Protamine (PRT) is routinely used to reverse heparin (H) anticoagulation during cardiopulmonary bypass (CPB). Previous studies have shown that PRT interacts with H to form ultra-large complexes that are immunogenic in mice (Chudasama 2010). Given the high levels of PRT and H used during CPB, we hypothesized that patients undergoing CPB will develop antibodies (Abs) to PRT/H complexes. To test this, we examined the serologic and clinical characteristics of 500 patients undergoing CPB from a recently completed prospective clinical trial (HIT 5801 study).
With IRB approval, we analyzed samples and clinical outcomes from patients at baseline, 3–7, and 30 days after CPB. Control subjects included healthy volunteers without diabetes, prior cardiac surgery, prior H exposure or chronic medications (n=101). PRT/H Abs were measured by enzyme linked-immunoassay (ELISA).
The mean A450nm for control subjects in the PRT/H ELISA was 0.39 ± 0.28, yielding a positive cut-off value (≥ mean A450nm +3 standard deviations (SD)) for the PRT/H ELISA of 1.2 (Fig A). Using this cut-off, PRT/H Abs were present in 2/101 (2%) of control subjects. New seroconversions were seen in 6/497 (1%) subjects at day 3–7 and 143/489 (29%) at day 30. The mean PRT/H reactivity of control subjects (mean A450nm ± SD; 0.39 ± 0.28) did not differ from that of patients at day 0 (0.28 ± 0.26) or day 3–7 (0.26 ± 0.37), but differed significantly from that at day 30 (1.06 ± 0.96, p < 0.0001). Serologic features of PRT/H Abs were examined on patients with high positivity in the screening ELISA (mean A450nm > 3.0, n = 32). These patients expressed high PRT/H Ab titers ranging from 1:1800–1:175,000 (mean 1:14,744). As seen in Fig B, high-titer PRT/H Abs bound to PRT alone and/or PRT/H complexes, but showed minimal binding to human platelet factor 4 (hPF4), hPF4/H complexes, or albumin (p < 0.0001 for PRT/H vs hPF4, PRT/H vs hPF4/H, and PRT/H vs albumin). Because Ab binding was significantly higher with PRT/H complexes than with PRT alone, we examined the effects of excess H (100 U/mL) on binding. All Abs with the exception of one, showed decreased binding in the presence of excess H (range: 8%-76%, mean inhibition = 45%). To determine the effects of Abs on platelet activation, a SRA was performed on patients (n=3) with high-titer Abs and control subjects. PRT/H seropositive plasma did not activate platelets in the presence of buffer or H (0.25 or 1 U/ml), but robustly activated platelets in the presence of PRT (1.25μg/mL) alone, with decreasing 14C release in the presence of PRT and increasing amounts of H (Fig C). Platelet activation in the presence of PRT was inhibited by IV.3, which blocks platelet activation via the platelet FcgRIIA.
There was a high incidence of PF4/H seroconversion after CPB (362/500 or 72%). While the majority of PRT/H seropositive patients (n=154) were also positive for PF4/H Abs (120/154 or 78%), the converse was not true. Of the 362 PF4/H seropositive patients, only 120 (33%) were positive for PRT/H. A minority of subjects were seronegative for both (104/500 or 21%). Results in one assay were not predictive of the other. In analyzing long-term outcomes (>30 days), there was a trend towards an association between PRT/H Ab positivity at baseline and event free survival (p = 0.07; hazard ratio (HR) = 1.65 (0.96–2.82); days to major adverse cardiac event). Seropositivity at other time points was not associated with decreased event free survival. There was no significant difference between platelet counts by Ab status.
Our studies are the first to comprehensively characterize a new class of H-dependent Abs, PRT/H Abs, in a large cohort of patients undergoing CPB. We show that PRT is a highly sensitizing drug, associated with formation of high-titer, antigen-specific Abs to PRT/H complexes that are capable of platelet activation. We show that the majority of seroconversions occurred after discharge and in the absence of circulating antigen (at day 30) was not associated with adverse clinical events. However, several features of PRT/H Abs, including high-titer expression and PRT-dependent platelet activation, suggest that additional studies are needed to determine the clinical significance of these Abs in the context of PRT re-exposure.
Arepally:Teva Pharmaceuticals: Research Funding.
Asterisk with author names denotes non-ASH members.