beta2-glycoprotein i is a cofactor for tissue plasminogen activator-mediated plasminogen activation

Abstract

Objective: Regulation of the conversion of plasminogen to plasmin by tissue plasminogen activator (tPA) is critical in the control of fibrin deposition. While several plasminogen activators have been described, soluble plasma cofactors that stimulate fibrinolysis have not been characterized. The purpose of this study was to investigate the effects of beta(2)-glycoprotein I (beta(2)GPI), an abundant plasma glycoprotein, on tPA-mediated plasminogen activation.

Methods: The effect of beta(2)GPI on tPA-mediated activation of plasminogen was assessed using amidolytic assays, a fibrin gel, and plasma clots. Binding of beta(2)GPI to tPA and plasminogen was determined in parallel. The effects of IgG fractions and anti-beta(2)GPI antibodies from patients with antiphospholipid syndrome (APS) on tPA-mediated plasminogen activation were also measured.

Results: Beta(2)-glycoprotein I stimulated tPA-dependent plasminogen activation in the fluid phase and within a fibrin gel. The beta(2)GPI region responsible for stimulating tPA activity was shown to be at least partly contained within beta(2)GPI domain V. In addition, beta(2)GPI bound tPA with high affinity (K(d) approximately 20 nM), stimulated tPA amidolytic activity, and caused an overall 20-fold increase in the catalytic efficiency (K(cat)/K(m)) of tPA-mediated conversion of Glu-plasminogen to plasmin. Moreover, depletion of beta(2)GPI from plasma led to diminished rates of clot lysis, with restoration of normal lysis rates following beta(2)GPI repletion. Stimulation of tPA-mediated plasminogen activity by beta(2)GPI was inhibited by monoclonal anti-beta(2)GPI antibodies as well as by anti-beta(2)GPI antibodies from patients with APS.

Conclusion: These findings suggest that beta(2)GPI may be an endogenous regulator of fibrinolysis. Impairment of beta(2)GPI-stimulated fibrinolysis by anti-beta(2)GPI antibodies may contribute to the development of thrombosis in patients with APS.

Figure 1. β₂GPI enhances t-PA–dependent Glu-plasminogen activation

(A) t-PA-dependent plasminogen activation was measured either alone (in the presence of BSA) or in the presence of 1 μM human β₂GPI (β₂GPI-n), recombinant β₂GPI (β₂GPI-r), β₂GPI domain V (β₂GPI-V), or β₂GPI domain V peptide (β₂GPI-p). Substrate hydrolysis was measured as relative fluorescence units (I_{360/465 nm}) as a function of time² (T)² (37). (B) Initial rates of plasmin generation were determined as in (A) in the presence of increasing concentrations of β₂GPI-n, β₂GPI-r, β₂GPI-V, or BSA.

Figure 2. Native β₂GPI and β₂GPI domain V enhance fibrinolysis in fibrin gels

Lysis of fibrin gels was assessed as described in Materials and Methods. (A) A representative experiment: (1) 10 nM t-PA + BSA; (2) 10 nM t-PA + 5 μM β₂GPI-V; (3) 10 nM t-PA + 5 μM native β₂GPI. (B) t-PA activity (U/ml) (as determined from the size of lytic areas) in the absence or presence of increasing amounts of native β₂GPI-n, β₂GPI-V, or β₂GPI peptide (β₂GPI-p), calculated from a standard curve.

Figure 3. Clot formation and lysis in the absence and presence of β₂GPI

(A) Plasma from a normal donor was divided into equal aliquots and subjected to chromatography on a column to which either anti-β₂GPI antibodies or preimmune-rabbit IgG had been conjugated. After passing through the former column, plasma was completely immunodepleted of β₂GPI as determined by immunoblotting. The flow-through from both columns was placed in 96 well microplates and clotted as described in Materials and Methods. Clots were monitored by measurement of A₄₀₅. Clotting led to an increase in A₄₀₅ which was maximal at approximately 30 minutes, followed by a progressive decrease as clots lysed. Normal plasma (black diamonds) did not undergo chromatography. (B) The identical experiment as depicted in (A) was performed, but β₂GPI-depleted plasma was analyzed either directly (squares, solid lines) or after the addition of 100 μM β₂GPI (triangles) or 200 μM β₂GPI (inverted triangles). The addition of β₂GPI to the β₂GPI-depleted plasma restored its fibrinolytic activity.

Figure 4. β₂GPI stimulates t-PA amidolytic activity

(A) t-PA amidolytic activity in the presence of increasing amounts of native β₂GPI and β₂GPI domain V. (B) The effect of 1 μMβ₂GPI on t-PA amidolytic activity in the presence of increasing concentrations of t-PA.

Figure 5. Binding of β₂GPI to t-PA

(A) Binding of β₂GPI to t-PA using a microplate assay was performed as described in Materials and Methods. (B) Inhibition of the binding of 40 nM β₂GPI to immobilized t-PA by fluid phase t-PA. β₂GPI binding is expressed as the percentage of β₂GPI bound in the presence of fluid-phase t-PA relative to that bound in its absence. (C) Binding of t-PA to β₂GPI using an optical biosensor. The concentration of t-PA used is depicted to the right of the curve. (D) Binding of t-PA to β₂GPI was inhibited by increasing concentrations of NaCl.

Figure 6. Anti-β₂GPI antibodies inhibit β₂GPI-mediated enhancement of plasminogen activation

Plasminogen activation was determined as described in Materials and Methods. Percent inhibition by anti-β₂GPI antibodies was calculated by comparing initial reaction rates in the absence or presence of antibodies. (A) IgG76, IgGC, IgG35 and IgG58 are anti-β₂GPI antibody-containing IgG fractions from patients with APS, BD4 and ID2 are monoclonal anti-β₂GPI antibodies, and IgG1 is normal human IgG. (B) Inhibition of t-PA-dependent plasminogen activation by increasing concentrations of anti-β₂GPI IgG (IgG76), or control IgG (IgG1). (C) Anti-β₂GPI antibodies were affinity purified from the plasma of 3 patients with APS and anti-β₂GPI antibodies (abGPI-11, abGPI-17, abGPI-27), and their ability to inhibit β₂GPI-dependent t-PA-mediated plasminogen activation determined. Each of the purified anti-β₂GPI antibody preparations inhibited plasminogen activation in a concentration-dependent manner. (D) The effect of IgG fractions from 40 normal controls, 22 APS patients with anti-β₂GPI, but not anti-t-PA antibodies (anti-β₂GPI⁺/anti-tPA⁻), or 21 APS patients with neither anti-β₂GPI nor anti-t-PA antibodies (anti-β₂GPI⁻/anti-tPA⁻) on t-PA-dependent plasminogen activation in the presence of β₂GPI. One outlying point in which an IgG fraction from a patient with anti-β₂GPI caused marked inhibition of plasminogen activation (~60% activity) was removed for this analysis.