Beta2-glycoprotein I (β2GPI) belongs to the complement control protein superfamily and circulates in plasma at a concentration of ∼4 μM (1). Like other members of the complement control protein superfamily that contain one or more characteristic short consensus repeats (SCRs) (2), β2GPI contains 5 SCRs. However, domain V of β2GPI (β2GPI-V) consists of an atypical SCR containing a lysine-rich sequence (CKNKEKKC) that imparts a positive charge to this domain and mediates the binding of β2GPI to phospholipid (3). Plasmin cleavage between Lys317 and Thr318 of the β2GPI domain V abolishes phospholipid binding (4).
Beta2-glycoprotein I is an important antigen in the antiphospholipid syndrome (APS), and anti-β2GPI antibodies are an independent risk factor for thrombosis and recurrent loss of pregnancy (5–7). Both procoagulant and anticoagulant activities may be regulated by β2GPI (7–9). Thrombin generation has been shown to be impaired in the plasma of β2GPI-deficient mice (10).
Impaired fibrinolysis may contribute to the development of thrombosis (11, 12). Plasmin plays a central role in the lysis of fibrin thrombi, and the conversion of plasminogen to plasmin by tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA) is precisely regulated (13). Tissue plasminogen activator binds fibrin with high affinity and may be of particular importance for the lysis of fibrin thrombi in the vasculature (13). Several stimulators of fibrinolysis, including insoluble proteins, protein aggregates, microfilaments, fibrin fragments, and type IV collagen, may stimulate fibrinolysis, primarily by promoting the formation of a ternary tPA–fibrin–plasminogen complex (14, 15). Annexin A2 is an endothelial cell coreceptor for plasminogen and tPA that accelerates tPA-dependent plasminogen activation (16, 17), and the annexin A2/S100A10 heterotetramer may be even more potent in this regard (18–20). However, to date, no soluble plasma cofactors that promote tPA-dependent plasminogen activation have been identified.
Several studies have examined the interactions of β2GPI with the fibrinolytic system. Lopez-Lira et al (21) and Yasuda et al (22) reported low-affinity binding of Glu-plasminogen to intact or plasmin-cleaved β2GPI, respectively. However, interactions of β2GPI with tPA have not been described. Here, we report our findings that β2GPI binds tPA with high affinity and enhances tPA activity and tPA-dependent plasminogen activation. Depletion of β2GPI from plasma impairs the lysis of plasma clots, and anti-β2GPI antibodies inhibit the ability of β2GPI to stimulate fibrinolysis. Given the abundance of β2GPI in plasma, these findings suggest that β2GPI may be an endogenous regulator of fibrinolysis and that impairment of fibrinolysis by anti-β2GPI antibodies may contribute to APS-associated thrombosis.
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The results of our studies demonstrate that β2GPI binds tPA with high affinity and stimulates tPA-dependent plasminogen activation. We found that β2GPI enhanced the catalytic efficiency (Kcat/Km) of tPA-dependent plasminogen activation by ∼20-fold, as a result of a decrease in the Km and an increase in the Vmax (Table 1). The enhancement of plasmin generation and the stimulation of fibrinolysis by β2GPI were also demonstrated in a fibrin gel (Figure 4). Moreover, the depletion of plasma β2GPI led to delayed clot lysis, which was restored upon repletion of purified β2GPI, suggesting that these findings are relevant to plasma.
The activity of full-length recombinant β2GPI expressed in 293T cells was similar to that of purified plasma β2GPI (Figure 1), suggesting that isolated plasma β2GPI was not denatured and, thus, that the activity of β2GPI was not attributable to nonspecific effects of denatured protein (30). The ability of recombinant β2GPI domain V to stimulate tPA-dependent plasminogen activation was ∼50% that of intact β2GPI, suggesting that β2GPI-V, as well as other regions of β2GPI, contribute to this activity. However, the β2GPI Gly274–Cys288 peptide neither enhanced tPA-dependent plasminogen activation nor significantly inhibited the binding of β2GPI to immobilized tPA. Thus, if this region is involved in the interaction of β2GPI with tPA, it likely requires conformational restraints imposed in the context of intact β2GPI. Additional studies using site-directed mutagenesis and β2GPI mutants lacking specific β2GPI domains may be of value in further defining the role of this region, if any, in the interactions of β2GPI with tPA.
Annexin A2 and the annexin A2/S100A10 heterotetramer are endothelial cell receptors for plasminogen and tPA (16, 31). Binding of tPA and plasminogen to annexin A2 enhances plasmin generation by facilitating the coassembly of these reactants and lowering the Km for their interaction (16, 17). Cesarman-Maus et al (32) have described anti–annexin A2 antibodies in patients with APS that inhibit the fibrinolytic activity of this complex. While investigators in our laboratory have demonstrated a high-affinity interaction between β2GPI and annexin A2 (33), we have not yet directly assessed the effects of β2GPI on the ability of annexin A2 to enhance tPA-mediated plasminogen activation, either in isolation or on cell surfaces. However, Lopez-Lira et al (21) reported an increase in plasmin generation on the surface of HMEC-1 cells, a human microvascular endothelial cell line, following the addition of β2GPI, and we have observed that fluid-phase annexin A2 and β2GPI stimulate tPA-mediated plasminogen activation in an additive manner (data not shown).
Additional studies will clearly be required to better characterize the effects of β2GPI on cell surface plasminogen activator activity and to better define the roles of specific receptors in these effects. Such studies will require a full consideration of the complex nature of the conformation-dependent interactions of β2GPI with phospholipids and proteins, such as apolipoprotein E receptor 2 (34), factor XI (35), annexin A2 (33), and lipoprotein(a) (36).
Lopez-Lira et al (21) reported that β2GPI binds Glu-plasminogen and stimulates streptokinase-mediated plasminogen activation. Our preliminary studies also suggest that there is low-affinity binding (Kd >1 μM) between Glu-plasminogen and β2GPI, although the nature of the interactions between these 2 proteins requires further investigation. However, tPA was not present in the system described by Lopez-Lira et al, and thus, our findings extend their results by demonstrating a direct interaction of β2GPI with tPA. Taken together, however, these results suggest that in the presence of fibrin, β2GPI may potentially promote fibrinolysis through multiple pathways, including 1) direct stimulation of tPA amidolytic activity, 2) lowering of the Km for tPA-mediated plasminogen activation, and 3) binding to fibrin and providing additional binding sites for plasminogen and tPA at the fibrin surface (21).
Yasuda et al (22) reported that nicked β2GPI, which has been cleaved by plasmin between Lys317 and Thr318 in domain V, but not intact β2GPI, binds with low affinity to Glu-plasminogen (Kd 0.37 μM). Those investigators also observed that nicked β2GPI suppressed plasmin generation in the presence of tPA, plasminogen, and fibrin. Taken together with our findings, these results suggest that intact β2GPI may stimulate tPA-mediated plasminogen activation and subsequently undergo cleavage by newly formed plasmin. In turn, cleaved (“nicked”) β2GPI, if generated in sufficient concentrations in plasma, might limit additional plasmin generation (22).
The pathogenic effects of many “antiphospholipid” antibodies may be mediated through interactions with β2GPI. Indeed, Takeuchi et al (11) observed diminished fibrinolysis in euglobulin fractions from APS patients, attributing this effect to impaired factor XII activation and activity. It has also been reported that β2GPI protects tPA from inhibition by plasminogen activator inhibitor type 1, an activity blocked by monoclonal antiphospholipid antibodies (12).
In this study, we demonstrated that monoclonal anti-β2GPI antibodies and affinity-purified anti-β2GPI antibodies from APS patients inhibit in a concentration-dependent manner the ability of β2GPI to enhance tPA-mediated plasminogen activation. Moreover, IgG fractions from APS patients with anti-β2GPI antibodies caused significantly more inhibition of tPA-mediated plasminogen activation than did those from control subjects or APS patients without anti-β2GPI antibodies. This activity was not due to anti-tPA antibodies (29) and, thus, appears to be attributable to anti-β2GPI antibodies, suggesting another potential mechanism by which heterogeneous antiphospholipid antibodies may contribute to the development of thromboembolic disease (7).
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Drs. Cai and McCrae had full access to all of the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis.
Study design. Bu, Cai, McCrae.
Acquisition of data. Bu, Gao, Xie, Zhang, He.
Analysis and interpretation of data. Bu, Zhang, McCrae.
Manuscript preparation. Bu, Cai, McCrae.
Statistical analysis. Bu, McCrae.