Microkinetic coagulation assays for human and zebrafish plasma

Abstract

Coagulation assays, prothrombin time (PT), and partial thromboplastin time (PTT) are tests to measure the clotting ability of plasma and used in evaluating patients suffering from bleeding disorders. These assays require 100 μl of human plasma. In zebrafish, dilute plasma with exogenously added human fibrinogen was used. Our objective is to create a microkinetic coagulation assay for human and zebrafish plasmas using 1 μl plasma under conditions similar to PT and PTTs. Here, we developed an assay using the Take3 plate with wells holding up to 6 μl, which can be loaded in a microplate reader for measuring the absorbance of fibrin formation. In this assay, we used 1 μl of citrated zebrafish or human plasma followed by the addition of either thromboplastin or Dade ACTIN or factor X activator from Russell viper venom as an activating agent and CaCl 2 . We found 4 or 3 μl of the final volume of reaction was optimal. Our results showed both zebrafish and human plasmas yielded kinetic PT, kinetic PTT, and kinetic Russel's viper venom time curves similar to previously established curves using dilute plasma. This kinetic coagulation was inhibited by heparin and was reduced significantly in coagulation factor deficient plasmas. These results validated our microkinetic coagulation assays. Moreover, we derived clotting times from these kinetic curves, which were identical to human PT, PTT, and Russel's viper venom time. In conclusion, we established a microkinetic assay that could measure blood coagulation activity in models like zebrafish and human blood samples obtained from a finger prick in adults or heel prick in infants.

High Mutational Heterogeneity, and New Mutations in the Human Coagulation Factor V Gene. Future Perspectives for Factor V Deficiency Using Recombinant and Advanced Therapies

“…Although the sequence alignment analyses indicate a lower homology in the case of the zebrafish, it must be noted that that is one of the teleost species where a greater number of orthologous genes are involved in hemostasis [ 79 , 80 ]. That is the reason why zebrafish has been postulated as a useful model for in vivo coagulation studies [ 81 , 82 ].…”

Characterization of zebrafish coagulation cofactors Fviii and Fv mutants and modeling hemophilia A and factor V deficiency

“…The plate was immediately placed in the Epoch 2 Microplate Spectrophotometer (Biotek) with double orbital shaking for 15 s and absorbance for the samples were recorded every 5 s for 10 min at 405 nm at room temperature. Negative controls were performed by replacing Dade ACTIN or Thromboplastin with 1X PBS [21].…”

Knockdown and Knockout of Tissue Factor Pathway Inhibitor in Zebrafish

“…Kinetic assays were performed on a Take3 Micro-Volume Plate (Biotek, Winooski, Vermont, United States) as described earlier. 25 Briefly, 1 µL zebrafish muscle thromboplastin for kinetic prothrombin time (kPT) or 1 µL of DADE ACTIN (Sigma, St. Louis, Missouri, United States) for kinetic partial thromboplastin time (kPTT) assay respectively were added to a microwell followed by Thrombosis and Haemostasis © 2022. Thieme.…”

“…Kinetic assays were performed on a Take3 Micro-Volume Plate (Biotek, Winooski, Vermont, United States) as described earlier. 25 Briefly, 1 µL zebrafish muscle thromboplastin for kinetic prothrombin time (kPT) or 1 µL of DADE ACTIN (Sigma, St. Louis, Missouri, United States) for kinetic partial thromboplastin time (kPTT) assay respectively were added to a microwell followed by 1 µL of zebrafish plasma, 1 µL of 1X PBS, and 1 µL of 100 mM CaCl 2 . The fibrin formation over time was detected by measuring the absorbance at 405 nm using Synergy H1 Hybrid Multi-Mode Microplate Reader (BioTek Instruments, Winooski, Vermont, United States).…”