Factor bb as a biomarker during breakthrough hemolysis in paroxysmal nocturnal hemoglobinuria Free

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a clonal disorder caused by loss of complement regulators on hematopoietic stem cells, rendering red blood cells (RBCs) vulnerable to complement-mediated lysis. A key feature of the alternative pathway (AP) of the complement cascade is its amplification loop; the AP C3 convertase C3bBb cleaves C3 to generate C3b, which can itself form new C3 convertase. C3b binding to C3bBb forms the AP C5 convertase, C3bBbC3b, which cleaves C5 and initiates terminal complement activation and membrane attack complex (MAC) formation.

Factor Bb (FBb), generated when C3b-bound Factor B is cleaved by Factor D, is a central component of both AP C3 and C5 convertases. Complement regulators such as Factor H limit AP activation and amplification by promoting C3bBb decay, releasing FBb into circulation.

Complement inhibitors (CI) protect PNH RBCs by blocking components of the complement cascade to prevent MAC formation. Around 10% of patients receiving CIs experience breakthrough hemolysis (BTH); a recurrence of intravascular hemolysis (IVH) marked by a rise in lactate dehydrogenase (LDH), often due to a complement amplifying event. Management of BTH can involve treating the trigger, transfusions, CI dose increases or additional doses.

As a key component of the AP, FBb is likely elevated during complement activation. Its measurement during BTH may offer insight into ongoing AP activity and could serve as a useful biomarker to support decision-making.

Methods Blood samples were collected from 66 patients with PNH (PNH white blood cell clones >10%) during routine clinic visits (baseline). Patients were grouped according to CI at sampling; untreated [n=16], eculizumab [n=8], ravulizumab [n=21], pegcetacoplan [n=16], iptacopan [n=16]). Some patients [n=11] were sampled at two timepoints following a change in treatment group. Those on CI had an LDH <1.5x the upper limit of normal (ULN).

EDTA blood samples were also collected during 17 BTH events in 10 patients (eculizumab n=1, ravulizumab n=2, pegcetacoplan n=12, iptacopan n=2), with paired clinic samples available for 9 patients. BTH was defined as new signs/symptoms of IVH with an LDH rise >1.5x the upper limit of normal (ULN). Samples were obtained as close as possible to symptom onset, and then for up to six days thereafter.

Plasma was separated by centrifugation and stored at –80°C. FBb levels were assayed using Quidel MicroVue Bb Plus Fragment EIA.

All patients were consented to the PNH Research Tissue Bank.

Results FBb levels correlated with markers of hemolysis, with a strong positive correlation with LDH (p<0.0001) and a weak negative correlation with hemoglobin levels (p<0.05) in both untreated patients and those on CI without BTH. FBb levels in untreated patients were significantly higher than those of patients on CI without BTH (mean 1.67 vs 0.95ug/ml, p<0.05). Patients receiving eculizumab and pegcetacoplan (mean 0.91ug/ml and 0.79ug/ml) had significantly lower FBb levels at baseline than untreated patients (p<0.05, p<0.0001); ravulizumab and iptacopan did not.

FBb levels rose significantly from baseline during BTH (mean 5.5ug/ml, p<0.001). The timing, magnitude of peak and rate of decline varied between patients, but levels typically peaked within 3 days of symptom onset and fell to baseline by day 5. Mean FBb levels measured on days 1, 2 and 3 were significantly higher than those on day 5 (p<0.01, p<0.01 and p<0.05 respectively). In contrast, LDH levels remained elevated >1.5x ULN in 15/17 BTH events throughout the 6 day sampling period, with mean levels on day 6 being higher than day 1 (p=0.44).

All 9 patients with paired samples showed an increase in FBb in the first 3 days of BTH, with an average maximum 7.33-fold rise from baseline (p<0.01).

Discussion FBb levels rise rapidly during BTH, typically peaking within the first 3 days of symptom onset. Levels correlate with established markers of IVH.

The use of LDH as a marker of hemolysis has limitations; its half-life of ~24 hours delays return to baseline after BTH, reducing reliability for tracking hemolysis. LDH is also non-specific and may be elevated in liver dysfunction or malignancy. FBb has a much shorter half-life (~1.5-2 hours) making it a more responsive marker of real-time AP activation. Its consistent rise during BTH, followed by return to baseline within days, supports its use as a sensitive biomarker. This may help assess ongoing BTH severity and guide clinical decisions.