Prothrombotic mechanisms in patients with congenital p.Cys89Tyr mutation in CD59

Highlights

• Congenital nonfunctioning CD59 has clinical manifestation in infancy that includes thrombosis.

• MAC deposition was increased, plasma hemoglobin and RBC-derived MP high.

• Platelets were activated and interacting with leukocytes.

Abstract

Background

Thrombosis is the prognostic factor with the greatest effect on survival in patients with paroxysmal nocturnal hemoglobinuria (PNH), who lack dozens of membrane surface proteins. We recently described a primary homozygous Cys89Tyr congenital nonfunctioning CD59 in humans with clinical manifestation in infancy, associated with chronic hemolysis, recurrent strokes, and relapsing peripheral demyelinating neuropathy. Here we investigated hypercoagulability mechanisms characterizing the syndrome.

Methods

Membrane attack complex (MAC) deposition (anti-SC5b-9) and free hemoglobin (colorimetric assay) were assessed. Platelet activation was identified (anti-CD61, anti-CD62P), and microparticles (MPs) of 0.5–0.9 μm, were characterized (Annexin V, anti-human GlyA, anti-CD15, anti-CD14, anti-CD61). Platelet-monocyte aggregation was assessed with FlowSight.

Findings

2/7 patients (29%) with homozygosity for Cys89Tyr and 6/12 (50%) with any of four described CD59 mutations had recurrent strokes. In plasma samples from four patients carrying identical mutations, MAC deposition was increased on RBCs (p < 0.0003), neutrophils (p < 0.009), and platelets (p < 0.0003). Free-plasma hemoglobin levels were abnormally high, up to 100 mg/dl. Patients with CD59 mutation had RBC-derived MP levels 9-fold higher than those in healthy controls (p < 0.01), and 2–2.5 fold higher than PNH patients (p < 0.09). Leukocyte-activated platelet aggregation was increased (p < 0.0062). Loss of CD59 was shown in the endothelium of these patients.

Interpretation

Nonfunctioning CD59 is a major risk factor for stroke and hypercoagulability. Uncontrolled hemolysis causes massive MP release and endothelial heme damage. MAC attack on unprotected endothelium and platelet activation and aggregation with leukocytes mediate additional mechanisms leading to vascular occlusion. It is suggested that CD59 loss represents a major arterial prothrombotic factor in PNH and additional diseases.

Introduction

Hemostasis is achieved via a balance of pro- and antithrombotic forces, is maintained by coagulation and fibrinolysis, and is influenced by blood physiology and plasma factors as well as factors derived from vessel walls and blood cells. Thrombosis is the prognostic factor with the greatest effect on survival in paroxysmal nocturnal hemoglobinuria (PNH) patients [1, 2]. Data from several retrospective studies in the pre-eculizumab era showed that the cause of death was related to thrombosis in 22.2–37.2% of PNH patients. The extremely high incidence of thrombosis in PNH, and its major impact on morbidity and mortality, underlines its clinical importance. The cumulative 10-year incidence of thrombosis in a retrospective study of 460 PNH patients with larger clones was 31–39% [1, 3, 4].

CD59 deficiency is a common finding in adult patients with PNH, which is characterized by clonal expansion of hematopoietic stem cells that have acquired a mutation in the PIGA gene (phosphatidylinositol glycan anchor biosynthesis, class A). PIGA encodes a GPI biosynthesis protein, phosphatidylinositol N-acetylglucosaminyltransferase subunit A [5, 6], and erythrocytes deficient in GPI-anchored membrane proteins, including CD59, undergo complement-mediated hemolysis.

We have originally described a primary homozygous Cys89Tyr CD59 mutation in Jewish patients of Sephardic ethnicity, that resulted in the amino acid substitution p.Cys89Tyr and thus a failure of CD59 protein function [7]. The Cys89Tyr mutation in CD59 was initially described with manifestation in infancy by chronic hemolysis and relapsing peripheral demyelinating disease resembling recurrent Guillain-Barre syndrome (GBS) or chronic inflammatory demyelinating polyneuropathy (CIDP). More recently, we have described two infants carrying the mutation who manifested with recurrent strokes [8], establishing the concept that primary Cys89Tyr mutation in CD59 leads to a thrombophilic state. In this work, we have tried to examine the possible thrombophilic mechanisms in primary CD59 Cys89Tyr mutation.