Thrombin Substrate, fluorescent (H-D-Phe-Pro-Arg-AFC)

Product Name: Thrombin Substrate, fluorescent (H-D-Phe-Pro-Arg-AFC) - 10 mg

Sequence One Letter Code: fPR-AFC

Sequence Three Letter Code: H-D-Phe-Pro-Arg-AFC

Chemical Formula:C30H34F3N7O5

Molecular Weight: 629.6

Purity: 95%

Form: Lyophilized

Storage Conditions: - 20 °C Protected from light

Research Area: peptide substrate

Source / Species: human

Conjugation: Conjugated

Conjugation Type: Fluorescent dyes

Code Nacres: NA.26

Application: H-D-Phe-Pro-Arg-AFC is a fluorogenic substrate developed for sensitive detection of thrombin activity in coagulation research. Thrombin, a central serine protease in the clotting cascade, cleaves the substrate at the Arg residue to release the AFC fluorophore, generating fluorescence measurable at Ex 380 nm and Em 500 nm. The substrate enables real-time kinetic analysis, inhibitor screening, and functional investigation of thrombin-dependent coagulation pathways.

Current Research: H-D-Phe-Pro-Arg-AFC is a fluorogenic peptide substrate designed for sensitive and quantitative measurement of thrombin (factor IIa) activity in coagulation research. The substrate consists of the tripeptide D-Phe–Pro–Arg linked to 7-amino-4-trifluoromethylcoumarin (AFC). Thrombin selectively cleaves the peptide bond at the C-terminal arginine residue, liberating free AFC and generating a measurable fluorescence signal at approximately Ex 380 nm and Em 500 nm. This design enables continuous, real-time monitoring of thrombin proteolytic activity in purified enzyme systems, plasma samples, or cell-based coagulation models. Biological Context Thrombin is a central serine protease in the coagulation cascade, responsible for: Conversion of fibrinogen to fibrin Activation of factors V, VIII, XI, and XIII Platelet activation via protease-activated receptors (PARs) Amplification of clot formation Because thrombin sits at a convergence point in both intrinsic and extrinsic coagulation pathways, precise measurement of its activity is critical in hemostasis and thrombosis research. Substrate Design and Mechanism The D-Phe–Pro–Arg sequence confers high specificity toward thrombin’s substrate-binding pocket. Incorporation of D-phenylalanine enhances resistance to non-specific proteolysis and improves selectivity. Upon enzymatic cleavage: Thrombin recognizes the Arg residue at the P1 position. The Arg–AFC bond is hydrolyzed. Free AFC is released into solution. Fluorescence intensity increases proportionally to enzymatic activity. This fluorogenic format allows sensitive detection without secondary reagents. Research Applications 1. Kinetic Characterization H-D-Phe-Pro-Arg-AFC supports determination of thrombin kinetic parameters (Km, Vmax, kcat) under defined buffer conditions. 2. Anticoagulant and Inhibitor Screening The substrate is widely used to evaluate thrombin inhibitors, including direct thrombin inhibitors (DTIs). Reduction in AFC release enables calculation of IC₅₀ or Ki values. 3. Coagulation Pathway Analysis In plasma or reconstituted coagulation systems, the substrate facilitates assessment of thrombin generation and amplification dynamics. 4. Functional Studies of Thrombin Signaling Beyond clot formation, thrombin activates cellular receptors such as PAR-1. This substrate enables correlation of enzymatic activity with downstream signaling events. Advantages High sensitivity fluorescence readout Real-time continuous kinetic monitoring Compatible with microplate fluorometers Suitable for purified enzyme and complex biological samples No coupled detection system required Experimental Considerations Assays should be conducted under physiological or defined ionic conditions appropriate for thrombin activity. Inclusion of selective thrombin inhibitors (e.g., hirudin analogs) can confirm specificity. Substrate concentration should be optimized relative to Km for accurate kinetic analysis.