Minor Role of Plasminogen in Complement Activation on Cell Surfaces

Abstract

Atypical hemolytic uremic syndrome (aHUS) is a rare, but severe thrombotic microangiopathy. In roughly two thirds of the patients, mutations in complement genes lead to uncontrolled activation of the complement system against self cells. Recently, aHUS patients were described with deficiency of the fibrinolytic protein plasminogen. This zymogen and its protease form plasmin have both been shown to interact with complement proteins in the fluid phase. In this work we studied the potential of plasminogen to restrict complement propagation. In hemolytic assays, plasminogen inhibited complement activation, but only when it had been exogenously activated to plasmin and when it was used at disproportionately high concentrations compared to serum. Addition of only the zymogen plasminogen into serum did not hinder complement-mediated lysis of erythrocytes. Plasminogen could not restrict deposition of complement activation products on endothelial cells either, as was shown with flow cytometry. With platelets, a very weak inhibitory effect on deposition of C3 fragments was observed, but it was considered too weak to be significant for disease pathogenesis. Thus it was concluded that plasminogen is not an important regulator of complement on self cells. Instead, addition of plasminogen was shown to clearly hinder platelet aggregation in serum. This was attributed to plasmin causing disintegration of formed platelet aggregates. We propose that reduced proteolytic activity of plasmin on structures of growing thrombi, rather than on complement activation fragments, explains the association of plasminogen deficiency with aHUS. This adds to the emerging view that factors unrelated to the complement system can also be central to aHUS pathogenesis and suggests that future research on the mechanism of the disease should expand beyond complement dysregulation.

Disturbed sialic acid recognition on endothelial cells and platelets in complement attack causes atypical hemolytic uremic syndrome

“…This activated the platelets slightly, as shown by increased P-selectin expression ( Figure 7B). In line with previous reports, 30,35 serum exposure further activated the platelets ( Figure 7B). Importantly, the difference in P-selectin expression between untreated and sialidase-treated platelets disappeared upon exposure to serum.…”

“…30 To remove sialic acid, 2 3 10 8 platelets in 200 mL citrate buffer 30 was incubated with sialidase (1500 U) for 30 minutes at 37°C. Platelets were washed first with citrate buffer and then modified Tyrode's buffer.…”

“…Platelets were washed first with citrate buffer and then modified Tyrode's buffer. 30 Assessment of sialic acid removal was determined as described above.…”

A critical role for plasminogen in inflammation

“…However, a more recent publication argues that plasminogen does not have a direct effect on complement proteins in aHUS and that plasmin only inhibits complement activation at concentrations much higher than normal blood concentrations of plasminogen. Plasminogen did not inhibit complement-mediated lysis of red blood cells or endothelial cells but did prevent platelet aggregation, and this proteolytic activity on thrombi may explain why plasminogen deficiency is seen in aHUS (Hyvärinen and Jokiranta, 2015).…”

Clinical Practice Guidelines for the Management of Atypical Hemolytic Uremic Syndrome in Korea

“…Four variants in PLG (c.112 A>G, p.Lys38Glu; c.2134 G>A, p.Gly712Arg; c.758 G>A, p.Arg253His; c.505 C>T, p.Pro169Ser) are associated with aHUS ( 42 ). Because plasmin is able to disintegrate formed platelet aggregates, reduced proteolytic activity of plasmin may result in a prethrombotic state of aHUS development ( 43 ).…”