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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.
Vaccine-induced immune thrombotic thrombocytopenia (VITT) and cerebral venous sinus thrombosis (CVST) have been recently described as rare complications following vaccination against SARS-CoV-2 with vector vaccines. We report a case of a young woman who presented with VITT and cerebral CVST 7 days following vaccination with ChAdOx1 nCov-19 (AstraZeneca). While the initial MRI was considered void of pathological findings, MRI 3 days later revealed extensive CVST of the transversal and sigmoidal sinus with intracerebral haemorrhage. Diagnostic tests including a platelet-factor-4-induced platelet activation assay confirmed the diagnosis of VITT. Treatment with intravenous immunoglobulins and argatroban resulted in a normalisation of platelet counts and remission of CVST.
Vector-based SARS-CoV-2 vaccines have been associated with vaccine- induced thrombosis with thrombocytopenia syndrome (VITT/TTS), but the causative factors are still unresolved. We comprehensively analyzed the ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Johnson and Johnson) vaccines. ChAdOx1 nCoV-19 contains significant amounts of host cell protein impurities, including functionally active proteasomes, and adenoviral proteins. A much smaller amount of impurities was found in Ad26.COV2.S. Platelet factor 4 formed complexes with ChAdOx1 nCoV-19 constituents, but not with purified virions from ChAdOx1 nCoV-19 or with Ad26.COV2.S. Vascular hyperpermeability was induced by ChAdOx nCoV-19 but not by Ad26.COV2.S. These differences in impurities together with EDTAinduced capillary leakage might contribute to the higher incidence rate of VITT associated with ChAdOx1 nCoV-19 compared to Ad26.COV2.S.
Platelet adhesion and spreading at the sites of vascular injury is vital to hemostasis. As an integral part of the innate immune system, platelets interact with opsonized bacterial pathogens through FcγRIIA and contribute to host defense. As mechanoscavangers, platelets actively migrate and capture bacteria via cytoskeleton-rich, dynamic structures, such as filopodia and lamellipodia. However, the role of human platelet FcγRIIA in cytoskeleton-dependent interaction with opsonized bacteria is not well understood. To decipher this, we used a reductionist approach with well-defined micropatterns functionalized with immunoglobulins mimicking immune complexes at planar interfaces and bacteriamimetic microbeads. By specifically blocking of FcγRIIA and selective disruption of the platelet cytoskeleton, we show that both functional FcγRIIA and cytoskeleton are necessary for human platelet adhesion and haptotaxis. The direct link between FcγRIIA and the cytoskeleton is further explored by single-particle tracking. We then demonstrate the relevance of cytoskeleton-dependent differential mobilities of FcγRIIA on bacteria opsonized with the chemokine platelet factor 4 (PF4) and patient-derived anti-PF4/polyanion IgG. Our data suggest that efficient capture of opsonized bacteria during host-defense is governed by mobility dynamics of FcγRIIA on filopodia and lamellipodia, and the cytoskeleton plays an essential role in platelet morphodynamics at biological interfaces that display immune complexes.
Divalent magnesium restores cytoskeletal storage lesions in cold-stored platelet concentrates
(2022)
Cold storage of platelet concentrates (PC) has become attractive due to the reduced risk of bacterial proliferation, but in vivo circulation time of cold-stored platelets is reduced. Ca2+ release from storage organelles and higher activity of Ca2+ pumps at temperatures < 15 °C triggers cytoskeleton changes. This is suppressed by Mg2+ addition, avoiding a shift in Ca2+ hemostasis and cytoskeletal alterations. We report on the impact of 2–10 mM Mg2+ on cytoskeleton alterations of platelets from PC stored at room temperature (RT) or 4 °C in additive solution (PAS), 30% plasma. Deformation of platelets was assessed by real-time deformability cytometry (RT-DC), a method for biomechanical cell characterization. Deformation was strongly affected by storage at 4 °C and preserved by Mg2+ addition ≥ 4 mM Mg2+ (mean ± SD of median deformation 4 °C vs. 4 °C + 10 mM Mg2+ 0.073 ± 0.021 vs. 0.118 ± 0.023, p < 0.01; n = 6, day 7). These results were confirmed by immunofluorescence microscopy, showing that Mg2+ ≥ 4 mM prevents 4 °C storage induced cytoskeletal structure lesion. Standard in vitro platelet function tests showed minor differences between RT and cold-stored platelets. Hypotonic shock response was not significantly different between RT stored (56.38 ± 29.36%) and cold-stored platelets with (55.22 ± 11.16%) or without magnesium (45.65 ± 11.59%; p = 0.042, all n = 6, day 1). CD62P expression and platelet aggregation response were similar between RT and 4 °C stored platelets, with minor changes in the presence of higher Mg2+ concentrations. In conclusion, increasing Mg2+ up to 10 mM in PAS counteracts 4 °C storage lesions in platelets, maintains platelet cytoskeletal integrity and biomechanical properties comparable to RT stored platelets.
Background: Hyperthyroidism is known to induce a hypercoagulable state. It stimulates plasma levels of procoagulative factors and reduces fibrinolytic activity. So far most of the data have been derived from patients with endogenous hyperthyroidism with a wide variability in the underlying pathogenesis and severity of the disease. Objectives: In this study we experimentally induced thyrotoxicosis in healthy volunteers to explore the effects of thyroxine excess on the plasma proteome. Using a shotgun proteomics approach, the abundance of plasma proteins was monitored before, during and after thyrotoxicosis. Methods: Sixteen healthy male subjects were sampled at baseline, 4 and 8 weeks under 250 µg/day thyroxine p.o., as well as 4 and 8 weeks after stopping the application. Plasma proteins were analyzed after depletion of 6 high-abundance proteins (MARS6) by LC-ESI-MS/MS mass spectrometry. Mass spectrometric raw data were processed using a label-free, intensity-based workflow. Subsequently, the linear dependence between protein abundances and fT<sub>4</sub> levels were calculated using a Pearson correlation. Results: All subjects developed biochemical thyrotoxicosis, and this effect was reversed within the first 4 weeks of follow-up. None of the volunteers noticed any subjective symptoms. Levels of 10 proteins involved in the coagulation cascade specifically correlated with fT<sub>4</sub>, supporting an influence of thyroid hormone levels on blood coagulation even at nonpathological levels. Conclusions: The results suggest that experimental thyrotoxicosis exerts selective and specific thyroxine-induced effects on coagulation markers. Our study design allows assessment of thyroid hormone effects on plasma protein levels without secondary effects of other diseases or therapies.
Inherited platelet disorders affecting the human platelet cytoskeleton result in increased bleeding risk. However, deciphering their impact on cytoskeleton-dependent intrinsic biomechanics of platelets remains challenging and represents an unmet need from a diagnostic and prognostic perspective. It is currently unclear whether ex vivo anticoagulants used during collection of peripheral blood impact the mechanophenotype of cellular components of blood. Using unbiased, high-throughput functional mechanophenotyping of single human platelets by real-time deformability cytometry, we found that ex vivo anticoagulants are a critical pre-analytical variable that differentially influences platelet deformation, their size, and functional response to agonists by altering the cytoskeleton. We applied our findings to characterize the functional mechanophenotype of platelets from a patient with Myosin Heavy Chain 9 (MYH9) related macrothrombocytopenia. Our data suggest that platelets from MYH9 p.E1841K mutation in humans affecting platelet non-muscle myosin heavy chain IIa (NMMHC-IIA) are biomechanically less deformable in comparison to platelets from healthy individuals.
Background: We assessed the effect of the uniform donor questionnaire (UDQ) on deferral rates in first-time and repeat donors. We focused on the introduced question about unprotected sexual contact with a new partner. Another goal was a stratified comparison of the deferral rates of the donor questionnaire (DQ) and UDQ. Methods: Data on donors and deferrals using the DQ and UDQ were collected at four blood establishments. The comparison included a 2-year period by questionnaire version. For the comparison of the questionnaires, an adjusted multinomial logistic regression was performed. Results: The analysis included 260,848 donations. First-time (FTD) and repeat donations (RD) showed higher deferral rates with the UDQ (FTD +5.4%, RD +1.4%). Deferral due to a new partner was 3.0% in first-time and 0.4% in repeat donors. The majority of these occurred in the youngest age groups. The most frequent deferral criterion was ‘disease' (5.1%). Conclusion: The regression revealed stronger predictors for deferral than the questionnaire version. Especially younger age carried a higher and independent risk for deferral. The additional deferrals of mainly young first-time donors due to a new sexual partner may identify those donors with potential heterosexual risk behavior who would otherwise not be identified.