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Despite >80 years of clinical experience with coagulation factor VIII (FVIII) inhibitors, surprisingly little is known about the in vivo mechanism of this most serious complication of replacement therapy for hemophilia A. These neutralizing antidrug alloantibodies arise in ∼30% of patients. Inhibitor formation is T-cell dependent, but events leading up to helper T-cell activation have been elusive because of, in part, the complex anatomy and cellular makeup of the spleen. Here, we show that FVIII antigen presentation to CD4+ T cells critically depends on a select set of several anatomically distinct antigen-presenting cells, whereby marginal zone B cells and marginal zone and marginal metallophilic macrophages but not red pulp macrophages (RPMFs) participate in shuttling FVIII to the white pulp in which conventional dendritic cells (DCs) prime helper T cells, which then differentiate into follicular helper T (Tfh) cells. Toll-like receptor 9 stimulation accelerated Tfh cell responses and germinal center and inhibitor formation, whereas systemic administration of FVIII alone in hemophilia A mice increased frequencies of monocyte-derived and plasmacytoid DCs. Moreover, FVIII enhanced T-cell proliferation to another protein antigen (ovalbumin), and inflammatory signaling-deficient mice were less likely to develop inhibitors, indicating that FVIII may have intrinsic immunostimulatory properties. Ovalbumin, which, unlike FVIII, is absorbed into the RPMF compartment, fails to elicit T-cell proliferative and antibody responses when administered at the same dose as FVIII. Altogether, we propose that an antigen trafficking pattern that results in efficient in vivo delivery to DCs and inflammatory signaling, shape the immunogenicity of FVIII., (© 2023 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

Development of neutralizing antibodies against biotherapeutic agents administered to prevent or treat various clinical conditions is a longstanding and growing problem faced by patients, medical providers and pharmaceutical companies. The hemophilia A community has deep experience with attempting to manage such deleterious immune responses, as the lifesaving protein drug factor VIII (FVIII) has been in use for decades. Hemophilia A is a bleeding disorder caused by genetic mutations that result in absent or dysfunctional FVIII. Prophylactic treatment consists of regular intravenous FVIII infusions. Unfortunately, 1/4 to 1/3 of patients develop neutralizing anti-FVIII antibodies, referred to clinically as "inhibitors," which result in a serious bleeding diathesis. Until recently, the only therapeutic option for these patients was "Immune Tolerance Induction," consisting of intensive FVIII administration, which is extraordinarily expensive and fails in ~30% of cases. There has been tremendous recent progress in developing novel potential clinical alternatives for the treatment of hemophilia A, ranging from encouraging results of gene therapy trials, to use of other hemostatic agents (either promoting coagulation or slowing down anti-coagulant or fibrinolytic pathways) to "bypass" the need for FVIII or supplement FVIII replacement therapy. Although these approaches are promising, there is widespread agreement that preventing or reversing inhibitors remains a high priority. Risk profiles of novel therapies are still unknown or incomplete, and FVIII will likely continue to be considered the optimal hemostatic agent to support surgery and manage trauma, or to combine with other therapies. We describe here recent exciting studies, most still pre-clinical, that address FVIII immunogenicity and suggest novel interventions to prevent or reverse inhibitor development. Studies of FVIII uptake, processing and presentation on antigen-presenting cells, epitope mapping, and the roles of complement, heme, von Willebrand factor, glycans, and the microbiome in FVIII immunogenicity are elucidating mechanisms of primary and secondary immune responses and suggesting additional novel targets. Promising tolerogenic therapies include development of FVIII-Fc fusion proteins, nanoparticle-based therapies, oral tolerance, and engineering of regulatory or cytotoxic T cells to render them FVIII-specific. Importantly, these studies are highly applicable to other scenarios where establishing immune tolerance to a defined antigen is a clinical priority., (Copyright © 2020 Lacroix-Desmazes, Voorberg, Lillicrap, Scott and Pratt.)

Historically, clinical trials of haemophilia with inhibitors (HwI) have been challenged by the small patient population. New approaches to clinical trial methodology and statistical modelling could potentially be used for study optimization. The aim of this work was to evaluate the impact of different trial designs and study conditions on the estimated drug potency and power, and compare traditional statistical methods with repeated time-to-event (RTTE) modelling in terms of power. Bleeding information from a clinical trial of 23 haemophilia patients with inhibitors treated on-demand was used to develop a baseline RTTE model using NONMEM. Clinical trial simulations for a hypothetical anti-haemophilic drug were performed, by adding a drug effect and a literature-derived placebo effect to the baseline RTTE model, using different trial designs (parallel-group, placebo-controlled parallel-group, crossover and placebo-controlled crossover designs) and study conditions, including sample size, study duration and doses. The precision and accuracy of the estimated drug potency (EC 50 ) and power for different trial designs, study conditions and statistical methods (RTTE modelling, t-test and negative binomial regression) were evaluated. The developed baseline RTTE model accurately described the clinical data. The crossover designs displayed up to four-fold higher precision of the estimated EC 50 and three-fold higher power relative to the parallel-group trial designs. Furthermore, RTTE modelling provided a higher power relative to the traditional statistical tests. We found that crossover designs in combination with RTTE modelling can reduce the required sample size and study duration, while ensuring high power and precise estimation of EC 50 , in clinical trials of HwI., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The role of tissue factor (TF) as the major initiator of hemostatic blood coagulation is well recognized. The ability to form an adequate hemostatic clot is essential to the normal healing of an injury by staunching bleeding, stabilizing the injured tissue, and serving as a scaffold for repair processes. Also, some molecules produced during hemostasis, particularly thrombin, have cytokine and growth factor-like activities that contribute to inflammation and repair. However, TF itself has activities as a regulator of cellular processes via direct signaling, as well as by facilitating activation of proteolytically activated receptors by activated factors VII and X. The importance of hemostasis in the host response to injury makes it very difficult to separate the hemostatic from nonhemostatic effects of TF on wound healing. The literature in this area remains sparse but suggests that TF influences the course and tempo of healing by cell signaling events that impact inflammation, epithelialization, and angiogenesis., Competing Interests: Disclosure The authors report no conflicts of interest in this work., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

There are some issues in the current factor (F)VIII replacement therapy for severe hemophilia A. One is mental and physical burden for the multiple intravenous infusions, and the other is difficulty in the hemostatic treatment for the patients with FVIII inhibitor. The development of novel drug with fully hemostatic effect, simply procedure, and long-acting reaction has been expected. Recently, FVIIIa-mimicking humanized recombinant bispecific antibody (ACE910) against FIXa and FX was developed. In the non-human clinical study, primate model of acquired hemophilia A demonstrated that the ACE910 was effective on both on-going and spontaneous bleedings. A phase I clinical study was conducted in healthy adults by single subcutaneous infusion of ACE910, followed by the patients' part study, Japanese patients with severe hemophilia A without or with inhibitor were treated with once-weekly subcutaneous injection of ACE910 at three dose levels for 12 successive weeks. There was no significant adverse event related to ACE910 in the clinical and laboratorial findings, and t1/2 of ACE910 was ∼30 days. The median annual bleeding rates were reduced very markedly dose-dependently, independently of inhibitor. Furthermore, among the patients with dose escalation, bleeding rate was decreased as ACE910 dose was increased. In conclusion, ACE910 would have a number of promising features: its high subcutaneous bioavailability and long half-life make the patients possible to be injected subcutaneously with a once-a-week or less frequency. In addition, ACE910 would provide the bleeding prophylactic efficacy, independently of inhibitor., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Unlabelled: ESSENTIALS: Anti-factor VIII (FVIII) inhibitory antibody formation is a severe complication in hemophilia A therapy. We genetically engineered and characterized a mouse model with complete deletion of the F8 coding region. F8(TKO) mice exhibit severe hemophilia, express no detectable F8 mRNA, and produce FVIII inhibitors. The defined background and lack of FVIII in F8(TKO) mice will aid in studying FVIII inhibitor formation., Background: The most important complication in hemophilia A treatment is the development of inhibitory anti-Factor VIII (FVIII) antibodies in patients after FVIII therapy. Patients with severe hemophilia who express no endogenous FVIII (i.e. cross-reacting material, CRM) have the greatest incidence of inhibitor formation. However, current mouse models of severe hemophilia A produce low levels of truncated FVIII. The lack of a corresponding mouse model hampers the study of inhibitor formation in the complete absence of FVIII protein., Objectives: We aimed to generate and characterize a novel mouse model of severe hemophilia A (designated the F8(TKO) strain) lacking the complete coding sequence of F8 and any FVIII CRM., Methods: Mice were created on a C57BL/6 background using Cre-Lox recombination and characterized using in vivo bleeding assays, measurement of FVIII activity by coagulation and chromogenic assays, and anti-FVIII antibody production using ELISA., Results: All F8 exonic coding regions were deleted from the genome and no F8 mRNA was detected in F8(TKO) mice. The bleeding phenotype of F8(TKO) mice was comparable to E16 mice by measurements of factor activity and tail snip assay. Similar levels of anti-FVIII antibody titers after recombinant FVIII injections were observed between F8(TKO) and E16 mice., Conclusions: We describe a new C57BL/6 mouse model for severe hemophilia A patients lacking CRM. These mice can be directly bred to the many C57BL/6 strains of genetically engineered mice, which is valuable for studying the impact of a wide variety of genes on FVIII inhibitor formation on a defined genetic background., (© 2015 International Society on Thrombosis and Haemostasis.)

In nonsevere haemophilia A (HA) patients the presence of an inhibitor may exacerbate the bleeding phenotype dramatically. There are very limited data on the optimal therapeutic approach to eradicate inhibitors in these patients. We aimed to describe inhibitor eradication treatment in a large cohort of unselected nonsevere HA patients with inhibitors. We included 101 inhibitor patients from a source population of 2,709 nonsevere HA patients (factor VIII 2-40 IU/dl), treated in Europe and Australia (median age 37 years, interquartile range (IQR) 15-60; median peak titre 7 BU/ml, IQR 2-30). In the majority of the patients (71 %; 72/101) the inhibitor disappeared; either spontaneously (70 %, 51/73) or after eradication treatment (75 %, 21/28). Eradication treatment strategies varied widely, including both immune tolerance induction and immunosuppression. Sustained success (no inhibitor after rechallenge with factor VIII concentrate after inhibitor disappearance) was achieved in 64 % (30/47) of those patients rechallenged with FVIII concentrate. In high-titre inhibitor patients sustained success was associated with eradication treatment (unadjusted relative risk 2.3, 95 % confidence interval 1.3-4.3), compared to no eradication treatment. In conclusion, in nonsevere HA patients most inhibitors disappear spontaneously. However, in 35 % (25/72) of these patients an anamnestic response still can occur when rechallenged, thus disappearance in these patients does not always equal sustained response. Treatment for those requiring eradication has to be decided case by case, as one single approach is unlikely to be appropriate for all.

There is no prospective evidence on inhibitor recurrence among haemophilia A patients with low titre inhibitors or history of inhibitors, and whether or how therapeutic choices affect the risk of recurrence. The aims of this study were to synthesise safety data in patients with moderate-severe haemophilia A and with low titre inhibitors or inhibitor history enrolled in the rAHF PFM (ADVATE) - Post-Authorization Safety Studies (ADVATE-PASS) international programme. The study was conducted in clinics participating to the ADVATE PASS programme. The patient population consisted of patients entering the studies with low titre (≤ 5 BU) inhibitors or a positive personal history of inhibitors. Patients on Immune Tolerance Induction at study entry were excluded. Primary outcome was new or recurrent inhibitor titre > 5 BU. Secondary outcomes were any increase of inhibitor titre not reaching 5 BU; any unexplained change in treatment regimen. Primary analysis was done by two-stage random effects meta-analysis. Secondary analysis was done by a hierarchical Bayesian random effects logistic model. A total of 219 patients from seven studies were included. Of these 214 (97.7 %) patients had been previously treated for more than 50 exposure days. Two hundred ten patients had positive history for inhibitors, nine a baseline measurable titre. No patient presented a primary outcome event (95 % confidence interval [CI] 0-1.6 %). Six patients with previous history developed a low titre recurrence (overall rate 2.2, 95 %CI 0-4.8 %). When any increase of inhibitor titre or any treatment change was accounted for, overall 3.7 % (95 % CI 0 %-8.0 %) of patients experienced the outcome. In conclusion, the observed rate of events does not support the definition of this population as at high risk for inhibitor development.

Background: Fibrocaps, a ready-to-use, dry-powder fibrin sealant containing human plasma-derived thrombin and fibrinogen, is being developed as an adjunct for surgical hemostasis., Materials and Methods: Safety and efficacy of Fibrocaps applied directly or by spray device, in combination with gelatin sponge, was compared with that of gelatin sponge-alone in two randomized, single-blind controlled trials: FC-002 US (United States) and FC-002 NL (the Netherlands). A total of 126 adult patients were randomized (Fibrocaps: n = 47 [FC-002 US], n = 39 [FC-002 NL]; gelatin sponge alone: n = 23 [FC-002 US], n = 17 [FC-002 NL). One bleeding site was treated during a surgical procedure (n = 125). Time to hemostasis (primary end point) was measured, with a 28-d safety follow-up. Four surgical indications included hepatic resection (n = 58), spinal procedures (n = 37), peripheral vascular procedures (n = 30), and soft tissue dissection (n = 1)., Results: Mean (standard deviation) time to hemostasis was significantly shorter after Fibrocaps treatment than after gelatin sponge alone (FC-002 US: 1.9 [1.3] versus 4.8 min [3.1], P < 0.001; FC-002 NL: 2.2 [1.3] versus 4.4 min [3.1], P = 0.004). The incidence of hemostasis was greater after Fibrocaps compared with that of gelatin sponge alone within 3 min (FC-002 US: 83% versus 35%, P < 0.001; FC-002 NL: 77% versus 53%, P = 0.11), 5 min (94% versus 61%, P = 0.001; 95% versus 71%, P = 0.022), and 10 min (100% versus 78%, P = 0.003; 100% versus 82%, P = 0.025). Adverse events were consistent with surgical procedures performed and patients' underlying diseases and generally similar between treatment arms; most were mild or moderate in severity. Non-neutralizing antithrombin antibodies were detected in 5% of Fibrocaps-treated patients on day 29., Conclusions: Fibrocaps had good safety and efficacy profiles, supporting continuing clinical development as a novel fibrin sealant., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Proteolytic cleavage of factor VIII (FVIII) to activated FVIIIa is required for participation in the coagulation cascade. The A2 domain is no longer covalently bound in the resulting activated heterotrimer and is highly unstable. Aspartic acid (D) 519 and glutamic acid (E) 665 at the A1-A2 and A2-A3 domain interfaces were identified as acidic residues in local hydrophobic pockets. Replacement with hydrophobic valine (V; D519V/E665V) improved the stability and activity of the mutant FVIII over the wild-type (WT) protein in several in vitro assays. In the current study, we examined the impact of mutations on secondary and tertiary structure as well as in vivo stability, pharmacokinetics (PK), efficacy, and immunogenicity in a murine model of Hemophilia A (HA). Biophysical characterization was performed with far-UV circular dichroism (CD) and fluorescence emission studies. PK and efficacy of FVIII was studied following i.v. bolus doses of 4, 10 and 40 IU/kg with chromogenic and tail clip assays. Immunogenicity was measured with the Bethesda assay and ELISA after a series of i.v. injections. Native secondary and tertiary structure was unaltered between variants. PK profiles were similar at higher doses, but at 4 IU/kg plasma survival of D519V/E665V was improved. Hemostasis at low concentrations was improved for the mutant. Immune response was similar between variants. Overall, these results demonstrate that stabilizing mutations in the A2 domain of FVIII can improve HA therapy in vivo.

JMI-thrombin is used as topical hemostatic agent. While earlier clinically available JMI were reported to produce immunologic responses upon repeated exposure, the improved JMI, Recothrom?, and Evithrom? are claimed to be less immunogenic. Recothrom, despite its reduced immunogenic nature, upon repeated administration may result in the generation of antibodies (Abs) and that may cross react with bovine and human thrombin. Therefore, groups of rabbits were challenged repeatedly with Recothrom, Evithrom, and JMI over a 9-month period. Pre-immune blood and antiserum were collected from each rabbit on different time point. To determine their relative cross reactivity, JMI, Recothrom, and Evithrom were evaluated by western blotting using the rabbit IgG fractions. The results suggest that anti-Recothrom Abs cross-react with Evithrom and JMI in a time dependent fashion. Anti-JMI Abs did not cross-react with Recothrom, and Evithrom. Also, anti-Evithrom did not show any cross-reactivity with Recothrom and JMI at any time.

We report a prospective trial of 55 previously untreated patients (PUPs) and minimally treated patients (MTPs) with severe/moderately severe haemophilia A (baseline factor VIII [FVIII] ≤2%) treated with a single FVIII replacement product. It was the objective of this study to evaluate the immunogenicity, efficacy, and safety of rAHF-PFM (Advate®). On-demand or prophylactic treatment regimens were determined at the discretion of the investigator. rAHF-PFM was also permitted for perioperative management. There were 633 bleeding episodes (BEs), including 517 treated, and 466 rated for efficacy. Haemostatic efficacy was considered excellent/good in 93% of 466 rated treatments. Of 517 treated BEs, 463/517 (90%) were managed with one (356/517 [69%]) or two infusions (107/517 [21%]). There were 27 surgeries. Intraoperative (n=22) and postoperative (n=25) haemostatic efficacies were considered excellent or good in 100% of rated surgeries. Related serious adverse events (SAEs) were inhibitor development in 16/55 (29.1%) subjects who received at least one infusion of rAHF-PFM. Non-serious, related adverse events (AEs) were few in number (14 in eight subjects). The odds ratio (OR [95% Confidence Interval, CI]) of developing inhibitors was significantly higher in subjects with a family history of inhibitor (4.95[1.29-19.06]), non-Caucasian ethnicity (4.18, [1.18-14.82]), and intensive treatment at high dose (4.5 [1.05-19.25]) within ≤20 exposure days (EDs). In conclusion, rAHF-PFM was safe and effective for the management and perioperative coverage of PUPs/MTPs with severe/moderately severe haemophilia A. This report supports previous findings from studies in which family history of inhibitor, non-Caucasian ethnicity, and high intensity treatment were associated with high risk of inhibitor development.

Background: This Phase 4, open-label study evaluated the immunogenicity and safety of a second exposure to recombinant human thrombin (rThrombin) in adult patients with previous exposure to rThrombin., Study Design: Topical rThrombin was applied as a hemostatic aid during a surgical procedure (day 1). Adverse events and clinical laboratory abnormalities were monitored to day 29 (study end). Immunogenicity samples were collected on days 1 and 29. Thirty-one patients were treated at 9 study sites; 30 patients completed the study., Results: Mean age was 59.5 years; 61.3% of patients were male. Study operations types included spinal (n = 23 of 31; 74.2%), arterial reconstruction or peripheral arterial bypass (n = 4; 12.9%), arteriovenous vascular access procedure (n = 3; 9.7%), and other (n = 1; 3.2%). A median of 10 mL rThrombin (1,000 IU/mL; range 5 to 60 mL) was prepared per patient. Median elapsed time since previous rThrombin exposure was 1.3 years (range 19 days to 3.3 years). Recombinant human thrombin was not observed to be immunogenic; no patients (n = 0 of 30, 0%; 95% CI 0.0%, 11.6%) became positive for anti-rThrombin product antibodies at day 29, approximately 1 month after the second exposure to rThrombin. The most commonly reported adverse events were procedural pain (n = 23 of 31, 74.2%), constipation (n = 8, 25.8%), and nausea (n = 8, 25.8%) All adverse events and clinical laboratory abnormalities were considered unrelated to treatment. For the majority of patients, maximal severity of any adverse event was mild or moderate., Conclusions: The immunogenicity and safety results of this Phase 4 rThrombin trial suggest that patients with known previous exposure may be safely re-exposed to topical rThrombin., (Copyright © 2011 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

Objective: To determine how the anti-inflammatory properties of aprotinin impact on postoperative complications in children undergoing the Fontan procedure., Methods: We included all patients between 14 months and 18 years (n=56) undergoing a Fontan operation at our institution between January 2005 and June 2009. The study group (n=29) included patients from January 2005 through December 2007 all of whom received aprotinin. The control group (n=27) included all patients from January 2008 through June 2009 who did not receive aprotinin. We reviewed all medical records and collected preoperative, intraoperative and postoperative data. Duration and volume of chest tube drainage were the primary outcome measures., Results: Of the 20% of patients who had postoperative arrhythmias, multivariate logistic regression analysis demonstrated only aprotinin was associated with significantly decreased postoperative arrhythmias (P=0.01). Renal function and fenestration or Fontan thrombosis did not differ significantly; there was no statistically significant difference in volume or duration of chest tube drainage. Median duration of chest tube drainage was 7 days in the aprotinin group and 8 days for patients who did not receive aprotinin (P=0.36)., Conclusion: The anti-inflammatory properties of aprotinin may be protective against postoperative arrhythmias. Aprotinin does not confer increased risks of prolonged chest tube drainage, renal dysfunction or thrombosis in patients undergoing the Fontan procedure.

Topical bovine thrombin has been associated with immune responses and anecdotal reports of coagulopathy. This open-label study assessed the impact on clinical hemostasis of human antibodies to bovine thrombin (aBT) or factor V/Va (aBV/Va) in response to topical bovine thrombin (THROMBIN-JMI) in patients both with and without preexisting anti-bovine antibodies. Noninferiority analysis assessed primary endpoint for mean shift from baseline activated partial thromboplastin time (aPTT) at 48 hours postsurgery; secondary endpoints included changes from baseline antibodies/titers and coagulation parameters through 8 weeks postsurgery. A total of 550 patients underwent surgery with THROMBIN-JMI utilized at investigator's discretion. Adjusted mean aPTT change in (+)aBT/(+)THROMBIN-JMI cohort was greater than (-)aBT/(-)THROMBIN-JMI cohort; 4.67-second upper confidence bound exceeded 4.5-second margin (based on assumed mean aPTT of 30 seconds) and noninferiority was not met. Post hoc analysis indicated noninferiority would have been met had noninferiority margin been set prior at relative 15% of actual baseline aPTT. Antibodies/titers were unchanged by THROMBIN-JMI exposure 48 hours postsurgery and unrelated to postsurgical changes in coagulation. Thus, THROMBIN-JMI exposure in patients with/without preexisting aBT or aBV/Va does not alter hemostasis.

Background: Previous studies of recombinant human thrombin (rThrombin) enrolled adult and adolescent patients. This phase 4, open-label, single-group study was conducted in pediatric patients undergoing synchronous burn wound excision and skin grafting to provide information regarding the safety and immunogenicity of rThrombin (primary and secondary endpoints) in this population., Methods: Topical rThrombin was applied as a hemostatic aid during a surgical procedure (day 1). Adverse events and clinical laboratory abnormalities were recorded during the study. Immunogenicity samples were collected on days 1 and 29 (study end). Study results were summarized with descriptive statistics., Results: Thirty patients enrolled and 28 completed the study. Mean age was 6.9 years (range, 0.9-17.8 years); 40.0% of patients were girls. Flame and scald were the most common burn types (33.3% each, n = 10/30). Mean graft size was 3.6% total body surface area. Procedural pain (50.0% patients), pruritus (43.3%), and anemia (30.0%) were the most commonly reported adverse events. All adverse events and clinical laboratory abnormalities were considered unrelated to treatment. No patients developed anti-rThrombin product antibodies at day 29., Conclusions: In pediatric patients undergoing burn wound excision and skin grafting, rThrombin was well tolerated and did not lead to the formation of anti-rThrombin product antibodies., (Copyright © 2011. Published by Elsevier Inc.)

Postoperative bleeding may occur from a number of etiologies. An uncommon cause of postoperative bleeding is immune-mediated coagulopathy resulting from reexposure to a topical hemostat containing bovine thrombin. Some patients may develop antibodies to bovine thrombin (and other bovine coagulation proteins present in the product) which cross-react with human coagulation proteins, resulting in a coagulopathy, and occasionally, in serious bleeding and death. Most of the clinical information on this coagulopathy is in the adult medical literature. This article reviews the literature on pediatric cases with this coagulopathy and summarizes clinical outcomes and effective therapies.

Background: Immunoassays that detect antibovine thrombin product antibodies are not widely available. However, knowing whether these antibodies are present preoperatively would be useful because re-exposure to bovine thrombin-containing products is contraindicated in patients with pre-existing antiproduct antibodies due to the risk of developing immune-mediated coagulopathies. In these exploratory analyses, we characterized one aspect of immune sensitization, the persistence of circulating antibodies after exposure to bovine thrombin product., Study Design: Elapsed time since a historical surgical procedure with documented or highly likely use of bovine thrombin product was determined for 204 patients enrolled in a recently completed trial. After study completion, baseline samples were assayed for antibovine thrombin product antibodies using validated immunoassays. Antibody data were sorted by time elapsed since the historical procedure. The proportion of patients with antibovine thrombin product antibodies and 95% confidence interval (CI) were determined for each 1-year period, providing an estimate for antibody persistence., Results: Antibovine thrombin product antibodies were detected in 20.7% of patients (23 of 111; 95% CI 14.2%, 29.2%) with ≤1 year since the historical surgical procedure; 6.8% of patients (3 of 44; 95% CI 1.68%, 18.9%) with 1 to <2 years; 16.1% of patients (5 of 31; 95% CI 6.62%, 33.1%) with 2 to <3 years; and 5.6% of patients (1 of 18; 95% CI 0.00%, 27.6%) with ≥3 years since the historical procedure., Conclusions: The proportion of patients with antibovine thrombin product antibodies ranged from 5.6% to 20.7% across the multiyear postoperative window. Clinicians should be aware that antibodies to bovine thrombin products may persist for years after exposure., (Copyright © 2010 American College of Surgeons. Published by Elsevier Inc. All rights reserved.)

It has been reported that patients exposed to topical bovine thrombin preparations may develop antibodies against bovine thrombin, factr V or various other proteins found in these preparations. Such antibodies can cross-react with human endogenous coagulation proteins and may lead to alterations in the coagulation laboratory parameters, hypersensitivity reactions, and severe bleeding or thrombosis. The underlying mechanisms for the coagulopathy are not fully understood yet. To better understand the cross-reactivity of anti-bovine thrombin antibodies with human corresponding coagulation proteins, bovine crude thrombin, and its purified versions, thrombin 4A (the old version of Thrombin-JMI before year 2008) and thrombin 4B (the current version of Thrombin-JMI on market), were used to generate relevant anti-bovine thrombin immunoglobulin G (IgGs) in rabbits. Using Western blotting, the cross-reactivity of each IgG with human alpha-thrombin and a recombinant version of human thrombin (Recothrom) was investigated. The results indicated that no cross-reactivity with either human alpha-thrombin or Recothrom was observed with both anti-bovine crude thrombin IgGs and thrombin 4B IgGs. However, anti-bovine thrombin 4A IgGs showed apparent cross-reactivity with human alpha-thrombin and Recothrom in a protein amount-dependent manner. Furthermore, the results revealed that the cross-reactivity of anti-bovine thrombin 4A IgGs with human alpha-thrombin and Recothrom was immunization time-dependent. The minimum concentration of 4A IgG required to exhibit cross-reactivity with human alpha-thrombin and Recothrom varied considerably among individual rabbits. These results indicate that rabbit anti-bovine thrombin IgGs can cross-react with human alpha-thrombin and Recothrom, suggesting that human antibodies against bovine thrombin may also cross-react with human recombinant thrombin. Thus, the patients who were previously exposed to bovine thrombin may also develop antibodies which can cross-react with human recombinant thrombin.

Background: We evaluated safety and immunogenicity observations pooled from 8 clinical trials of recombinant human thrombin (rThrombin), an active topical hemostatic agent., Study Design: Recombinant thrombin was applied with an absorbable gelatin sponge or spray applicator during a surgical procedure (day 1). Adverse events and laboratory parameters were monitored until study end (day 29). Immunogenicity was evaluated after study completion on plasma samples collected at baseline and on day 29., Results: Studies included 583 rThrombin-treated patients (median age, 59 years; 54% men). Surgical procedures included: spinal, 33% of patients; hepatic resection, 14%; peripheral arterial bypass, 23%; arteriovenous graft formation for hemodialysis access, 18%; and skin graft after burn wound excision, 12%. Adverse events reported for >or= 10% patients included incision site pain, procedural pain, nausea, constipation, pyrexia, anemia, insomnia, vomiting, and pruritus. Five of 552 patients developed antibodies to rThrombin (0.9%; 95% CI, 0.3 to 2.1; day 29); antibodies did not neutralize the biologic activity of native human thrombin. At baseline, 12 patients had pre-existing, antibodies recognizing rThrombin (12 of 552; 2.2%; 95% CI, 1.1 to 3.8); these patients had no previous exposure to rThrombin and their antibody titer did not increase >or= 1.0 unit (>or= 10-fold) at day 29., Conclusions: Results from 8 clinical trials collectively demonstrated that rThrombin is well tolerated in numerous surgical settings when used as a topical adjunct to hemostasis. Adverse events and changes in laboratory parameters were consistent with commonly reported postoperative events. Less than 1% of patients developed antibodies to rThrombin; the antibodies did not neutralize native human thrombin.

Surgical hemostasis may be achieved by using a number of physical, chemical, or biologic methods. One such method is with topical thrombin; however, one member of that class of drugs, bovine-derived thrombin, is associated with potentially serious consequences such as development of immune-mediated coagulopathy. This case report describes a 61-year-old man with peripheral artery disease who presented with a nonhealing ulcer between his toes. Previous exposure to bovine thrombin was unknown but was considered likely because of his extensive surgical history that included procedures in which topical thrombin is commonly used. The patient was admitted and underwent lower extremity revascularization during which he received his first documented exposure to bovine-derived thrombin. By postoperative day 9, he developed a 2.7-cm retroperitoneal hematoma that had progressed to 9.6 cm by postoperative day 13. Evacuation of the hematoma was performed, during which the patient received his second known exposure to topical bovine thrombin. Based on a plasma mixing study on postoperative day 25, presence of factor V and thrombin inhibitors was suspected. A hematology consultation determined that the patient had developed an immune-mediated coagulopathy manifested as exaggerated laboratory coagulation values that continued even after discontinuation of oral anticoagulation, treatment with multiple transfusions of fresh frozen plasma, and intravenous vitamin K administration. The patient was discharged, after no further bleeding episodes had occurred, on postoperative day 29. Although determining previous exposure to bovine-derived thrombin or presence of antibodies can be difficult, a surgeon's index of suspicion should be raised in patients experiencing coagulopathy if they have previously undergone vascular, cardiac, or spinal procedures in which they were most likely exposed to topical thrombin.

Background: The immunogenicity and safety of recombinant human thrombin (rThrombin) were evaluated in this phase 3b, open-label, single-group, multisite study of 209 adult vascular and spinal operation patients at high risk for preexisting anti-bovine thrombin product antibodies., Study Design: Patients received rThrombin applied as a topical hemostat during a surgical procedure (day 1). Immunogenicity samples were collected at baseline and approximately 1 month after operation (day 29) and were analyzed after study participation., Results: Mean patient age was 61.5 years; median number of previous surgical procedures was 5.0 (range, 1 to 25). Operation types included spinal (n = 89 of 209 [43%]), arterial reconstruction (including peripheral arterial bypass; n = 75 of 209 [36%]), and arteriovenous vascular access procedures (n = 45 of 209 [22%]). All patients had confirmed or highly likely previous bovine thrombin exposure; at baseline, 15.6% of patients (n = 32 of 205) had preexisting anti-bovine thrombin antibodies. Of 200 patients with complete immunogenicity evaluations, 31 had preexisting anti-bovine thrombin antibodies (15.5%), and 4 had preexisting anti-rThrombin product antibodies (2.0%). None of the 200 patients became antibody positive for rThrombin antibodies on day 29 (seroconversion or > or = 10-fold increase in titer). Adverse events and laboratory results were consistent with a surgical population with substantial comorbidities. Patients with preexisting antibodies to bovine thrombin were older (p = 0.04) and had undergone more surgical procedures previously (p < 0.001) than patients without preexisting antibodies., Conclusions: Results of this study confirm the low immunogenicity of rThrombin and suggest that rThrombin can be used safely as an aid to hemostasis in patients with or without preexisting anti-bovine thrombin antibodies. A sizeable proportion of this vascular and spinal operation patient population (15.6%) had preexisting anti-bovine thrombin antibodies; these patients are at risk for immune responses after reexposure to bovine thrombin.

This study evaluated the safety, immunogenicity, and hemostatic effect of recombinant human Thrombin (rThrombin), in patients undergoing skin grafting for burns. This was a phase 2 multiple site, single-arm, open-label study in patients receiving partial- or full-thickness autologous grafts. rThrombin was applied using a spray applicator to newly excised wounds of 1 to 4% body surface area at 5 minutes intervals for up to 20 minutes, after point source bleeding was stopped. Adverse events, skin graft survival, and formation of anti-rThrombin antibodies were measured at baseline and Day 29. There were no deaths or study drug discontinuations. Adverse events occurred in 63 of 72 patients (88%), and were typical of sequelae of skin grafting. Hemostasis was achieved within 20 minutes after application of rThrombin in 65 of 71 patients (91.5%). Skin graft failure occurred in 4 patients (6%). At the day 29 evaluation, for those patients who returned, 88.9% had > or =90% graft survival. One patient (1 of 70, 1.4%) had specific, low titer antibodies to rThrombin at baseline, but no increase in titer posttreatment; a second patient (1 of 62, 1.6%), developed antibodies to rThrombin at day 29. None of the antibodies neutralized native human thrombin. In excised burn wounds, hemostasis at 20 minutes was achieved in 91.5% of patients and skin graft survival was excellent. There was a low rate of antibodies to rThrombin at baseline (1.4%) and a low rate of anti-rThrombin antibody formation at day 29 (1.6%). rThrombin was well tolerated when administered with a pump spray.

Background: Topical bovine thrombin has been used to accelerate attainment of hemostasis in the surgical setting for >60 years, and its immunogenicity has been widely reported. Although the development of antibodies is inherent in the introduction of any non-self-therapeutic protein such as bovine-sourced thrombin, there are questions about the relationship between the presence of antibodies to constituents of the therapeutic protein preparation and the occurrence of clinically relevant adverse events (AEs)., Objective: This review examines the proposed mechanisms for the immunogenicity of topical bovine thrombin preparations and summarizes available evidence from randomized clinical trials, observational studies, and case reports to explore possible relationships between the reported immunogenicity of topical bovine thrombin and the occurrence of AEs., Methods: A search of MEDLINE (1966-August 2008) for studies published in English was conducted using the Medical Subject Heading terms surgery, antibodies, and hemorrhage, as well as equivalent key words for bovine, adverse events, and thrombin. For inclusion in the review, studies had to report clinical or laboratory safety data for patients exposed to topical bovine thrombin during surgery., Results: The evidence suggests that patients with repeated perioperative exposure to topical bovine thrombin have a 3- to 10-fold greater risk for development of antibodies to topical bovine thrombin than do patients with no history of surgery-related exposure to this agent. Early case reports associated the development of anti-bovine protein antibodies with bleeding and/or thrombotic complications. However, in one prospective, randomized controlled trial comparing topical bovine thrombin with topical recombinant human thrombin, 99.5% of patients in each treatment arm developed postoperative AEs. In an-other, 54% and 55% of patients in the respective treatment arms developed postoperative AEs. In a prospective, randomized controlled trial that compared topical bovine thrombin and plasma-derived human thrombin, 95.5% of patients in each treatment arm developed postoperative AEs., Conclusions: Repeated perioperative exposure to topical bovine thrombin may increase both the prevalence and titers of antibodies to >or=1 protein contained in nonhomogeneous topical bovine thrombin preparations. However, the evidence reviewed does not support a definitive association between preoperative or postoperative generation of anti-bovine protein antibodies and an increased risk of AEs in surgical patients treated with topical bovine thrombin.

Objective: To review published evidence suggesting a link between topical bovine thrombin (TBT) and important adverse events (AEs)., Research Design and Methods: English language articles and abstracts were obtained from MEDLINE using combinations of text and MeSH terms for thrombin, bovine thrombin and their trade names. References from summary articles were also retrieved. Published case reports, review articles, and retrospective, prospective or observational studies involving either immunogenicity or AEs were selected for further assessment. Retrieved articles were evaluated separately as AE case reports, quantitative studies of antibodies, or quantitative studies of AEs., Main Outcome Measures: Presence of case causal information, temporal pattern of case report publication, reproducibility of aggregate data findings, and study design features., Results: The major limitations of reviewed publications were insufficient information regarding TBT and other exposures, and designs in which linkage between laboratory immune phenomena and AEs could not be evaluated. While immunogenicity studies did support an increased risk for post-TBT antibodies, there was no consistent evidence that this led to an increased AE risk or severity. Common evidentiary deficiencies included case reports from high incidence environments, studies of combination or mixture products, biased study designs, lack of patient-level exposure data, inadequate control groups and insufficient sample sizes. The best designed study (a randomized, controlled comparison of TBT to a recombinant bovine product) documented post-TBT antibody production, but no important efficacy or AE differences. An examination of publication dates for case reports showed a peak between 1992 and 1994 followed by a substantial drop. Since 1997 the number of published AE case reports has continued to decline., Conclusions: TBT increases the risk for antibody elevations in patients. A careful review of published evidence does not show that either TBT itself or any associated elevations in anti-bovine antibodies are risk factors for clinically important AEs.

Thrombin is a common hemostatic drug used in surgical practice for over 100 years because of its simplicity and efficacy. Thrombin converts fibrinogen to fibrin, activates platelets, and induces vascular contraction. It is available in multiple forms, including human thrombin, bovine thrombin, and, most recently, human recombinant thrombin. Over 100 case reports of adverse reactions to bovine thrombin include hemorrhage, thrombosis, and substantial immune reaction when used on cardiovascular surgery patients. Approximately 30% of patients exposed to bovine thrombin develop cross-reacting antibodies. Thirty percent of patients with anticlotting factor antibodies develop abnormal coagulation that can be detected by prothrombin time, partial thromboplastin time, or thrombin time, which makes anticoagulation monitoring difficult. Patients with multiple elevated antibodies prior to surgery are also more likely to sustain adverse events. Animal studies confirm these immunological responses seen in humans. With the available clinical and laboratory data, a less immunogenic yet biologically effective thrombin should be available for use in our surgical patients.

Recombinant human thrombin (rhThrombin) is being developed as an alternative to thrombin products purified from pooled human or bovine plasma, which are currently marketed for topical hemostasis. Preclinical studies of rhThrombin were conducted prior to its evaluation as a topical adjunct to surgical hemostasis in clinical trials. No overt clinical pathology or signs were observed in cynomolgus monkeys following implantation of a gelatin sponge containing either rhThrombin or bovine thrombin to a surgical liver wound, and similar gross and microscopic wound healing characteristics were observed over an eight-week recovery period with either compound. Repeated subcutaneous injections of rhThrombin or bovine thrombin to cynomolgus monkeys produced no treatment-related effects. Whereas no monkeys demonstrated anti-rhThrombin antibody seroconversion, specific anti-bovine antibodies were detected in all tested monkeys exposed to bovine thrombin. Addition of rhThrombin or bovine thrombin to mouse fibroblast cells resulted in expected detachment and shape change. Topical application of rhThrombin to rabbits did not cause irritation to the eye, normal skin, or abraded skin. These studies showed that topical, subcutaneous, or implanted rhThrombin was minimally immunogenic, safe, and well tolerated in nonclinical models, and supported the clinical evaluation of rhThrombin in a variety of surgical settings.

Thrombin, the key enzyme of the coagulation cascade, is involved in inflammation. It was proposed recently that thrombin activity may play an important role in allergic inflammation. Interferon-gamma (IFN-gamma) is a potent Th1-related cytokine secreted by activated T cells and is usually downregulated in allergic inflammation. We recently demonstrated that thrombin enhances interleukin-10 (IL-10) in peripheral blood mononuclear cells (PBMC). Thus, we hypothesized that thrombin may promote a Th2 profile. We here report that human alpha- thrombin downregulates IFN-gamma expression at both protein and mRNA levels in activated PBMCs. The use of proteolytically inactive thrombin and of the specific thrombin receptor agonist peptide, SFLLRN, shows that this downregulation is thrombin specific and requires thrombin proteolytic activity. The addition of an anti- IL-10 monoclonal antibody (mAb) to thrombin-treated PBMCs abolishes IFN-gamma downregulation, suggesting that thrombin exerts its effect through IL-10, a Th2-related cytokine. Furthermore, IFN-gamma reduction was accompanied by increased IL-4 release, as well as by an increase in the proinflammatory cytokine IL-1. In conclusion, the observation that thrombin affects the production of IFN-gamma (Th1 profile) and IL-4 (Th2 profile) provides further evidence for the role played by thrombin in modulating Th1/Th2 cytokine balance, which could be particularly relevant in allergic inflammation.

Aprotinin (Trasylol) is a serine protease inhibitor, isolated from bovine lung that initially was marketed for the treatment of pancreatitis. In the mid 1980s, reports of its ability to decrease hemorrhaging after cardiopulmonary bypass surgery introduced the drug to the realm of cardiac surgery. Unfortunately, its introduction into this arena was followed by the publication of multiple studies and case reports that blamed aprotinin for poor outcomes in the form of early graft closure. More than 17 years have passed since the initial article describing the use of aprotinin during cardiopulmonary bypass, and with time there has been a significant increase in scientific knowledge and clinical experience. Interestingly, modern literature does not support the dogma that aprotinin is a procoagulant. Aprotinin increases the activated partial thromboplastin time (aPTT), as well as the kaolin- and celite-activated clotting time (ACT), regardless of heparin. Aprotinin, because of its ability to inhibit kallikrein, has been found to decrease thrombin antithrombin III complexes, fibrin-split products, fibrinopeptide 1+2, prothrombin fragments, and all markers of thrombin formation. Some authors have suggested that it may have a synergistic effect with heparin to ensure graft patency. Anticoagulation monitoring during the use of aprotinin also has been developed based on early studies. Aprotinin administration does influence the results of various ACT tests, and consequently different methods of testing anticoagulation have been developed. Researchers have demonstrated that the celite ACT is not "artificially" prolonged in the presence of heparin and aprotinin, rather the kaolin ACT is "artificially" shortened. This article will review the scientific literature with regard to aprotinin's anticoagulatory effects and review the current recommendations for hemostasis monitoring during the use of aprotinin.

Arteriovenous (AV) graft use as hemodialysis access remains highly prevalent, with a consequent high thrombosis rate. The magnitude of this problem requires that all potentially modifiable risk factors for graft thrombosis be thoroughly investigated. During graft surgery, topical bovine thrombin is often administered, which can lead to the development of antibovine thrombin antibodies and subsequent hemostatic changes. A recent study correlated the presence of plasma antibovine thrombin antibodies with graft thrombosis in hemodialysis patients. We therefore hypothesized that perioperative topical bovine thrombin exposure would lead to the development of antibovine thrombin antibodies and graft thrombosis. We screened 314 hemodialysis patients and identified 73 patients who had 74 grafts placed for whom complete data on perioperative topical bovine thrombin exposure and subsequent graft outcomes was available. Sixty-one of these patients were available for retrospective measurement of antibovine thrombin antibodies, antihuman thrombin antibodies, and the thrombin activation markers thrombin activatible fibrinolysis inhibitor (TAFI) and thrombin precursor protein (TpP). In these grafts, there was no significant association between topical bovine thrombin exposure and primary assisted patency (P = 0.37). The presence of antibovine thrombin antibodies (P = 0.13), antihuman thrombin antibodies (P = 0.10), and increased TAFI (P = 0.18) were associated with trends toward reduced primary assisted patency which did not reach significance. There was a correlation between antibovine thrombin antibodies and antihuman thrombin antibodies (r = 0.30, P < 0.0001) and between TAFI and TpP trade mark (r = 0.30, P < 0.0001), but no significant correlation between topical bovine thrombin exposure and elevated levels of antibovine thrombin antibodies, antihuman thrombin antibodies, TAFI or TpP trade mark. We conclude that perioperative topical bovine thrombin exposure is not associated with subsequent graft thrombosis.

Background: Hypersensitivity reactions to aprotinin have been reported in adult cardiac surgical patients undergoing initial and re-exposure to the medication. This study describes the incidence and impact of aprotinin hypersensitivity reactions in children undergoing cardiothoracic surgery., Methods and Results: In this retrospective review of our entire experience with aprotinin (n=865), 681 first exposures, 150 second exposures, and 34 third or higher exposures were examined. Reactions were classified as mild (generalized cutaneous erythema, Type A) or severe (unexplained cardiopulmonary instability after aprotinin exposure, Type B). Records of patients sustaining a reaction were reviewed to assess the impact of the reaction on outcome and to survey reaction management strategies. Reactions occurred in 7 of 681 first exposures (1.0%), of which 2 were Type A and 5 were Type B. In second exposures, there were reactions in 2 of 150 (1.3%), of which both were Type B. In 34 third or higher exposures, there was only 1 reaction (2.9%), which was Type B. Reactions were no more likely on second, third, or higher exposure than on initial exposure. Skin testing had a negative predictive value of 98.9% and a positive predictive value of 20%. Anti-aprotinin IgE was undetectable in 7 of 8 reactor cases tested. No adverse sequelae were attributed to aprotinin reaction., Conclusions: The risk of hypersensitivity reactions to aprotinin is low in children undergoing cardiothoracic surgery, even with multiple exposures to the medication. Reactions are more likely with re-exposure, and risk increases with multiple exposures. Neither skin testing nor assays for IgE identified reactors.

Exposure to topical bovine thrombin during surgery frequently results in the development of antibodies to multiple protein and carbohydrate antigens. We investigated the frequency of increased levels of antibodies to cardiolipin and beta(2)-glycoprotein I (beta(2)-GPI) in two groups of patients, one exposed to bovine thrombin during cardiovascular surgery (n = 151) and a "control" group undergoing cardiovascular surgery but without exposure to bovine thrombin (n = 11). Anticardiolipin antibody levels were increased before surgery in 10 of the 151 patients exposed to topical thrombin (6.6%). Four to 8 weeks after surgery, 84 patients (55.6%) had increased anticardiolipin antibody levels (P <.0001). In the control group, an increased anticardiolipin antibody level was present in a single patient before and after surgery (9%). Increased levels of antibodies to bovine and human beta(2)-GPI were also observed after surgery in the patients exposed to topical thrombin (37.7% and 38.2%, respectively). Increased anticardiolipin levels correlated with higher levels of antibody to bovine, but not human, beta(2)-GPI. In addition, increased levels of anticardiolipin antibody were associated with higher levels of antibodies to bovine factor V and prothrombin, as well as human factor V. Antibody binding on an enzyme-linked immunosorbent assay conducted to detect anticardiolipin antibody was dependent on the presence of anionic phospholipid, indicating that binding was not linked to the fetal bovine serum in the blocking buffer alone. Seven of 8 patients with delayed thromboembolic complications had increased anticardiolipin IgG antibody levels after surgery, but this association was not statistically significant. Nevertheless, our findings support the recommendation that the clinical safety of these commonly used hemostatic agents should be reassessed.

Background: Topical thrombin spray is frequently used as a hemostatic agent in surgery, particularly following cardiovascular, orthopedic and neurologic surgery. Acquired coagulation factor inhibitors to thrombin and factor V may develop following gynecologic surgery with repeat topical thrombin use., Case: A 45-year-old woman underwent total abdominal hysterectomy/bilateral salpingo-oophorectomy. Her past medical history included idiopathic thrombocytopenic purpura and Marfan's syndrome and past surgery included splenectomy and aortic valve replacement with a three-vessel coronary artery bypass graft. She was converted from Coumadin (DuPont Pharmaceuticals, Wilmington, Delaware) to heparin preoperatively, and heparin was stopped six hours before surgery, with normalization of her prothrombin time (PT) and partial thromboplastin time (PTT). Topical bovine thrombin spray was applied to the surgical field in anticipation of early resumption of heparin anticoagulation. Seven days after surgery, after recovering from some acute bleeding problems, the patient began to have elevation of her PT in spite of being off Coumadin for 10 days. Her PT and international normalized ratio (INR) continued to rise, reaching peak values of 31.4 and 6.99, respectively. A mixing study did not correct the problem, confirming the presence of coagulation inhibitors. The patient was given intravenous immunoglobulins and corticosteroids, and her PT/PTT and INR values slowly normalized., Conclusion: Acquired coagulation factor may develop following gynecologic surgery with topical thrombin use. This potential complication should be strongly considered in any patient prior to using topical bovine thrombin, especially those with a prior exposure or history of surgical procedures in which bovine thrombin is commonly used.

Background: Aprotinin, a serine proteinase inhibitor, reduces bleeding during cardiac surgery. As aprotinin is derived from bovine lung, it has antigenic properties. This investigation examined the incidence of anaphylactic reactions in patients reexposed to aprotinin and the relation to preformed antiaprotinin immunoglobulin (Ig)G and IgE antibodies., Methods: This prospective observational study conducted at five centers in Germany evaluated patients undergoing repeat cardiac surgery reexposed to aprotinin between 1995 and 1996. Antiaprotinin IgG and IgE antibody measurements, using a noncommercial enzyme-linked immunosorbent assay and an immunofluorescence assay, respectively, were performed preoperatively and postoperatively. An anaphylactic reaction was defined as major changes from baseline within 10 min of aprotinin administration of systolic pressure 20% or greater, heart rate 20% or greater, inspiratory pressure greater than 5 cm H2O, or a skin reaction., Results: In 121 cases (71 adults, 46 children), a mean aprotinin reexposure interval of 1,654 days (range, 16-7,136 days) was observed. Preoperative antiaprotinin IgG (optical density ratio > 3) and IgE antibodies (radioallergosorbent test [RAST] score < 3) were detected in 18 and 9 patients, respectively. High concentrations of each (IgG, optical density ratio > 10; IgE, RAST score > or = 3) were detected in five patients. Three patients (2.5%; 95% confidence interval, 0.51-7.1%) experienced an anaphylactic reaction after aprotinin exposure, followed by full recovery; these patients had reexposure intervals less than 6 months (22, 25, and 25 days) and the highest preoperative IgG concentrations of all patients (P < 0.05). Assay sensitivity was 100%, as no anaphylactic reactions occurred in IgG-negative patients (95% confidence interval, 0.0-3.1%); assay specificity was 98%. Preoperative IgE measurements were quantifiable in two of three reactive patients and in three nonreacting patients., Conclusions: Quantitative detection of antiaprotinin IgE and IgG lacks specificity for predictive purposes; however, quantitation of antiaprotinin IgG may identify patients at risk for developing an anaphylactic reaction to aprotinin reexposure.

A randomized, controlled clinical study of the management of diffuse bleeding with CoStasis surgical hemostat, a new hemostat containing bovine thrombin and collagen with the patient's own plasma, included patients undergoing cardiac, hepatic, iliac, and general surgery. Sera from 92 patients treated with CoStasis and 84 control patients were collected preoperatively and at a post surgical follow-up of 8 weeks. Among the control group, 57 patients were treated with Instat collagen sponge in noncardiac indications. Results showed that antibody responses in the CoStasis clinical study were similar to the reported literature for all antigens screened and were not associated with any adverse reactions. The bovine thrombin preparations in CoStasis and other commercially available thrombins were compared with the use of SDS-PAGE and Western blot analyses. Within this clinical study, CoStasis was shown to be a safe and effective hemostatic product containing bovine thrombin and bovine collagen and no pooled human blood products., (Copyright 2001 John Wiley & Sons, Inc.)

After the injection treatment of a femoral pseudoaneurysm, an anaphylactic reaction occurred in a patient undergoing hemodialysis who previously had repeated exposure to thrombin. Before injecting bovine thrombin in patients with a history of prior exposure, we recommend that they undergo skin prick testing to detect possible allergy.

Unlabelled: Acute hypersensitivity reactions are serious complications of reexposure to aprotinin. Previous contact via infusions or fibrin tissue adhesives can induce specific antibodies. In this study, we aimed to elucidate the preoperative prevalence of aprotinin-specific antibodies in patients scheduled for cardiac operations. Sera of 520 consecutive cardiosurgical patients were collected preoperatively and screened retrospectively for aprotinin-specific IgG using a standard enzyme-linked immunosorbent assay (ELISA). Positive sera were analyzed also for aprotinin-specific IgA (ELISA) and IgE (fluorescence enzyme immunoassay). The histories of all patients were reviewed with focus on aprotinin preexposure. Of 520 patients, 22 (4%) had specific IgG. Only three of these had a documented aprotinin preexposure. Of 448 patients exposed to aprotinin intraoperatively, 15 had preformed specific antibodies. The only patient presenting with severe anaphylaxis was positive for both IgG and IgE, and had a recent IV preexposure in cardiovascular surgery. The presence of aprotinin-specific IgG alone seems not to induce adverse reactions on exposure. Exposure history alone is not sensitive enough to identify patients with aprotinin-specific antibodies., Implications: Anaphylaxis on IV reexposure to aprotinin is a medical emergency. The clinical significance of preformed aprotinin-specific IgG remains questionable, whereas preformed IgE was present in the only patient who suffered from severe anaphylaxis on reexposure to aprotinin. Preformed antibodies are not reliably predicted by exposure history.

Objective: Most commercially available fibrin sealants contain aprotinin in doses of 1500 kallikrein inactivator units per milliliter. They are used in many operative disciplines. An elevated risk of hypersensitivity reactions exists at reexposure to aprotinin. Our aim was to examine the immunogenic potency of aprotinin as a fibrin sealant content., Methods: We investigated 49 children with operatively treated congenital heart disease. All patients received aprotinin only topically as contained in fibrin sealant. Serum samples were drawn preoperatively, 1 week, 2 weeks, 6 weeks, and approximately 1 year after operation. They were analyzed for aprotinin-specific immunoglobulin G antibodies with a standard enzyme-linked immunosorbent assay and a fluorescence enzyme immunoassay for aprotinin-specific immunoglobulin E antibodies., Results: At 1 week, 2 weeks, 6 weeks, and 1 year, we found prevalences of 8% (2 of 26), 8% (2 of 24), 6% (3 of 49), and 0% for aprotinin-specific Immunoglobulin E, and for aprotinin-specific immunoglobulin G 8% (2 of 26), 17% (4 of 24), 39% (19 of 49), and 12% (5 of 41). The doses of aprotinin given did not differ significantly in antibody-negative and antibody-positive patients; no significant factors could predict the immune response., Conclusions: Our findings show the existence of a subgroup of patients who had aprotinin-specific antibodies develop after topical aprotinin application. Any use of aprotinin must be carefully documented. If aprotinin use is planned in patients who previously underwent a surgical procedure, preexposure to aprotinin in any form must be sought to avoid unexpected anaphylactic reactions. The necessity itself and alternatives for aprotinin as a stabilizing agent in fibrin sealants merit consideration.

We report a case of severe anaphylactic shock during a cardiac operation that occurred as a consequence of aprotinin readministration in the presence of preformed aprotinin-specific antibodies. Both immunoglobulin G (3 hours) and immunoglobulin E (5 minutes) antibody levels dropped early after the clinical event. Despite their possibly limited clinical significance, we still recommend the conductance of specific antibody screening tests before readministration of aprotinin.

High-dose aprotinin is now routinely used in cardiac surgery to reduce postoperative blood loss and transfusion requirements, although several cases of anaphylactic reactions to the proteinase inhibitor have been reported. As part of a multi-centre study to evaluate the immunological response to aprotinin after first exposure 61 cardiac surgical patients were treated with the Hammersmith regimen. Patients with previous aprotinin exposure were excluded from the study. To determine specific IgG and IgE antibodies blood samples were taken pre-operatively, within 3 to 4 weeks and 6 to 7 months after operation. Determinations were made by using Western Blot and ELISA methods. Fifty-six patients were followed up for a 6-month period, 26 (46.4%) of them developed IgG antibodies to aprotinin determined by Western Blot, whereas only 14 (26.8%) patients with IgG antibodies were found by the ELISA. IgE antibodies were not found in any of the patients. On hospital admission and 6 months post-operatively additional intradermal prick tests were performed. No clear-cut positive reaction to the skin test was found in any patient.

Since its commercial release, Zyderm collagen implant has been used to treat more than 200,000 subjects in the United States for soft-tissue contour defects and more than 250,000 patients internationally (including the United States). Approximately 3 percent of subjects' skin tested with Zyderm collagen experience localized hypersensitivity reactions to collagen, whereas approximately 1 percent of treated patients demonstrate symptoms of hypersensitivity at treatment sites. Of the latter treatment responses reported since the conclusion of clinical trials with Zyderm, 56 percent occurred following the first treatment, 28 percent following the second, 10 percent following the third, and 6 percent following subsequent exposures. The data indicate that most patients receive a median of three treatments (mean = 4.4) with Zyderm collagen, but most patients who are likely to develop sensitivity to Zyderm collagen appear to respond immunologically to the test implant or first treatment exposure. Examining these treatment responses, 45 percent of the patients reported an onset of symptoms within 10 days and 22 percent at more than 30 days following the last treatment with Zyderm collagen. Erythema was the sole symptom in 24 percent of cases, whereas erythema plus induration comprised an additional 42 percent. Antibodies against Zyderm collagen were detected in the sera of 88 percent of these subjects using an ELISA, but no reactivity was observed against human collagen. Sera from patients reporting only systemic symptoms were not found to have anticollagen antibodies. These data suggest that the relative risk of a hypersensitivity reaction to Zyderm collagen does not increase with multiple exposures, since patients who are going to develop an immune response to bovine collagen react with greatest frequency to initial injections of collagen. In animal models, Zyderm collagen was shown to be less immunogenic than other medical devices which are composed of bovine collagen. Specifically, comparative studies were conducted in which Zyderm collagen and hemostatic agents were implanted in the guinea pig subcutaneum: sera from animals treated with collagen-derived hemostatic devices possessed significant levels of anti-implant antibodies (titers greater than 640), whereas animals treated with Zyderm collagen mounted minimal responses (titers less than 40). Additional studies were conducted in which implant materials were compared in a guinea pig parietal (bony defect) model and in a rabbit hemostasis model: in both, Zyderm collagen demonstrated lower immunogenicity than commercial bovine collagen hemostatic agents. Histologic results from these studies showed Zyderm