Plasminogen activator inhibitor from human endothelial cells. Purification and partial characterization

Abstract

An inhibitor of plasminogen activator was purified to apparent homogeneity from human umbilical vein endothelial cell conditioned medium. The purification was achieved by a speedy and simple two-step procedure, without the use of denaturants. The purified protein was a single-chain glycoprotein with apparent molecular mass of 48 kDa.The purified inhibitor had a specific activity of 8500 U/mg protein and the activity could be stimulated about fourteenfold by treatment with denaturants.An antiserum to the purified inhibitor was raised in rabbits. It recognised plasminogen activator inhibitor from platelets and plasma as well as from cultured endothelial cells. The immunoglobulin fraction of the antiserum neutralised the functional activity of the inhibitor from all these sources.The activity of the fibrinolytic system depends on the cleavage of the zymogen plasminogen to form plasmin by plasminogen activators. Two major immunologically distinct types of plasminogen activator, tissue-type (tPA) and urokinase (u-PA) have been identified (reviewed in [l]). t-PA is present in normal resting plasma at a molecular mass of about 110 kDa [2 -41, in contrast to the 65-kDa form isolated from tissues [5]. The high-molecular-mass t-PA in plasma is now known to be an equimolar complex of t-PA with a plasminogen activator inhibitor (PAI) [3, 41. Inhibitors active against both t-PA and u-PA have been discovered in bovine [6] and human [7 -91 endothelial cells, in platelets [lo, 111 and in plasma [3, 12 -141. Immunological cross-reactivity between the bovine endothelial and human platelet inhibitor has been established [15] and this type of PA1 is now designated PAI-1 [16].The bovine endothelial cell PA1 is in an inactive or latent form that is converted to an active form in the presence of denaturants such as SDS and guanidinium hydrochloride [17]. This inhibitor has been purified using SDS/polyacrylamide gel electrophoresis (SDS-PAGE) as the final preparative step; thus the purified product was active [18].In the study reported here, we aimed to purify PA1 from human endothelial cells in the absence of denaturants. T h s Correspondence to

Purification and Characterization of Active and Stable Recombinant Plasminogen‐Activator Inhibitor Accumulated at High Levels in Escherichia coli

“…The use of low pH to retain PAI-1 activity was reported for the endothelial cell product [27]. The same observation has been confirmed for PAI-1 from HT-1080 cells [53] and it is noteworthy that pHvalues of pH 5.5-6 are used in several procedures that yield partially active PAI-1 [32,36,50,541.…”

“…The success of the purification protocol, while due to the quality of starting material, need not be limited to such high-level accumulation systems. Use of a salt gradient to elute PAI-1 and inclusion, if necessary, of a gel-filtration step would allow purification from a source with a lower level of PAI-1 in the starting material [27], without undue extension of the time taken for the procedure. The purified PAI-1 had a mean molecular mass of 42774Da by mass spectroscopy, very close to the calculated mass of 42770 Da [17,501.…”

“…Proteins were transferred onto nitrocellulose and PAI-1 was detected as described previously [27], except that 0.6 mM 5-bromo-4-chloro-3-indolyl phosphate-p-toluidine, 0.5 mM nitroblue tetrazolium in 200 mM Tris/HCl, pH 9.5, 10 mM MgCL was used as the substrate to detect alkaline phosphatase [46].…”

“…The purification protocol was based on the procedure for endothelial PAI-1 [27] and used ion-exchange chromatography at pH 5.6. The original method used a gradient of NaCl for elution but, in the interests of more rapid purification, a batch elution with 1 M NaCl was used for recombinant PAI-1.…”

“…Purification of active PAI-1 ng. PAI-1 was bound onto nitrocellulose under vacuum, the Soluble PAI-1 obtained in the 30000Xg supernatant from 5x300 ml cultures was purified in a single step by ion-exchange chromatography, based on a previously described protocol [27]. Samples containing PAI-1 were loaded onto a column of CM-50 Sephadex (Pharmacia) of 5-cm diameter, 120 ml bed volume, equilibrated with 20 mM sodium acetate, pH 5.6, containing 200 mM NaCl and 0.01 % Tween-20 and run at 130mlh.…”