| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
J. Biol. Chem., Vol. 278, Issue 34, 32020-32026, August 22, 2003
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
From the
Department of Haematology, Imperial
College London, United Kingdom, ¶Division of
Hematology, Stanford University School of Medicine, Stanford, California
94305-5156, and ||Academic Unit of Molecular
Vascular Medicine, University of Leeds School of Medicine, Leeds LS1 3EX,
United Kingdom
Received for publication, May 22, 2003
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
- and B
-chains of fibrinogen, respectively. FPA is cleaved
first, leading to spontaneous polymerization of the fibrin monomers. This is
shortly followed by proteolysis of the B-chain, which is associated with
lateral aggregation of the fibrin protofibrils to produce thicker fiber
bundles (3,
4). To ensure a more stable
clot structure, activated factor XIII (factor XIIIa), a transglutaminase,
covalently cross-links specific glutamine and lysine side chains of the
protofibrils, resulting in increased resistance of the clot to chemical,
physical, and proteolytic insults
(5,
6).
Factor XIII is a heterologous tetramer with a molecular mass of 324,000
Daltons. It consists of two A-subunits that contain the active site of the
transglutaminase and two B-subunits that serve a carrier function for the
hydrophobic A-subunit in the aqueous environment of human plasma
(5,
6). Thrombin activates factor
XIII by cleavage of a 37 amino acid activation peptide from the factor XIII
A-subunits (7). Consequently,
the carrier B-subunits dissociate from the activated A-subunits to completely
unmask the active site (8).
Activation of factor XIII is closely controlled by the presence of its
substrate fibrin(ogen). The presence of fibrin(ogen) accelerates activation of
factor XIII 
80-fold (9).
This acceleration is caused by an enhancing effect of fibrin on both thrombin
cleavage of the factor XIII activation peptide and dissociation of the factor
XIII A- and B-subunits. It has been shown that residues 242–424 in the
C-domain of fibrinogen regulate the dissociation of the B-chains from
the thrombin-cleaved A-subunits of factor XIII
(10). The enhancement effect
of fibrinogen on factor XIII activation can be readily inhibited by specific
inhibitors of fibrin polymerization
(11,
12). However, the process by
which polymerizing fibrin enhances the activation of factor XIII is poorly
understood.
Approximately 10% total fibrinogen exists as a variant known as
'. This fibrinogen variant occurs by alternative splicing of mRNA
resulting in the deletion of four amino acids from the C terminus of A
with a substitution of an additional 20 amino acid residues
(13). The
'-region is thought to bind factor XIII zymogen and thrombin
(14) but the functional
consequences of this for factor XIII activation are as yet uncertain
(15).
In addition to activating factor XIII and proteolytic cleavage of FPA and
FPB from fibrinogen, thrombin is also responsible for numerous other
proteolytic interactions. It activates factors V, VIII, XI, and
thrombin-activatable fibrinolysis inhibitor
(2,
16). Furthermore, thrombin
cleaves the protease (activated) receptors, resulting in platelet activation
and aggregation (17). These
functions of thrombin are all procoagulant (ultimately promoting clot
formation). However, thrombin is also able to behave as an anticoagulant by
activating protein C when bound to thrombomodulin. Activated protein C cleaves
and thereby inhibits both activated factors V and VIII. Additionally, thrombin
is directly inhibited by formation of an irreversible complex with
antithrombin. As substrates of thrombin participate in either procoagulant or
anticoagulant functions, thrombin is critical for regulating hemostasis. The
unique specificity of thrombin toward its substrates is thought to arise by
combination of insertion loops flanking the upper and lower faces of the
active site (Leu-46/Asn-57 and Leu-144/Gly-155), which occlude and restrict
the active site and two exosites (I and II) on opposite faces of the active
cleft. Binding of substrates, cofactors, and inhibitors to either exosite is
important for overcoming restricted access to the active site. The
sodium-binding site defined by a cylindrical channel shaped by three
-strands appears to be important for the procoagulant (fast) form of
thrombin (18).
The aim of this study was to determine how thrombin specifically recognizes factor XIII in preference to its many other substrates and how activation can be enhanced by fibrin. For this purpose, we have utilized a library of 53 thrombin mutants encompassing a total of 78 surface exposed, charged, and polar residues mutated to alanine. This library has previously been used to identify residues of thrombin involved in the interaction with many of its substrates including protein C, thrombin-activatable fibrinolysis inhibitor, fibrinogen (19), antithrombin (20), factor V (21), and factor VIII (22). Here, we have used the library to investigate which residues of thrombin are involved in direct factor XIII activation and in the fibrin-enhanced reaction. Knowledge of these residues has enabled us to draw general conclusions about how the activities of thrombin are directed.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
To assess the activity of thrombin mutants for either fibrinogen-enhanced or fibrin-enhanced activation of factor XIII, microtiter plates were coated with either (i) 0.12 nM fibrinogen (Calbiochem and Merck Biosciences Ltd, Nottingham, United Kingdom) for 1 h or (ii) 0.12 nM fibrinogen for 1 h, which was preincubated with 46 nM thrombin for 20 min in the presence of 5 mM CaCl2 at 37 °C to form fibrin. Residual thrombin was removed by thorough washing using 40 mM Tris-HCl, 750 mM NaCl, pH 8.3, followed by a final wash with 40 mM Tris-HCl, 140 mM NaCl, pH 8.3. For both fibrinogen-enhanced and fibrin-enhanced factor XIII activation, 6.7 nM factor XIII and 18 nM thrombin were incubated in activation buffer containing 0.1 mM 5-(biotinamido)pentylamine for 8 min at room temperature. The reaction was stopped by the addition of 200 mM EDTA, pH 8.3. The amount of factor XIII that was activated by thrombin was determined by measuring the amount of 5-(biotinamido)pentylamine cross-linked to the fibrin(ogen) using alkaline phosphatase streptavidin conjugate followed by incubation with p-nitrophenol substrate (and measuring at 405 nm). When activation of factor XIII by thrombin was performed in the presence of thrombomodulin, increasing molar concentrations of rabbit thrombomodulin (Hematologic Technologies Inc.) were preincubated with 1.8 nM human thrombin prior to the addition of 6.7 nM factor XIII (in the absence and presence of fibrin, see above).
Factor XIII activation peptide cleavage was determined by reverse-phase
HPLC using a Pepmap 0.46 x 250 mm C18 column (Applied
Biosystems, Warrington, United Kingdom) and the AKTA basic chromatography
system (Amersham Biosciences). Application and elution buffers for HPLC were
phosphate- and acetonitrile-based as previously described
(11) with elution using an
8.5–40% acetonitrile gradient. Purified factor XIII was dialyzed against
reaction buffer (9.47 mM sodium phosphate, 137 mM NaCl,
2.5 mM KCl, 0.1% polyethylene glycol, pH 7.4) prior to use. In the
absence of fibrinogen, 1.8 µm factor XIII was incubated at 37 °C with
4.1 nM of the thrombin mutant under investigation. 100-µl
samples were then removed at five intervals between 1 and 220 min (dependent
on the expected activity of the mutant in question). The reactions were
stopped by the addition of 10 µl of 3 M perchloric acid, the
precipitate was removed by centrifugation, and 100 µl of sample was loaded
onto the C18 column. To investigate factor XIII activation peptide
release in the presence of fibrinogen, 1.8 µM factor XIII and
3.1 µM fibrinogen were incubated at 37 °C with 1.83
nM of the thrombin mutant in question. The reaction mixture was
immediately aliquoted into 100-µl volumes to avoid subsampling difficulties
following fibrin formation. Reactions were stopped at five intervals between 1
and 160 min by the addition of 10 µl of 3 M perchloric acid. The
precipitate was removed by centrifugation, and 100 µl of the reaction was
loaded onto the C18 column. The relative amounts of activation
peptides released were determined by measurement of the areas under the
respective peaks using Unicorn Analysis software (Amersham Biosciences).
Catalytic efficiencies were calculated by fitting of the data from time course
experiments to Equation 1 (9),
 | (Eq. 1) |
Purified fibrinogen fragment D was also studied as an enhancer of factor
XIII activation. The fragment D preparation (Calbiochem) was first
characterized with respect to its ability to bind factor XIII as it is
sensitive to proteolytic cleavage at its C terminus where a factor XIII
binding site is located. Binding analysis of factor XIII to both fragment D
and fibrinogen was carried out with surface plasmon resonance using a BIAcore
3000 system (BIAcore UK, Stevenage, United Kingdom). Fragment D at 75 µg/ml
and fibrinogen at 50 µg/ml were covalently coupled to an activated
carboxymethyl dextran-coated biosensor chip (CM5) using the manufacturer's
recommended protocol for kinetic analysis. The conditions employed were
similar to those employed elsewhere
(25) for the study of
tissue-type plasminogen activator and plasminogen binding to the
C-domain of fibrinogen. However, the running buffer contained 5
mM EDTA rather than 0.1 mM phenylmethylsulfonyl
fluoride, and the chip was regenerated using buffer containing 750
mM NaCl. The association and dissociation data were recorded after
subtraction of signal in the reference cell, which was activated and blocked
in the absence of ligand. Each data set was generated using factor XIII in a
range of concentrations between 7.8 and 125 nM and was fitted to
the 1:1 (Langmuir) binding model using the BIAevaluation software supplied
with the equipment. Fibrinogen and fragment D were found to be able to bind
factor XIII with KDs of 210 ± 69
nM (n = 6) and 198 ± 54 nM (n =
6), respectively. In the reactions to study enhancement of factor XIII
activation, a 2-fold molar excess of the isolated fragment D was used (as
fibrinogen contains two D-domains). Specifically, 1.8 µM factor
XIII and either 2.25 µM fibrinogen or 4.5 µM
fragment D were incubated at 37 °C with 1.83 nM wild-type (WT)
thrombin. The reaction mixture was immediately aliquoted into 100-µl
volumes. The reactions were then stopped at five intervals (between 1 and 16
min for fibrinogen and between 1 and 80 min for fragment D) by the addition of
10 µl of 3 M perchloric acid followed by HPLC, as explained
above.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
These three variants, E229A, R233A, and R178A/R180A/D183A, together with W50A were assessed further using increasing amounts of thrombin to confirm the role of these residues in the activation of factor XIII (data not shown). Each mutant required 4–8-fold increases in concentration to achieve the activity attained by WT thrombin.
The above results in Table I, obtained with the microtiter plate-screening assay, employed an arbitrary cutoff of 50% WT activity to select the thrombin mutants with reduced ability to activate factor XIII. To obtain a more reliable and quantitative assessment of activity reduction, the kinetic parameter kcat/Km was determined for WT and mutant thrombins. This was achieved by determination of factor XIII activation peptide release using HPLC analysis. As factor XIII was isolated from normal pooled plasma, its activation peptide was heterogeneous, containing the polymorphic forms Val-34 and Leu-34 (see Fig. 1A). Rather than sum the relative amounts of each form, their individual relative release rates were determined for each thrombin preparation (see time courses of release in Figs. 1, C and D). The results of catalytic efficiency determinations are illustrated in Fig. 2. The kcat/Km values obtained for the activation of Val-34 and Leu-34 forms of the factor XIII using WT thrombin (0.18 ± 0.008 and 0.33 ± 0.049 µM –1 s–1, respectively) conform to prior reports using plasma with human thrombin as an activator (11). Each thrombin mutant with <50% WT activity selected from Table I for kinetic analysis had greatly reduced kcat/Km with the R233A mutant being particularly affected (kcat/Km of 0.011 ± 0.0008 and 0.018 ± 0.001 µM – 1 s–1 for cleavage of the Val-34 and Leu-34 forms of activation peptide, respectively) (Fig. 2). The position of the mutated residues on the surface of thrombin (see below) together with their limited number suggests that activation of factor XIII in the absence of cofactor is due primarily to direct (and inefficient) interactions of factor XIII within the long extended active site cleft between the S1 specificity pocket and exosite II.
|
|
Preliminary screening experiments for fibrinogen-enhanced factor XIII activation used microtiter plates coated with fibrinogen and utilizing pentylamine incorporation into the resultant fibrin by activated factor XIII as a reporter. The rate of factor XIII activation was greatly enhanced by the presence of fibrinogen, requiring only 8 min and 50% less thrombin to achieve the equivalent amount of factor XIII activity generated in1hin the absence of fibrinogen. More thrombin residues were found to influence fibrinogen-enhanced factor XIII activation when compared with direct factor XIII activation alone (Table II). Thrombin variants with 50% or less activities compared with WT were H66A, Y71A, N74A (which map to exosite I of thrombin), and R89A/R93A/E94A (which map to exosite II) in addition to the previously characterized mutants W50A, R178A/R180A/D183A, E229A, and R233A. Dose-response experiments for these mutant thrombins showed that 4–8-fold increased amounts of the mutants were required to attain WT thrombin activity, confirming the validity of the screening assay (results not shown).
|
All of the mutants that resulted in reduced fibrinogen-enhanced activation of factor XIII were studied further in experiments in which factor XIII activation was carried out on the surface of preformed fibrin. Fibrinogen coated onto microtiter plates was preincubated with WT thrombin to form fibrin. Thrombin was subsequently removed by washing with 40 mM Tris-HCl, 750 mM NaCl, pH 8.3. Complete removal of thrombin was confirmed by the absence of activation of factor XIII in the blank sample. Fibrin-enhanced factor XIII activation by the mutants was then assessed. The results (Table II) show that mutants with impaired direct activation of factor XIII (Trp-50, Arg-178/Arg-180/Asp-183, Glu-229, and Arg-233) have reduced activity in this assay as might be expected because of their essential role in factor XIII activation. Furthermore, mutation of residues His-66, Tyr-71, and Asn-74 as well as reducing fibrinogen-enhanced factor XIII activation directly reduces fibrin-enhanced factor XIII activation (19 ± 2.4, 36 ± 5.3, and 49 ± 8.8% (mean ± S.D.), respectively of WT thrombin activity) (Table II). The mutation of residues Arg-89, Arg-93, and Glu-94 affects fibrinogen-enhanced activation of factor XIII (36 ± 8.5% WT thrombin activity) more than fibrin-enhanced factor XIII activation (68 ± 9.3% of WT activity). This probably reflects their important role in fibrinopeptide cleavage.
Thrombin mutants H66A, Y71A, and N74A therefore influenced both fibrinogen- and fibrin-enhanced factor XIII activation in the microtiter plate assays and were subsequently assessed further by HPLC analysis of the activation peptide release in the presence of fibrinogen. The activation experiments generated activation peptides from both factor XIII (Val-34 and Leu-34 forms of the activation peptide) and fibrinogen (FPA and FPB). These peptides were completely resolved on HPLC (Fig. 1B) and estimates for the catalytic efficiencies for each of these cleavage reactions were obtained (Table III). The presence of fibrinogen enhanced the release of the Val-34 and Leu-34 factor XIII activation peptides by WT thrombin to kcat/Km of 2.29 ± 0.21 and 2.84 ± 0.38 µM –1 s–1, respectively. Each of the three mutants, H66A, Y71A, and N74A, had reduced enhancement effects, and Y71A was particularly reduced with kcat/Km values of 0.038 ± 0.002 and 0.11 ± 0.016 µM –1 s–1 for cleavage of the Val-34 and Leu-34 activation peptides, respectively. These data confirm reduced fibrin-enhanced factor XIII activation of the three mutants. However, it is evident from Table III that the mutants also had impaired ability to convert fibrinogen to fibrin. The kcat/Km for WT thrombin cleavage of FPA is 4.54 ± 0.58 µM –1 s–1 and is lower for all of the mutants falling to 0.023 ± 0.0004 µM –1 s–1 for Y71A. There was a parallel reduction in the catalytic efficiencies associated with FPB release (Table III). These data show that residues His-66, Tyr-71, and Asn-74 have a role in FPA and FPB cleavage as well as fibrin-enhanced FXIII activation.
|
The residues His-66, Tyr-71, and Asn-74 form part of a known extensive surface cluster (anion-binding exosite I) used for recognition of thrombomodulin (see below) (19). If His-66, Tyr-71, and Asn-74 have a direct role in enhancing factor XIII activation, thrombomodulin would be predicted to specifically inhibit fibrin-enhanced activation on the surface of a preformed clot. This was investigated using the above microtiter plate-based functional assays for factor XIII activity in the absence and presence of thrombomodulin. In Fig. 3, the results for the addition of increasing molar concentrations of thrombomodulin to thrombin prior to the addition of factor XIII and 5-(biotinamido)pentylamine (using casein-coated microtiter plates to assess activated factor XIII activity) are illustrated (upper line). Thrombomodulin does not inhibit direct activation of factor XIII by thrombin, even at 100-fold molar excess. In contrast, fibrin-enhanced activation of factor XIII (using preformed fibrin as enhancer) clearly shows a dose-related inhibition of factor XIII activation by thrombomodulin (Fig. 3, lower line) with 10-fold excess of thrombomodulin producing maximum inhibition. At 100-fold molar excess of thrombomodulin, factor XIII activity was not completely inhibited in this assay and this presumably reflects residual non-enhanced direct activation of factor XIII by thrombin.
|
We then compared purified fragment D and fibrinogen as enhancers of thrombin cleavage of factor XIII activation peptides by HPLC analysis. Fragment D was studied at twice the molar concentration as fibrinogen contains two integral D-domains. The results in Fig. 4 demonstrate no enhancement of factor XIII activation by isolated fragment D, despite its retained ability to bind to factor XIII (see "Materials and Methods"). This finding confirms the importance of the polymerization reaction in the enhancement of factor XIII activation.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
Common recognition residues on thrombin for procoagulant (factor XIII) and
anticoagulant (protein C and antithrombin) substrates raises the possibility
of competition between the substrates for thrombin during normal hemostasis
in vivo. However, it is unlikely that such a substrate competition is
important in vivo because of the important enhancing role of
cofactors in these reactions of thrombin. Thus, protein C can be activated
directly in vitro by thrombin, but in vivo this direct
activation is likely to be essentially negligible as the catalytic efficiency
of the reaction is very low
kcat/Km = 5.6 x
102 M–1 s–1
(27). It is only when thrombin
forms a complex with thrombomodulin, which facilitates ternary complex
formation, that the Km is decreased to around the
concentration of protein C in plasma and that the catalytic efficiency is
accelerated 10,000-fold
(kcat/Km = 5.9 x
106 M–1
s–1)
(27), allowing the reaction to
take place at physiological conditions in vivo. Similarly, thrombin
inhibition by antithrombin is not highly efficient
(kcat/Km = 1.4 x
104 M–1
s–1)
(28). Formation of a ternary
complex of thrombin and antithrombin with heparin or heparan sulfate rapidly
accelerates inhibition 2,000–3,000-fold
(kcat/Km = 3 x
107 M–1
s–1)
(29,
30). It is also unlikely that
thrombin directly activates factor XIII in vivo without a cofactor as
there has to be a mechanism for localizing the activation onto the surface of
the clot. As has been previously shown, fibrin(ogen) provides the enhancing
role in factor XIII activation
(9,
11,
12) and this is further
supported by our data. To date, the molecular mechanism of
fibrin(ogen)-enhanced factor XIII activation has been uncertain. Both thrombin
and factor XIII have been reported to bind to the '-chain of
fibrinogen located on the fragment D-domain of fibrinogen. It is unlikely that
a mechanism based on simultaneous binding of factor XIII and thrombin to the
same site on fibrinogen will be dominant in enhancement, because
polymerization is essential for fibrin-enhanced factor XIII activation
(12). Indeed, we show that
purified fragment D with a functional binding site for factor XIII is unable
to enhance factor XIII activation. Rather, we propose that the fibrin polymer
formed following thrombin cleavage of fibrinogen and subsequent polymerization
acts in a similar way as thrombomodulin and heparin/heparan sulfate as a
cofactor to initiate ternary complex formation. Under optimum conditions,
ternary complex formation with fibrin, thrombin, and factor XIII will increase
the catalytic efficiency 80-fold
(26). However, the precise
mechanism of cofactor-enhanced activation clearly differs in a unique way for
factor XIII as fibrinogen is initially a substrate of thrombin. It is
subsequent to the initial encounter of thrombin and fibrinogen that a cofactor
surface is generated. Identification here of the residues of thrombin involved
in fibrin-enhanced activation and those involved in direct factor XIII
activation, together with the finding that isolated fragment D is unable to
enhance factor XIII activation, enables a plausible model for this ternary
complex formation and enhanced activation to be proposed. Such a model draws
upon binding sites for thrombin and factor XIII on the separate and
complementary domains of fibrinogen that are brought together in the
polymerization reaction. One possibility, not directly tested herein, is that
thrombin bound to the fragment E domain after the release of fibrinopeptides
is brought together with factor XIII prebound to the fragment D-domain during
polymerization. This model explains much published data on the influence of
polymerization on the enhanced activation of factor XIII. It also provides an
explanation for localized cross-linking by factor XIIIa on the surface of the
newly generated clot.
If we consider that in vivo most of these reactions of thrombin are only likely to occur when their cofactor is present and that the residues of thrombin involved in substrate recognition of factor XIII, protein C, and antithrombin are common, it can be inferred that the activity of thrombin will be primarily directed by the location at which it is generated and the nature and local concentration of the cofactor. One of the first actions of thrombin in hemostasis is likely to be cleavage of fibrinopeptides with subsequent fibrin formation, and this will be followed closely by factor V and factor VIII activation (32, 33). Each of these reactions proceeds without a cofactor. At the site of vessel damage, thrombin bound on the newly forming fibrin clot on the hemostatic plug is positioned as a consequence of the polymerization process to initiate and enhance factor XIII activation. Factor XIIIa is then able to cross-link fibrin to form a more stable clot structure. Should thrombin migrate away from the clot in areas of vessel damage to intact regions of the vessel wall, it will bind to thrombomodulin and heparan sulfate, initiating the inhibition of further thrombin generation via protein C activation and its direct inhibition by antithrombin. Indeed, the present work suggests that in the undamaged vessel (on the fringe of the hemostatic plug), there will be direct competition by thrombomodulin for the cofactor interaction site of thrombin used for fibrin-enhanced factor XIII activation. As the affinity of thrombin for thrombomodulin greatly exceeds its affinity for fibrin, down-regulation of both thrombin and factor XIII activation will be favored. This model of cofactor-directed specificity and competition can also be expected to have general applicability and govern other functions of thrombin, such as platelet activation via thrombin glycoprotein 1b binding (34) and enhanced protease receptor cleavage (31).
|
FOOTNOTES |
|---|
To whom correspondence may be addressed: Academic Unit of Molecular Vascular
Medicine, University of Leeds School of Medicine, Leeds General Infirmary,
Leeds LS1 3EX, United Kingdom. Tel.: 44-113-392-6202; Fax: 44-113-242-3811;
E-mail:
medhp{at}medphysics.leeds.ac.uk
(H. P.) or Dept. of Hematology, Imperial College London, Du Cane Rd., London
W12 ONN, United Kingdom. Tel.: 44-208-383-2295; Fax: 44-208-383-2296; E-mail:
d.lane{at}imperial.ac.uk
(D. A. L.).
1 The abbreviations used are: FPA and FPB, fibrinopeptides A and B,
respectively; HPLC, high pressure liquid chromatography; WT, wild-type.
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  T. Myles and L. L. K. Leung Thrombin Hydrolysis of Human Osteopontin Is Dependent on Thrombin Anion-binding Exosites J. Biol. Chem., June 27, 2008; 283(26): 17789 - 17796. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. A. Lane, H. Philippou, and J. A. Huntington Directing thrombin Blood, October 15, 2005; 106(8): 2605 - 2612. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Xu, L. A. Bush, A. O. Pineda, S. Caccia, and E. Di Cera Thrombomodulin Changes the Molecular Surface of Interaction and the Rate of Complex Formation between Thrombin and Protein C J. Biol. Chem., March 4, 2005; 280(9): 7956 - 7961. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. T. B. Crawley, J. K. Lam, J. B. Rance, L. R. Mollica, J. S. O'Donnell, and D. A. Lane Proteolytic inactivation of ADAMTS13 by thrombin and plasmin Blood, February 1, 2005; 105(3): 1085 - 1093. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
