Regular Article Alpha 2-macroglobulin enhances prothrombin activation and thrombin potential by inhibiting the anticoagulant protein C/protein S system in cord and adult plasma Gerhard Cvirn * , Siegfried Gallistl, Martin Koestenberger, Joerg Kutschera, Bettina Leschnik, Wolfgang Muntean Ludwig Boltzmann Research Institute for Pediatric Hemostasis and Thrombosis, Graz, Austria Department of Pediatrics, University of Graz, Graz, Austria Received 5 September 2001; accepted 25 February 2002 Accepting Editor: H. H. Watzke Abstract Protein S (PS) is a vitamin K-dependent plasma protein and serves as a cofactor for the anticoagulant activities of activated protein C (APC). We investigated the effects of different PS concentrations on prothrombin activation and thrombin generation in cord and adult plasma containing APC and different amounts of alpha 2-macroglobulin (a2-M). Prothrombin activation was assessed by monitoring the time-course of prothrombin fragment 1 + 2 (F1 + 2) generation. Thrombin generation curves were determined by means of a subsampling technique using the chromogenic substrate S-2238. We demonstrate a dose-dependent inhibition of the anticoagulant action of PS by a2-M: suppression of F1 + 2 and thrombin generation due to addition of PS was stronger in plasma containing low amounts of a2-M than in plasma with elevated a2-M levels. Since no complex formation between a2-M and PS was observed by means of SDS-PAGE, we attribute decreased anticoagulant action of PS at high a2-M levels to enhanced complex formation between APC and a2-M. Thereby, APC is subtracted from its cofactor PS, resulting in suppressed formation of the anticoagulant APC/PS complex. Thus, our data suggest that a2-M, besides its well- known anticoagulant effects, also acts as a procoagulant by suppressing the formation of the anticoagulant APC/PS complex. Our findings have implications particularly on thrombin generation and inhibition in cord plasma, since a2-M levels in newborns are elevated over adult values and the antithrombotic APC/PS pathway is up-regulated at birth. Therefore, elevated levels of a2-M might restrict the up-regulation of the APC/PS pathway. D 2002 Published by Elsevier Science Ltd. Keywords: Alpha 2-macroglobulin; Protein S; Protein C; Thrombin potential; Prothrombin activation; Cord and adult plasma 1. Introduction Alpha 2-macroglobulin (a2-M) is a broad-spectrum pro- tease binding protein. It is known to bind, i.e., a-thrombin [1] and activated protein C (APC) [2]. The proteinases are physically trapped and thus their active sites are excluded from natural substrates. It has been suggested that a2-M may provide protection from thromboembolic events in antith- rombin (AT)-deficient children due to complex formation with free a-thrombin [3]. We have recently shown that in cord and adult plasma containing physiological amounts of AT no anticoagulant effect of added a2-M due to complex formation with free thrombin was observed [4]. To the contrary, a2-M exhibits procoagulant activity at physiological AT levels due to complex formation with APC, associated with enhanced thrombin potential (TP) [5–7], and prothrombin activation [8]. These results are in good agreement with the findings of Nicolaes et al. [9] who have shown the effect of APC on TP in normal and APC-resistant individuals. Thus, a2-M seems to play an important role in the regulation of the APC activity present in plasma. Protein S (PS), a vitamin K-dependent plasma protein, binds to APC and serves as a cofactor for the anticoagulant 0049-3848/02/$ - see front matter D 2002 Published by Elsevier Science Ltd. PII:S0049-3848(02)00042-7 Abbreviations: a2-M, alpha 2-macroglobulin; APC, activated protein C; AT, antithrombin; TP, thrombin potential; PS, protein S; F1+2,prothrombin fragment 1+2; FVa, activated factor V; FVIIIa, activated factor VIII; aPTT, activated partial thromboplastin time; PCI, protein C inhibitor. * Corresponding author. Univ. Klinik fu ¨r Kinder- und Jugendheilkunde, Gerinnungslabor, Auenbruggerplatz 30, A-8036 Graz, Austria. Tel.: +43- 316-385-4031; fax: +43-316-385-4024. E-mail address: gerhard.cvirn@klinikum-graz.at (G. Cvirn). Thrombosis Research 105 (2002) 433 – 439