Figure 2.
Plasma clot formation and clot turbidity are unaffected by UHRA. Clot formation in diluted human plasma titrated with varying amounts of UHRA or protamine was investigated using a turbidimetric assay, as described in “Methods.” (A) Turbidity curves (A405nm) were obtained on the addition of TF and CaCl2 (20 mM) to plasma. (B) Lag time (sec) characterizing the time taken for initial protofibril formation during clotting were determined from A. Significant prolongation of lag time was observed by 50 µg/mL protamine (***P < .001). Remarkably, even at 1000 µg/mL UHRA, no significant change in lag time was observed. (C) Maximum absorbance values of plasma clots determined from A formed with UHRA remain unchanged, whereas significant (**P < .005) changes are recorded for clots formed in 50 µg/mL protamine. This indicated that UHRA neither inhibits nor alters fibrin polymerization in plasma. (D) Turbidity curves (A405nm) were obtained on recalcification (20 mM) of plasma. (E) Lag times observed in the presence of UHRA or protamine. No significant change in lag time was observed with UHRA, whereas impaired plasma clotting caused prolongation of lag times at all protamine concentrations studied (*P < .015). (F) Maximum absorbance of plasma clots containing UHRA or protamine. No statistically significant differences in final absorbance values were recorded for the UHRA-containing and polycation-free systems, whereas significant changes (**P < .01) were observed in the 50 µg/mL protamine system. Moreover, no clot was formed at 100 at 200 µg/mL protamine, demonstrating its potent intrinsic anticoagulation activity. A and D report the average turbidity obtained from 3 separate experiments. Absorbance measured at 3-minute intervals is depicted. Results are expressed as the mean ± SE of 9 measurements from 3 independent experiments. Unpaired 2-tailed t tests were performed to determine the significance.