We have developed a model of the extrinsic blood coagulation system that includes the stoichiometric anticoagulants. The model accounts for the formation, expression, and propagation of the vitamin ...K-dependent procoagulant complexes and extends our previous model by including: (a) the tissue factor pathway inhibitor (TFPI)-mediated inactivation of tissue factor (TF)·VIIa and its product complexes; (b) the antithrombin-III (AT-III)-mediated inactivation of IIa, mIIa, factor VIIa, factor IXa, and factor Xa; (c) the initial activation of factor V and factor VIII by thrombin generated by factor Xa-membrane; (d) factor VIIIa dissociation/activity loss; (e) the binding competition and kinetic activation steps that exist between TF and factors VII and VIIa; and (f) the activation of factor VII by IIa, factor Xa, and factor IXa. These additions to our earlier model generate a model consisting of 34 differential equations with 42 rate constants that together describe the 27 independent equilibrium expressions, which describe the fates of 34 species. Simulations are initiated by “exposing” picomolar concentrations of TF to an electronic milieu consisting of factors II, IX, X, VII, VIIa, V, and VIIII, and the anticoagulants TFPI and AT-III at concentrations found in normal plasma or associated with coagulation pathology. The reaction followed in terms of thrombin generation, proceeds through phases that can be operationally defined as initiation, propagation, and termination. The generation of thrombin displays a nonlinear dependence upon TF, AT-III, and TFPI and the combination of these latter inhibitors displays kinetic thresholds. At subthreshold TF, thrombin production/expression is suppressed by the combination of TFPI and AT-III; for concentrations above the TF threshold, the bolus of thrombin produced is quantitatively equivalent. A comparison of the model with empirical laboratory data illustrates that most experimentally observable parameters are captured, and the pathology that results in enhanced or deficient thrombin generation is accurately described.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
In vertebrate hemostasis, factor Va serves as the cofactor in the prothrombinase complex that results in a 300,000-fold increase in the rate of thrombin generation compared with factor Xa alone. ...Structurally, little is known about the mechanism by which factor Va alters catalysis within this complex. Here, we report a crystal structure of protein C inactivated factor Va (A1·A3-C1-C2) that depicts a previously uncharacterized domain arrangement. This orientation has implications for binding to membranes essential for function. A high-affinity calcium-binding site and a copper-binding site have both been identified. Surprisingly, neither shows a direct involvement in chain association. This structure represents the largest physiologically relevant fragment of factor Va solved to date and provides a new scaffold for the future generation of models of coagulation cofactors.
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Previously, we reported modeling synovial sarcomas in mice by conditionally expressing the human t(X;18) translocation-derived SYT-SSX2 fusion protein in Myf5-expressing myoblasts. Using a ...tamoxifen-inducible CreER system in mice, we show here that sporadic expression of SYT-SSX2 across multiple tissue types leads to exclusive formation of synovial sarcoma-like tumors, whereas its widespread expression is lethal. Certain clinical and histologic features of tumors in this new model suggest additional nonmyoblast origin for synovial sarcoma. CreER-based sporadic expression both avoids the severe early developmental phenotypes associated with widespread SYT-SSX2 expression and better models natural pathogenesis of cancers in which transformed cells usually arise within an environment of largely normal cells. Furthermore, this strategy may recapitulate multiple potential cellular origins within a single model system.
Extraction and purification of intact chromosomes are critical sample preparation steps for transchromosomic research and other applications. The commonly used sample preparation methods lead to too ...few chromosomes with chromosome deactivation and degradation. In this paper, a “mild” chromosome extraction process that combines a chemical and mechanical lysis approach is introduced for the preparation of intact chromosomes that can readily be used for downstream processing. Metaphase cells are treated by chemical lysis buffer and pushed through a microfluidic pinched flow device. Cells are ruptured, and chromosomes are released by a combination of shear stress and chemical reagents. Chromosomes are released intact from the cell membrane into the solution. Simulations and experiments are performed to optimize the microfluidic device geometry and operation parameters. Cell rupture and chromosome release are found to be improved by the shear stress in the pinched flow device. Simulation results indicate that the maximum shear stress appears in the channel constriction region, and the narrow channel maintains constant shear stress. It is concluded that the constriction design, narrow channel width, and operation flow rate have a significate influence on chromosome release. Utilizing an optimized device, near-complete cell lysis is achieved and 4 times as many chromosomes are released (8% in control experiments to 25% in optimized pinched flow devices). Sample treatment time can also be reduced utilizing this combined chemical-mechanical chromosome release method.
In this paper, we use a spiral channel inertial focusing device for isolation and purification of chromosomes, which are highly asymmetric. The method developed is proposed as a sample preparation ...process for transchromosomic research. The proposed microfluidics-based chromosome separation approach enables rapid, label-free isolation of bioactive chromosomes and is compatible with chromosome buffer. As part of this work, particle force analysis during the separation process is performed utilizing mathematic models to estimate the expected behavior of chromosomes in the channel and the model validated with experiments employing fluorescent beads. The chromosome sample is further divided into subtypes utilizing fluorescent activated cell sorting , including small condensed chromosomes, single chromosomes, and groups of two chromosomes (four sister chromatids). The separation of chromosome subtypes is realized based on their shape differences in the spiral channel device under high flow rate conditions. When chromosomes become aligned in the shear flow, the balance between the inertial focusing force and the Dean flow drag force is determined by the chromosome projection area and aspect ratio, or shape difference, leading to different focusing locations in the channel. The achieved results indicate a new separation regime in inertial microfluidics that can be used for the separation of non-spherical particles based on particle aspect ratios, which could potentially be applied in fields such as bacteria subtype separation and chromosome karyotyping.
The inactivation of factor Va is a complex process which includes bond cleavage (at three sites) and dissociation of the A2N·A2C peptides, with intermediate activity in each species. Quantitation of ...the functional consequences of each step in the reaction has allowed for understanding of the presentation of disease in individuals possessing the factor V polymorphism factor VLEIDEN. APC cleavage of membrane-bound bovine factor Va (Arg306, Arg505, Arg662) leads to the dissociation of fragments of the A2 domain, residues 307−713 (A2N·A2C + A2C-peptide), leaving behind the membrane-bound A1·LC species. Evaluation of the dissociation process by light scattering yields invariant mass loss estimates as a function of APC concentration. The rate constant for A2 fragment dissociation varies with APC, reaching a maximal value of k = 0.028 s-1, the unimolecular rate constant for A2 domain fragment dissociation. The APC binding site resides in the factor Va light chain (LC) (K d = 7 nM), suggesting that the membrane-bound LC·A1 product would act to sequester APC. This inhibitory interaction (LC·A1·APC) is demonstrated to exist with either purified factor Va LC or the products of factor Va inactivation. Utilizing these experimental data and the reported rates of bond cleavage, binding constants, and product activity values for factor Va partial inactivation products, a model is developed which describes factor Va inactivation and accounts for the defect in factor VLEIDEN. The model accurately predicts the rates of inactivation of factor Va and factor VaLEIDEN, and the effect of product inhibition. Modeled reaction progress diagrams and activity profiles (from either factor Va or factor VaLEIDEN) are coincident with experimentally derived data, providing a mechanistic and kinetic explanation for all steps in the inactivation of normal factor Va and the pathology associated with factor VLEIDEN.
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The inactivation of factor Va was examined on primary cultures of human umbilical vein endothelial cells (HUVECs), either after addition of activated protein C (APC) or after addition of ...alpha-thrombin and protein C (PC) zymogen. Factor Va proteolysis was visualized by Western blot analysis using a monoclonal antibody (alpha HVaHC No. 17) to the factor Va heavy chain (HC), and cofactor activity was followed both in a clotting assay using factor V-deficient plasma and by quantitation of prothrombinase function. APC generation was monitored using the substrate 6-(D-VPR)amino-1-naphthalenebutylsulfonamide (D-VPR-ANSNHC4 H9), which permits quantitation of APC at 10 pmol/L. Addition of APC (5 nmol/L) to an adherent HUVEC monolayer (3.5 x 10 cells per well) resulted in a 75% inactivation of factor Va (20 nmol/L) within 10 minutes, with complete loss of cofactor activity within 2 hours. Measurements of the rate of cleavage at Arg and Arg in the presence and absence of the HUVEC monolayer indicated that the APC-dependent cleavage of the factor Va HC at Arg was accelerated in the presence of HUVECs, while cleavage at Arg was dependent on the presence of the HUVEC surface. Factor Va inactivation proceeded with initial cleavage of the factor Va HC at Arg, generating an Mr 75 000 species. Further proteolysis at Arg generated an Mr 30 000 product. When protein C (0.5 micro mol/L), alpha-thrombin (1 nmol/L), and factor Va (20 nmol/L) were added to HUVECs an APC generation rate of 1.56 +/- 0.11 x 10 mol/min per cell was observed. With APC generated in situ, cleavage at Arg on the HUVEC surface is followed by cleavage at Arg, generating Mr 75 000 and Mr 30 000 fragments, respectively. In addition, the appearance of two novel products derived from the factor Va HC are observed when thrombin is present on the HUVEC surfacethe HC is processed through limited thrombin proteolysis to generate an Mr 97 000 fragment, which is further processed by APC to generate an Mr 43 000 fragment. NH2 -terminal sequence analysis of the Mr 97 000 fragment revealed that the thrombin cleavage occurs in the COOH-terminus of the intact factor Va HC since both the intact HC as well as the Mr 97 000 fragment have the same sequence. Our data demonstrate that the inactivation of factor Va on the HUVEC surface, initiated either by APC addition or PC activation, follows a mechanism whereby cleavage is observed first at Arg followed by a second cleavage at Arg. The latter cleavage is dependent on the availability of the HUVEC surface. This mechanism of inactivation of factor Va is similar to that observed on synthetic phospholipid vesicles. (Arterioscler Thromb Vasc Biol. 1997;17:2765-2775.)
The products of cleavage of bovine factor Va by activated protein C (APC) in the presence and absence of phospholipid (25% phosphatidylserine, 75% phosphatidylcholine, PCPS) were evaluated using ...sedimentation velocity/equilibrium methods in the analytical ultracentrifuge and by immunoprecipitation using an antibody directed against the light chain of the factor Va molecule. The molecular weight and sedimentation coefficient of the associated heavy and light chains of factor Va, 173,000 (7.9 S) is reduced to 132,000 (7.1 S) by APC cleavage at Arg505 and Arg662. Complete cleavage of the factor Va heavy chain (with APC-PCPS) at Arg505, Arg662 and Arg306 results in a drastic change in the molecular weight observed for the product. Two products are resolved with sedimentation coefficients of 3.3 and 6.3 S with estimated molecular weights of 48,000 and 114,000, respectively. Immunoprecipitation studies showed that the products of factor Va cleavage at Arg505 and Arg662 (A1A2N·A2C·LC) are mostly noncovalently associated and consequently immunoprecipitated with an antibody directed against the light chain of the factor Va molecule. In contrast, for factor Va cleaved at Arg505, Arg662, and Arg306 the precipitated complex consisted of the A1 domain (residues 1–306) and the light chain (residues 1537–2183) of factor Va (A1·LC). The fragments corresponding to residues 307–505 (A2N) and 506–662 (A2C) are found in the supernatant. The combined mass of these two products (48,000) is similar to the estimated mass of the 3.3 S fragment estimated from sedimentation velocity/equilibrium studies; while the combined mass of the 1–306 + 1537–2183 products corresponds to 114,000, the estimated mass of the 6.3 S fragment. These data lead to the conclusion that cleavages at Arg306, Arg505, and Arg662 of the factor Va molecule resulted in the dissociation of the entire A2 domain as two noncovalently associated fragments (A2N·A2C). Enzyme kinetic and light scattering data suggest that the complete inactivation of the factor Va molecule involves not only cleavage at Arg306 but also the dissociation of the A2 domain. These data also suggest that the complete APC inactivation of the factor Va molecule is analogous to the spontaneous inactivation of factor VIIIa, which occurs via the dissociation of the A2 domain.
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GEOZS, IJS, IMTLJ, KILJ, KISLJ, NLZOH, NUK, OILJ, PNG, SAZU, SBCE, SBJE, UILJ, UL, UM, UPCLJ, UPUK, ZAGLJ, ZRSKP
Self-Association of Human Protein S Pauls, Jonathan E. D; Hockin, Matthew F; Long, George L ...
Biochemistry (Easton),
05/2000, Volume:
39, Issue:
18
Journal Article
Peer reviewed
Protein S functions as a cofactor with activated protein C in the down-regulation of the blood coagulation cascade. In vitro studies have historically produced conflicting data with regard to the ...extent of various protein S activity in clotting assays which typically involve adding CaCl2 to initiate reactions. We report here that protein S reversibly self-associates in the absence of Ca2+. Sedimentation experiments showed a transition in sedimentation velocity from 7.2 to 4.2 S with a transition midpoint (T m) of 0.42 mM Ca2+ for intact protein S. Studies of thrombin cleaved (Arg70) protein S revealed similar results with a transition in sedimentation velocity from 7.9 to 4.4 S with a T m of 0.42 mM Ca2+. This transition is reversible with the addition of 10 mM EDTA. Sedimentation equilibrium data suggest at a minimum, a monomer−dimer−trimer association. Sedimentation velocity experiments were also performed on mixtures of protein S and prothrombin which showed no heterodimer formation in either Ca2+ or EDTA solutions. These data suggest that previous interpretations of protein S structure and function may have been confounded by the self-associative behavior of protein S in non-Ca2+ solutions.
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