Methylene blue enhances lateral association of fibrin resulting in rapid gelation and thick fiber formation.

Methylene blue (MB) has been suggested as a therapeutic alternative for heparin reversal in patients sensitive to protamine. We investigated the impact of MB on the assembly and structure of thrombin induced fibrin and plasma gels. MB (1,600 micrograms/ml) reduced the thrombin clotting time (TCT) of plasma by 30% and of purified fibrinogen by 46%. Above 1,600 micrograms/ml, TCTs were prolonged due to MB mediated fibrinogen precipitation. The presence of 5 mM CaCl2 masked the effect of MB in both purified and plasma systems and lowered the threshold for MB-induced purified fibrinogen precipitation to 800 micrograms/ml. MB shortened the lag phase prior to thrombin-induced turbidity increase, and enhanced final gel turbidity. The fibrin fiber mass/length ratio increased from 5.2 to 13.1 X 10(13) dalton/cm in purified fibrin gels and from 3.2 to 10.4 X 10(13) dalton/cm in plasma gels as the MB concentration increased from 0 to 200 micrograms/ml. Due to the photooxidant effect of MB on fibrinogen, rapid time-dependent loss of fibrinogen clottability was obvious at low MB concentrations (50 to 400 micrograms/ml). At high MB concentrations, intense MB light absorption partially protected fibrinogen within the sample. Accurate measurements could only be made, however, when MB was added just prior to thrombin and the assays were performed in the dark. While erythrocytes may reduce the impact of MB photooxidation in whole blood, plasma samples must be shielded from light if reproducible results are to be obtained.