MASP2 levels are elevated in thrombotic microangiopathies: association with microvascular endothelial cell injury and suppression by anti-MASP2 antibody narsoplimab

Abstract

Involvement of the alternative complement pathway (AP) in microvascular endothelial cell (MVEC) injury characteristic of a thrombotic microangiopathy (TMA) is well documented. However, the role of the lectin pathway (LP) of complement has not been explored. We examined mannose-binding lectin associated serine protease (MASP2), the effector enzyme of the LP, in thrombotic thrombocytopenic purpura, atypical hemolytic uremic syndrome and post-allogeneic hematopoietic stem cell transplantation (alloHSCT) TMAs. Plasma MASP2 and terminal complement component sC5b-9 levels were assessed by enzyme-linked immunosorbent assay (ELISA). Human MVEC were exposed to patient plasmas, and the effect of the anti-MASP2 human monoclonal antibody narsoplimab on plasma-induced MVEC activation was assessed by caspase 8 activity. MASP2 levels were highly elevated in all TMA patients versus controls. The relatively lower MASP2 levels in alloHSCT patients with TMAs compared to levels in alloHSCT patients who did not develop a TMA, and a significant decrease in variance of MASP2 levels in the former, may reflect MASP2 consumption at sites of disease activity. Plasmas from 14 of the 22 TMA patients tested (64%) induced significant MVEC caspase 8 activation. This was suppressed by clinically relevant levels of narsoplimab (1·2 μg/ml) for all 14 patients, with a mean 65·7% inhibition (36.8-99.4%; P < 0·0001). In conclusion, the LP of complement is activated in TMAs of diverse etiology. Inhibition of MASP2 reduces TMA plasma-mediated MVEC injury in vitro. LP inhibition therefore may be of therapeutic benefit in these disorders.

Keywords: MASP; complement; hemolytic uremic syndrome; lectin pathway; thrombotic microangiopathy.

Fig. 1

Plasma mannose‐binding lectin‐associated serine protease 2 (MASP2) levels in the setting of allogeneic hematopoietic stem cell transplantation. Plasma MASP2 levels were measured in 39 healthy controls, nine individuals developing a non‐thrombotic thrombocytopenic purpura (TTP) type of thrombotic microangiopathy (TMA) post‐alloHSCT, and 80 individuals undergoing an alloHSCT who did not develop a TMA but were assessed at the mean time‐point for TMA development in the former group (day 100 ± 28 days).

Fig. 2

Longitudinal assessment of mannose‐binding lectin‐associated serine protease 2 (MASP2) plasma levels in five individuals developing a persistent thrombotic microangiopathy (TMA) post‐alloHSCT. The large dots represent plasma levels at the time of TMA recognition, in addition to levels obtained at regularly scheduled visits (five times/year) for all individuals post‐transplant. Four of the five patients received anti‐C5 monoclonal antibody (mAb) eculizumab at the time of TMA diagnosis (the exception is patient 052).

Fig. 3

Acute thrombotic microangiopathy (TMA) plasmas induce caspase 8 activation in human microvascular endothelial cell (MVEC), which is blocked by anti‐mannose‐binding lectin‐associated serine protease 2 (MASP2) monoclonal antibody (mAb) narsoplimab. Cultures of primary human neonatal dermal microvascular endothelial cells were exposed to plasmas from patients with acute thrombotic thrombocytopenic purpura (TTP) or non‐TTP types of TMA not associated with transplantation for 24 h in the presence or absence of anti‐MASP2 mAb narsoplimab (1·2 μg/ml). Caspase 8 activity was analyzed in cell lysates.

Fig. 4

Interaction among components of the complement cascade and the coagulation system, and points of intervention with anti‐complement therapeutics. Multiple points of interaction among the three principal pathways of the complement cascade, the classical (CP), alternative (AP) and lectin (LP) pathways, are illustrated. They include pre‐initiation, initiation and amplification phases ultimately leading to a terminal phase involving inflammatory, lytic and procoagulant outcomes. Points susceptible to blockade by narsoplimab, the anti‐mannose‐binding lectin‐associated serine protease 2 (MASP2) monoclonal antibody (mAb) and eculizumab, the anti‐C5 mAb, are illustrated. In terms of specific interactions of the LP with the coagulation cascade, MASP2 can directly cleave C4 as well as C3, activating prothrombin [7]. MASP1 and MASP2 contribute to in‐vitro fibrin clot formation, and MASP1, the exclusive activator of MASP2 [7], is essential for obstructive thrombosis in a murine model of arterial injury [1]. Plasmin and thrombin can cleave C3 and C5, directly activating complement, at least in vitro [4] Reciprocal interactions with thrombin‐induced complement activation in vivo may require platelet involvement, as the amount of thrombin generated in vivo is insufficient to directly induce C activation, at least in the fluid phase [34, 35]. Complement components also have a direct effect on platelets, which express complement receptors [C1qR, C5aR, C3aR, P‐selectin (which acts as a C3b receptor)] and complement regulatory molecules [C1‐I, CD55, CFH, CD46 (MCP), CD59] [3, 5, 36]. C3a and C5a directly activate resting platelets and potentiate platelet activation induced by multiple agonists, and sublytic concentrations of sC5b‐9 are potent platelet agonists, leading to platelet storage granule secretion and release of procoagulant.