Studies were conducted on the effect of heparin or 6-aminohexanoic acid (6-AH) on the activation of glutamic plasminogen (Glu-Plg) by streptokinase in the presence of different concentrations of buffer, NaCl and divalent cations. Heparin and 6-AH inhibited streptokinase-mediated activation of Glu-Plg using 10 mM Tris-HCl buffer pH 7.4. This inhibition was partially reversed by the addition of 0.2-1.0 mM of Mg ions. Increasing the ionic strength of Tris-HCl buffer from 10 to 50 mM or addition of 50-150 mM of NaCl to 50 mM Tris-HCl pH 7.4 inhibited the activation of Glu-Plg by streptokinase while decreasing the % inhibition by heparin over the control samples. Double reciprocal plot of the activation of Glu-Plg by streptokinase using 50 mM Tris-HCl pH 7.4 containing 100 mM NaCl showed that the addition of heparin lowered Vmax by 50% without affecting Km. To determine whether the inhibitory effect of heparin was specifically directed towards Glu-Plg or streptokinase, the ratios of the initial rate of plasmin generation in the presence of heparin over the controls were plotted against the inverse of the volume fraction of Glu-Plg or streptokinase after serial dilutions. The results indicated that the dilutions of streptokinase but not of Glu-Plg influenced the ratios, suggesting an interaction of heparin with streptokinase. Addition of 6-AH reversed the inhibitory effect of NaCl on the activation of Glu-Plg by streptokinase and the results of the near UV CD spectra of Glu-Plg showed that addition of 6-AH enhanced the spectra in this region with an increase in the ellipticity which was not affected by addition of NaCl.

Studies were conducted on the mechanism of the stimulatory effect of 6-aminohexanoic acid (6-AH) during the in vitro activation of human glutamic plasminogen (Glu-Plg) by streptokinase or by tissue plasminogen activator (t-PA) and the possible role of the addition of physiological concentrations of NaCl to the buffer solution. Enhancement by 6-AH was investigated by measuring the rate of plasmin generation using chromogenic substrate H-D-glu-phe-lys-pNA (S-2403). Control studies using plasmin showed that the addition of 6-AH at concentrations below 20 mM did not significantly affect the initial rate of the amidolytic activity of plasmin with or without the addition of NaCl to 0.05 M Tris buffer (pH 7.4). On the other hand, addition of NaCl to the buffer slowed down the initial rate of activation of Glu-Plg by streptokinase or by t-PA while increasing the percent enhancement by 6-AH when compared with the controls. The ratios of the initial rates of plasmin generation in the presence or in the absence of 6-AH were plotted against the inverse of the volume fraction of Glu-Plg, streptokinase or t-PA after serial dilutions. The results showed that when the activation reactions were performed in 50 mM of Tris buffer (pH 7.4), the enhancements by 6-AH were related to its interaction with streptokinase or t-PA, while using the same Tris buffer containing 0.6 % NaCl, the enhancements by 6-AH were related to its interaction with both Glu-Plg and streptokinase or t-PA. However, upon increasing the NaCl to 0.9%, the results showed that the enhancements by 6-AH of the activation of Glu-Plg by streptokinase or t-PA were related to its interaction with Glu-Plg. The results suggested that changes in the concentrations of NaCl play a regulatory role during the activation process.

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