Generation of the combined prothrombin activation peptide (F1-2) during the clotting of blood and plasma.

Aronson, David L.; Stevan, L; Ball, A. P.; Franza, B. R.; Finlayson, J. S.

doi:10.1172/jci108902

Cited by 118 publications

(50 citation statements)

References 20 publications

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“…Despite the short incubation time, the sensor yielded a 220% fluorescence increase in serum compared with the 270% fluorescence gain obtained in buffer at 750 nM thrombin concentration. We found that the background signals were noticeably larger in undoped serum compared to buffer; given that the FBS used in the experiment was harvested from calves in utero, we suspect that some activation of the blood-clotting cascade may have occurred, thereby producing detectable levels of thrombin in the serum (33). Similar background thrombin signal in serum has also been observed in related work (34).…”

Section: Resultssupporting

confidence: 76%

In vitro selection of structure-switching, self-reporting aptamers

Plakos²,

Lou³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

119

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We describe an innovative selection approach to generate selfreporting aptamers (SRAs) capable of converting target-binding events into fluorescence readout without requiring additional modification, optimization, or the use of DNA helper strands. These aptamers contain a DNAzyme moiety that is initially maintained in an inactive conformation. Upon binding to their target, the aptamers undergo a structural switch that activates the DNAzyme, such that the binding event can be reported through significantly enhanced fluorescence produced by a specific stacking interaction between the active-conformation DNAzyme and a small molecule dye, N-methylmesoporphyrin IX. We demonstrate a purely in vitro selection-based approach for obtaining SRAs that function in both buffer and complex mixtures such as blood serum; after 15 rounds of selection with a structured DNA library, we were able to isolate SRAs that possess low nanomolar affinity and strong specificity for thrombin. Given ongoing progress in the engineering and characterization of functional DNA/RNA molecules, strategies such as ours have the potential to enable rapid, efficient, and economical isolation of nucleic acid molecules with diverse functionalities.G-quadruplex | systematic evolution of ligands by exponential enrichment | sensor | binding induced folding | molecular recognition N ucleic acid-based aptamers (1, 2) can be selected in vitro against a wide range of targets (3-8), chemically modified and synthetically produced, thereby making them a promising class of molecules for many applications including diagnostics (9, 10), in vivo imaging (11,12), and targeted therapeutics (13,14). Unlike most conventional affinity reagents (e.g., antibodies), aptamers can also be engineered to perform complex molecular functions beyond binding. For example, our group has recently demonstrated the use of an aptamer that undergoes target-binding-induced conformational change for continuous, real-time detection of a small molecule in undiluted blood serum (15). However, the selection process required to directly isolate aptamers with the desired function poses a significant technical challenge. One common approach entails selection for an aptamer on the basis of target binding, followed by postmodification and optimization through rational design in order to link the molecular binding capability to the desired functions (16,17). Unfortunately, such approaches rely upon prior knowledge of the threedimensional aptamer structure and require lengthy optimization steps that often compromise aptamers' affinity and specificity for their targets (18)(19)(20).Alternately, one can directly isolate aptamers with the desired function by combining a specifically designed nucleic acid library and a suitable selection process. For example, pioneering work by Nutiu and Li (21) described the isolation of structure-switching aptamers using a DNA library that was designed to incorporate two short randomized stretches sandwiching a central fixedsequence motif, with binding sites for two PCR...

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Section: Resultssupporting

confidence: 76%

In vitro selection of structure-switching, self-reporting aptamers

Plakos²,

Lou³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

119

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“…Therefore a-thrombin plays a central role in the mechanism of blood coagulation. Incidentally, the concentrations of oc-thrombin used in this study were less than the ones found in spontaneously clotting human blood (circa 140 nM; Aronson et al, 1977). In addition, this enzyme also exerts hormone-like effects on a large number of cell types (Shuman, 1986).…”

Section: Discussionmentioning

confidence: 65%

Synergism between the contractile effect of epidermal growth factor and that of des‐Arg⁹‐bradykinin or of α.‐thrombin in rabbit aortic rings

deBlois

Drapeau

Petitclerc

et al. 1992

British J Pharmacology

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1 Rabbit aortic rings were used to test the possible contractile effects of growth factors and their interaction with other stimuli. A rapid potentiation of kinin-induced contraction by epidermal growth factor (EGF) has been previously observed in this preparation. 2 EGF (5-1500 ng ml ') and the isoform BB of platelet-derived growth factor (PDGF-BB; 1-126ngml ) exerted modest but sustained contractile effects in rabbit aortic rings. 3 EGF pretreatment (100ngml-1) potentiated the contractile responses to des-Arg9-bradykinin (desArg9-BK), an agonist of the B. receptors for kinin found in this preparation, and to human a-thrombin but not to several other contractile stimuli. The interaction appeared also relatively selective for the growth factor, because PDGF-BB pretreatment potentiated neither des-Arg9-BK nor a-thrombininduced contraction. 4 EGF, applied on a contraction plateau induced by des-Arg9-BK or x-thrombin, exerted a synergistic contractile effect, with a time course and a half-maximal concentration for EGF-induced contraction similar to the ones recorded in resting tissues (between 67 and 220 ng ml-', depending on the series of experiments). 5 The direct or synergistic contractile effects of EGF were not modified by the removal of the endothelium or by treatment with indomethacin. However, the tyrosine kinase inhibitors, erbstatin or genistein, inhibited the synergistic effect of EGF with des-Arg9-BK. The small direct contractile effect of EGF was significantly reduced by genistein. The synergistic effect of EGF with a-thrombin was comparatively more resistant to the tested tyrosine kinase inhibitors. 6 An inhibitor of the catalytic activity of a-thrombin, D-Phe-Pro-Arg-CH2Cl, prevented the contractile effect of x-thrombin in the aortic rings. In this system, a tetradecapeptide derived from a recently cloned a-thrombin receptor was a contractile stimulus at and above 10 gLM. Consistent with the hypothesis that this peptide could behave as an ox-thrombin receptor agonist, its contractile effect was potentiated by EGF pretreatment. Pharmacological evidence was provided to show that the receptors for o-thrombin were distinct from the B, receptors for kinins. Together, these findings suggest that a model of a cleavable receptor recently elaborated to account for a-thrombin effects on human platelets is valid in blood-free vascular smooth muscle preparations such as the rabbit isolated aorta. 7 The synergism between EGF and kinin-or a-thrombin-induced contractions constitutes a novel mode of myotropic action for growth factors. The synergism is probably dependent on the tyrosine kinase activity of receptors for EGF. These combinations of stimuli could occur in various types of vascular disease and account for abnormal vascular reactivity often associated with atheroma lesions or vascular wound healing.

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“…Affected members of family VIII possessed the other major subtype of inherited antithrombin deficiency. This disorder is produced by a discrete molecular 22 antithrombin deficient subjects derived from these kindreds were studied in detail (Table I; Fig. 1).…”

Section: Resultsmentioning

confidence: 99%

“…After 90 min of incubation, the clot was removed, heparin (final concentration I U/ml) was added, and the suspension was stirred for an additional 5 min. Subsequently, F1+2 was isolated from the sera by DEAE-cellulose batch adsorption-elution and hydroxylapatite chromatography as described by Aronson et al (22). Examination of a series of fractions eluted from hydroxylapatite with a potassium phosphate concentration of 0.35-0.45 M, revealed that >90% of the resultant material was F1+2 as judged by the sodium dodecyl sulfate (SDS) gel electrophoretic technique of Laemmli (23).…”

Section: Methodsmentioning

confidence: 99%

Elevated factor Xa activity in the blood of asymptomatic patients with congenital antithrombin deficiency.

Bauer¹,

Goodman²,

Kass³

et al. 1985

J. Clin. Invest.

116

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The presence of congenital antithrombin deficiency has been consistently shown to predispose patients to venous thrombosis. We have utilized the prothrombin fragment F1+2 radioimmunoassay to quantitate factor Xa activity in the blood of 22 asymptomatic individuals with this clinical disorder not receiving antithrombotic therapy. The mean level of F1+2 was significantly elevated in these patients as compared to normal controls (3.91 vs. 1.97 nM, P < 0.001). The metabolic behavior of 131I-F,+2 was found to be similar in antithrombin-deficient subjects and normal individuals. The hemostatic system hyperactivity as measured by the F1+2 assay could be specifically corrected by raising the plasma antithrombin levels of the above asymptomatic individuals into the normal range. This study provides the first demonstration that the prethrombotic state can be biochemically defined as an imbalance between the production and inhibition of factor Xa enzymatic activity within the human circulation. It is known that antithrombin and a1-proteinase inhibitor (PI) are the major inhibitors of factor Xa in human plasma in the absence of heparin. To further evaluate the mechanism by which antithrombin functions as an inhibitor of factor Xa in humans, we studied five patients who exhibited severe congenital deficiencies of a,-PI. Our results indicated that the plasma of these subjects showed virtually identical decreases in plasma antifactor Xa activity in the absence of heparin when compared to antithrombin-deficient individuals, but the plasma F1+2 levels in the a,-PI deficient population were not significantly different than normal. This data suggests that a,-PI does not function as a major inhibitor of factor Xa in vivo, and that a tonically active heparin-dependent mechanism exists in humans for accelerating the neutralization of this enzyme by antithrombin.

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Generation of the combined prothrombin activation peptide (F1-2) during the clotting of blood and plasma.

Cited by 118 publications

References 20 publications

In vitro selection of structure-switching, self-reporting aptamers

In vitro selection of structure-switching, self-reporting aptamers

Synergism between the contractile effect of epidermal growth factor and that of des‐Arg⁹‐bradykinin or of α.‐thrombin in rabbit aortic rings

Elevated factor Xa activity in the blood of asymptomatic patients with congenital antithrombin deficiency.

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Generation of the combined prothrombin activation peptide (F1-2) during the clotting of blood and plasma.

Cited by 118 publications

References 20 publications

In vitro selection of structure-switching, self-reporting aptamers

In vitro selection of structure-switching, self-reporting aptamers

Synergism between the contractile effect of epidermal growth factor and that of des‐Arg9‐bradykinin or of α.‐thrombin in rabbit aortic rings

Elevated factor Xa activity in the blood of asymptomatic patients with congenital antithrombin deficiency.

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Synergism between the contractile effect of epidermal growth factor and that of des‐Arg⁹‐bradykinin or of α.‐thrombin in rabbit aortic rings