XIVth International Fibrinogen Workshop 11
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27. F i b r i n o g e n b - c h a i n c r o s s - l i n k i n g b y tissue
transglutaminases
O. V. Mitkevich, J. R. Shainoff 1, G. B. Smejkal 1,
P. M. Dibello 1, M. J. I m 1
Institute of Experimental Cardiology, Russian
Cardiology Research Center, Moscow, 121552, Russia;
IResearch Institute, The Cleveland Clinic Foundation,
Cleveland, OH 44195, USA
Prior characterizations of cross-linking of fibrinogen by
tissue-transglutaminase (TG) had shown that it cross-links
fibrinogen a- and g-chains in patterns differing from the
cross-linking catalyzed by blood coagulation factor XlIIa
(XlIIa). However, little attention had been paid to the possi-
bility that TG might differ further by forming previously
unknown b-chain cross-links. We purified polyclonal anti-
human b-chain antibodies to remove all traces of compo-
nents cross-reacting with a- and g-chains, and used them to
probe Western blots of SDS-electropherograms of partially
cross-linked fibrinogen. We found, surprisingly, that
fibrin(ogen) b-chains do undergo cross-linking by TG. As
anticipated, no b-chain cross-linking was observed with
XlIIa. The b-chain cross-linking catalyzed by TG is slow com-
pared to the rapid formation homologous a2 and an multi-
mers by TG, but it is not slow compared to the formation of
the high molecular weight hybrid aJg multimers that form
late in the cross-linking reaction. Guinea pig liver TG incor-
porates b-chains into hybrid multimers at a rate approxi-
mately one-half of the rate of formation of a/g multimers. But
the TG isolated as the Gah GTP-binding protein from failed
human hearts behaved differently from guinea pig liver TG
by principally (90%) forming what we believe to be intra-b-
chain cross-links instead of high molecular weight multi-
chain products (10%): The intra-chain cross-linking was
indicated by a shift to faster electrophoretic mobility without
degradation of the chains. The conversion to the fast migrat-
ing chains (coinciding with mobility of reference g-chains)
proceeded at a rate comparable to the g-chain cross-linking
by the Gah TG. These modes of cross-linking by the two TGs
were essentially the same for fibrinogen and fibrin. Work is
in progress to confirm these findings by isolating and identi-
fying the loci of the cross-links in the derivatives. The intra-
b-chain cross-links formed by human Gah TG might serve as
clear markers for assessing the contribution of TG to the
formation of cross-linked fibrinogen in the course of athero-
genesis.
28. A new model of fibrinogen a n d fibrin
i n t r a m o l e c u l a r a n d e x t r a m o l e c u l a r association
sites a n d fibrin c r o s s l i n k i n g : a p p l i c a t i o n to s t u d y o f
dysfibrinogenemia
M. W. Mosesson, K. R. Siebenlist
Sinai Samaritan Medical Center, Univ Wisc Med Schl,
Milwaukee Clin Campus, Milwaukee, WI, USA
Crosslinking of fibrinogen at its C-terminal g-chain crosslink-
ing site occurs in the presence of factor XIIIa due to self-asso-
ciation at a constitutive D domain site ('gXL'). C-terminal
regions of fibtinogen Aa chains, mainly the C-terminal -100
residues comprising the 'aC' region, enhance self-association
© Pearson Professional L td 1996
and the crosslinking rate at the gXL site. In addition, there is a
constitutive self-association site ('D:D') situated at the outer
end of each D domain that contributes to linear molecular
alignment along fibril strands. Upon thrombin-catalyzed con-
version of fibrinogen to fibrin, FPA release exposes an N-ter-
minal site ('EA'; GPRV) that then interacts with a constitutive
site in each D domain ('Da') to drive the fibrin 'D:E' assembly
process. The 'aC' domain, which tends to be tethered to the
fibrinogen E domain, is released following cleavage of FPB,
and promotes fibrin assembly by enhancing lateral fibril asso-
ciations. Thus, these association sites participate in either fib-
rin(ogen) self-association and/or polymerization, and collec-
tively define a new and more detailed model of fibrin
assembly. Defects at any of the association sites result in dis-
tinctive abnormalities in the assembly/crosslinking process.
This has been exemplified by recent studies of dysfibrinogen-
emia that can be explained in terms of the model, and which
in turn provide examples of the abnormalities that ensue
from each type of defect. We will present examples of dysfib-
rinogens illustrating one or more association site defects
including fibrinogens Dusart (aC), Birmingham (EA), Cedar
Rapids/Tokyo II (D:D).
29. T h r o m b o p h i l i a associated w i t h
dysfibrinogenemia (fibrinogen Cedar Rapids
(g R275C)) a n d a concurrent heterozygous factor V
Leiden defect
M. W. M o s e s s o n 1, K. R. Siebenlist 1, J. D. Olson 2
ISinai Samaritan Medical Center, Univ Wisc Med Schl,
Milwaukee Clin Campus, Milwaukee, WI; 2 Univ Iowa,
Iowa City, Iowa, USA
Fibrinogen Cedar Rapids (g R275C) is a heterozygotic dys-
fibrinogenemia that is associated with severe pregnancy-
associated thrombophilia in three second-generation family
members. Like fibrinogen Tokyo II (g R275C) (Mosesson et
al, J Clin Invest 1995; 96:1053), which is not associated with
thrombophilia, fibrinogen Cedar Rapids is characterized by
defective fibrin assembly, normal fibrin D:E assembly, and
normal fibrinogen and fibrin factor XIIIa-mediated
crosslinking. Fibrin formed from Cedar Rapids fibrin forms
a network structure that is more highly branched than nor-
mal fibrin. Investigation of Cedar Rapids family members
indicated that each affected subject was also heterozygotic
for the factor V Leiden defect. One first-generation parent
who was normal with respect to factor V Leiden but
expressed the abnormal Cedar Rapids gene, gave no history
of thrombophilia, nor did 9 of her siblings. The other parent
was normal with respect to fibrinogen Cedar Rapids, but
was heterozygotic for the factor V Leiden defect; neither he
nor 13 of his siblings gave any history of thrombophilia.
One third generation heterozygotic male subject is clinically
normal at present but expresses both fibrinogen Cedar
Rapids and the factor V Leiden defect. These findings sug-
gest that co-expression of factor V Leiden and fibrinogen
Cedar Rapids is causally associated with thrombophilia,
although the mechanism by which this situation comes
about is not clear.
Fibrinolysis (1996) 10, SuppL 4, 1-26