Enhancement of fibrinolysis by inhibiting enzymatic cleavage of precursor α2-antiplasmin: a rebuttal


Jofre Tenorio-Laranga, Division of Pharmacology and Toxicology, University of Helsinki, PO Box 56 (Viikinkaari 5E), Helsinki 00014, Finland.
Tel.: +358 9 191 59460; fax: +358 9 191 59471.
E-mail: jofre.tenorio-laranga@helsinki.fi


See also Lee KN, Jackson KW, Christiansen VJ, Dolence EK, McKee PA. Enhancement of fibrinolysis by inhibiting enzymatic cleavage of precursor α2-antiplasmin. J Thromb Haemost 2011; 9: 987–96; Lee KN, Jackson KW, Christiansen VJ, McKee PA. Enhancement of fibrinolysis by inhibiting enzymatic cleavage of precursor α2-antiplasmin: reply to a rebuttal. This issue, pp 1268–9.

Lee et al. [1] reported that there is no prolyl oligopeptidase (PREP) activity in citrated mammalian plasma, concluding that PREP is not present in circulating blood. We agree with Lee et al. [1] in that the true properties of circulating PREP have not been identified. However, our laboratory has previously demonstrated [2], like some others earlier [3], that there is a significant PREP activity in EDTA plasma. In our studies, the activity attributed to PREP represents 40% of the total PREP-like activity in plasma. The other 60% is attributed to the fibroblast activated protein (FAP or seprase). In addition, we have demonstrated that citrate inhibits PREP strongly and irreversibly [2].

The aim of this letter is to emphasize that (i) PREP is indeed present in circulating blood and (ii) it is essential to use a proper anticoagulant in blood samples.

Mammalian PREP is a specific endopeptidase that cleaves peptides shorter than 30-mer at the carboxy side of an internal proline [4]. The biological relevance of PREP activity lies in its participation in the processing (sometimes activation) or degradation of bioactive peptides, or both [5]. PREP is mainly a cytoplasmic enzyme, but a membranous form has also been identified [6]. Nevertheless, PREP-like activity has been described in different body fluids, including serum or plasma and cerebrospinal fluid. There are earlier reports of low PREP-like activity in plasma from patients with depression [7,8] and increased activity in plasma from maniac and schizophrenic subjects [9].

All studies measuring PREP-like activity in plasma samples have basically been carried out using two very similar substrates, succinyl-Gly-Pro-AMC or Z-Gly-Pro-AMC. There are two circulating enzymes capable of cleaving these substrates, PREP and FAP [2,3,10]. Therefore, the PREP-like activity from plasma samples is the sum of two different proteases. Both activities can be analyzed separately by using a specific PREP inhibitor. Indeed, before FAP was identified as being responsible for the partial PREP-like activity found in plasma, this activity was called Z-Pro-Polinal insensitive peptidase (ZIP). In a previous report, we have measured the PREP activity from EDTA plasma using the specific PREP inhibitor Z-Pro-Prolinal (ZPP) at 50 nm [2]. This ZPP concentration fully inhibits PREP but it has no effect on the FAP activity. Therefore, we consider that a significant reduction of the PREP-like activity in the EDTA plasmas, in the presence of ZPP 50 nm, indicates that there is another enzyme besides FAP. It is true that citrate strongly inhibits PREP, and this PREP inhibition is irreversible [2]. To substantiate this effect, we collected plasma samples from six healthy volunteers after informed consent. From each volunteer we obtained two samples, one in the presence of EDTA and the other in the presence of citrate. We measured PREP activity and we observed that it was strongly reduced in citrated plasma (Fig. 1). Therefore, for the plasma PREP assay, the nature of the anticoagulant is crucial, and when citrate is used as by Lee et al. 2011 [1], there is no or very low PREP activity left in plasma. Accordingly, in those conditions no conclusions on circulating PREP levels can be drawn.

Figure 1.

 PREP activity from paired plasma samples obtained with different anticoagulants (EDTA or citrate). ***P value < 0.001 (t-test).

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.