Crosstalk between coagulation and complement activation promotes cardiac dysfunction in arrhythmogenic right ventricular cardiomyopathy.

Aims: We previously found that complement components are upregulated in the myocardium of patients with arrhythmogenic right ventricular cardiomyopathy (ARVC), and inhibiting the complement receptor C5aR reduces disease severity in desmin knockout (Des-/- ) mice, a model for ARVC. Here, we examined the mechanism underlying complement activation in ARVC, revealing a potential new therapeutic target. Methods: First, immunostaining, RT-PCR and western blot were used to detect the expression levels of complement and coagulation factors. Second, we knocked out the central complement component C3 in Des-/- mice (ARVC model) by crossing Des-/- mice with C3-/- mice to explore whether complement system activation occurs independently of the conventional pathway. Then, we evaluated whether a targeted intervention to coagulation system is effective to reduce myocardium injury. Finally, the plasma sC5b9 level was assessed to investigate the role in predicting adverse cardiac events in the ARVC cohort. Results: The complement system is activated in the myocardium in ARVC. Autoantibodies against myocardial proteins provided a possible mechanism underlying. Moreover, we found increased levels of myocardial C5 and the serum C5a in Des-/-C3-/- mice compared to wild-type mice, indicating that C5 is activated independently from the conventional pathway, presumably via the coagulation system. Crosstalk between the complement and coagulation systems exacerbated the myocardial injury in ARVC mice, and this injury was reduced by using the thrombin inhibitor lepirudin. In addition, we found significantly elevated plasma levels of sC5b9 and thrombin in patients, and this increase was correlated with all-cause mortality. Conclusions: These results suggest that crosstalk between the coagulation and complement systems plays a pathogenic role in cardiac dysfunction in ARVC. Thus, understanding this crosstalk may have important clinical implications with respect to diagnosing and treating ARVC.

Interference of thrombin in immunological assays for hirudin specific antibodies.

Recombinant hirudins (desirudin, lepirudin) are direct thrombin inhibitors administered as anticoagulants for heparin-induced thrombocytopenia (HIT) and venous thromboembolism (VTE) prophylaxis. Although these small polypeptides are widely used, concern exists over reports of antigenicity. In the largest study of r-hirudin immunogenicity to-date, we evaluated the prevalence, quantity and specificity of IgG immune responses to desirudin (15 mg SC q12h for as long as clinically required) in 245 surgical and medically-ill subjects enrolled in DESIRABLE, a multicenter, open-label, clinical trial of hospitalized patients requiring VTE prophylaxis. Sera obtained before and 30 days after desirudin administration were analyzed for IgG anti-desirudin by immunoenzymetric assay using immobilized desirudin to bind desirudin-reactive antibody and peroxidase conjugated monoclonal-anti-human IgG Fc to detect bound IgG antibody. Of 245 study subjects, 19 (7.7%) were antibody "responders" (>2-fold increase in IgG antibody levels with >50% inhibition by desirudin 30 days post-treatment). There were no differences between responders and non-responders in incidence of clinical outcomes or bleeding-related adverse events. Forty-six patients had detectable desirudin-reactive IgG antibody prior to treatment, with no significant increase in antibody levels after exposure and no increase in clinical events. The origin of pre-existing hirudin-reactive IgG antibody requires further investigation involving suspected anti-thrombin-thrombin interactions. These results indicate a low potential for immunogenicity, with <8% of patients developing IgG antibodies after desirudin administration for VTE prophylaxis. In contrast to reports on lepirudin, production of anti-hirudin antibodies to desirudin has no apparent effect on clinical events.

The occurrence of antibodies to heparin-platelet factor 4 in cardiac and thoracic surgical patients receiving desirudin or heparin for postoperative venous thrombosis prophylaxis.

INTRODUCTION: This randomized, exploratory study compared the incidence of heparin-dependent antibodies associated with subcutaneous (SC) desirudin or heparin given for deep-vein thrombosis prophylaxis following cardiac and thoracic surgery. MATERIALS AND METHODS: Adult patients scheduled for elective cardiac or thoracic surgery received desirudin 15 mg SC twice daily or unfractionated heparin 5000 units SC thrice daily. Duration of thrombosis prophylaxis was determined by the treating physician. Primary outcome measure was the incidence of new antibody formation directed against platelet factor 4 (PF4)/heparin complex. Secondary outcomes included bleeding and thrombotic complications. Blood was tested for anti-PF4/heparin antibodies at baseline, after surgery prior to study drug administration, postdrug day (PDD) 2, PDD 7, and at 1 month. Doppler studies were done before discharge. RESULTS: Of 120 patients, 61 received desirudin, 59 received heparin. New PF4/heparin antibodies occurred in 10.2% and 13.6% of desirudin- and heparin-treated patients, respectively. Among desirudin patients with no heparin exposure, none (0/36) developed PF4/heparin antibodies versus 17.1% with heparin exposure. Incidence of deep venous thrombosis was 4.9% and 3.4% in the desirudin and heparin groups, respectively. Two heparin-group patients developed pulmonary embolism. Two patients per group had bleeding events; no patients required re-exploration for bleeding complications. Median chest tube output was similar with desirudin (900 mL) and heparin (692 mL) as was blood transfusion requirements of more than 2 units (5/61, desirudin; 2/59 heparin). CONCLUSIONS: The incidence of thrombotic events was low in both groups. There were no safety concerns, and desirudin was not associated with anti-PF4/heparin antibodies.

Microscale characterization of the binding specificity and affinity of a monoclonal antisulfotyrosyl IgG antibody.

Sulfation is a potentially important post-translational modification of proteins and has been demonstrated in a number of polypeptides, notably in gastrointestinal hormones. In contrast to phosphorylation, however, the investigation of sulfation patterns in tissues and on purified proteins has been complicated by the absence of specific immunoreagents (antibodies) for this modification as well as the chemical lability of the sulfate group. Here, we investigate the properties of a novel mAb against sulfated tyrosyl groups (anti-Tyr(SO(3)H) antibody) using CE and a panel of sulfated and nonsulfated peptides and proteins. The data show that the anti-Tyr(SO(3)H) antibody is completely specific for compounds containing sulfated tyrosyls. Affinity electrophoresis experiments allowed us to estimate dissociation constants for sulfated hirudin fragment (56-65), gastrin-17, and cholecystokinin octapeptide (CCK8) in the 1-3 microM range. The affinity of the antibody toward complement 4 protein that contains three sulfotyrosines was analyzed by surface plasmon resonance technology and modeled according to a bivalent-binding model which yielded a K(d1) of 20.1 microM for the monovalent complex. The same binding was studied by CE and found to be in the micromolar scale albeit with some uncertainty due to complex separation patterns. The work illustrates the amount of information on antibody-antigen interactions that may be obtained with microelectrophoretic methods consuming minute quantities of material. Furthermore the specificity of this antibody could be confirmed in one operation using an array of sulfated and nonsulfated compounds.

The direct thrombin inhibitor hirudin.

This review discusses the pharmacology and clinical applications of hirudin, a bivalent direct thrombin inhibitor (DTI). Besides the current major indication for hirudin--anticoagulation of patients with heparin-induced thrombocytopenia (HIT)--the experience with hirudin in other indications, especially acute coronary syndromes, are briefly presented. Hirudins have been formally studied prior to their regulatory approval; however, important information on their side effects and relevant preventative measures only became available later. Therefore, current recommendations and dosing schedules for hirudin differ considerably from the information given in the package inserts. Drawbacks of hirudin and important precautions for avoiding potential adverse effects are discussed in detail in the third part of this review.

Bivalirudin.

Bivalirudin is a direct thrombin inhibitor (DTI) frequently used for anticoagulation in the setting of invasive cardiology, particularly percutaneous coronary intervention (PCI). Bivalirudin has a unique pharmacologic profile: unlike other marketed DTIs, it undergoes predominant non-organ elimination (proteolysis), and has the shortest half-life (approximately 25 min). Its affinity for thrombin is intermediate between that of lepirudin (highest) and argatroban (lowest)--this helps explain why it interferes with functional clotting assays to an extent intermediate between that achieved by these two other DTIs. This effect is best known for the PT (INR)--higher affinity for thrombin corresponds to lower molar DTI requirements to prolong the APTT; in turn, lower concentrations required for APTT prolongation (and, presumably, in-vivo effect) result in reduced PT (INR) prolongation. Bivalirudin is primarily used for its first FDA-approved indication, namely anticoagulation during percutaneous transluminal coronary angioplasty ("balloon angioplasty"), the most frequent type of PCI. Bivalirudin is also indicated for PCI with provisional use of glycoprotein IIb/IIIa antagonist therapy, and for patients with, or at risk of, heparin-induced thrombocytopenia (HIT), or HIT with thrombosis syndrome (HITTS), undergoing PCI. The bivalirudin development program has used a "quadruple" endpoint comprising a "triple" efficacy endpoint plus major bleeding - this approach anticipated the subsequent emphasis on strategies to improve clinical outcomes through bleeding reduction. Besides summarizing the key trials evaluating bivalirudin use for acute coronary syndrome (especially employing PCI), we review also the studies of bivalirudin as anticoagulant for "on-" and "off-pump" cardiac surgery, including both HIT and non-HIT situations.

Bivalirudin anticoagulation for a patient with hypercoagulable immune syndromes undergoing mitral valve surgery.

Unfractionated heparin has been a near universal anticoagulant for cardiac surgery; however it is contraindicated in heparin-induced thrombocytopenia type II. Alternative anticoagulants such as bivalirudin (a direct thrombin inhibitor) are being utilized. Bivalirudin was successfully used in an immunologically complex patient (diagnoses of heparin-induced thrombocytopenia type II, systemic lupus erythematosus, antiphospholipid syndrome, and dialysis-dependent renal failure) requiring cardiopulmonary bypass. Thrombotic events are common in antiphospholipid syndrome patients undergoing cardiac surgery utilizing high-dose heparin. This may represent unrecognized heparin-induced thrombocytopenia type II. Our patient did not experience perioperative thrombotic or bleeding complications. The possible cross-reactivity between heparin induced thrombocytopenia type II and antiphospholipid syndrome has not been investigated.

Lepirudin in patients with heparin-induced thrombocytopenia - results of the third prospective study (HAT-3) and a combined analysis of HAT-1, HAT-2, and HAT-3.

OBJECTIVES: To assess efficacy and safety of lepirudin in patients with heparin-induced thrombocytopenia (HIT) in a prospective study (HAT-3) as well as in a combined analysis of all HAT study data. PATIENTS/METHODS: Patients with laboratory-confirmed HIT were treated with lepirudin in three different aPTT-adjusted dose regimen and during cardiopulmonary bypass (CPB). Endpoints were new thromboembolic complications (TEC), limb amputations, and death and major bleeding. A historical control group (n = 120) was used for comparison. RESULTS: After start of lepirudin in 205 patients treated in HAT-3, the combined endpoint occurred in 43 (21.0%). Thirty (14.6%) patients died, 10 (4.9%) underwent limb amputation, and 11 (5.4%) new TECs occurred. Major bleeding occurred in 40 patients (19.5%) (seven during CPB surgery). Combining all prospective HAT trials (n = 403), after start of lepirudin treatment, the combined endpoint occurred in 82 patients (20.3%), with 47 deaths (11.7%), 22 limb amputations (5.5%), 30 new TECs (7.4%), and 71 (17.6%) major bleedings. Compared with the historical control group (log-rank test), the combined endpoint after start of treatment was reduced (29.7% vs. 52.1%, P = 0.0473), primarily because of reduction in new thromboses (11.9% vs. 32.1%, P = 0.0008). Mean lepirudin maintenance doses ranged from 0.07 to 0.11 mg kg(-1) h(-1). Major bleeding was more frequent in the lepirudin-treated patients (29.4% vs. 9.1%, P = 0.0148). CONCLUSIONS: The rate of new TECs in HIT patients is low after start of lepirudin treatment. The rate of major bleeding of 17.6% might be reduced by reducing the starting dose to 0.1 mg kg(-1) h(-1).

Antihirudin antibodies following low-dose subcutaneous treatment with desirudin for thrombosis prophylaxis after hip-replacement surgery: incidence and clinical relevance.

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

Competitive immunoassay for recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection.

A competitive immunoassay based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been developed for the determination of recombinant hirudin (r-hirudin) in biological mixtures. Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of (r-hirudin), specific and reproducible analysis methods are demanded. The work involved the development of separation conditions allowing for routine analysis of plasma samples. In this study, r-hirudin was labeled with fluorescein isothiocyanate (FITC), and FITC-labeled r-hirudin was purified using high-performance liquid chromatography. The purified product was then mixed with the sample followed with the addition of anti-hirudin antibody. Free, antibody-bound, and tagged r-hirudin could be separated within 5 min by CE analysis using uncoated fused-silica capillary with high reproducibility. The developed method can be used to determine r-hirudin with good precision and a detection limit lower than 20 nM. This result demonstrates the feasibility of the CE-LIF immunoassay method for the determination of r-hirudin in plasma samples.

Delayed-type hypersensitivity and cross-reactivity to heparins and danaparoid: a prospective study.

BACKGROUND: Delayed-type hypersensitivity (DTH) reactions in patients receiving heparin may occur with both unfractionated (UFHs) and low molecular weight heparins (LMWHs). Skin testing is a clue to detect tolerated heparin or heparinoid preparations for further treatment. OBJECTIVE: To study in vivo cross-reactivity between LMWHs, UFHs, and danaparoid by skin testing in patients with suspected DTH to heparin. METHODS: Patients who fulfilled the criteria for the diagnosis of suspected heparin allergy were involved in a prospective study after informed consent. Patients presented with or had a history of typical erythematous plaques at the heparin injection sites. Skin testing was performed by subcutaneous injections of heparin (300-500 IU anti-Xa activity) and danaparoid (375 IU, eight patients). Desirudin (27,000 IU) was tested in three patients. We read skin reactions after 24, 48, and 96 hours and after 7 days. RESULTS: Fourteen female and 4 male patients were included in our series. Erythematous plaques had been reported or developed after 14-35 days in patients during first-time heparin treatment and after 2-10 days in reexposed patients. Positive skin test results were seen in 15 of 18 (83.3%) patients. Of these, 11 (73.3%) showed cross-reactivity between heparins and/or danaparoid. Six patients reacted to LMWHs only, nine patients to both LMWHs and UFHs. Danaparoid was tolerated in six of eight patients; desirudin was tolerated in all three patients tested. CONCLUSIONS: DTH to heparins is characterized by considerable cross-reactivity between LMWHs, UFHs, and danaparoid. UFHs may be tolerated even if LMWHs are not. Subcutaneous testing of a panel of heparins, danaparoid, and desirudin (hirudin) is recommended to determine acceptable treatment options for patients allergic to specific heparins.

Antithrombin inhibits lipopolysaccharide-induced tissue factor and interleukin-6 production by mononuclear cells, human umbilical vein endothelial cells, and whole blood.

OBJECTIVE: To investigate the effects of antithrombin on lipopolysaccharide (LPS)-induced tissue factor and interleukin-6 (IL-6) production in three different in vitro cellular systems: whole blood, human umbilical vein endothelial cells, and mononuclear cells. DESIGN AND SETTING: Laboratory in vitro study of the effects of antithrombin on procoagulant activity and cytokine release by LPS-stimulated endothelial and peripheral blood cells. SUBJECTS: In vitro whole blood, isolated human umbilical vein endothelial cell, and mononuclear cell cultures. INTERVENTIONS: Addition of antithrombin to LPS-treated whole blood, human umbilical vein endothelial cells, and mononuclear cells. MEASUREMENT AND MAIN RESULTS: Citrated whole blood, isolated human umbilical vein endothelial cells, or mononuclear cells were stimulated with LPS for 4-6 hrs in the presence or absence of antithrombin. Tissue factor activity was estimated by a tissue factor-dependent clotting or chromogenic assay and IL-6 was measured by specific ELISA. Antithrombin was found to inhibit tissue factor and IL-6 production in all three systems in a dose-dependent manner (0-40 IU/mL). Flow-through fractions of immunoadsorbed antithrombin concentrate were found to be ineffective. Five different batches of the same antithrombin concentrate were tested and the inhibitory activity was found to be consistent throughout all batches. Up to 40 microM of recombinant hirudin, a specific thrombin inhibitor, did not inhibit the production of tissue factor or IL-6 in either of the three cell systems, suggesting that the observed inhibition by antithrombin was not due solely to its ability to inhibit thrombin. CONCLUSIONS: Apart from the inhibition of thrombin and other activated clotting factors, antithrombin may also down-regulate the cellular expression of proinflammatory cytokines. Consequently, antithrombin concentrates may have value in the treatment of sepsis-induced disseminated intravascular coagulation.

Allosteric effects of a monoclonal antibody against thrombin exosite II.

We previously isolated a monoclonal antithrombin IgG from a patient with multiple myeloma [Colwell et al. (1997) Br. J. Haematol. 97, 219-226]. Using a panel of 55 surface mutants of recombinant thrombin, we now show that the epitope for the IgG most likely includes Arg-101, Arg-233, and Lys-236 in exosite II. The IgG affects the rate at which thrombin cleaves various peptide p-nitroanilide substrates with arginine in the P1 position, increasing the kcat for substrates with a P2 glycine residue but generally decreasing the kcat for substrates with a P2 proline. The allosteric effect of the IgG is altered by deletion of Pro-60b, Pro-60c, and Trp-60d from the 60-loop of thrombin, which lies between exosite II and the catalytic triad. The effect of the IgG, however, does not depend on the presence or absence of sodium ions, a known allosteric regulator of thrombin. The IgG does not affect the conformation of thrombin exosite I as determined by binding of a fluorescent derivative of hirudin54-65. These results provide evidence for a direct allosteric linkage between exosite II and the catalytic site of thrombin.

Weak allergenicity of recombinant hirudin CGP 39393 (REVASC) in immunocompetent volunteers. The European Hirudin in Thrombosis Group (HIT Group).

BACKGROUND: As a result of their polypeptidic nature, hirudins could theoretically elicit an immunologic response in humans. METHODS: The present open survey evaluates the allergenic potential of recombinant hirudin CGP 39393 (REVASC) after repeated intravenous or subcutaneous exposures in immunocompetent volunteers with no known previous exposure to hirudins. Clinical signs and symptoms of allergic manifestations and surrogate markers of allergy (i.e. response to skin tests) were collected before and after each administration of CGP 39393. Hirudin-specific immunoglobulin (Ig)G or IgE antibodies were measured. RESULTS: Two hundred and sixty-three healthy volunteers were eligible, of whom 12.2% had a history of allergy and 18.3% had a high level of total IgE. No signs or symptoms of allergy directly attributable to CGP 39393 were reported, either during or immediately after a first challenge. Irritative skin reactions, to either the prick or the intradermal skin test, were observed in eight volunteers 28-56 days after the challenge. Three out of 200 volunteers exposed to a second course of CGP 39393 showed signs and symptoms of an allergic reaction. In all but one subject with a pruritic erythema it was possible to rule out a causative role for CGP 39393 (0.50%, 95% confidence limits [CL] 0.01-2.75). Three other volunteers displayed a suspect or a positive immediate-type skin reaction to the follow-up intradermal skin test without any signs or symptoms of allergy. Another asymptomatic volunteer (0.80%, 95% CL: 0.02-4.41) developed a low [i.e. 1+ on the radio-allergosorbent test (RAST) scale] but measurable titre of hirudin-specific IgE antibodies after the second exposure to CGP 39393. A third exposure in five volunteers was clinically uneventful. A positive reaction to the prick test and to the intradermal skin test was observed in one individual. CONCLUSIONS: Recombinant hirudin CGP 39393 appears to be a weak allergen. Repeated exposures are safe in fully immunocompetent subjects, including those with a history of previous allergies and high levels of total IgE. Type I allergic reactions are rare (i.e. less than 1%) after a second exposure and are limited to the skin. Routine skin tests are not needed to identify patients at risk of developing type I allergic reactions. Hirudin-specific IgE antibodies are rarely seen and then at very low titres.

Preparation of antibodies to a synthetic C terminus of hirudin and identification of an antigenic site.

Hirudin is a 65 amino acid anticoagulant peptide produced in the leech. The single polypeptide is cross-linked by three disulfide linkages in the NH2 terminal half of the molecule. A peptide corresponding to the COOH terminus (residues 45-65) was synthesized utilizing lysine 47 as a specific residue to conjugate to thyroglobulin as a carrier for raising antibodies in mice. Using an enzyme-linked immunosorbent assay (ELISA) technique, it was found that the major antigenic domain(s) was located between residues 52-65. The COOH terminal residues Ile-59, Tyr-63, and Leu-64 are crucial for maintaining the antigenic structure. The NH2 terminal region (residues 45-52) that is proximal to the carrier protein, however, was not immunoreactive. A possible mechanism by which antibodies recognize the COOH terminal region of the synthetic peptide and the strategy for raising such antibodies are discussed.