Fibrinogen Milano XII: a dysfunctional variant containing 2 amino acid substitutions, Aα R16C and γ G165R

Abstract

Fibrinogen Milano XII was detected in an asymptomatic Italian woman, whose routine coagulation test results revealed a prolonged thrombin time. Fibrinogen levels in functional assays were considerably lower than levels in immunologic assays. Polymerization of purified fibrinogen was strongly impaired in the presence of calcium or ethylenediaminetetraacetic acid (EDTA). Two heterozygous structural defects were detected by DNA analysis: A␣ R16C and ␥ G165R. As seen previously with other heterozygous A␣ R16C variants, thrombin-catalyzed release of fibrinopeptide A was 50% of normal. Additionally, the release of fibrinopeptide B was delayed. Immunoblotting analysis with antibodies to human serum albumin indicated that albumin is bound to A␣ 16 C. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) analysis of plasmin digests of fibrinogen Milano XII in the presence of calcium or EDTA showed both normal and novel D1 and D3 fragments. Further digestion of abnormal D3 fragments by chymotrypsin resulted in degradation products of the same size as the fragments derived from normal fibrinogen. SDS-PAGE analysis under reducing conditions showed no difference between normal fibrinogen and fibrinogen Milano XII or between their plasmic fragments. Circular dichroism analysis revealed a shift in the mean residual ellipticity and a significant reduction of the ␣-helix content in the variant D3 fragment. It is concluded that the A␣-chain substitution is mainly responsible for the coagulation abnormalities, whereas the substitution in the ␥-chain induced a conformational change in the D3 fragment. IntroductionFibrinogen, a soluble plasma glycoprotein of 340 kd, is made up of 2 copies of 3 different polypeptide chains (A␣ 2 , B␤ 2 ,␥ 2 ), linked together with 29 interchain and intrachain disulfide bridges. The molecule is organized in a dimeric fashion consisting of a central E domain containing the amino termini of all 6 polypeptide chains and 2 outer D domains. In the final stage of blood coagulation, thrombin cleaves the fibrinopeptides A and B in a sequential manner from the amino termini of the A␣-and B␤-chains. Cleavage occurs between residues R16 (single-letter amino acid abbreviations) and G17 of the A␣-chain and residues R14 and G15 of the B␤-chain, exposing the A and B polymerization sites. Resultant monomers join together to form 2-stranded, halfstaggered protofibrils. It has been shown that protofibrils result from longitudinal D-D interactions and noncovalent contacts between the A and the a sites. The a site is formed by residues 329, 330, 340, and 364 in the carboxy-terminal part of the ␥-chain. 1,2 Subsequently, the growing fibrils aggregate in a lateral fashion to form fibers that increase progressively in thickness and develop branch points. The evolving network is finally stabilized with covalent bonds by the activated factor XIII, resulting in a clot resistant to mechanical disruption.Dysfibrinogenemia is a heritable disorder characterized by structural mutations in any of the 3 polypeptide cha...

Dysfibrinogenemia in childhood: two cases of congenital dysfibrinogens

“…Similar results were obtained by Lane et al[24], Lee et al[25], Stü cki et al[6], Reber et al[26], Loreth et al[27], and Bolliger-Stü cki et al[28] (all fibrinogens with the Aa 16 Arg ! Similar results were obtained by Lane et al[24], Lee et al[25], Stü cki et al[6], Reber et al[26], Loreth et al[27], and Bolliger-Stü cki et al[28] (all fibrinogens with the Aa 16 Arg !…”

Short fungal fractions of β-1,3 glucans affect platelet activation

“…TNF-␣ (20 ng/ml) was then added and after incubation for 30 min at 37°C, nonadherent cells were removed and the wells were washed with HBSS (5). To determine platelet adhesion to fibrinogen, fibrinogen was isolated from a plasma pool from HDs as previously (4). A 96-well plate was incubated with 200 l/well of fibrinogen (3.6 mg/ml) for 2 h at 37°C and overnight at 4°C.…”

Measurement of functional fibrinogen levels using the Thrombelastograph

“…It has been suggested that in children, younger than 12 months, not all of the fibrinogen is completely functional [2,3]. In addition, adults with certain genetic variants have dysfunctional fibrinogen [4][5][6][7][8][9]. The TEG measures functional fibrinogen as opposed to fibrinogen antigen levels, which are measured by immunoassay.…”