Fibrocaps for surgical hemostasis: two randomized, controlled phase II trials.

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.

Salmon fibrin glue in rats: antibody studies.

Fibrin sealants and topical thrombin preparations are often used for haemostatic and sealing applications in clinical practice. Some of these preparations contain coagulation factors from bovine sources. To minimize the risk of infection and immunogenicity connected with mammalian blood products, proteins derived from the plasma of farmed Atlantic salmon have been considered as an alternative to these mammalian sources. The purpose of this study is to characterize the immunogenicity of salmon fibrin glue in an animal model focusing on crossreactivity of IgG antibodies to host endogenous counterparts. After two immunizations with salmon fibrin glue, rats developed antibodies of IgG and IgM type to both fibrin glue components. Weak crossreactivity to endogenous fibrinogen and thrombin was seen in a subset of rats after the second application of salmon proteins. Coagulation tests showed that salmon fibrin application has no effect on coagulation profiles in mammalian hosts, consistent with previous reports that found no evidence of significant crossreactivity with host proteins. These studies support the potential suitability of salmon fibrin glue for the development of preparations with clinical impact. Before human use can be considered, however, additional data about safety of this preparation in other animal models, including large animal studies, should be obtained.

Characterization of the biological effect of fish fibrin glue in experiments on rats: immunological and coagulation studies.

Fibrin glues (FG) of human or bovine origin are widely used for haemostasis and wound healing. In addition FGs are studied in many biomedical areas like cell therapy or tissue engineering. As any mammalian plasma products FG-s pose risk of transmission of bacteria, viruses, or prions and may compromise patient homeostasis. In this study, we examined coagulation parameters and immunological status of rats treated with salmon-derived FG. We evaluated the changes in thrombin time, prothrombin activity, and presence of antibodies on 46 Wistar rats. This study shows that salmon-derived FG, injected intraperitoneally, does not cause coagulation disturbances in the peripheral blood. After a first challenge with salmon-derived FG there were low but detectable amounts of antibodies revealed by ELISA and immunoblot. After a second administration there was substantial elevation of antibodies to FG components and other copurifying plasma proteins. Antibody reactivity to human Factor Va, revealed in three animals, was not associated with FG application. Taken together, blood immunological and coagulation parameters support the suitability of salmon-derived FG in the development of fibrin sealants for medical use.

Comparison of structure, strength and cytocompatibility of a fibrin matrix supplemented either with tranexamic acid or aprotinin.

Fibrin sealants are used as hemostats, sealants, tissue adhesives, and as matrix for substances/cells in a number of surgical and tissue engineering procedures. Main characteristics of fibrin are high tensile strength, adhesive strength, biocompatibility, and resorption. A major adverse event would be premature fibrin lysis and recurrent bleeding. This must be prevented by fibrinolysis inhibitors. The most common fibrinolysis inhibitors used are aprotinin and tranexamic acid (t-AMCA). Comparison of commercially available fibrin sealants utilizing aprotinin or t-AMCA revealed a lower sealing efficacy in an in vivo lung resection model for a t-AMCA containing product. Therefore, we compared the influence of t-AMCA and aprotinin on structure, mechanical properties, and cytocompatibility of a fibrin matrix. In our experiments, we found that substitution of aprotinin with t-AMCA reduced the tensile strength and formation of fibrin fibers and affected viability of a fibroblast cell-line. In conclusion, t-AMCA negatively affects physical and biological properties of fibrin relevant for clinical application as well as tissue regeneration.

Stability, sterility, coagulation, and immunologic studies of salmon coagulation proteins with potential use for mammalian wound healing and cell engineering.

Fibrin sealants made by polymerization of fibrinogen activated by the protease thrombin have many applications in hemostasis and wound healing. In treatments of acute injury or surgical wounds, concentrated fibrin preparations mimic the initial matrix that normally prevents bleeding and acts as a scaffold for cells that initiate tissue repair. However risks of infectious disease, immunogenic reaction, and the high cost of purified human or other mammalian blood proteins limit widespread use of these materials. Purified coagulation proteins from Atlantic salmon represent a potentially safer, equally effective, and less costly alternative in part because of the low ambient temperature of these farmed animals and the absence of endogenous agents known to be infectious in mammalian hosts. This study reports rheologic measurements of lyophilized salmon fibrinogen and thrombin that demonstrate stability to prolonged storage and gamma irradiation sufficient to reduce viral loads by over five orders of magnitude. Coagulation and immunologic studies in rats and rabbits treated intraperitoneally with salmon fibrin show no deleterious effects on coagulation profiles and no cross reactivity with host fibrinogen or thrombin. The results support the potential of salmon fibrin as an alternative to mammalian proteins in clinical applications.

Delivery of high dose VEGF plasmid using fibrin carrier does not influence its angiogenic potency.

Delivery of DNA mixed with a degradable matrix carrier was supposed to improve transgene expression. Using a rabbit hind-limb ischemia model, we tested the angiogenic potency of plasmid encoding human vascular endothelial growth factor (pSG5-VEGF165) entrapped in fibrin sealant. Animals were injected intramuscularly with 500 microg of pSG5-VEGF165 or control plasmid, dissolved in saline (PBS) or fibrin glue. After 14 days, presence of delivered constructs and expression of transgene was confirmed in injected muscles of all animals. There were no significant differences in the levels of human VEGF mRNA and protein between VEGF-PBS and VEGF-fibrin groups (Mann-Whitney test). Accordingly, pSG5-VEGF165 regardless of the way of delivery, induced similar increases in capillary density within treated muscles (ANOVA). Control plasmid did not show any effects. In conclusion, injection of pSG5-VEGF165 into ischemic adductor muscle leads to synthesis of human VEGF and increases the number of capillaries. Fibrin carrier does not influence its angiogenic potential.

[Aseptic meningitis as a complication caused by an allergic reaction after microvascular decompression: two case reports].

We report here two cases of patients complicated with aseptic meningitis after microvascular decompression (MVD). The first case, a 56-year-old female complained of headache with high fever 18 days after the MVD for right trigeminal neuralgia. The amount of cells in cerebrospinal fluid (CSF) had so much increased that bacterial meningitis was suspected. However, there was no improvement after antibiotics therapy, so immune globulin was injected and the meningitis gradually improved. Eosinophilia remained in peripheral blood and the symptoms improved rapidly after the steroid therapy. Because of this, we suspected that meningitis was caused by an abnormal allergic reaction. The second case, a 30-year-old male complained of headache with mild fever 15 days after MVD for left hemifacial spasm. The amount of cells in CSF increased, so bacterial meningitis was suspected. Eosinophilia remained in peripheral blood and the steroid therapy proved very effective for the meningitis. Because of this, we suspected that meningitis was caused by an abnormal allergic reaction. We suspected that the two patients suffered from aseptic meningitis caused by allergic reaction, and the antigen for this abnormal allergic reaction was the foreign materials used for MVD. The materials were Dacron for prostesis, Goatex or Lyodula for dural plasty, fibrin glue for preventing CSF leakage. We ascertained that the abnormal allergic reaction was caused by human fibrinogen in the second case. It is important to be aware of such allergic reaction to fibrin glue in the post-operative stage after MVD.

Acquired FV inhibitors: a needless iatrogenic complication of bovine thrombin exposure.

BACKGROUND: FV inhibitors are a largely preventable iatrogenic coagulopathy in which the frequency is increasing in clinical practice. STUDY DESIGN AND METHODS: Three cases associated with our institution are reported. A systematic review of the MEDLINE database was performed, and reference lists were reviewed to identify relevant publications. RESULTS: One hundred twenty-six cases of FV inhibitors have been reported in the world's literature. Eighty-seven have been reported in the last decade, of which two thirds are due to exposure to bovine thrombin. Bovine thrombin-associated FV antibodies develop in 40 to 66 percent of cardiac surgery patients and in 20 percent of neurosurgery patients. Thirty-three percent of reported patients developed bleeding complications. Inhibitors persisted on average 2.3 months. Standard coagulation assays do not reliably predict clinical manifestations. Multimodality therapy, including immunosuppression, is useful for treatment of symptomatic patients. CONCLUSIONS: FV inhibitors are a common complication of bovine thrombin exposure that can have devastating clinical consequences. Transfusion medicine specialists and hematologists can play a critical role in reducing the incidence of FV inhibitors by educating the medical community about safer alternative fibrin sealants.

Immunochemical analysis of polyspecific antibodies in patients exposed to bovine fibrin sealant.

BACKGROUND: Patients exposed to bovine thrombin preparations in fibrin sealant often develop antibodies to bovine coagulation proteins, which cause significant bleeding by cross-reacting with human homologues. Recipients of our left ventricular assist system (LVAS) routinely are exposed to fibrin sealant; therefore, we determined whether they developed antibodies. METHODS: We compared sera from 6 LVAS recipients exposed to fibrin sealant (THROMBOGEN, Johnson & Johnson, Arlington, TX ) during LVAS placement to that of 5 nonexposed LVAS recipients. Pre-LVAS and weekly post-LVAS sera were tested for immunoglobulin (Ig)G, IgA, and IgM reactivity to THROMBOGEN by enzyme-linked immunosorbent assay. Peak IgG and IgA reactive sera were characterized by immunoblotting. RESULTS: All patients exposed to THROMBOGEN developed antibodies: 5 developed IgG, 4 IgA, and 3 IgM. In contrast, nonexposed patients did not develop antibodies. Only some antibody reactivity was contributed by antithrombin or antifactor V antibodies. Silver stain sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of THROMBOGEN showed more than 18 bands, many of which were recognized in Western blot by positive patient sera. CONCLUSIONS: We found both IgG and IgA polyspecific antibody responses in patients exposed to bovine thrombin preparations.

Management of a patient with a mechanical aortic valve and antibodies to both thrombin and factor V after repeat exposure to fibrin sealant.

We describe a patient who developed a markedly prolonged PT, PTT, and thrombin time 13 days after repeat exposure to fibrin sealant during coronary artery bypass grafting and aortic valve replacement. Evaluation revealed an inhibitor to bovine thrombin that cross-reacted with human thrombin. In addition an inhibitor to human coagulation factor V was identified. Despite coagulation abnormalities there was no evidence of bleeding. Nevertheless, effective anticoagulation was required to minimize the thrombotic complications associated with the patient's prosthetic valve. We elected to take a conservative approach and not utilize pharmacologic anticoagulation until there was diminution in the effect of the acquired inhibitors. We report on our patient's course and review the available literature addressing the management of patients demonstrating inhibitors to blood coagulation factors after repeat exposure to fibrin sealants.

Fibrin sealant, aprotinin, and immune response in children undergoing operations for congenital heart disease.

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.

Antigenic properties of fibrinogen component of Hemaseel HMN subjected to the antiviral severe dry heat treatment.

The experiments in this work were focused on determination of the extent to which fibrinogen component of fibrin glue (Hemaseel HMN, Haemacure Biotech.) retained the conformation of the parent molecule after dry heat antiviral treatment (one hour at 100 degrees C). For this purpose antigenic structure of the fibrinogen component, heated and non-heated, was compared to that of control fibrinogens, i.e. the isolated one and the fibrinogen present in fresh human blood plasma. The analytic parameters CI50 and CIs were calculated from the competitive inhibition data obtained in ELISA systems using antisera to fibrinogen, plasmic fragments D and E, and to polypeptide chains A alpha, B beta, and gamma. These immunochemical analyses indicated that there was a modified expression of some fibrinogen epitopes probably resulting from unfolding of the A alpha chain and the better exposure of the E domain to the hydrated environment induced upon a heating procedure. Our data show that dry heat treatment of fibrinogen component is not associated with a significant overall conformational change of the molecule.

Antibodies formed against fibrin glue components and their circulatory relevance.

To elucidate the safety of fibrin glue application, antibody formation against fibrinogen and thrombin was studied in rabbits. The pathophysiology of developed antibodies was tested by an intravenous challenge with both components and by related blood pressure measurements. All fibrin glue-treated animals developed antibodies against both components. The quantity of antibodies against thrombin (20.4 mg/mL) was higher than the quantity against fibrinogen (0.38 mg/mL). The intravenous challenge with both components resulted in a significant and long-lasting blood pressure decrease to about 50% of the control values, indicating that these antibodies can induce circulatory disorders by antigen-antibody reaction, activation of the complement cascade, and liberation of histamine. Since rabbits promptly produce antibodies against antigenic material and humans do not, the results of this investigation suggest that fibrin glue should be used with caution in humans.

Fibrin glue in surgery: frequent development of inhibitors of bovine thrombin and human factor V.

We report on a 34-year-old woman whose plasma showed a marked prolongation of thrombin time (TT) (> 200 s) using bovine thrombin. The patient had previously been exposed twice to topical bovine thrombin contained in fibrin glue during cardiac surgery. TT was normal when human thrombin was used as reagent. The patient's purified IgG reacted with bovine prothrombin and bovine thrombin in immunoblotting studies but showed virtually no cross-reaction with human thrombin. In addition, following surgery, factor V clotting activity (FV:C) was reduced to 9% of normal. The inhibitor of bovine thrombin persisted over a period of more than a year, while the level of FV:C progressively returned to normal within this time period. Development of thrombin and FV:C inhibitors was also investigated in plasma of 34 consecutive patients who had undergone either cardiac surgery or neurosurgery with use of fibrin glue containing bovine thrombin. Eleven of 24 patients after cardiac surgery and two of 10 patients after neurosurgery presented with TT > or = 25 s (normal plasma 15 s). Two patients had been re-exposed to fibrin glue during cardiac re-operation and showed markedly prolonged TT (> 60 s). All 13 patients who had acquired a thrombin inhibitor also had low FV:C activity (10-60% of normal plasma), whereas FV:C activity remained in the normal range in the 21 patients with normal TT. Our findings indicate that development of inhibitors of bovine thrombin as well as co-immunization to factor V occurs frequently and is associated with the amount of applied fibrin glue and with the type of operation. Re-exposure to fibrin glue seems to enhance formation of inhibitors of bovine thrombin and human factor V.

Immunization by bovine thrombin used with fibrin glue during cardiovascular operations. Development of thrombin and factor V inhibitors.

Brief case histories of three patients aged 58, 38, and 44 years are reported. All underwent cardiovascular operations. Subsequently hemostasis test abnormalities developed between the seventh and eighth postoperative days after exposure to bovine thrombin used with fibrin glue. These were characterized by an increased activated partial thromboplastin time (64 to 147 seconds), prothrombin time (19 to 24 seconds), bovine thrombin time (> 120 seconds) and a markedly reduced factor V level (< 10% in two patients and 16% in the third patient). A patient plasma dilution of 1 in 200 with a normal plasma pool was necessary to correct bovine thrombin time. No fast-acting or progressive inhibitor against factor V could be detected by coagulation tests, and fresh frozen plasma perfusion had no effect. Plasmapheresis was performed preventatively to avoid bleeding, and factor V levels stabilized at around 50% after two to four exchanges. Immunologic studies showed that the inhibitors were directed not only against bovine factors but also against human ones. Therefore factor V decrease could have been the result of rapid clearance from the circulation of complexes formed with a nonneutralizing inhibitor that is not detected by clotting tests. These antibodies were purified by standard methods and immunoaffinity. Fast immunization could be explained by a prior sensitization to bovine thrombin exposure during previous operations. It is suggested that bovine thrombin used with fibrin glue contains small amounts of factor V and may be responsible for these abnormalities. This is in agreement with previous literature reports. However, these described neutralizing factor V inhibitors, which were easily detected.

Evaluation of bovine fibrin sealant in the dog.

Fibrin sealant (human fibrinogen-bovine thrombin) is an effective biodegradable hemostatic agent. However, there is a risk of transmission of infectious viral disease. A new bovine fibrinogen-thrombin sealant (BFTS) was tested for tissue and immune responses in intrathoracic aorta graft in dogs. Intrathoracic aorta replacement was performed in three dogs with a porous Dacron graft presealed with BFTS. Dogs were immunized preoperatively with four dermal applications of BFTS at 9-day intervals. Cellular and humoral immunity to BFTS were determined with lymphocyte blastogenesis test and enzyme-lined immunosorbent assay, respectively. Dogs were necropsied 3 weeks after aortic replacement. Histopathological examination showed that all dogs had a mild inflammatory reaction to the BFTS sealed graft, as expected in response to an inert foreign body. Assay of cellular and humoral immunity to BFTS revealed a low lymphocyte response and a moderate immunoglobulin G (Ig G) response, with no evidence of immediate Ig E (type I) or delayed (type IV) hypersensitivity reaction. We conclude that BFTS causes no adverse tissue response or immunologic reaction when used as a hemostatic agent in the dog even after multiple applications of the material.