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Poly(L-lysine) (PLL; Mr=15,000-40,000) or human albumin was conjugated with Sepharose 4B-CNBr to obtain PLL-Sepha- rose 4B or albumin Sepharose 4B. When human citrated plasma was passed through a column (3×1 cm) of PLL-Sepharose 4B, it was observed that eluted plasma had a prolonged PTT time and further analysis showed that it was completely deficient in factors II, VII, IX, and X. Some loss of factors V, VIII, XI and XII was also noticed. Plasma passed through a column of albumin under identical conditions suffered no detectable loss of factors II, VII, IX and X. Loss of factors V, VIII, XI and XII was also reduced. Factors II, VII, IX and X that adsorbed to PLL-Sepharose 4B column did not elute from the column even after extensive washing (100 ml of 0.15 M NaCl containing 0.018 M sodium citrate in 5 ml aliquots). Immunoelectrophoretic examination of the eluate showed that it was essentially free of albumin indicating that the washing of the gel was satisfactory. Also, no other protein was detectable in the wash. Addition of 0.75 M NaCl containing 0.018 M sodium citrate to the washed gel resulted in the elution of factors II, VII, IX and X. Addition of the high salt eluate (after adjustment of ionic strength to 0.15 M NaCl with distilled water) to the plasma previously made deficient by passage through the PLL-Sepharose 4B column resulted in normalization of the PTT time. Subsequent analysis of the high salt eluate revealed that the recovery of various factors was: II=88%, VII=81%, IX=79% anl X=92%. Preliminary studies indicated also that the eluate from PLL-Sepharose 4B was apparently devoid of activated factor X. The possibility of other activated blood clotting factors being present in the PLL-Sepharose 4B eluate is currently under investigation.

Use Of Poly(L-Lysine)-Sepharose 4B For Isolation Of Vitamin K Dependent Factors From Human Plasma