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Rapid and sensitive techniques for identification and analysis of 'reactive-centre' mutants of C1-inhibitor proteins contained in type II hereditary angio-oedema plasmas.

ABSTRACT: Novel procedures for structural analysis of the 'reactive-centre' residues, particularly the P1 residue, of the dysfunctional C1-inhibitor proteins found in the plasmas of type II hereditary angio-oedema (HAE) patients are described. C1-inhibitor is adsorbed directly from plasma on to Sepharose-anti-(C1 inhibitor) beads. The P1 residue of C1 inhibitor is arginine and hence a potential cleavage site for trypsin. Thus trypsin digestion of the immobilized protein, followed by SDS/PAGE of the released fragments, identifies P1 residue mutations. Pseudomonas aeruginosa elastase digestion of the immobilized protein, followed by purification of the released C-terminal peptide (by h.p.l.c.) and N-terminal sequence analysis defines the new P1 residue (or other mutations in the reactive-centre region). The techniques are both rapid and highly sensitive, requiring only 400 microliters of plasma. In addition, they permit accurate assessment of the level of normal (functional) inhibitor in a subclass of type II HAE plasmas, those containing P1-residue mutant proteins.

SUBMITTER: Aulak KS

PROVIDER: S-EPMC1149599 | biostudies-other | 1990 Nov

REPOSITORIES: biostudies-other

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Project description:BackgroundHereditary angioedema (HAE) is characterized by unpredictable attacks of debilitating subcutaneous and mucosal edema. Gastrointestinal attacks are painful, of sudden onset and often mistaken for acute abdomen leading to unnecessary surgery. The purpose of this study was to analyze symptom presentation of gastrointestinal angioedema in pediatric and adult HAE patients.MethodsInformation collected during the clinical development of ecallantide for treatment of acute HAE attacks included affected anatomic location, accompanying symptoms, medical history, and pain assessments. Efficacy endpoints included Treatment Outcome Score (TOS, maximum score = 100; minimally important difference = 30), a point-in-time measure of treatment response, and time to treatment response.ResultsForty-nine percent of 521 HAE attacks only involved abdominal symptoms. The most commonly reported abdominal symptoms were distension (77%), cramping (73%) and nausea (67%). The most common pain descriptors were tender, tiring-exhausting, aching, cramping and sickening. White blood cell counts were elevated (>10 × 10(9)/L) in 23% of attacks (mean ± SD: 15.1 ± 11.27 × 10(9)/L). A high proportion of patients reported a history of abdominal surgery, including appendectomy (23%), cholecystectomy (16.4%), and hysterectomy (8.2%). Mean TOS at 4 hours post ecallantide was 77 ± 33 versus 29 ± 65 for placebo. Median time to significant symptom resolution was 165 minutes (95% CI 136, 167) for ecallantide versus >4 hours (95% CI 161, >4 hours) for placebo. Anaphylactic reactions occurred in 6 of the 149 treated patients.ConclusionsHAE should be considered in the differential diagnosis of patients with recurrent discrete episodes of severe, unexplained crampy abdominal pain associated with nausea.Trials registrationThe data used in the analysis were gathered across multiple clinical trials conducted during the clinical development program for ecallantide. All of the studies were conducted using Good Clinical Practices (GCP) and in accordance with the ethical principles that have their origins in the Declaration of Helsinki. Each site that participated in the clinical trials obtained the appropriate IRB or Ethics Committee approval prior to enrolling any patients. All patients provided written informed consent prior to undergoing any study-related procedures. Pediatric patients provided written assent and their parents or guardians gave written informed consent.The following trials have been registered at http://www.clinicaltrials.gov: EDEMA2 (identifier NCT01826916); EDEMA3 (identifier NCT00262080); EDEMA4 (identifier NCT00457015); and DX-88/19 (identifier NCT00456508).

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Rapid and sensitive techniques for identification and analysis of 'reactive-centre' mutants of C1-inhibitor proteins contained in type II hereditary angio-oedema plasmas.

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