SARS-CoV-2 Neutralization in Convalescent Plasma and Commercial Lots of Plasma-Derived Immunoglobulin.

INTRODUCTION: Patients with primary or secondary immunodeficiency (PID or SID) face increased insecurity and discomfort in the light of the COVID-19 pandemic, not knowing if and to what extent their comorbidities may impact the course of a potential SARS-CoV-2 infection. Furthermore, recently available vaccination options might not be amenable or effective for all patients in this heterogeneous population. Therefore, these patients often rely on passive immunization with plasma-derived, intravenous or subcutaneous immunoglobulin (IVIG/SCIG). Whether the ongoing COVID-19 pandemic and/or the progress in vaccination programs lead to increased and potentially protective titers in plasma-derived immunoglobulins (Ig) indicated (e.g., for humoral immunodeficiency) remains a pressing question for this patient population. PURPOSE: We investigated SARS-CoV-2 reactivity of US plasma-derived IVIG/SCIG products from the end of 2020 until June 2021 as well as in convalescent plasma (CP) from May 2020 to August 2020 to determine whether potentially neutralizing antibody titers may be present. METHODS: Final containers of IVIG/SCIG and CP donations were analyzed by commercial ELISA for anti-SARS-CoV-2 S1-receptor binding domain (RBD) IgG as well as microneutralization assay using a patient-derived SARS-CoV-2 (D614G) isolate. Neutralization capacities of 313 single plasma donations and 119 plasma-derived IVIG/SCIG lots were determined. Results obtained from both analytical methods were normalized against the WHO International Standard. Finally, based on dense pharmacokinetic profiles of an IVIG preparation from previously published investigations, possible steady-state plasma levels of SARS-CoV-2 neutralization capacities were approximated based on currently measured anti-SARS-CoV-2 potencies in IVIG/SCIG preparations. RESULTS: CP donations presented with high variability with regards to anti-SARS-CoV-2 reactivity in ELISA as well as in neutralization testing. While approximately 50% of convalescent donations were not/low neutralizing, approximately 10% were at or above 600 IU/mL. IVIG/SCIG lots derived from pre-pandemic plasma donations did not show neutralizing capacities for SARS-CoV-2. Lots produced between December 2020 and June 2021 entailing plasma donations after the emergence of SARS-CoV-2 showed a rapid and constant increase in anti-SARS-CoV-2 reactivity and neutralization capacity over time. While lot-to-lot variability was substantial, neutralization capacity increased from a mean of 21 IU/mL in December 2020 to 506 IU/mL in June 2021 with a maximum of 864 IU/mL for the most recent lots. Pharmacokinetic extrapolations, based on non-compartmental superposition principles using steady-state reference profiles from previously published pharmacokinetic investigations on IVIG in PID, yielded potential steady-state trough plasma levels of 16 IU/mL of neutralizing SARS-CoV-2 IgG based on the average final container concentration from May 2021 of 216 IU/mL. Maximum extrapolated trough levels could reach 64 IU/mL based on the latest maximal final container potency tested in June 2021. CONCLUSIONS: SARS-CoV-2 reactivity and neutralization capacity in IVIG/SCIG produced from US plasma rapidly and in part exponentially increased in the first half of 2021. The observed increase of final container potencies is likely trailing the serological status of the US donor population in terms of COVID-19 convalescence and vaccination by at least 5 months due to production lead times and should in principle continue at least until Fall 2021. In summary, the data support rapidly increasing levels of anti-SARS-CoV-2 antibodies in IVIG/SCIG products, implicating that a certain level of protection could be possible against COVID-19 for regularly substituted PID/SID patients. Nevertheless, more research is still needed to confirm which plasma levels are needed to provide protection against SARS-CoV-2 infection in immune-compromised patients.

People with deficiencies in their immune system often have an insufficient antibody response to antigens such as bacteria, viruses, or vaccines. These patients therefore often receive antibodies from healthy people to replace the missing antibodies and build a first line of defense against infections. These antibodies (also called immunoglobulins [Ig]) are prepared from plasma, the liquid fraction of the blood without cells, of healthy donors. This plasma is then split up during pharmaceutical production into its protein components. One of these is immunoglobulin G (IgG), which is the protein family that neutralizes/inactivates infectious agents as well as marks these infectious agents so they can be recognized by other parts of the immune system. With the ongoing COVID-19 pandemic and the severe to fatal outcomes for certain patient groups, especially people with impaired immunity, these patients and their physicians are interested in whether their antibody replacement therapy also confers protection against SARS-CoV-2 infection. We analyzed the capability of plasma-derived Ig lots to (i) recognize SARS-CoV-2 protein by ELISA method as well as (ii) neutralize SARS-CoV-2 by neutralization studies using the actual virus under biosafety level 3 (BSL-3) conditions. Here we show increasing anti-SARS-CoV-2 activity over time of manufactured Ig lots produced between December 2020 and June 2021. The most recent lots had a neutralizing activity of up to 864 IU/mL. Considering that the US represents Octapharma's main plasma source, the progress in vaccination levels together with the evolution of the COVID-19 pandemic in this country suggests that the intravenous or subcutaneous immunoglobulin (IVIG/SCIG) neutralization capacities against SARS-CoV-2 might still increase and could potentially reach a level where antibody plasma concentrations in the patient confer immune protection.

Octanorm [cutaquig®], a new immunoglobulin (human) subcutaneous 16.5% solution for injection (165 mg/mL) - Biochemical characterization, pathogen safety, and stability.

Octanorm (marketed as cutaquig® in Canada and US [2018] and registered in several European countries [2019]) is a new immunoglobulin subcutaneous 16.5% liquid for the treatment of patients with primary immune deficiency (PID) and secondary immune deficiency (SID) depending on country's specific indications. Octanorm contains ≥96% human IgG and is characterized by especially low concentrations of polymers and aggregates, IgA and IgM, a physiological osmolality along with a low isoagglutinin titer. The Octanorm manufacturing process is based on the well-established IVIG octagam® 5% and 10% process, but yields a higher immunoglobulin concentration of 16.5% in the final product. Octanorm shows a distribution of immunoglobulin G subclasses closely proportional to native human plasma and comprises a broad spectrum of antibodies against infectious agents. Potential procoagulant activity is not detectable. IgG functionality and physico-chemical integrity have been demonstrated by state-of-the-art-methods. The virus safety of Octanorm is ensured via a combination of three validated independent methods as part of the manufacturing process. Substantial prion depletion during the manufacturing process has also been demonstrated. Compared with other commercially available subcutaneous immunoglobulin (SCIG) 20% products, Octanorm 16.5% shows a lower viscosity, which is a valuable feature that allows for a more comfortable infusion experience.

Biochemical characterization, stability, and pathogen safety of a new fibrinogen concentrate (fibryga®).

Fibryga® is a new lyophilized fibrinogen concentrate for intravenous use for the treatment of congenital fibrinogen deficiency. fibryga® is produced from pooled human plasma and the final product is characterized by high purity, integrity, and pathogen safety. Functional activity of fibrinogen was demonstrated by cross-linking studies and thromboelastometry; integrity of the fibrinogen molecule was demonstrated by size exclusion chromatography and the detection of only trace amounts of activation markers in the final product. Pathogen safety of fibryga® was proved by downscaling studies for the two dedicated pathogen inactivation/removal steps, i.e. solvent detergent treatment and nanofiltration. Fibryga® is stable for at least three years when stored at room temperature. In conclusion, the performed studies demonstrated that fibryga® meets the requirements for a state-of-the-art fibrinogen concentrate, such as a satisfactory activity profile combined with a favorable pathogen safety profile and stability.

Thawing of Pooled, Solvent/Detergent-Treated Plasma octaplasLG®: Validation Studies Using Different Thawing Devices.

BACKGROUND: The aim of this study was to perform validation of the thawing process for solvent/detergent-treated plasma octaplasLG® using different thawing devices. Optimized settings for temperature and thawing time should be defined based on the results of both temperature measurements and extensive biochemical characterization studies. METHODS: octaplasLG units were thawed using water bath systems (i.e. MB-13A, QuickThaw® DH4), dry tempering systems (i.e. plasmatherm, SAHARA-III), and microwave oven (i.e. transfusio-therm® 2000). Optimized thawing conditions were defined. Subsequently, using the selected thawing conditions, octaplasLG units were thawed and tested on product release parameters. RESULTS: The fastest thawing was observed for the microwave oven. All octaplasLG units thawed by different devices and optimized thawing conditions were clear and free of solid and gelatinous particles, indicating no protein denaturation or overheating. In addition, no significant differences were found in the coagulation and inhibition activity and hemostatic potency of octaplasLG when thawed by the different devices tested. All parameters after thawing were within the product release specification levels. CONCLUSION: Our study demonstrated that octaplasLG can be thawed using all above listed devices without any negative influence on the plasma quality, presupposed that optimized settings defined for this plasma product are used.

Biochemical characterization and stability of immune globulin intravenous 10% liquid (Panzyga®).

Panzyga® is a new glycine-formulated immune globulin intravenous 10% liquid for the treatment of patients suffering from immunodeficiencies and autoimmune diseases. Panzyga® is a high purity, native and functional IgG product with an IgG subclass distribution equivalent to normal plasma. The levels of hemagglutinins and accompanying plasma proteins (including IgA and IgM) are low. Potential procoagulant activity is not detectable. Functional activity of the IgG was demonstrated by opsonophagocytosis and receptor binding assays. Dynamic light scattering measurements and fluorescent dye binding were used to characterize the integrity of the IgG molecule. Panzyga® is stable under refrigerated storage for at least two years regarding all assessed physicochemical and functional parameters; it can also be stored at room temperature for at least twelve months within its total shelf-life.

Pathogen Safety of a New Intravenous Immune Globulin 10% Liquid.

BACKGROUND: The manufacturing process of a new intravenous immune globulin (IVIG) 10% liquid product incorporates two dedicated pathogen safety steps: solvent/detergent (S/D) treatment and nanofiltration (20 nm). Ion-exchange chromatography (IEC) during protein purification also contributes to pathogen safety. The ability of these three process steps to inactivate/remove viruses and prions was evaluated. OBJECTIVES: The objective of this study was to evaluate the virus and prion safety of the new IVIG 10% liquid. METHODS: Bovine viral diarrhea virus (BVDV), human immunodeficiency virus type 1 (HIV-1), mouse encephalomyelitis virus (MEV), porcine parvovirus (PPV), and pseudorabies virus (PRV) were used as models for common human viruses. The hamster-adapted scrapie strain 263K (HAS 263K) was used for transmissible spongiform encephalopathies. Virus clearance capacity and robustness of virus reduction were determined for the three steps. Abnormal prion protein (PrPSc) removal and infectivity of the samples was determined. RESULTS: S/D treatment and nanofiltration inactivated/removed enveloped viruses to below detection limits. IEC supplements viral safety and nanofiltration was highly effective in removing non-enveloped viruses and HAS 263K. Overall virus reduction factors were: ≥9.4 log10 (HIV-1), ≥13.2 log10 (PRV), ≥8.2 log10 (BVDV), ≥11.7 log10 (MEV), ≥11.6 log10 (PPV), and ≥10.4 log10 (HAS 263K). CONCLUSION: Two dedicated and one supplementing steps in the manufacturing process of the new IVIG 10% liquid provide a high margin of pathogen safety.

Universal pooled plasma (Uniplas(®)) does not induce complement-mediated hemolysis of human red blood cells in vitro.

BACKGROUND: Pooling of plasma of different blood groups before large scale manufacturing of Uniplas(®) results in the formation of low levels of soluble immune complexes (CIC). The aim of this study was to investigate the level and removal of CIC during Uniplas(®) manufacturing. In addition, an in vitro hemolysis assay should be developed and investigate if Uniplas(®) does induce complement-mediated hemolysis of human red blood cells (RBC). MATERIALS AND METHODS: In-process samples from Uniplas(®) (universal plasma) and Octaplas(LG)(®) (blood group specific plasma) routine manufacturing batches were tested on CIC using commercially available ELISA test kits. In addition, CIC was produced by admixing heat-aggregated immunoglobulins or monoclonal anti-A/anti-B antibodies to plasma and removal of CIC was followed in studies of the Uniplas(®) manufacturing process under down-scale conditions. The extent of RBC lysis was investigated in plasma samples using the in-house hemolysis assay. RESULTS: Levels of CIC in Uniplas(®) are within the normal ranges for plasma and comparable to that found in Octaplas(LG)(®). Down-scale experiments showed that both IgG/IgM-CIC levels are significantly removed on average by 40-50% during Uniplas(®) manufacturing. Uniplas(®) does not induce hemolysis of RBCs in vitro. Hemolysis occurs only after spiking with high titers of anti-A/anti-B antibodies and depends on the antibody specificity (i.e. titer) in the plasma sample. CONCLUSION: The results of this study confirm the safety of Uniplas(®) regarding transfusion to patients of all ABO blood groups.

A retrospective study of Octaplex in the treatment of bleeding in patients with haemophilia A complicated by inhibitors.

The nonactivated prothrombin complex concentrate (PCC) Octaplex (Octapharma PPGmbH, Vienna, Austria) has been used successfully for the treatment of congenital and acquired coagulation factor deficiencies and associated bleeding. The aims of this study were to assess retrospectively whether Octaplex is an effective treatment option for haemophilia A patients with high-titre inhibitors of factor VIII (FVIII) and to investigate the impact of Octaplex on thrombin generation in vitro and ex vivo. Retrospective data were collected from 15 haemophilia A patients with FVIII inhibitors who had been treated with Octaplex. Mild bleeds were treated for a median of 1 day with a median dose of 77 IU/kg and moderate bleeds for 3 days with 57 IU/kg. The physician's overall satisfaction with Octaplex, taking into account efficacy, safety and cost in comparison with other treatment options, was assessed for each bleed. The overall rating was good, very good or excellent for 29 of 41 (71%) bleeds. No adverse drug reactions were reported. In in-vitro studies of thrombin generation with normal plasma samples, experimental inhibition of FVIII activity prolonged the lag phase, diminished the peak thrombin concentration and decreased the area under the concentration-time curve, as expected. Marked improvement in thrombin generation parameters was achieved by adding 0.5-3 IU factor IX/ml PCC into the samples. The same held true when using plasma samples from haemophilia A patients with FVIII inhibitors. These results demonstrate that Octaplex overcomes inhibition of FVIII in in-vitro and ex-vivo assays of thrombin generation, and that Octaplex is an effective treatment option for haemophilia A patients with FVIII inhibitors.

Exposure of NK cells to intravenous immunoglobulin induces IFN gamma release and degranulation but inhibits their cytotoxic activity.

The mechanisms underlying the modulation of Natural Killer (NK) cell functions by intravenous immunoglobulin (IVIg) are poorly understood. Using an ex vivo whole blood assay system we demonstrate that IVIg suppresses NK cell cytotoxicity. This was paralleled by IVIg-induced degranulation of CD56(bright), CD16(positive) NK cells, reduced expression of CD16 and elevated IFN gamma release. To assess whether these findings also occur in vivo we analyzed whole blood before and after IVIg therapy of patients. Following IVIg treatment the number of NK cells in peripheral blood dropped significantly. We observed reduced CD16 expression, elevated IFN gamma-amounts in plasma, reduced NK cell cytotoxicity, and granzyme B release into the plasma, confirming our in vitro data. These effects on the functions of NK cells describe a novel immunomodulatory effect of IVIg. The in vitro assays employed here could represent informative test systems to monitor effects of in vivo IVIg treatment at an individual level.

Prion safety of transfusion plasma and plasma-derivatives typically used for prophylactic treatment.

Reports about transfusion-related transmissions of variant Creutzfeldt-Jakob disease have urged the need for more information regarding the risk for prion contaminated units in the blood supply and the safety of transfusion plasma and biopharmaceuticals derived from this precious raw material. According to a possible epidemiological model, the risk in many European countries is the same or lower than that of human immunodeficiency virus. Comprehensive investigations have shown that the prion safety margin of both single-donor and pooled solvent/detergent treated transfusion plasma is high. Furthermore, prophylactic treatment using plasma-derivatives poses a very low risk in terms of prion disease despite extensive lifetime exposure.

Thrombin generation capacity is impaired in methylene-blue treated plasma compared to normal levels in single-donor fresh-frozen plasma, a licensed solvent/detergent-treated plasma (Octaplas) and a development product (Uniplas).

Impaired capacity of thrombin generation (TG) was found in single-donor fresh-frozen plasma (FFP) units subjected to medical device treatment by a combination of methylene-blue dye and subsequent white-light exposure (MB plasma, MBP) compared to normal levels in non-MB-treated single-donor FFP, the licensed plasma product Octaplas, and a product under development (Uniplas; working title) applicable independently from the recipient's blood group. This result held true for MBP units obtained from two different European sources revealing a more significant TG impairment using the local inactivation system in blood banks ("in-house") than an industrial MB treatment. Supplementation of functional fibrinogen to physiological levels did not normalise the altered TG capacity in MBP, whereas addition of Octaplas did. No clear-cut correlation between coagulation factor levels in MBP and hampered TG capacity was found, suggesting a composite effect of impairment. A thorough elucidation and evaluation regarding the possible clinical impact of these findings seems prudent.

A biochemical comparison of a pharmaceutically licensed coagulation active plasma (Octaplas) with a universally applicable development product (Uniplas) and single-donor FFPs subjected to methylene-blue dye and white-light treatment.

The strive for more standardised and highly efficacious products is one of the important mainstays in modern haemotherapy. Coagulation active plasma for transfusion is the product of choice when treating hereditary or acquired isolated or complex coagulopathies, when no specific concentrate is available. The aim of this study was to perform an extensive biochemical comparison of the pharmaceutically licensed coagulation active plasma named Octaplas with an identical, but universally applicable, development product (Uniplas, working title) and single-donor fresh-frozen plasma (FFP) units subjected to a medical device treatment using a combination of methylene-blue dye and subsequent white-light exposure (MB plasma). Our study showed that there are differences in the biochemical characteristics between Octaplas and MB plasma, while Uniplas revealed the same quality as Octaplas. The variability of selected plasma proteins in the 20 individual MB plasma units tested was high compared to Octaplas/Uniplas. Beyond the reported decreased levels of protein S and plasmin inhibitor found in Octaplas/Uniplas, and the significant loss of fully functional fibrinogen in MB plasma and its impact on selected global coagulation parameters, the latter product additionally revealed several coagulation factor activities outside the ranges given for normal single-donor FFP. It is important for plasma prescribers to be aware of the major inherent differences between Octaplas and MB plasma.

Effect of oriented or random PEGylation on bioactivity of a factor VIII inhibitor blocking peptide.

Peptides as therapeutic substances are efficient agents in the treatment of several diseases. However, they often have to be chemically modified in order to be suited as therapeutic agent. Conjugation to large carrier molecules is often required. A critical step is the identification of available sites for chemical reaction, without influencing bioactivity. Peptide 238/S1 with the sequence NH(2)-PYWKWQYKYD-COOH previously selected from a combinatorial decapeptide library, has the ability to block inhibitory antibodies against blood clotting factor VIII (FVIII) and therefore, it constitutes a lead for developing a drug to treat patients suffering from development of such antibodies. The aims of this study were (i) to identify sites of the peptide, which are suited for modification without losing bioactivity and (ii) to find out the influence of molecular size of polyethylene glycol (PEG) for bioactivity of the peptide. The contribution of each amino acid residue to biological functionality was investigated by mutational analysis. This method confirmed that the N-terminus is crucial for activity, whereas both lysine residues could be exchanged by other L-amino acids. Using mutational analysis it was possible to identify peptides with higher reactivity compared to the wild type 238/S1. PEGylation experiments demonstrated that conjugation of the peptide to PEG 20,000 resulted in a loss of reactivity, while PEG 5,000 could maintain the bioactivity when conjugated in a site directed manner. The peptide lost its neutralization properties when PEG was coupled to the N-terminus, again indicating that this part of the peptide is important for functionality.

Mapping of FVIII inhibitor epitopes using cellulose-bound synthetic peptide arrays.

Epitope mapping using antibodies against factor VIII (FVIII) has been performed using blotting techniques with truncated and/or digested FVIII molecules. Here, we focused on the precise mapping of affinity purified IgG from patients with an immune response against blood clotting FVIII using synthetic peptide arrays on cellulose membranes comprising the entire sequence of FVIII. The aim was to elucidate the epitope profile from different inhibitors and possibly detect new epitopes, which have not been described before. The epitope patterns from five patients showed reactivity with all domains in the FVIII molecule, but were different between various patients. These results included epitopes usually buried within the folded protein. However, in competition assays using FVIII as competitive agent in a mixture with inhibitor IgG, the most immunogenic regions were located in the FVIII light chain. Our results show that the C1 domain was the region with highest immunogenicity in all patients. Here, we demonstrate that the SPOT method is very well suited for the precise location of epitopes in the core of the protein, which usually cannot be detected by other methods.

Combinatorial peptides directed to inhibitory antibodies against human blood clotting factor VIII.

The development of antibodies against blood clotting factor VIII is a major complication affecting 20-30% of hemophilia A patients receiving replacement with FVIII concentrates. This study investigated generating peptides acting as broadly neutralizing agents to block factor VIII antibodies. These peptides were selected from dual positional scanning decapeptide libraries on cellulose membranes. From this library comprising 6.8 x 10(12) peptides we selected 468 peptides for further screening rounds. Finally we identified two decapeptides with the ability to block 8 out of 10 inhibitory antibodies from sera of patients with FVIII inhibitors demonstrated by competition assays. Sequence alignment of the peptides showed similarity with several domains in the FVIII molecule demonstrating the mimotope nature of the selected peptides. Our results show the efficiency of the combinatorial library approach and show the potential of combinatorial peptides to compete out polyclonal inhibitor IgG from a broad range of patients' sera. Combinatorial peptides could be novel and highly effective drug candidates for alternative treatment in patients with factor VIII inhibitors.

Stability of solvent/detergent-treated plasma and single-donor fresh-frozen plasma during 48 h after thawing.

The aim of this study was to compare the quality of solvent/detergent (SD) treated plasma, Octaplas, and single-donor fresh-frozen plasma (FFP) units during 48-h storage after thawing. Octaplas bags of different blood groups and individual FFP units were thawed and stored at either +4 degrees C or at room temperature (RT) for 48 h. Samples drawn during the observation period were investigated on various coagulation factor and protease inhibitor activities using standard coagulation and chromogenic assays. The generation of FVIIa was followed as a marker of coagulation factor activation. All investigated coagulation factors and protease inhibitors were stable for at least 8h during storage of Octaplas at +4 degrees C. FVIII levels started to decline earlier in FFP than in Octaplas at both storage temperatures. Stored Octaplas OD660 values were more stable during the storage period than FFP OD660 values, whereas VWF multimeric patterns were comparably stable in both types of plasma. In conclusion, this stability study has demonstrated that thawed Octaplas maintains its high quality, even with a time safety margin, for 8 h at +4 degrees C and for 6 h at RT. In general, there was more variability in coagulation factor levels among different FFP units compared with different Octaplas batches.

Quantification of antithrombin isoform proportions in plasma samples of healthy subjects, sepsis patients, and in antithrombin concentrates.

Antithrombin (AT) circulates in plasma in two isoforms, AT-alpha (90-95%) and AT-beta (5-10%). AT isoform proportions were measured in plasma samples of 17 healthy subjects and 26 posttraumatic or postoperative septic patients, as well as in 4 commercially available AT concentrates. Total AT was immune-purified from plasma and concentrates. Micellar electrokinetic chromatography was used to analytically separate and quantify the isoforms. Compared with plasma samples of healthy donors, septic plasmas revealed significantly reduced AT activity (p < 0.001) and beta-isoform content (p < 0.05). AT-beta correlated inversely with urea and creatinine serum concentrations (p < 0.01), indicating a relationship between better renal function and higher beta-isoform content. beta-Isoform neither correlated with age, gender, and 28-day mortality, nor with plasma concentrations of various inflammatory and organ function parameters. The commercial AT concentrate, which is equivalent to the current WHO standard, had an AT-beta content close to that found in plasma of healthy subjects. The availability of this novel quantitative AT isoform assay allows, for the first time, a closer look at the role of AT isoforms in hemostasis and sepsis pathophysiology.

Adverse effect of heparin on antithrombin action during endotoxemia: microhemodynamic and cellular mechanisms.

A recent clinical sepsis trial reported a significant reduction in 90-day mortality by antithrombin (AT) exclusively in the subgroup of patients without simultaneous heparin prophylaxis. Patients additionally receiving heparin did not benefit from AT treatment. Herein, we studied the microhemodynamic and cellular mechanisms of this adverse effect of heparin on AT actions by the use of intravital microscopy and granulocyte culturing. In Syrian golden hamsters normotensive endotoxemia was induced by 2 mg/kg endotoxin (LPS, E. coli) i.v. In a first group of animals, AT (AT, 250 IU/kg i.v., n = 6) was given 5 min before LPS administration. A second group of animals (Heparin + AT, n = 5) received AT (250 IU/kg i.v.) combined with unfractionated heparin (sodium heparin, 100 IU/kg/24 h, i.v.). Additional animals (LMWH + AT, n = 5) received AT (250 IU/kg i.v.) combined with LMWH (nadroparin 47.5 IU anti-Xa/kg, s.c., 2 h before LPS). LPS-treated animals, which received only saline, served as controls (control, n = 6). Using dorsal skinfold fold preparations, LPS-induced microvascular leukocyte-endothelial cell interaction (LE) and alteration of functional capillary density (FCD) were studied by intravital video fluorescence microscopy. In controls, LPS induced a massive increase in LE with a maximum at 8 h and an impressive decrease in FCD over a 24-hour period. Both LPS effects were effectively prevented by AT treatment (p < 0.01), whereas Heparin + AT and LMWH + AT animals showed microcirculatory alterations comparable to that in controls. In additional in vitro chemotaxis assays. AT blocked neutrophil chemotaxis, an effect reversed by both unfractionated heparin and LMWH. Thus, our study elucidates a relevant in vivo and in vitro unfractionated heparin and LMWH adverse effect in the microcirculatory actions of AT during endotoxemia. These results indicate that heparin should be avoided to permit AT to modulate LPS-induced inflammatory responses.