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Background: Heparins are usually produced from animal tissues. It is now possible to synthesize heparins. This provides the abilities to overcome shortages of heparin, to optimize biological effects, and to reduce adverse drug effects. Heparins interact with platelet factor 4 (PF4), which can induce an immune response causing thrombocytopenia. This side effect is called heparin-induced thrombocytopenia (HIT). We characterized the interaction of PF4 and HIT antibodies with oligosaccharides of 6-, 8-, 10-, and 12-mer size and a hypersulfated 12-mer (S12-mer)., Methods: We utilized multiple methodologies including isothermal calorimetry, circular dichroism spectroscopy, single molecule force spectroscopy (SMFS), enzyme immunosorbent assay (EIA), and platelet aggregation test to characterize the interaction of synthetic heparin analogs with PF4 and anti-PF4/heparin antibodies., Results: The synthetic heparin-like compounds display stronger binding characteristics to PF4 than animal-derived heparins of corresponding lengths. Upon complexation with PF4, 6-mer and S12-mer heparins showed much lower enthalpy, induced less conformational changes in PF4, and interacted with weaker forces than 8-, 10-, and 12-mer heparins. Anti-PF4/heparin antibodies bind more weakly to complexes formed between PF4 and heparins ≤ 8-mer than with complexes formed between PF4 and heparins ≥ 10-mer. Addition of one sulfate group to the 12-mer resulted in a S12-mer, which showed substantial changes in its binding characteristics to PF4., Conclusions: We provide a template for characterizing interactions of newly developed heparin-based anticoagulant drugs with proteins, especially PF4 and the resulting potential antigenicity., (© 2019 The Authors. Journal of Thrombosis and Haemostasis published by Wiley Periodicals, Inc. on behalf of International Society on Thrombosis and Haemostasis.)

Essentials At low pH and low salt concentrations: Maximal conformational change of PF4 upon complexation with heparin occurs. Changing physicochemical conditions may become an approach to better discriminate the signal of platelet-activating- and nonactivating PF4/H Abs in antigen tests., Background: Enzyme immunosorbent assays (EIA) are widely used to detect human antiplatelet factor 4/heparin antibodies (aPF4/H Abs) to rule out heparin-induced thrombocytopenia. EIAs cannot differentiate between clinically relevant, platelet-activating, and nonrelevant, nonplatelet-activating Abs and only ~50% of patients' sera testing positive by EIA contain antibodies that activate platelets. Recently, we have shown platelet-activating aPF4/H Abs bind more strongly to PF4/H complexes than nonplatelet-activating antibodies. Antigen-antibody interactions are known to depend on electrostatic interactions governed by pH, heat, and ionic strength. We tested whether changes in pH and ionic strength can improve the specificity of EIAs detecting aPF4/H Abs., Methods: We investigated first the conformational change of PF4 when binding to heparin under various pH and salt conditions using circular dichroism spectroscopy, and then the binding of aPF4/H Abs to PF4/H complexes by EIA., Results: Maximal conformational change of PF4 on complexation with heparin was identified at low pH and low salt concentrations. EIA tested with a large number of sera at 50 mmol/L NaCl, pH 6.0 shows a potential to increase the specificity for the detection of platelet-activating aPF4/H Abs., Conclusion: Changing physicochemical conditions may become an approach to better discriminate the signal of platelet-activating and nonactivating PF4/H Abs in antigen tests., (© 2019 International Society on Thrombosis and Haemostasis.)

The immune response in heparin-induced thrombocytopenia (HIT) is puzzling: heparin-naive patients can develop IgG antibodies and clinical HIT as early as day 5, and evidence for an anamnestic response on heparin reexposure is lacking. We assessed daily serum samples by anti-PF4/heparin enzyme-immunoassay (EIA) in patients receiving heparin thromboprophylaxis. Of 435 patients, 56.1% showed an increase in EIA optical density (OD) of more than or equal to 15%, with more than 90% starting between days 4 and 14. After reaching maximum reactivity by days 10 to 12, ODs declined despite heparin continuation, including in 2 patients with clinical HIT. Individual IgG/A/M classes showed identical time of onset (median, day 6). Most (58.7%) antibody-positive patients developed all 3 Ig classes; only 11.3% lacked IgG response. IgG/A/M increase usually occurred simultaneously (+/- 1 day) with no general tendency for IgM precedence. Consistent with the transient immune response, none of the IgG-EIA-positive (OD > 0.5) patients at discharge developed clinically evident thrombosis during extended low-molecular-weight heparin thromboprophylaxis. The rapid onset of the anti-PF4/heparin immune response, its transience, and the simultaneous appearance of antibodies of different classes with no IgM precedence suggest short-term activation of B cells that have previously undergone Ig-class switching even without previous pharmacologic heparin exposure.

Background: Minimal invasive breast biopsy is standard care for the diagnosis of suspicious breast lesions. There are different vacuum biopsy (VB) systems in use. The aim of the study was to determine the differences between the 8-gauge and the 11-gauge needle with respect to a) diagnostic reliability, b) complication rate and c) subjective perception of pain when used for vacuum-assisted breast biopsy., Methods: Between 01/2000 and 09/2004, 923 patients at St. Josefs-Hospital Wiesbaden underwent VB using the Mammotome (Ethicon Endosurgery, Hamburg). Depending on preoperative detection, the procedure was performed under sonographic or mammographic guidance under local anaesthesia. All patients included in the study were followed up both clinically and using imaging techniques one week after the VB and a second time after a median of 41 months. Excisional biopsy on the ipsilateral breast was an exclusion criteria. Subjective pain scores were recorded on a scale of 0 - 10 (0 = no pain, 10 = unbearable pain). The mean age of the patients was 53 years (30 - 88)., Results: 123 patients were included in the study in total. 48 patients were biopsied with the 8-gauge needle and 75 with the 11-gauge needle. The use of the 8-gauge needle did not show any significant differences to the 11-gauge needle with regard to diagnostic reliability, complication rate and subjective perception of pain., Conclusion: Our data show that there are no relevant differences between the 8-gauge and 11-gauge needle when used for VB. Under sonographic guidance, the use of the 8-gauge needle is recommended for firm breast tissue due to its sharp scalpel point and especially for complete removal of benign lesions. We did not find any advantages in the use of the larger 8-gauge needle compared to the 11-gauge needle in the mammography setting. The utilisation costs of the 8-gauge needle are somewhat higher.

Objective: Heparin-induced thrombocytopenia (HIT) is a prothrombotic drug reaction caused by antibodies that recognize positively charged platelet factor 4 (PF4), bound to the polyanion, heparin. The resulting immune complexes activate platelets. Unfractionated heparin (UFH) causes HIT more frequently than low-molecular-weight heparin (LMWH), whereas the smallest heparin-like molecule (the pentasaccharide, fondaparinux), induces anti-PF4/heparin antibodies as frequently as LMWH, but without exhibiting cross-reactivity with these antibodies. To better understand these findings, we analyzed the molecular structure of the complexes formed between PF4 and UFH, LMWH, or fondaparinux., Methods and Results: By atomic force microscopy and photon correlation spectroscopy, we show that with any of the 3 polyanions, but in the order, UFH>LMWH>>fondaparinux--PF4 forms clusters in which PF4 tetramers become closely apposed, and to which anti-PF4/heparin antibodies bind. By immunoassay, HIT antibodies bind strongly to PF4/H/PF4 complexes, but only weakly to single PF4/heparin molecules., Conclusions: HIT antigens are formed when charge neutralization by polyanion allows positively charged PF4 tetramers to undergo close approximation. Whereas such a model could explain why all 3 polyanions form antibodies with similar specificities, the striking differences in the relative size and amount of complexes formed likely correspond to the observed differences in immunogenicity (UFH>LMWH approximately fondaparinux) and clinically relevant cross-reactivity (UFH>LMWH>>fondaparinux).

Bivalirudin is a synthetic antithrombin sharing a sequence of 11 amino acids with the recombinant hirudin lepirudin. We investigated whether antilepirudin antibodies recognize epitopes on bivalirudin. Antilepirudin antibody-positive sera of 43 patients, treated with lepirudin for heparin-induced thrombocytopenia, were analyzed. Lepirudin- and bivalirudin-coated microtiter plates were used for antibody testing in an enzyme-linked immunosorbent assay (ELISA) system. Of the 43 sera-containing antibodies binding to lepirudin, 22 (51.2%) contained antibodies that also recognized bivalirudin. Binding of these antibodies to bivalirudin was inhibited by more than 70% by preincubation with high doses of bivalirudin. However, if lepirudin-coated microtiter plates were used, high concentrations of bivalirudin inhibited only 2 of the 43 positive sera by more than 30%. Therefore antihirudin antibodies must be polyspecific. The clinical consequences of this cross-reactivity are unknown but bivalirudin, targeted by antibodies of patients treated with lepirudin previously, could potentially boost antibody titers in such patients or even trigger an immune response by itself. Clinically significant antibody formation in response to bivalirudin monotherapy has not been observed, however. Yet, as lepirudin and antilepirudin antibodies have recently been implicated in severe anaphylactic reactions, caution is warranted when using bivalirudin in patients previously treated with lepirudin.

Recombinant hirudin has been found to be immunogenic in patients treated with lepirudin following heparin-induced thrombocytopenia (HIT). We assessed the incidence of immunoglobulin G (IgG) antihirudin antibodies by enzyme-linked immunosorbent assay in 112 patients enrolled in a dose-finding study with desirudin. Patients received desirudin subcutaneously following orthopedic hip surgery at 10 mg twice a day (n = 17), 15 mg twice a day (n = 75), and 20 mg twice a day (n = 20). Of 112 patients, 11 (9.8%) developed antihirudin antibodies independently of the dose. The rate of immunization did not differ from that observed in HIT patients treated with lepirudin (P =.113). Plasma concentrations of desirudin did not differ between antihirudin antibody-positive and -negative patients. Antihirudin antibodies had no impact on incidences of deep vein thrombosis and/or pulmonary embolism, allergic reactions, and hemorrhage. However, the total number of immunized patients observed was low and so infrequent (but severe) effects of antihirudin antibodies cannot be excluded.

The present experiments were designed to assess the renal functional response to alterations in nitric oxide formation in animals with different forms of nephropathy. To address this issue, the effects of Nomega-nitro-L-arginine methyl ester (L-NAME) or L-arginine were assessed in animal models exhibiting arterial hypertension due to chronic nitric oxide inhibition (L-NAME, 50 mg/l in drinking water for 12 weeks) or diabetes mellitus (streptozotocin, 60 mg/kg IP). Vehicle-treated, age-matched animals served as controls. Following 12 weeks of pretreatment, mean arterial pressure (MAP), renal hemodynamics, urinary albumin, and electrolyte excretion were determined in standard clearance experiments prior to and following infusion of L-NAME (50 microg/kg/min), l-arginine (5 mg/kg/min), or saline vehicle. In control animals, L-NAME resulted in an increase in MAP and renal vascular resistance and a decline in glomerular filtration rate and renal plasma flow, as expected. L-arginine had no effect on renal hemodynamics. In nitric oxide-depleted hypertensive animals, L-NAME had no additional effect on MAP or renal hemodynamics. Infusion of L-arginine reduced elevated MAP but did not reverse changes in renal hemodynamics. Diabetic rats demonstrated glomerular hyperfiltration and proteinuria. No significant changes in MAP or renal hemodynamics were observed following infusion of L-NAME or L-arginine, respectively. However, L-NAME increased urinary albumin excretion in the absence of hemodynamic changes. The effects of nitric oxide on vascular tone were shown to be dependent on the vascular bed and the underlying disease. Variations in local nitric oxide formation and susceptibility may account for the differential response of the systemic and renal vasculature and contribute to the degree of renal functional impairment observed in different systemic diseases.