During short-terra culture monocytes (M) only, not granulocytes or lymphocytes, generate thromboplastin (TP) activity which is absent Immediately after isolation. To clarify the mechanism whereby M generate TP, J approaches were applied: I. To investigate the presence of hidden” TP, freshly Isolated M were subjected either to mechanical disruption, to enzymatic stripping or to dellpldation, followed by reconstitution of the lipoproteins with exogenous phospholipids. No TP activity could be detected after any of these treatments. II. Using an antiserum against human brain TP apoprotein which cross-reacted with monocyte TP, it was shown that intact and disrupted freshly Isolated M lack TP related antigen whereas cultured M display binding of anti-TP antibodies. However, M cultured in the presence of protein synthesis Inhibitors and cultured lymphocytes, lacked TP antigen. III. Using several agents known to affect cellular processes, TP generation was shown to be dependent on;protein and RNA synthesis, energy metabolism, cAMP metabolism, assemblage of microtubules and divalent cations. Inhibition of prostaglandin synthesis by indomethacln did not arrect TP generation. M from 3 patients with COD displayed normal TP generation. These two observations suggest that prostaglandin synthesis and oxygen radicals are not involved Is TP generation. In conelusion, our data indicates that de novo synthesis of TP apoprotein Is responsible for the in vitro generation of TP activity by monocytes.
Bacterial expression and protein purification of mini-fluorescence-activating proteins
