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Blood, Vol. 95 No. 3 (February 1), 2000:
pp. 943-951
HEMOSTASIS, THROMBOSIS, AND VASCULAR BIOLOGY
From the Departments of Pathology, Biochemistry, and Medicine;
Queen's University, Kingston, Ontario, Canada.
Previous studies have shown that thrombin generation in vivo caused
a 92% decrease in factor IX (F.IX) activity and the appearance of a
cleavage product after immunoblotting that comigrated with activated
F.IX (F.IXa). Under these conditions, the fibrinolytic system was
clearly activated, suggesting plasmin may have altered F.IX. Thus, the
effect(s) of plasmin on human F.IX was determined in vitro. Plasmin (50 nM) decreased the 1-stage clotting activity of F.IX (4 µM) by 80%
and the activity of F.IXa (4 µM) by 50% after 30 minutes at
37°C. Plasmin hydrolysis of F.IX yields products of 45, 30, 20, and
14 kd on reducing sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) and 2 products of 52 and 14 kd under
nonreducing conditions. Plasmin-treated F.IX did not bind the active
site probe, p-aminobenzamidine, or form an SDS-stable complex with
antithrombin. It only marginally activated human factor X in the
presence of phospholipid and activated factor VIII. Although
dansyl-Glu-Gly-Arg-chloromethyl ketone inactivated-F.IXa inhibited the
clotting activity of F.IXa, plasmin-treated F.IX did not. Plasmin
cleaves F.IX after Lys43, Arg145, Arg180, Lys316, and Arg318, but F.IXa
is not appreciably generated despite cleavage at the 2 normal
activation sites (Arg145 and Arg180). Tissue plasminogen activator-catalyzed lysis of fibrin formed in human plasma results in
generation of the 45- and 30-kd fragments of F.IX and decreased F.IX
clotting activity. Collectively, the results suggest that plasmin is
able to down-regulate coagulation by inactivating F.IX.
(Blood. 2000;95:943-951)
The processes of clot formation and lysis consist of a
series of enzymatic reactions that involve the controlled activation of
inactive zymogens and cofactors.1-4 The blood-clotting
reactions result in the generation of thrombin, which mediates clot
formation by catalyzing the conversion of soluble fibrinogen to
insoluble fibrin. Fibrinolysis occurs upon generation of plasmin, which cleaves insoluble fibrin to fibrin degradation products. Experimental evidence indicates that a hemostatic balance exists between coagulation and fibrinolysis under normal conditions5,6 and that this balance is perturbed in response to injury7 and during
disease.8-10 Components of the coagulation and fibrinolytic
processes may not only regulate their respective pathways but may also
interact with each other. For example, in the presence of
thrombomodulin, thrombin activates a plasma carboxypeptidase (thrombin
activable fibrinolysis inhibitor) whose resultant activity
downregulates fibrinolysis.11 Conversely, plasmin has been
shown in vitro to inactivate factor (F.)IX,12 activate
F.VII13 and F.XII,14 and initially activate and
then subsequently inactivate F.V15 and
F.VIII.16 Plasmin's action on F.V, F.VII, F.VIII, F.IX, and F.XII may profoundly alter the levels of thrombin generated and
significantly influence the resultant rate of coagulation.
The infusion of activated F.X (F.Xa) and
phosphatidylcholine/phosphatidylserine (PCPS) vesicles results in
thrombin generation in vivo.17-19 These animal models have
been used to study the coagulation and fibrinolytic reactions that
occur subsequent to thrombin generation in vivo. At elevated doses of
F.Xa and PCPS, the regulation of hemostasis may be compromised, and the
resultant phenotype is remarkably similar to the human disorder,
disseminated intravascular coagulation (DIC).20 A study of
the individual coagulation factors indicated that the clotting activity
of F.IX decreased by 92% in this experimental model after F.Xa/PCPS
infusion.21 In addition, a lower apparent molecular mass
cleavage product of F.IX approximating the size of F.IXa was observed
by immunoblotting following F.Xa/PCPS infusion. This result was
unexpected because the F.IXa-like species produced as a result of
thrombin generation would have been expected to complex with plasma
antithrombin (AT) into a larger product. These results
suggest that, in response to thrombin generation in vivo, either F.IXa
was generated from the action of its physiologic activators F.IX is synthesized in the liver as a single-chain glycoprotein
(Mr 57 000), and the zymogen circulates in human plasma at approximately 5 µg/mL (0.09 µM).24 The complete
nucleotide sequence of the F.IX gene and amino acid sequence of the
mature protein has been determined.25 F.IX is activated in
a 2-step process by either F.XIa22 or TF and
F.VIIa.23 Initial cleavage occurs at Arg145 to yield a
2-chain inactive "intermediate" of 46 kd (F.IX Materials
Methods
Hydrolysis of F.IX and F.IXa by plasmin.
Purified F.IX or F.IXa (4 µM) was treated at 37°C with 50 nM of
plasmin in 50 mM of HEPES per 0.15 mol/L NaCl, pH 7.4 (HBS) containing
5 of mM CaCl2 (HBS/Ca). After varying times of incubation, 2 aliquots were removed from the same reaction. One aliquot was added
to an equal volume of SDS-PAGE sample buffer as previously described.38 The other aliquot was added to VFK-CMK (2.2 µM final concentration) in HBS and incubated on ice for 10 minutes prior to assay for F.IX clotting activity (see below). VFK-CMK (2.2 µM) completely inactivated plasmin activity toward both F.IX and the
plasmin peptide substrate S-2251, but it had no effect on the F.IX/IXa
coagulant activity at the concentrations of F.IX/IXa assayed.
Factor IX clotting assay.
This was performed using an aPTT clotting assay and F.IX-deficient
human plasma as previously described.38 NHP was used as the
standard, assigning 1 unit of F.IX clotting activity per milliliter of plasma.
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
SDS-PAGE was carried out according to the method of
Neville.39 Approximately 4 µg of F.IX protein was loaded
per lane using 5%-to-15% or 5%-to-20% linear polyacrylamide
gradient gels. The plasmin cleavage products of F.IX were visualized
after 12 to 16 hours of staining at 22°C with 0.0016% Coomassie
Blue R-250 in 5% acetic acid and 7.5% ethanol and destaining for 2 to
3 hours in 18% methanol and 9% acetic acid.
Binding of pAB to F.IX treated with F.XIa or plasmin.
The binding of the fluorescent active site probe pAB to F.IX treated
with either F.XIa or plasmin was performed according to Lin et
al.40 F.IX (4 µM) was incubated with either 500 nM of
F.XIa or 50 nM of plasmin and 4 µM of pAB in HBS, pH 7.4. The reactions were performed at 37°C using a Perkin Elmer Luminescence Spectrometer (Model LS50B; Montreal, Quebec) with excitation and emission wavelengths of 336 nm and 376 nm, respectively, and a 350-nm
cutoff filter in the emission beam.
Effect of plasmin on the interaction of F.IX and F.IXa with AT.
F.IX (4 µM) was incubated in HBS/Ca at 37°C with either no
additions for 60 minutes (control) or with 50 nM of plasmin for 60 minutes (F.IXp) or with 500 nM of F.XIa for 2 hours (F.IXa). In some
cases, F.IXa and F.IXp (4 µM of each) were further treated at
37°C with plasmin (50 nM for 60 minutes) and F.XIa (500 nM for 2 hours); samples were designated F.IXa/p and F.IXp/a, respectively. The
plasmin was inactivated as described above. The F.IX samples were then
either diluted for the clotting assay, prepared directly for
nonreducing SDS-PAGE, or further incubated with AT (5 µM) and heparin
(2 mg/mL) for 30 minutes at 37°C and prepared for nonreducing
SDS-PAGE.
Activation of human factor X.
Factor VIII (60 U/mL) was activated with thrombin (1 nM) for 5 minutes
at 37°C according to Astermark et al.41 Thrombin was
inactivated by the addition of DAPA to a final concentration of
1µM. F.IX (4 µM) was treated at 37°C in HBS/Ca with either 50 nM of plasmin for 60 minutes or with 500 nM of F.XIa for 2 hours. The
plasmin was inactivated as described above. F.IXa or plasmin-treated
F.IX (0.3 nM of each) was incubated with human F.X (600 nM), F.VIIIa
(0-10 U/mL), and PCPS vesicles (30 µM) according to Nishimura et
al.42 Assay of F.Xa generated was carried out following the
addition of S-2222 to 0.4 mM and measurement of the initial rates of
p-nitoraniline produced at 405 nm and 37°C in a 96-well plate
(Dynatech Immulon, VWR Scientific, Mississauga, Ontario) using a
SpectroMax 250 plate reader (Molecular Devices, Sunnyvale, CA). The
amount of F.Xa in the different reactions was calculated from a
standard curve using purified human F.Xa.
Effect of plasmin-cleaved F.IX or dEGR-F.IXa on the clotting
activity of F.IXa.
F.IX (4 µM) was treated at 37°C in HBS/Ca with either 50 nM of
plasmin for 60 minutes or with 500 nM of F.XIa for 2 hours. The plasmin
was inactivated as described above. Half of the F.IXa reaction was
further treated with a 5-fold molar excess of dEGR-CMK (20 µM) for 20 minutes at 37°C and incubated for 30 minutes at 37°C to
inactivate any free dEGR-CMK. The F.IX samples were assayed at 0.01 to
10 000 pmol/L in the clotting assay using a fixed concentration of
F.IXa (10 pmol/L).
NH2-terminal sequencing of the plasmin cleavage
products of F.IX.
F.IX (4 µM) was incubated with plasmin (50 nM) for 2 or 30 minutes in
HBS/Ca at 37°C, and the plasmin was inactivated as described above.
The fragments from the 2 plasmin-digested F.IX samples were subjected
to dialysis, reducing SDS-PAGE, and blotting as previously
described.38 Amino acid sequencing was performed by Dr
Teng-Song Chen at The Hospital For Sick Children (Toronto, Ontario)
directly from the blotted material using a Porton Gas-Phase Microsequencer (Model 2090; Tarzana, CA) with online
phenylthiohydantoin (PTH) analysis. The PTH-amino acids were identified
by HPLC and compared with a chromatogram containing all the amino
acids, excluding cysteine.
tPA-catalyzed lysis of fibrin clots formed in human plasma and F.IX
immunoblotting.
NHP was added to the wells of a Dynatech Immulon plate that contained
separated aliquots of thrombin, CaCl2, and tPA. The final
concentrations of thrombin, CaCl2, and tPA were 6 nM, 2 mM,
and 0 to 10 nM, respectively. The plate was incubated at 37°C, and
the absorbance at 405 nm was measured every 2 minutes for 74 minutes
using a SpectroMax 250 plate reader. The material in the wells was then
solubilized and prepared for reducing SDS-PAGE according to
Neville.39 In cases where the fibrin clot had lysed completely, an aliquot was added to 2.2 µM of VFK-CMK prior to measurement of its F.IX clotting activity. In some cases, purified F.IX
(4 µM) was digested with plasmin (50 nM) for various times (2-60 minutes) at 37°C and "spiked" into NHP before reducing
SDS-PAGE to serve as a positive control. The proteins were transferred to an Immobilon-P membrane according to Towbin et al43 and
probed sequentially with rabbit anti-human F.IX IgG and then goat
anti-rabbit IgG conjugated to horseradish peroxidase. Detection of F.IX
and its cleavage products was accomplished using the chemiluminescence reagents and exposing the blots to Kodak X-OMAT AR film before development in a Kodak M35A processor (Eastman Kodak, Rochester, NY).
The effect of plasmin on the procoagulant activity of F.IX and
F.IXa
SDS-PAGE analysis of plasmin cleavage of F.IX and F.IXa
The interaction of F.IXa and plasmin treated F.IX with pAB
The interaction of F.IX derivatives with AT and heparin F.IX, F.IXa, plasmin-cleaved F.IX (F.IXp), F.IXa cleaved with plasmin (F.IXa/p), and plasmin-treated F.IX that was subsequently exposed to F.XIa (F.IXp/a) were compared with respect to their nonreducing SDS-PAGE staining patterns before and after incubation with AT and heparin (Figures 3A and B), respectively. Aliquots from these same reactions were also analyzed with respect to their F.IX clotting activities (Figure 3C).
Activation of human factor X by F.IXa and plasmin-treated F.IX Plasmin-cleaved F.IX and F.IXa were also compared with respect to their ability to activate human F.X with increasing amounts of F.VIIIa. As illustrated in Figure 4, F.X activation by plasmin-treated F.IX was only marginally affected by increasing the concentration of F.VIIIa compared with F.IXa. At the greatest level of F.VIIIa assayed (10 U/mL), plasmin-treated F.IX possessed approximately 5% of the F.X activating potential, as was observed with equivalent amounts of F.IXa (Figure 4).
The effect of dEGR-F.IXa and plasmin-treated F.IX on the clotting activity of F.IXa The effect of increasing amounts of dEGR-F.IXa and plasmin-treated F.IX on the clotting activity of a fixed amount of F.IXa was also studied. The results indicate that when dEGR-F.IXa was present at 10, 100, 1000, and 10 000 pmol/L, the clot times of 10 pmol/L of F.IXa progressively increased from 63.6 seconds to 65.6 seconds, 71.1 seconds, 84.9 seconds, and 98.2 seconds, respectively. In contrast, when plasmin-treated F.IX was assayed at 10, 100, 1000, and 10 000 pmol/L, the clot times of 10 pmol/L of F.IXa remained for the most part unchanged from 63.6 seconds to 63.8 seconds, 63.5 seconds, 61.4 seconds, and 53.1 seconds, respectively. The shortened clot time of 10 pmol/L of F.IXa (53.1 seconds) upon assay with 10 000 pmol/L of plasmin-treated F.IX may reflect the presence of a low level of uncleaved F.IX that manifested its increased coagulant activity only upon assay at this elevated concentration. Collectively, the overall lack of a large competitive prolongation of the F.IXa aPTT clot times with increasing amounts of plasmin-treated F.IX suggests that the products of plasmin-cleaved F.IX failed to block the proper assembly of F.IXa into the tenase complex.NH2-terminal sequencing of the plasmin cleavage products of F.IX The data from the NH2-terminal sequencing of the plasmin cleavage products of F.IX are shown in Table 1, and a schematic diagram of the plasmin cleavage sites within the F.IX molecule is presented in Figure 5.
tPA-catalyzed lysis of fibrin in human plasma and F.IX immunoblotting To test the potential relevance of the cleavage and inactivation of F.IX by plasmin in vitro, a series of tPA-induced fibrin clot lysis experiments were performed in NHP, and the reactions were subjected to F.IX immunoblotting. The time courses of clot lysis induced by tPA (0-10 nM), which occurred subsequent to fibrin formation in NHP catalyzed by thrombin in the presence CaCl2, are illustrated in Figure 6A. These same reactions were also subjected to F.IX immunoblotting to probe for the presence of plasmin-like fragments of F.IX (Figure 6B). Combining NHP with thrombin and CaCl2 resulted in maximal fibrin formation after 18 minutes (Figure 6A, closed squares). Inclusion of 0.2 nM or 0.5 nM of tPA with NHP, thrombin, and CaCl2 did not significantly change the time course of the turbidity profile from that observed with NHP, thrombin, and CaCl2 (Figure 6A, compare downward and upward closed triangles, respectively, with closed squares). Addition of 1 and 2 nM of tPA to NHP, thrombin, and CaCl2 increased the maximal turbidity at all times by 5% to 8% from that observed in the absence of tPA or with 0.2 and 0.5 nM of tPA (Figure 6A, compare open squares and open circles, respectively, with closed squares and downward and upward closed triangles). Significant fibrin clot lysis was observed only when 5 and 10 nM of tPA were each combined with NHP, thrombin, and CaCl2 (Figure 6A, downward and upward open triangles, respectively).
It has been proposed that, in normal hemostasis, a balance exists
between coagulation and fibrinolysis and, thus, thrombus formation and
dissolution is finely regulated at the point of vascular
injury.5,6 It has also been suggested that imbalance between these 2 systems may lead to pathologic thrombosis or
bleeding.7-10 Both experimental44,45 and
clinical46,47 observations support this proposal. In
clinical DIC, the course is frequently complicated by bleeding
consequent to the significant consumption and proteolysis of vital
clotting factors during its development.48 Plasmin is a
likely candidate as an instigator of the proteolysis observed. Experimental19,49 and clinical50,51 evidence
have confirmed its excessive generation in fulminating DIC and,
predictably, its specificity for fibrin would be compromised if the
capacity of its natural inhibitor,
Submitted February 4, 1999; accepted September 29, 1999.
Supported by grants from The Medical Research Council of Canada (MA-7667) and The Bayer/Canadian Red Cross Society Research and Development Fund (grant 9610).
A.R.G. is a Distinguished Research Professor of The Heart & Stroke Foundation of Ontario.
A preliminary report of these studies was presented at the XVIth Congress of The International Society of Thrombosis and Hemostasis, Florence, Italy, June 6-12, 1997.
Reprints: John A. Samis, Department of Pathology, Botterell Hall, Rm A222, Queen's University, Kingston, ON, Canada, K7L 3N6; e-mail: samisj{at}post.queensu.ca.
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.
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  K. Nogami, K. Nishiya, E. L. Saenko, M. Takeyama, K. Ogiwara, A. Yoshioka, and M. Shima Identification of Plasmin-interactive Sites in the Light Chain of Factor VIII Responsible for Proteolytic Cleavage at Lys36 J. Biol. Chem., March 13, 2009; 284(11): 6934 - 6945. [Abstract] [Full Text] [PDF]
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K. Nogami, M. Shima, T. Matsumoto, K. Nishiya, I. Tanaka, and A. Yoshioka Mechanisms of Plasmin-catalyzed Inactivation of Factor VIII: A CRUCIAL ROLE FOR PROTEOLYTIC CLEAVAGE AT Arg336 RESPONSIBLE FOR PLASMIN-CATALYZED FACTOR VIII INACTIVATION J. Biol. Chem., February 23, 2007; 282(8): 5287 - 5295. [Abstract] [Full Text] [PDF]
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 M. Nesheim Thrombin and Fibrinolysis Chest, September 1, 2003; 124(3_suppl): 33S - 39S. [Abstract] [Full Text] [PDF]
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J. Rohlena, J. A. Kolkman, R. C. Boertjes, K. Mertens, and P. J. Lenting Residues Phe342-Asn346 of Activated Coagulation Factor IX Contribute to the Interaction with Low Density Lipoprotein Receptor-related Protein J. Biol. Chem., March 7, 2003; 278(11): 9394 - 9401. [Abstract] [Full Text] [PDF]
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 J. R. Toomey, R. E. Valocik, P. F. Koster, M. A. Gabriel, M. McVey, T. K. Hart, E. H. Ohlstein, A. A. Parsons, and F. C. Barone Inhibition of Factor IX(a) Is Protective in a Rat Model of Thromboembolic Stroke Stroke, February 1, 2002; 33(2): 578 - 585. [Abstract] [Full Text] [PDF]
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A. R. Zeibdawi and E. L. G. Pryzdial Mechanism of Factor Va Inactivation by Plasmin. LOSS OF A2 AND A3 DOMAINS FROM A Ca2+-DEPENDENT COMPLEX OF FRAGMENTS BOUND TO PHOSPHOLIPID J. Biol. Chem., June 1, 2001; 276(23): 19929 - 19936. [Abstract] [Full Text] [PDF]
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M. Kalafatis and K. G. Mann The Role of the Membrane in the Inactivation of Factor Va by Plasmin. AMINO ACID REGION 307-348 OF FACTOR V PLAYS A CRITICAL ROLE IN FACTOR Va COFACTOR FUNCTION J. Biol. Chem., May 18, 2001; 276(21): 18614 - 18623. [Abstract] [Full Text] [PDF]
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Top
Abstract
Introduction
F.XIa
2
)
consisting of an N-terminal 18-kd light-chain disulphide linked to a
C-terminal 28-kd heavy chain. The physiologic importance of cleavage at
Arg145 relates to an increased binding affinity between F.IX
-carboxyglutamic
acid (GLA) residues, 2 human epidermal growth factor (EGF)-like
domains, and a linker (L) region.
