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Protamine is administered as protamine sulfate to reverse the anticoagulant effect of heparin following cardiopulmonary bypass surgery. Immunogenicity of protamine has been recognized for decades in several patient groups including vasectomized men, diabetic patients on protamine-containing insulin and patients undergoing cardiopulmonary bypass surgery. Anti-protamine/heparin antibodies are a newly described class of heparin-dependent antibodies found in about 30% of patients exposed to protamine and heparin during cardiac surgery. A subset of seropositive patients especially who tested positive for platelet-activating anti-protamine/heparin immunoglobulin G (IgG) antibodies before surgery have prolonged postoperative thrombocytopenia with an increased risk for arterial occlusions. Studies presented in this thesis shed light on potential approaches that may prevent antibody-mediated platelet activation by anti-protamine/heparin antibodies. Two approaches are presented in this thesis, partially desulfated heparin (ODSH) and low molecular weight protamine (LMWP). Our studies demonstrated the ability of ODSH to inhibit anti-protamine/heparin antibody-mediated platelet destruction in the NOD/SCID mouse model by: i) reduction of antibody binding to preformed protamine/heparin complexes, as shown by enzyme immunoassay, ii) interfering with the binding of protamine/heparin complexes to platelets as shown by flow cytometry and fluorescence microscopy, and iii) inhibition of antibody-mediated platelet activation. Interestingly, ODSH was also able to block ongoing platelet destruction by displacing pre-bound complexes from the platelet surface. In addition, our data suggest the use of synthesized LMWP as a substitute for protamine in heparin reversal. The in vitro investigations showed that synthesized LMWP efficiently neutralizes heparin using the activated partial thromboplastin time. Anti-protamine/heparin antibodies have low binding properties to LMWP/heparin complexes as indicated in enzyme immunoassay. The ability of platelet-activating anti-protamine/heparin antibodies to induce platelet activation in the functional assay was significantly reduced in the presence of LMWP/heparin compared to protamine/heparin complexes. Owing to findings obtained in our studies, both approaches might be a promising future option to reduce anti-protamine/heparin antibody-mediated adverse effects.
Protamine (PRT) is a positively charged protein, which is widely used in medicine as an adjunct to certain preparations of insulin and as a rapidly-acting antidote for heparin, particularly to neutralize the effects of high heparin concentrations needed for anticoagulation during cardiac surgical procedures using cardiopulmonary bypass. It has been demonstrated that PRT and heparin form multimolecular complexes and that these complexes have high immunogenicity in a mouse model. Studies in this thesis provide new insights into the pathophysiology of anti-PRT/heparin antibodies. The results of study I showed that the administration of PRT combined with heparin is responsible for high immunoglobulin G (IgG) immunization after cardiac surgery. A subset of these antibodies was able to induce platelet activation in a way similar to that observed by heparin-induced thrombocytopenia (HIT). Using an animal model, we demonstrated that anti-PRT/heparin antibodies are capable of platelet destruction in the presence of PRT and heparin. Moreover, our data suggests that platelet-activating anti-PRT/heparin antibodies at surgery are potentially associated with postoperative thrombocytopenia and an increased risk for thromboembolic events. In study II, the immune response against PRT/heparin complexes was investigated. This study showed a relatively fast development of IgG with no general preceding IgM formation. In addition, patients undergoing liver transplantation developed anti-PRT/heparin antibodies without previous exposure to PRT. These results suggest that a previous contact with the antigen(s) itself or other antigens with molecular mimicry induced this immune response. In fact, we were able to identify Neutral Protamine Hagedorn (NPH) insulin and core histones (DNA-binding proteins) as potentially antigenic candidates for a previous immunization. Furthermore, the findings of study III demonstrate the ability of anti-PRT/heparin antibodies to activate platelets in the presence of NPH insulin in a heparin-dependent way suggesting that diabetic patients may have an enhanced risk for thromboembolic complications if treated with NPH insulin and possibly while receiving prophylactic heparin. These observations justify further clinical investigations to assess the impact of the interaction between anti-PRT/heparin antibodies and PRT-mimicking antigens, such as NPH insulin or histones.
Streptococcus pneumoniae (S. pneumoniae, pneumococci) and Staphylococcus aureus (S. aureus) belong to the Gram-positive, facultative pathogenic bacteria. They are typical commensals of the human upper respiratory tract and most people get colonized at least once during their life. Nevertheless, these potentially pathogenic bacteria are able to spread from the site of colonization to invade into deeper tissues and the blood circulation. Thereby, severe local and invasive infections like bacteremia and life-threatening sepsis can be caused. Once reaching the bloodstream, bacteria get in contact with platelets. Platelets are small, anucleated cells and the second most abundant cell type in the circulation. The role of platelets in hemostasis is well known. Circulating resting platelets sense vessel injury independent of its cause. Platelets bind to injured endothelium and exposed molecules of the underlying extracellular matrix, get activated and release intracellular adhesion proteins and different modulatory molecules. This in turn initiates activation and binding of nearby platelets resulting in closure of vascular injury by formation of small thrombi. Despite being pivotal in maintenance of the endothelial barrier they got increasingly recognized as cells with important immune functions. Platelets excert functions of the immune response by either, i) interacting with immune cells of different pathways of the immune response, ii) releasing immunomodulatory molecules stored in their granules or iii) interacting with invading pathogens via direct or indirect binding.
The basis for this study were results demonstrating direct binding of different S. aureus proteins to platelets resulting in platelet activation. The identified proteins in the mentioned study are the S. aureus proteins Eap, AtlA-1, CHIPS and FlipR. Severe invasive infections with S. pneumoniae are quite often associated with development of thrombocytopenia or disseminated vascular dissemination. This frequent observation hints towards either a direct or indirect interplay of platelets with pneumococci. Hence, this study aims to analyze potential interactions and aims to decipher involved factors on both the platelet- and bacterial site.
A screening of recombinant pneumococcal surface proteins identified proteins belonging to the group of lipoproteins, sortase-anchored proteins and choline-binding proteins to directly activate human platelets. Besides these surface proteins also the intracellular pneumococcal pneumolysin (Ply) induced highly increased values for the platelet activation marker P-selectin. Since Ply is a major virulence factor of
S. pneumoniae the primary focus was set on involvement of this pore forming toxin on platelet activation. Surprisingly, our data revealed Ply induced platelet activation to be a false positive result based on formation of large Ply pores in the platelet membrane. In fact, it was clearly demonstrated that Ply lyses platelets even at low concentrations and thereby rendering them non-functional. Lysis of platelets could be inhibited by the addition of pharmaceutical immunoglobulin preparations as well as antibodies specifically targeting Ply. Inhibition of Ply also resulted in fully rescued platelet function either in washed platelets or in whole blood as shown by thrombus formation. Next to pneumococci also S. aureus expresses pore forming toxins, namely α-hemolysin (Hla) and different pairs of bicomponent pore forming leukocidins. Whereas the different tested leukocidins did not affect platelets, Hla acted in a two-step mechanism on human platelets. The results confirm previous data on Hla induced platelet activation via Hla resulting in e.g., reversible platelet aggregation or surface expression of activation markers. Nevertheless, platelet activation by Hla is followed by dose- and time-dependent lysis of platelets resulting in loss of platelet function and abrogated thrombus formation. Platelet lysis by Hla could neither be rescued with specific monoclonal anti-Hla antibodies nor with pharmaceutical IgG preparations containing anti-Hla IgGs. Taken together, the presented data reveal new pathomechanisms involving disturbance of platelets by bacterial pore forming toxins. Platelet lysis as well as impaired platelet function play an important role in development of severe complications during invasive infections. In life threatening infections caused by S. pneumoniae the usage of antibody formulations containing antibodies targeting Ply might be a promising approach for the prevention or even intervention and improvement of clinical outcome.