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HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Center for Molecular Medicine, Jichi Medical
School, Tochigi, Japan; Toshiba Hospital, Tokyo, Japan; Department of
Cell Biology, The Tokyo Metropolitan Institute of Medical Science,
Tokyo Metropolitan Organization for Medical Research, Tokyo, Japan;
Department of Cell and Developmental Biology, University of
Pennsylvania, School of Medicine, Philadelphia, PA; and Department of
Pediatrics, Kawasaki Medical School, Kurashiki, Japan.
The authors have identified a 12-residue carboxyl-terminal
extension of Lys-Ser-Pro-Met-Arg-Arg-Phe-Leu-Leu-Phe-Cys-Met in a dysfibrinogen derived from a woman heterozygotic for this abnormality and associated with severe bleeding. This extension is due to a T-to-A
mutation that creates AAG encoding Lys at the stop (TAG) codon, thus
translating 36 base pairs in the noncoding region of the B Fibrinogen is a 340-kd plasma protein that
participates in the final step of blood coagulation. Fibrinogen is
composed of 2 identical molecular halves, each molecular half being
composed of 3 nonidentical polypeptides, A The 3 chains are encoded by 3 independent genes clustered on chromosome
4. During the synthesis of fibrinogen, individual chains are
translated, processed, and assembled, and the mature fibrinogen
molecule is eventually secreted into blood circulation. Although
different assembly pathways have been proposed,9,10 a
molecular half, A The fibrinogen-to-fibrin conversion resulting in a fibrin network
consists of highly ordered molecular interactions, including binding
with thrombin, thrombin-catalyzed cleavage of fibrinopeptides A and B,
formation of staggered double-stranded fibrin protofibrils, and their
lateral associations. In these molecular interactions, several binding
modes have been proposed, and some of them have been confirmed by
electron microscope and x-ray crystallographic analyses.1-2,5-7,11
A hereditary dysfibrinogen is a fibrinogen molecule that is unable to
exert its physiologic functions because of a structural alteration(s)
determined at the gene level.12 Among 60 or more structural alterations so far identified at molecular level, those with
a Cys substitution are rather frequent, although the status of the Cys
in those dysfibrinogens has been elucidated only in some
cases.13 In this paper, we describe the presence of 2 types of end-linked fibrinogen homodimers formed by a single or pair of
disulfide bridges between the Cys residues in the carboxyl-terminal extension of a mutant B Fibrinogen was purified from normal and patient-citrated plasma.
Human Studies on purified fibrinogen
Lysylendopeptidase mapping of the fibrinogen B Amino acid composition and sequence analysis
Permeation and compaction studies Permeation study was performed according to the methods of Nair and Dhall17 with a slight modification as described previously.16 Compaction experiments were performed by a minor modification of a previously described method.16 Percent compaction was expressed as volume percent of the original volume (0.75 mL) of the clot.Scanning electron microscopy of Osaka VI clots Specimens for scanning electron microscopy (SEM) were prepared in 30-µL Plexiglas microdialysis cells perforated for solvent perfusion.18 Fibrinogen (1 mg/mL) was incubated with -thrombin (0.16 NIH U/mL) in a microdialysis cell in 50 mmol/L
Tris-HCl, pH 7.4, containing 0.1 mol/L NaCl at 25 °C for 45 minutes
or overnight. Specimens were washed 3 times with the rinse buffer (50 mmol/L cacodylate buffer, pH. 7.4) to remove excess salt, fixed for 2 hours in 2% glutaraldehyde in the rinse buffer, and then rinsed 3 times. The samples were dehydrated in a gradient series of ethanol concentrations through 100% over a period of 1.5 hours. The clots were
critical point-dried with CO2 in an HCP-2 (Hitachi, Tokyo, Japan), mounted, and finally coated with about 12.5 nm of
gold-palladium with an Hitachi Ion Sputter, E-1030. Specimens were
observed and photographed using an Hitachi SEM-S4100 scanning
electron microscope.
Electron microscopy of individual molecules Fibrinogen samples were prepared by spraying a dilute solution (25 µg/mL) of molecules in a volatile buffer (25 mmol/L ammonium formate, pH 7.4) containing 35% (vol/vol) glycerol onto freshly cleaved mica.8 Shadowing was performed with platinum at an angle of 8 degrees and carbon at 90 degrees on a rotary stage in a vacuum evaporator, VE-2000 (Vacuum Device, Ibaraki, Japan). The specimens were examined in a JEM-1010 transmission electron microscope (JEOL, Tokyo, Japan) at 80 kV and a magnification of 50 000.
Description of the patient The patient was a 36-year-old Japanese woman with a history of massive postpartum hemorrhage on each occasion of her previous 2 childbirths, for which 1300 mL and 2500 mL, respectively, of fresh blood had been transfused to control the bleeding. For her third childbirth, she delivered a baby uneventfully with the aid of 2.0 g of human fibrinogen fraction infused just before the delivery. Among the blood coagulation and hemostatic tests, the prothrombin time and the activated partial thromboplastin time were both moderately prolonged: 17.7 seconds (control 13.3 seconds) and 44.0 seconds (control 36.0 seconds), respectively. The thrombin time and the ancrod time were markedly prolonged: 50.8 seconds and 78.8 seconds (control 13.3 seconds and 16.8 seconds), respectively. The plasma fibrinogen concentration evaluated by the thrombin time method was less than 40 mg/100 mL, whereas that by the turbidimetric methods was in the normal range. Although the presence of a mild hepatic dysfunction seemed to account at least partly for the prolonged prothrombin time and activated partial thromboplastin time, decreased levels of plasma fibrinogen determined by the 2 different methods suggested the presence of a congenital dysfibrinogenemia. These data prompted us to conduct further investigations, including determination of structural defects and their relevance to the postpartum hemorrhages. The plasma of 2 of 3 daughters also gave prolonged thrombin and ancrod times.Abnormality of purified fibrinogen The purified patient-derived fibrinogen has 2 B -chain species,
denoted as B' and B, as evidenced by SDS-PAGE (Figure 1A). The
B-chain species corresponded to the normal B chain, whereas the
B'-chain species was found to have a molecular weight higher by
about 2000 than the normal B chain. Under nonreducing
conditions, the patient-derived fibrinogen also migrated as a doublet,
one at the position for normal fibrinogen denoted as Fbg and the other at a position equivalent to a 2-fold higher molecular weight protein, Fbg'. No trimer or higher molecular weight proteins were seen. This was
the first dysfibrinogen that could be seen as a doublet band with no
other high molecular weight species. In the second SDS-PAGE run under
reducing conditions, the Fbg' species was found to have B' chains
alone, suggesting that Fbg' exists as a homodimer consisting of 2 fibrinogen molecules with the B' chains only. On the other hand, Fbg
was found to consist of normal B-chain species only (Figure 1B). The
A and  chains appeared to be normal.
The thrombin time was moderately prolonged (27 seconds, control 14.4 seconds), and Ca++ partially corrected the delay. Profiles
of
Identification of an aberrant peptide in lysylendopeptidase digests
of Pe-B chain derived from the patient's fibrinogen, there was an
aberrant peptide peak, K34', and a peak with a wide shoulder, K16'
(Figure 3). They were not present in the
mapping profiles of digests of normal fibrinogen. By amino acid
sequence analyses, K16' was found to comprise the B (454-461)
segment with an additional Lys residue linked to its carboxyl terminus,
whereas no amino acids were detectable at each cycle of the normal
counterpart, K16 (Table 1). Because the
K16 peak was absent in the mapping profile of fibrin Pe- chain
digests, normal K16 peptide is the amino-terminal B (1-21) segment
whose amino-terminal amino acid is pyroglutamic acid, which is not
detectable by sequence analysis. These data suggested that the
patient-derived K16' contains 2 peptides, the B (1-21) peptide and
an aberrant peptide corresponding to the 454-to-461 residues that is
linked with an additional Lys residue at its carboxyl-terminal Gln461.
By sequence analysis of the neighboring K17' peptide, the normal
carboxyl-terminal B (454-461) segment was identified as
expected.
On the other hand, K34' was found to have a sequence of
Ser-Pro-Met-Arg-Arg-Phe-Leu-Leu-Phe-Cys(PeCys)-Met, which
seemed to be equivalent to those translated by a 36-base noncoding
region following the stop codon (TGA). Because K16' was found to have Lys at its carboxyl terminus, one base exchange that creates a codon
for Lys was expected. Indeed, a single T-to-A mutation at nucleotide
1464 of the B Although there was an extra Cys residue in this extended peptide
segment, no free sulfhydryl groups were detected by a sulfhydryl titration experiment of the patient's fibrinogen using a fluorogenic titrant, N-pyrenyl-maleimide (data not shown). Amino acid
sequence analysis of a whole fibrinogen molecule also showed that no
albumin was linked to the extra Cys residue of the aberrant B Ultrastructure of individual fibrinogen molecules The patient's fibrinogen molecules were rotary shadowed with platinum and examined by transmission electron microscopy (TEM), and the results from the several preparations are summarized in Figure 4. In our TEM images, the carboxyl-terminal and chains were separately observed in the D
region at higher magnifications, but additional small nodules
corresponding to the C domain19 were not observed. Of
1860 images of the patient's fibrinogen, about 52% had a simple
trinodular structure, 35% had a dimeric form connected at the D
regions, and 13% appeared to be multimers or irregular aggregates
(Figure 4B). No trimers in any forms were observed, but all the
aggregates were composed of even numbers of fibrinogen
molecules.
Interestingly, many of the dimers appeared to be end-linked, in which the 2 molecules were arranged with the ends of 2 molecules near each other, ie, with the molecular backbones at an angle, straight, or in parallel. In some dimers, the 2 D regions appeared to be fused into one elongated nodule. The straight form of dimer had some resemblance to the end-to-end joined fibrinogen dimers, which were covalently cross-linked by factor XIIIa.20,21 About 25% of the dimers were in the end-linked bilayer form in which the 2 molecules were aligned next to each other in parallel and appeared to be linked at D regions at both ends. In contrast to the Osaka VI fibrinogen molecule, most of the control fibrinogen molecules manifested simple trinodular structures with only a few aggregates. The presence of dimeric fibrinogen molecules may well disturb the fibrin monomer assembly and thus produce the abnormal fibrin network structure to be described next. Scanning electron micrographs of Osaka VI-fibrin clots When examined by SEM, the patient's fibrin clots were very different in appearance from control clots (Figure 5). The patient's clots obtained after 45 minutes of incubation with thrombin revealed spongelike gels composed of thinner fibers and large pores, as compared with the normal fibrin architecture (Figure 5A,B). Free fiber ends were often observed at the boundary between the networks and the pores so that the clots were very porous. When the gels were fully incubated with thrombin for 17 hours, however, a significantly different nature of appearance was commonly observed. Completely gelled Osaka VI clots were found to be composed of highly branched thin fibers creating a lacelike, uniform mesh structure (Figure 5C). There were very few free fiber ends or thick bundles, but large pores or open areas bounded by lacelike networks were frequently observed (Figure 5D). Many fibers were estimated to be about 15 to 25 nm in diameter and 210 to 300 nm in length between the branch points when calculated from the images at higher magnifications. On the contrary, the fibers and fiber bundles of normal fibrin clots were of a uniform thicker size with very few fiber ends. Branching structures seemed to be regularly spaced.
Compaction and permeation studies As shown in Figure 6, the patient's fibrin was considerably more compressible than normal fibrin, indicating that the patient's fibrin network was made up of fragile fibers, such as thin fibers with larger pores, a finding that is consistent with the appearance of the SEM images (Figure 5D).
Permeation studies were also conducted on Osaka VI fibrin. However, less satisfactory data were obtained because channeling along the walls or the other irregularities of flow through the fibrin gels were often observed soon after the flow experiment was started. From these results, we considered the Osaka VI clots as particularity fragile gels in which enlargement of the pores inside the networks leading to the destruction of the network easily occurred.
We have identified a unique 12-residue extension at the carboxy
terminus of the B A variety of Cys mutants have been reported, but no free sulfhydryl
groups have so far been reported in any of these mutant molecules.12 In some molecules, the Cys substitution is
linked with a single Cys as shown in fibrinogen Osaka II13
and, in another, with serum albumin as in fibrinogens
Dusart22 and Nijmegen and IJmuiden.23 There is
a unique Cys residue at A Among the TEM images of the patient-derived fibrinogen, we were able to
see 2 types of end-linked dimers, ie, an end-to-end-linked dimer and a
bilayer dimer in addition to individual trinodular fibrinogen molecules
and some aggregates. Although some of the end-to-end-linked dimers
resemble factor XIIIa cross-linked dimers,20,21 most of
those dimers appear to be arranged with a variety of angles between the
molecules, indicating that the disulfide-linked abnormal fibrinogen
dimers are certainly different from the rigid, factor XIIIa
cross-linked fibrinogen dimers. On the other hand, the end-linked bilayer dimer seems to have a unique structure in which the D regions
of 2 fibrinogen molecules seem to be connected at both ends of both
molecules (Figure 7). The 22-residue
peptide backbone length, 80 Å at maximum, may be long enough to form
the end-linked bilayer dimer. Although there might be high structural
tension in maintaining the alignment of the 2 molecules in parallel,
these dimers were clearly discernible. Because only even numbers of molecules were observed in aggregates in TEM images of the patient's fibrinogen, the aggregates are most likely to be produced by
noncovalent interactions of the dimers. If we consider the
aggregates as derivatives of dimers, the ratio of dimers to
single fibrinogen molecules, (A
Theoretically, both of the autosomal loci of genomic DNA for
fibrinogen could be operative, and thus 3 molecular species would be
synthesized in the hepatocytes of the heterozygous patients. They are
the fibrinogen molecules consisting of (1) two normal, (2) one normal
and one abnormal, and (3) two abnormal molecular halves. In this
patient's fibrinogen, however, we could see only homodimeric
fibrinogens, consisting of normal-normal or abnormal-abnormal molecular
halves, as deduced from SDS-PAGE profiles. Namely, the band (Fbg) was
found to have normal B In the first step of biosynthesis of fibrinogen, the formation of
A The structure of the fibrin network is usually influenced by the
substances coexisting in the reaction mixture.29 However, some of the fibrin networks of abnormal fibrinogens, such as Dusart, Caracas II, and Marburg fibrins,30-33 were found to be
composed of thinner and highly branched fibers due to the presence of
covalently bound albumin or extra sugar on the molecules. These
abnormal fibrinogens were noted to have defects in fibrin fiber
assembly. The major defect of fibrinogen Osaka VI was observed in the
step of lateral association of the double-stranded protofibrils.
Formation of double-stranded protofibrils seemed to proceed at normal
speed up to a certain level, as evidenced by normal release of
fibrinopeptide B and normal t-PA-catalyzed plasmin generation.
Nevertheless, we presumed that the presence of end-linked dimers may
have affected longitudinal elongation of the protofibrils together with
their lateral association. The reasons for this conclusion are as
follows: (1) the normal half-staggered arrangement of fibrin monomer in an overlapping manner could hardly be formed at the sites of the end-linked dimers, especially at the sites of end-linked bilayer dimers
that are aligned. This disturbance may well yield many branch
points; (2) steric constraints in the end-linked dimers may result in a
decrease or a loss of the conformational freedom required for the
longitudinal alignment of the fibrin dimers and thus increase the
possibility to have a fiber end or a bent form that would tend to make
branching junctions; and (3) the disulfide bond between the extended
regions of B Schematic models of Osaka VI fibrin assembly are shown in Figure 7. The Osaka VI fibrin clots were transparent and very fragile and have some resemblance to the Caracas II clots32 or the "fine clots" formed under high ionic strength conditions, because they have similar properties of thinner and more highly branched fibers than normal clots.34 However, these 3 networks were completely different on the basis of fiber thickness and branch-point density in the network and, especially, the network strength toward physical pressure. Each Osaka VI fibrin fiber seemed to form a twisted fiber with only a few protofibrils, though our estimation of the fiber width (< 20 nm) from the images of SEM may contain many latent errors produced by critical-point drying and metal vapor coating. The uniformity of the fiber width and the lacelike structure was distinctive to the Osaka VI fibrin networks. One of the most characteristic features of the fibrinogen Osaka VI clots is their fragility. More branching by itself might tend to make the clots very stiff, like fibrinogen Dusart clots. However, the Osaka VI clots have many points of weakness throughout because of the defective assembly of the protofibrils (Figure 7). There are many places where the bonding will be weak because the molecules cannot assemble with normal interaction. This type of network may be more easily damaged to form many channels or large pores inside the gels when fluids pass through the gels. Because of this breakage, we could not get any significant values in the permeation experiments of the Osaka VI fibrin, whereas "fine clots" exhibited lower permeation than normal clots (data not shown), and higher permeation was reported in the case of Caracas II fibrin.32 Compaction, the collapsibility of the network under constant centrifugal forces, was also higher than that of normal clots, indicating that there are many large pores inside the gels. The weakness of the Osaka VI fibrin clot may well explain the massive postpartum bleeding in this proband.
We thank Sumiko Murakami, Shinji Adachi, Kiyomi Inose, and Chandrasekar Nagaswami for their technical assistance and Michiko Takano for her secretarial assistance.
Submitted May 12, 2000; accepted July 27, 2000.
Supported in part by Scientific Research Grants-in-Aid for Scientific Research 11470250 and for International Scientific Research Program, Joint Research Grants 09044329, 10044316, and 11694308 from the Ministry of Education, Science and Culture of the Government of Japan, and National Institutes of Health HL 30954.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Michio Matsuda, Division of Cell and Molecular Medicine, Center for Molecular Medicine, Jichi Medical School, Yakushiji 3311-1, Tochigi, 329-0498, Japan; e-mail: thmichi{at}jichi.ac.jp.
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© 2000 by The American Society of Hematology.
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  A. A. Amelot, M. Tagzirt, G. Ducouret, R. L. Kuen, and B. F. Le Bonniec Platelet Factor 4 (CXCL4) Seals Blood Clots by Altering the Structure of Fibrin J. Biol. Chem., January 5, 2007; 282(1): 710 - 720. [Abstract] [Full Text] [PDF]
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 K. C. Lounes, J. B. Lefkowitz, A. H. Henschen-Edman, A. I. Coates, R. R. Hantgan, and S. T. Lord The impaired polymerization of fibrinogen Longmont (B{beta}166Arg{right-arrow}Cys) is not improved by removal of disulfide-linked dimers from a mixture of dimers and cysteine-linked monomers Blood, August 1, 2001; 98(3): 661 - 666. [Abstract] [Full Text] [PDF]
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| Copyright © 2000 by American Society of Hematology Online ISSN: 1528-0020 | |||||||||
-chain extension lead to fragile clot structure
Top
Abstract
Introduction
one (Fbg) being apparently equivalent
to normal fibrinogen and the other (Fbg') being about twice as large as
the other in terms of molecular size. Thus, the Fbg' species seems to
exist as a fibrinogen dimer. On reducing SDS-PAGE, the B
Cys) and its association with abnormal fibrin polymerization and thrombophila.
J Clin Invest.
1993;91:1637-1643.