Newer synthetic peptide substrates in coagulation testing: some practical considerations for automated methods

Area of Science:

• Biochemistry
• Biotechnology
• Clinical Chemistry

Background:

• Over 100 chromogenic and fluorogenic peptide substrates exist for coagulation and related parameter evaluation.
• Many current substrates have poor physical properties (insolubility, adsorption) and can interfere with assays.
• Some substrates inhibit serine protease generation during activation in global assays.

Purpose of the Study:

• To develop synthetic chromogenic substrates with improved physical and biochemical characteristics.
• To address limitations of existing substrates, including insolubility and assay interference.
• To create more reliable tools for evaluating coagulation and related parameters.

Main Methods:

• Utilized modified amino acids (CHG, CHT, Nleu) and molecular manipulations to enhance substrate properties.
• Synthesized and characterized substrates for Factor Xa, thrombin, and plasmin.
• Evaluated substrate solubility, autoamidolysis, and performance in laboratory assays using normal and pathologic plasma samples.

Main Results:

• Developed novel substrates with superior or identical biochemical characteristics compared to existing ones.
• New substrates demonstrated increased solubility (>5 x 10(-4) M), reduced autoamidolysis, and no solution opacity.
• Achieved comparable results (R > 0.9) in assays for Factors Xa, IIa, kallikrein, and plasmin using these substrates.

Conclusions:

• Modified amino acids and molecular design yield synthetic substrates with desirable physical and biochemical properties.
• These enhanced substrates offer improved performance and reliability in coagulation and related diagnostic assays.
• Thorough biochemical and physical screening of new substrates is crucial before clinical application.