Parallel imaging of coagulation pathway proteases activated protein C, thrombin, and factor Xa in human plasma

Abstract

Using a collection of natural and unnatural amino acids, we synthesized a set of fluorescent activity-based probes for the fast, direct, and simultaneous detection of coagulation factors in human plasma.

Fluorescent Activity-Based Probe To Image and Inhibit Factor XIa Activity in Human Plasma

“…Previous studies reported that the fXIa S1 pocket is wide, deep, highly conserved and able to interact exclusively with positively charged arginine, 7,30,31 which is also observed in the case of other serine proteases from the coagulation cascade. 33 We found that in addition to arginine, the fXIa S1 subsite could also accommodate an unnatural phenylalanine derivative with a guanidine group in the para position, that is, L-Phe(guan) and several other amino acid residues (mostly phenylalanine derivatives). Our HyCoSuL screening showed that fXIa exhibited extremely narrow substrate specificity at the P2 position.…”

“…Protein C, factor X, and prothrombin were purified from fresh frozen plasma and then activated as previously described. 33 Expression and Purification of Recombinant Human Factor XI. Full-length cDNA for human factor XI was ligated into the cloning site of a mammalian expression vector (pCEP4) containing the cytomegalovirus promoter.…”

“…Synthesis of the Inhibitor and ABPs. Irreversible inhibitor, biotin-labeled, and fluorescently labeled probes for fXIa were synthesized and purified as described previously for APC, thrombin, and fXa by Modrzycka et al 33 Determination of Inhibition Kinetics (k obs /I) for the Inhibitor and Fluorescently Labeled ABP. The k obs /I parameters for APC, thrombin, fXa, and fXIa were measured under pseudo-first-order conditions.…”

“…Detection of fXIa in Human Plasma. Human plasma (collected in tubes containing anticoagulant EDTA) was isolated from whole blood as described elsewhere 33 and incubated with a fluorescently labeled probe at probe concentrations ranging from 1 to 20 μM. Incubation was carried out in assay buffer for 60 min at 37 °C.…”

Green-Emitting 4,5-Diaminonaphthalimides in Activity-Based Probes for the Detection of Thrombin

“…The tert -butyl ester group was easily hydrolyzed by trifluoroacetic acid (TFA), yielding 4a and 4b . On the basis of the structure of the previously obtained thrombin-selective substrate 36 we designed two fluorescent activity-based probes, namely, 5a and 5b , that contained fluorophores 4a and 4b , respectively, and one reference biotin-labeled probe 5c that contained a commercially available biotin tag. First, using the solid-phase approach, a recognition element was synthesized by elongating the peptide on 2-chlorotrityl resin.…”