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Reply to the fibrinogenolytic pathway of fibrinogen catabolism. A comment
LETTER
REPLY
TO THE
States
TO THE
A.
OF FIBRINOGEN
Sherman
of Laboratory bledicine, Barnes Hospital Washington University School of Medicine, St. Louis, Ilissouri 63110
(Received
19.4.1974.
1974 Inc.
EDITORS-IN-CHIEF
FIBRINOGENOLYTIC PATHWAY CATABOLIS?I. A CONNENT. Laurence
Division
ti, pp. 901-9Oj, Pergamon Press,
Vol.
THRO?lBOSIS RESEARCH Printed in the United
Accepted
by Editor
A.L.
Plaza,
Copley)
In their reply to Mosesson's letter on the Fibrinogenolytic Pathway of fibrinogen catabolismi, Collen, et al', have raised specific questions regarding the concept that fibrinogenolysis is a major catabolic pathway of fibrinogen in rabbits and that there is suggestive evidence that such a pathway occurs in man. Collen, et al, had criticisms in two specific areas, one being that the observed heterogeneity of fibrinogen may have been due to in vitro proteolytic digestion occurring during the fractionating procedure, and secondly, "the coagulationfibrinolysis balance in humans and in rabbits operates at similar rates" may not be a valid assumption and, thus, animal evidence of in vivo fibrinogenolysis may not be applicable to man. germaine.
Several comments regarding these criticisms would seem
First of all, in the several in vivo studies we have conducted of
multiple pathways of fibrinogen catabolism in animals3&,
there has been clear
internal evidence that the transformations observed are not the result of the in vitro processing procedure.
As may be seen in the enclosed figure from our
first publication3, in this experiment radioactive labeled high molecular weight fibrinogen fraction l-2 was injected into rabbits and, with time, the radioactive label appeared in the lower molecular weight high solubility of fraction I-8. 901
ON THE FIBRINOGENOLYTIC
902
PATHWAY
Sample analysis was conducted by ethanol fractionation on the samples,
If the
appearance of radioactivity in the lower molecular weight material was an in vitro artifact occurring during the fractionating procedure, than the same specific activity should have been observed in I-8 as in I-2, in the sample drawn from the animal at zero time (5 min after injection).
Such was not.the case.
Only small
amounts of contaminant radioactivity appeared in 1-8, but rather the specific activity increased in samples drawn later in time strongly indicates that the process was an in vivo phenomenon.
The degree of contamination noted in the
zero time sample has been a reproducible constant phenomenon both in vivo and in vitro and would indicate either in complete separation, or that a slight degree of in vitro proteolysis occurs.
However, the results also indicated that these
were the limits of in vitro proteolysis and that the higher levels of specific activity observed on the subsequent days' samples were indeed an in vivo phenomenon Collen, et al, additionally drew an unwarranted conclusion from a subsequent work4 which indicated that a fibrin derivative ;was also a catabolic pathway. They made the assumption that our data indicated that a quantitatively large amount of fibrinogen was catabolized through a thrombin mediated pathway and noted that Nossel's data suggested that such was not the case in man.
Therefore, they .
concluded "if in vivo coagulation in man operates at a much lower rate than in rabbits, it is possible and even likely that in vivo fibrinogenolysis may also be much slower".
This is not a valid assumption for the reason that we did not
indicate that a quantitatively large amount of rabbit fibrinogen was mediated via a thrombin pathway.
In fact, as was noted in the paper, we did not feel
it was possible to quantitate the amount catabolized via this route.
However,
based on the data in that paper, a very rough estimate would suggest that no more than five to seven percent of fibrinogen catabolism is catabolized via this route.
Such a value is not greatly different than the two to three percent
argued from the Nossel data and cannot be used to support the contention of marked differences in fibrinogen catabolism between rabbit and man.
Collen,
ON THE FIBRINOGENOLYTIC
METABOLISM OF S-35
FIBRINOGEN
PATHWAY
903
FRACTION I-2
Fibrinogen :
k
lnfechon of S -35 Fibrinogen Fraction Z-2
I
I
I
I
I
I
I
0
1
2
DA?S
4
5
6
FIG.
1
Clearance of 35S-fibrinogen fraction I-2.
Fraction specific radioactivities
are plotted for serial samples after injection of 35S-fibrinogen fraction I-2.
The fractions represented are the 8 percent ethanol precipitable LSF
(open circles) and the 8 percent ethanol soluble HSF (closed circles).
et al, have also commented on a lack of effect of heparin on fibrinogen turnover in normal man, implying the absence of fibrin formation.
If, as noted, this
pathway is only two to seven percent of total turnover, measurement of total turnover is unlikely to demonstrate any significant change with heparinization. It is important on viewing all of the data to be aware that the biologic significance of a catabolic pathway is not necessarily related to the quantitative amounts of fibrinogen catabolized by that route.
The fact that any amounts of
fibrinogen may be demonstrated to be catabolized via a fibrinogenolytic route may make this of significant biologic importance even though it may not be quantitatively the major pathway of catabolism.
Obviously, as Collen, et al, suggests,
ON THE
904
FIBRINOGENOLYTIC
PATHWAY
further in vivo studies in man need to be conducted and, indeed, such are in progress. Lastly, while we have indicated the striking biochemical similarity of these in vivo fibrinogen derivatives to in vitro products of plasmin and thrombin activity, other enzymes may be involved, but such hypothetical enzymes must have similar actions to plasmin and thrombin. Mosesson
Both the observations of Harpel and
on modification of plasmin activity by cys macroglobulin and Merskey's6
speculations on hemostatic derangements caused by noncoagulation enzymes must be considered.
However, the in vitro studies of ourselves and Mosesson7's are
strong evidence that whatever the mechanism, that human plasma fibrinogen is heterogeneous in nature.
The fibrinogenolytic mechanism and possibly the fibrin
monomer pathways are concepts based on significant in vitro data in man and in vivo evidence in animals.
Their potential importance relates only partly to their
quantitative role in overall catabolism but to the fact of such enzymatic activity occurring at all under physiological conditions.
Such then permits a view of
pathology as an exaggeration of normal physiological mechanisms, a concept in keeping with a variety of diseases in other areas of medicine.
REFERENCES
1.
Mosesson, M.W. The fibrinogenolytic pathway of fibrinogen catabolism. Thromb. Res. 2:185, 1973.
2. Collen, D., Semeraro, N., and Verstraete, M. pathway of fibrinogen catabolism.
The fibrinogenolytic Thromb. Res. &:491, 1974.
3.
Sherman, L.A., Fletcher, A.P., and Sherry, S. In vivo transformation between fibrinogen varying ethanol solubilities: a pathway of fibrinogen catabolism. J. Lab. Clin. Med. -73~574, 1969.
4.
Sherman, L.A. Fibrinogen turnover: demonstration of multiple pathways 79:70, 1972. of catabolism. J. Lab. Clin. Med. -
5.
Harpel, P.C. and Mosesson, M.W. Degradation of human fibrinogen by plasma cu,-macroglobulin-enzyme complexes. J. Clin. Invest. =:2175,
6.
Merskey, C.
Defibrination syndrome or ...? Blood g:599,
1973.
1973.
Vo1.4,No.6
ON THE
FIBRINOGENOLYTIC
PATHWAY
903
7.
Sherman, L.A., Fletcher, A.P., and Sherry, S. Isolation and characteriza. tion of the clottable low molecular weight fibrinogen derived by limited plasmin hydrolysis of human fraction I-4. Biochemistry 8:1515, 1969.
8.
Mosesson, M.W., Finlayson, J.S., Umfleet, R.A., and Galanakis, D. Human fibrinogen heterogeneities. I. Structural and related studies of plasma fibrinogens which are high solubilities catabolic intermediates. J. Biol. Chem. 247:5210, 1972.
REPLY
TO THE
States
TO THE
A.
OF FIBRINOGEN
Sherman
of Laboratory bledicine, Barnes Hospital Washington University School of Medicine, St. Louis, Ilissouri 63110
(Received
19.4.1974.
1974 Inc.
EDITORS-IN-CHIEF
FIBRINOGENOLYTIC PATHWAY CATABOLIS?I. A CONNENT. Laurence
Division
ti, pp. 901-9Oj, Pergamon Press,
Vol.
THRO?lBOSIS RESEARCH Printed in the United
Accepted
by Editor
A.L.
Plaza,
Copley)
In their reply to Mosesson's letter on the Fibrinogenolytic Pathway of fibrinogen catabolismi, Collen, et al', have raised specific questions regarding the concept that fibrinogenolysis is a major catabolic pathway of fibrinogen in rabbits and that there is suggestive evidence that such a pathway occurs in man. Collen, et al, had criticisms in two specific areas, one being that the observed heterogeneity of fibrinogen may have been due to in vitro proteolytic digestion occurring during the fractionating procedure, and secondly, "the coagulationfibrinolysis balance in humans and in rabbits operates at similar rates" may not be a valid assumption and, thus, animal evidence of in vivo fibrinogenolysis may not be applicable to man. germaine.
Several comments regarding these criticisms would seem
First of all, in the several in vivo studies we have conducted of
multiple pathways of fibrinogen catabolism in animals3&,
there has been clear
internal evidence that the transformations observed are not the result of the in vitro processing procedure.
As may be seen in the enclosed figure from our
first publication3, in this experiment radioactive labeled high molecular weight fibrinogen fraction l-2 was injected into rabbits and, with time, the radioactive label appeared in the lower molecular weight high solubility of fraction I-8. 901
ON THE FIBRINOGENOLYTIC
902
PATHWAY
Sample analysis was conducted by ethanol fractionation on the samples,
If the
appearance of radioactivity in the lower molecular weight material was an in vitro artifact occurring during the fractionating procedure, than the same specific activity should have been observed in I-8 as in I-2, in the sample drawn from the animal at zero time (5 min after injection).
Such was not.the case.
Only small
amounts of contaminant radioactivity appeared in 1-8, but rather the specific activity increased in samples drawn later in time strongly indicates that the process was an in vivo phenomenon.
The degree of contamination noted in the
zero time sample has been a reproducible constant phenomenon both in vivo and in vitro and would indicate either in complete separation, or that a slight degree of in vitro proteolysis occurs.
However, the results also indicated that these
were the limits of in vitro proteolysis and that the higher levels of specific activity observed on the subsequent days' samples were indeed an in vivo phenomenon Collen, et al, additionally drew an unwarranted conclusion from a subsequent work4 which indicated that a fibrin derivative ;was also a catabolic pathway. They made the assumption that our data indicated that a quantitatively large amount of fibrinogen was catabolized through a thrombin mediated pathway and noted that Nossel's data suggested that such was not the case in man.
Therefore, they .
concluded "if in vivo coagulation in man operates at a much lower rate than in rabbits, it is possible and even likely that in vivo fibrinogenolysis may also be much slower".
This is not a valid assumption for the reason that we did not
indicate that a quantitatively large amount of rabbit fibrinogen was mediated via a thrombin pathway.
In fact, as was noted in the paper, we did not feel
it was possible to quantitate the amount catabolized via this route.
However,
based on the data in that paper, a very rough estimate would suggest that no more than five to seven percent of fibrinogen catabolism is catabolized via this route.
Such a value is not greatly different than the two to three percent
argued from the Nossel data and cannot be used to support the contention of marked differences in fibrinogen catabolism between rabbit and man.
Collen,
ON THE FIBRINOGENOLYTIC
METABOLISM OF S-35
FIBRINOGEN
PATHWAY
903
FRACTION I-2
Fibrinogen :
k
lnfechon of S -35 Fibrinogen Fraction Z-2
I
I
I
I
I
I
I
0
1
2
DA?S
4
5
6
FIG.
1
Clearance of 35S-fibrinogen fraction I-2.
Fraction specific radioactivities
are plotted for serial samples after injection of 35S-fibrinogen fraction I-2.
The fractions represented are the 8 percent ethanol precipitable LSF
(open circles) and the 8 percent ethanol soluble HSF (closed circles).
et al, have also commented on a lack of effect of heparin on fibrinogen turnover in normal man, implying the absence of fibrin formation.
If, as noted, this
pathway is only two to seven percent of total turnover, measurement of total turnover is unlikely to demonstrate any significant change with heparinization. It is important on viewing all of the data to be aware that the biologic significance of a catabolic pathway is not necessarily related to the quantitative amounts of fibrinogen catabolized by that route.
The fact that any amounts of
fibrinogen may be demonstrated to be catabolized via a fibrinogenolytic route may make this of significant biologic importance even though it may not be quantitatively the major pathway of catabolism.
Obviously, as Collen, et al, suggests,
ON THE
904
FIBRINOGENOLYTIC
PATHWAY
further in vivo studies in man need to be conducted and, indeed, such are in progress. Lastly, while we have indicated the striking biochemical similarity of these in vivo fibrinogen derivatives to in vitro products of plasmin and thrombin activity, other enzymes may be involved, but such hypothetical enzymes must have similar actions to plasmin and thrombin. Mosesson
Both the observations of Harpel and
on modification of plasmin activity by cys macroglobulin and Merskey's6
speculations on hemostatic derangements caused by noncoagulation enzymes must be considered.
However, the in vitro studies of ourselves and Mosesson7's are
strong evidence that whatever the mechanism, that human plasma fibrinogen is heterogeneous in nature.
The fibrinogenolytic mechanism and possibly the fibrin
monomer pathways are concepts based on significant in vitro data in man and in vivo evidence in animals.
Their potential importance relates only partly to their
quantitative role in overall catabolism but to the fact of such enzymatic activity occurring at all under physiological conditions.
Such then permits a view of
pathology as an exaggeration of normal physiological mechanisms, a concept in keeping with a variety of diseases in other areas of medicine.
REFERENCES
1.
Mosesson, M.W. The fibrinogenolytic pathway of fibrinogen catabolism. Thromb. Res. 2:185, 1973.
2. Collen, D., Semeraro, N., and Verstraete, M. pathway of fibrinogen catabolism.
The fibrinogenolytic Thromb. Res. &:491, 1974.
3.
Sherman, L.A., Fletcher, A.P., and Sherry, S. In vivo transformation between fibrinogen varying ethanol solubilities: a pathway of fibrinogen catabolism. J. Lab. Clin. Med. -73~574, 1969.
4.
Sherman, L.A. Fibrinogen turnover: demonstration of multiple pathways 79:70, 1972. of catabolism. J. Lab. Clin. Med. -
5.
Harpel, P.C. and Mosesson, M.W. Degradation of human fibrinogen by plasma cu,-macroglobulin-enzyme complexes. J. Clin. Invest. =:2175,
6.
Merskey, C.
Defibrination syndrome or ...? Blood g:599,
1973.
1973.
Vo1.4,No.6
ON THE
FIBRINOGENOLYTIC
PATHWAY
903
7.
Sherman, L.A., Fletcher, A.P., and Sherry, S. Isolation and characteriza. tion of the clottable low molecular weight fibrinogen derived by limited plasmin hydrolysis of human fraction I-4. Biochemistry 8:1515, 1969.
8.
Mosesson, M.W., Finlayson, J.S., Umfleet, R.A., and Galanakis, D. Human fibrinogen heterogeneities. I. Structural and related studies of plasma fibrinogens which are high solubilities catabolic intermediates. J. Biol. Chem. 247:5210, 1972.