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Related Experiment Videos

Ultrasensitive fluorogenic substrates for serine proteases

S Butenas1, M E DiLorenzo, K G Mann

  • 1Department of Biochemistry, Health Science Complex, University of Vermont, Burlington 05405, USA.

Thrombosis and Haemostasis
|November 19, 1997
PubMed
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New fluorogenic substrates (PNS-substrates) enable sensitive and selective quantification of key serine proteases in blood coagulation and fibrinolysis, including plasmin, factor XIa, and thrombin, at picomolar and femtomolar levels.

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Serine proteases play critical roles in blood coagulation and fibrinolysis.
  • Accurate quantification of these enzymes is essential for understanding hemostasis and developing therapeutics.
  • Existing assays may lack the required sensitivity or specificity for certain proteases.

Purpose of the Study:

  • To develop novel fluorogenic substrates (PNS-substrates) for sensitive and selective quantification of serine proteases.
  • To evaluate the performance of these substrates against key enzymes in coagulation and fibrinolysis.
  • To establish structure-efficiency correlations for optimizing substrate design.

Main Methods:

  • Synthesis and evaluation of over 100 fluorogenic PNS-substrates.

Related Experiment Videos

  • Assessing substrate hydrolysis by various serine proteases including Lys-plasmin, factor XIa, thrombin, activated protein C (APC), urokinase-type plasminogen activator (uPA), factor Xa, and factor VIIa.
  • Determining kinetic parameters such as kcat and KM to assess enzyme specificity and efficiency.

    • Main Results:

    • PNS-substrates with Lys in the P1 position are specific for Lys-plasmin, enabling quantification at 1 pM.
    • Substrates targeting factor XIa achieved high hydrolysis rates (kcat up to 170 s-1) and allowed quantification at 10 fM.
    • Highly specific thrombin substrates were developed, with some showing >10,000-fold selectivity over factor Xa, enabling quantification as low as 20 fM.
    • Sensitive quantification limits were established for APC (1 pM), uPA (1 pM), factor VIIa/tissue factor complex (1 pM), factor Xa (0.4 pM), and factor VIIa (40 pM).

    Conclusions:

    • PNS-substrates represent a versatile platform for developing direct and highly sensitive assays for serine proteases.
    • These substrates offer significant improvements in sensitivity and specificity compared to existing methods.
    • The developed assays have potential applications in clinical diagnostics and drug discovery related to hemostasis and thrombosis.