Rapid disappearance of protamine in adults undergoing cardiac operation with cardiopulmonary bypass

The immune response in heparin-induced thrombocytopenia is initiated by and directed to large multimolecular complexes of platelet factor 4 (PF4) and heparin (H). We have previously shown that PF4:H multimolecular complexes assemble through electrostatic interactions and, once formed, are highly immunogenic in vivo. Based on these observations, we hypothesized that other positively charged proteins would exhibit similar biologic interactions with H. To test this hypothesis, we selected 2 unrelated positively charged proteins, protamine (PRT) and lysozyme, and studied H-dependent interactions using in vitro and in vivo techniques. Our IntroductionHeparin-induced thrombocytopenia (HIT) is an immune-mediated disorder caused by antibodies that recognize multimolecular complexes of platelet factor 4 (PF4), a positively charged platelet protein, and heparin (H), a negatively charged carbohydrate. We, and others, have shown that PF4 and H complexes assemble primarily through nonspecific electrostatic interactions governed by principles of colloidal chemistry. [1][2][3][4][5] In colloidal systems, molecules of opposite charge "aggregate" or grow in size due to effects of charge neutralization. Particle interactions are frequently dependent on stoichiometric ratios of the 2 compounds, with the largest complexes occurring at molar ratios of the compounds leading to charge neutralization. When either compound is in molar excess, charge restabilization occurs and repulsive forces predominate, leading to reduced complex size and/or complex disassembly.Studies to date indicate that PF4/H multimolecular complex formation is central to the pathogenesis of HIT. The characteristic bell-shaped curve seen with HIT antibody binding over a range of H concentrations coincides with H-dependent formation of multimolecular complexes. 2,3 HIT antibody binding, as gauged by serologic assays or functional studies of platelet activation, is optimal when multimolecular complexes form at or near equimolar ratios of PF4:H. However, antibody binding is markedly reduced with increasing H concentrations, a phenomenon that can be directly attributed to loss of complex formation. [2][3][4] Recent studies from our laboratory indicate that similar H-dependent changes affect the immunogenicity of PF4/H complexes in vivo. 5,6 Our studies demonstrate that PF4/H complexes are immunogenic over a certain range of H concentrations associated with multimolecular complex formation and that the immune response is attenuated when PF4 or H is given alone or when H is in molar excess of PF4. 5 H and H-like molecules bind several positively charged proteins in addition to PF4. 7 These H-binding proteins (HBPs) are structurally and functionally diverse, and include, to name a few, nuclear proteins (protamine), enzymes (C1 esterase and lysozyme), adhesion molecules (fibronectin and vitronectin) growth factors (fibroblast growth factor), and lipid-binding proteins (apolipoprotein E and lipoprotein lipase). To date, it appears that a majority of HBP-H interactions a...

Section: Discussionmentioning

confidence: 99%

Heparin modifies the immunogenicity of positively charged proteins

Chudasama

Espinasse²,

Hwang³

et al. 2010

“…Protamine has a short half-life (;5 minutes). 23,24 As protamine is not a circulating endogenous protein like PF4, it is unlikely that a mechanism similar to the one observed in delayed-onset HIT 25 (ie, cross-reactivity of PF4-heparin antibodies with PF4 bound to glycosaminoglykans 26 ) is also the cause for prolonged thrombocytopenia in patients with protamine-heparin antibodies. It is also unlikely that the antibodies cross-react with epitopes of proteins other than protamine.…”

Section: Blood 11 April 2013 X Volume 121 Number 15 Clinical Relevamentioning

confidence: 99%

Anti–protamine-heparin antibodies: incidence, clinical relevance, and pathogenesis

Bakchoul¹,

H²,

Amiral³

et al. 2013

Key Points• Immunization against protamine/heparin complexes was frequently observed in patients undergoing cardiac surgery.• Platelet-activating antiprotamine-heparin antibodies are a potential risk factor for early postoperative thrombosis and thrombocytopenia.Protamine, which is routinely used after cardiac surgery to reverse the anticoagulant effects of heparin, is known to be immunogenic. Observing patients with an otherwise unexplained rapid decrease in platelet count directly after protamine administration, we determined the incidence and clinical relevance of protamine-reactive antibodies in patients undergoing cardiac-surgery. In vitro, these antibodies activated washed platelets in a FcgRIIa-dependent fashion. Using a nonobese diabetic/severe combined immunodeficiency mouse model, those antibodies induced thrombocytopenia only when protamine and heparin were present but not with protamine alone. Of 591 patients undergoing cardiopulmonary bypass surgery, 57 (9.6%) tested positive for anti-protamine-heparin antibodies at baseline and 154 (26.6%) tested positive at day 10. Diabetes was identified as a risk factor for the development of anti-protamine-heparin antibodies. In the majority of the patients, these antibodies were transient and titers decreased substantially after 4 months (P < .001). Seven patients had platelet-activating, anti-protamine-heparin antibodies at baseline and showed a greater and more prolonged decline in platelet counts compared with antibody-negative patients (P 5 .003). In addition, 2 of those patients experienced early arterial thromboembolic complications vs 9 of 584 control patients (multivariate analysis: odds ratio, 21.58; 95% confidence interval, 2.90-160.89; P 5 .003). Platelet-activating antiprotamine-heparin antibodies show several similarities with anti-platelet factor 4-heparin antibodies and are a potential risk factor for early postoperative thrombosis. (Blood. 2013;121(15):2821-2827

“…3,4 However, pharmacokinetic studies have demonstrated that protamine is cleared rapidly from human plasma (half life 7.4 minutes), so that repeated doses may be necessary to prevent rebound heparin anticoagulant effects. 10,11 Animal studies have shown that although protamine fully neutralizes the thrombininhibitory activity of low molecular weight heparins (LMWH), it can only partially neutralize their anti-factor Xa (FXa) activities. 12,13 Therefore, the clinical efficacy of protamine for reversing LMWH-induced anticoagulation remains unclear.…”

Section: Introductionmentioning

confidence: 99%

Protamine sulfate down-regulates thrombin generation by inhibiting factor V activation

Áinle¹,

Preston²,

Jenkins³

et al. 2009

114

Protamine sulfate is a positively charged polypeptide widely used to reverse heparininduced anticoagulation. Paradoxically, prospective randomized trials have shown that protamine administration for heparin neutralization is associated with increased bleeding, particularly after cardiothoracic surgery with cardiopulmonary bypass. The molecular mechanism(s) through which protamine mediates this anticoagulant effect has not been defined. In vivo administration of pharmacologic doses of protamine to BALB/c mice significantly reduced plasma thrombin generation and prolonged tail-bleeding time (from 120 to 199 seconds). Similarly, in pooled normal human plasma, protamine caused significant dose-dependent prolongations of both prothrombin time and activated partial thromboplastin time. Protamine also markedly attenuated tissue factor-initiated thrombin generation in human plasma, causing a significant decrease in endogenous thrombin potential (41% ؎ 7%). As expected, low-dose protamine effectively reversed the anticoagulant activity of unfractionated heparin in plasma. However, elevated protamine concentrations were associated with progressive dose-dependent reduction in thrombin generation. To assess the mechanism by which protamine mediates down-regulation of thrombin generation, the effect of protamine on factor V activation was assessed. Protamine was found to significantly reduce the rate of factor V activation by both thrombin and factor Xa. Protamine mediates its anticoagulant activity in plasma by downregulation of thrombin generation via a novel mechanism, specifically inhibition of factor V activation. (Blood. 2009;114: 1658-1665) IntroductionProtamine sulfate is a 5-kDa cationic polypeptide derived from salmon sperm that can bind negatively charged unfractionated heparin (UFH). 1 Although the mechanisms of the molecular interaction between protamine and heparin are not well defined, this binding serves to neutralize the antithrombin-mediated anticoagulant properties of heparin. Moreover, the resultant protamineheparin complex is rapidly cleared by the reticuloendothelial system. 2 Consequently, for more than 30 years, protamine has been widely used to reverse the anticoagulant effects of UFH. Protamine is considered the treatment of choice for patients who develop significant bleeding complications while on UFH. 3,4 Furthermore, protamine is routinely administered postoperatively to reverse the high concentrations of UFH required for patients undergoing cardiac surgery and cardiopulmonary bypass (CPB). 5 As a result, estimates suggest that more than 2 million heparinized patients are managed with protamine each year. 2 Clinical use of protamine is, however, associated with several important adverse side effects, including potentially life-threatening systemic arterial hypotension and pulmonary artery hypertension. 1 Paradoxically, in vitro studies have also suggested that protamine may possess intrinsic anticoagulant properties. [6][7][8] Administration of excess protamine in the neutralization of UFH has b...