

Structures at 6. The decision of immobilizing prethrombin-2 and not fragment-1 was based on the observation that prethrombin-2 showed significant more nonspecific binding compared to fragment After initial binding tests to confirm interactions between fragment-1 and prethrombin-2, a single-cycle kinetic assay was set up to examine dose-response dependent.

Selective labeling of the unpaired Cys residues with Alexa Fluor C2-maleimide AF as the donor and Alexa Fluor C2-maleimide AF as the acceptor Thermo Fisher Scientific was achieved and probed by limited proteolysis with thrombin, as described recently Briefly, experiments were carried out with pulsed interleaved excitation PIE 42 , which reports the status of both donor and acceptor fluorophores by sorting molecules on the basis of relative donor:acceptor stoichiometry S and apparent FRET efficiency E.

Suited bandpass filters were inserted to eliminate the respective excitation wavelength and minimize spectral crosstalk. The fluorescence was detected with two avalanche photodiodes using Time-correlated Single Photon Counting with the TimeHarp board. Data were stored in the Time-tagged Time-resolved Mode.

Signals from single molecules were observed as bursts of fluorescence. Bursts with more than 35 counts were searched with the all photon burst search APBS algorithm while integration time was set to 0. The number of independent Gaussians was determined according to the corrected Akaike information criterion AICc.

Data recording and initial data analysis were performed using the SymphoTime Software 6. Briefly, prothrombin 0. Each experiment was repeated at least three times using three different batches of proteins. Conversion of prothrombin to thrombin was also monitored using a colorimetric assay that continuously reports the amount of thrombin that is generated upon cleavage by the prothrombinase complex.

All reagents and relevant data are available from the authors upon request. Please contact nicola. Page, M. Serine peptidases: classification, structure and function.

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Sun, W. Prothrombin deficiency results in embryonic and neonatal lethality in mice. Pozzi, N. The linker connecting the two kringles plays a key role in prothrombin activation. Mann, K. Methods Enzymol 80 Pt C , — Haynes, L. Prothrombin activation by platelet-associated prothrombinase proceeds through the prethrombin-2 pathway via a concerted mechanism. J Biol Chem , —55 Whelihan, M.

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The prothrombinase-catalyzed activation of prothrombin proceeds through the intermediate meizothrombin in an ordered, sequential reaction. J Biol Chem , —84 Bradford, H. Membrane binding by prothrombin mediates its constrained presentation to prothrombinase for cleavage.

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Substitution of Gly to Ala in the substrate binding pocket of prothrombin Perija leads to the loss of thrombin proteolytic activity. Thromb Haemost 87 , —7 Conformational selection in trypsin-like proteases.