Protamine-induced immune thrombocytopenia

Abstract

Background: Protamine is widely used to reverse the anticoagulant effects of heparin. Although mild thrombocytopenia is common in patients given protamine after cardiac procedures, acute severe thrombocytopenia has not been described. We encountered a patient who experienced profound thrombocytopenia and bleeding shortly after administration of protamine and performed studies to characterize the responsible mechanism.

Study design and methods: Patient serum was studied for antibodies that recognize protamine, heparin-protamine complexes, and platelets (PLTs) treated with protamine using flow cytometry, enzyme-linked immunosorbent assay, and serotonin release from labeled PLTs.

Results: A high-titer immunoglobulin G antibody was detected in patient serum that recognizes protamine in a complex with heparin or PLT surface glycosaminoglycans (GAGs) and activates PLTs treated with protamine at concentrations achieved in vivo after protamine infusion. The antibody is distinctly different from those found in patients with heparin-induced thrombocytopenia on the basis of its failure to recognize heparin in a complex with PLT factor 4 (PF4) and to release serotonin from labeled PLTs in the absence of protamine.

Conclusions: Findings made suggest that the patient's antibody is specific for conformational changes induced in protamine when it reacts with heparin or a PLT surface GAG. Development of severe thrombocytopenia after treatment of this patient with protamine defines a previously undescribed mechanism of drug-induced immune thrombocytopenia. Patients given protamine who produce this type of antibody may be at risk of experiencing thrombocytopenia if given the drug a second time while antibody is still present.

Figure 1. An IgG antibody present in the patient’s serum reacted with normal Group O platelets when protamine is present

Results shown (flow cytometry) are typical of four independent studies that gave comparable results. Protamine sulfate was used at 12.5 µg/ml in both the primary reaction and in buffer used for washing prior to adding FITC-labeled secondary antibody.

Figure 2. A protamine concentration of about 12.5 µg/ml was optimal for protamine-dependent binding of patient’s antibody to platelets (filled squares)

Results with normal serum and protamine were negative (filled squares). Values shown (flow cytometry) are averages of triplicate determinations. Brackets denote 1.0 S.D.

Figure 3. The protamine-dependent patient antibody reacted with platelets at dilutions up to 1:800

Filled squares indicate reactions (flow cytometry) obtained with protamine at 12.5 µg/ml; filled diamonds indicate reaction in the absence of protamine.

Figure 4. Reactions of patient’s antibody (diluted 1:50) with complexes of heparin:protamine prepared at various ratios

The strongest reactions were obtained with complexes prepared using protamine sulfate 31 µg/ml and heparin 9 international units/ml (filled diamonds). No reactions were obtained with either of two normal sera (filled squares and triangles). Values shown are averages of triplicate determinations. Brackets denote 1.0 S.D.

Figure 5. The patient’s antibody caused release of serotonin from platelets labeled with ¹⁴C-serotonin when protamine was present at 5–125 µg/ml

¹⁴C-release is shown as counts/minute (CPM), and the equivalent percent release obtained with the normal serum (filled squares) was 0%. The equivalent percent release obtained with the patient’s serum (filled diamonds) ranged from 6% (0 µg/ml protamine) to 43% (125 µg/ml protamine). Results shown are typical of two independent studies.