Inactivation of ADAMTS13 by plasmin as a potential cause of thrombotic thrombocytopenic purpura

ORIGINAL ARTICLE

Inactivation of ADAMTS13 by plasmin as a potential causeof thrombotic thrombocytopenic purpura

H. B . FEYS ,* N . VANDEPUTTE ,* R . PALLA ,� F . PEYVANDI ,� K . PEERL INCK ,� H. DEC KMYN, *

H . R . L I JNEN� and K . VANHOORELBEKE**Laboratory for Thrombosis Research, Katholieke Universiteit Leuven Campus Kortrijk, Kortrijk, Belgium; �Angelo Bianchi Bonomi Haemophilia

and Thrombosis Centre, Department of Medicine and Medical Specialities, University of Milan, IRCCS Maggiore Hospital, Mangiagalli and

Regina Elena Foundation, Milan, Italy; and �Centre for Molecular and Vascular Biology, Katholieke Universiteit Leuven, Leuven, Belgium

To cite this article: Feys HB, Vandeputte N, Palla R, Peyvandi F, Peerlinck K, Deckmyn H, Lijnen HR, Vanhoorelbeke K. Inactivation of ADAMTS13

by plasmin as a potential cause of thrombotic thrombocytopenic purpura. J Thromb Haemost 2010; 8: 2053–62.

Summary. Background: ADAMTS13 deficiency causes accu-

mulation of unusually large von Willebrand factor molecules,

which cross-link platelets in the circulation or on the endothelial

surface. This process of intravascular agglutination leads to the

microangiopathy thrombotic thrombocytopenic purpura

(TTP). Most TTP patients have acquired anti-ADAMTS13

autoantibodies that inhibit enzyme functionand/or clear it from

the circulation. However, the reason for ADAMTS13 defi-

ciency is not always easily identified in a subset of patients.

Objectives: To determine the origin of ADAMTS13 deficiency

in a case of acquired TTP. Methods: Western blotting of

ADAMTS13 in plasmas from acute and remission phases was

used. Results: The ADAMTS13 deficiency was not caused by

mutations or (detectable) autoantibodies; however, an abnor-

mal ADAMTS13 truncated fragment (100 kDa) was found in

acute-phase but not remission-phase plasma. This fragment

resulted from enzymatic proteolysis, as recombinant ADAM-

TS13 was also cleaved when in the presence of acute-phase

but not remission-phase plasma. Inhibitor screening showed

that ADAMTS13 was cleaved by a serine protease that could

be dose-dependently inhibited by addition of exogenous

a2-antiplasmin. Examination of the endogenous a2-antiplasmin

antigen and activity confirmed deficiency of a2-antiplasmin

function in acute-phase but not remission-phase plasma. To

investigate the possibility of ADAMTS13 cleavage by plasmin

in plasma, urokinase-type plasminogen activator was added

to an (unrelated) congenital a2-antiplasmin-deficient plasma

sample to activate plasminogen. This experiment confirmed

cleavage of endogenous ADAMTS13 similar to that observed

in our TTP patient. Conclusion: We report the first acquired

TTP patient with cleaved ADAMTS13 and show that plasmin

is involved.

Keywords: ADAMTS13, plasmin, TTP.

Introduction

Thrombotic thrombocytopenic purpura (TTP) is a rare but

severe thrombotic disorder characterized by disseminated

platelet rich-thrombi blocking the microcirculation of multiple

organs. TTP may be idiopathic or secondary to certain

diseases, drug toxicity, pregnancy or hematopoietic stem cell

transplantation [1].

During the past decade, significant advances in our under-

standing of TTP pathophysiology have been made, owing to

the identification of the vonWillebrand factor (VWF)-cleaving

protease �a disintegrin and metalloprotease with thrombospon-

din-1 motifs� (ADAMTS13) in 2001 [2]. When this enzyme is

absent or dysfunctional, ultralarge VWF molecules are not

cleaved, and accumulate in the circulation or on the vessel wall.

These abnormal VWF multimers are very adhesive and can

spontaneously agglutinate platelets, explaining the widespread

thromboses.

In contrast to secondary TTP, idiopathic disease is fre-

quently accompanied by severely reducedADAMTS13 activity

levels (< 5% of normal), often caused by inhibiting autoan-

tibodies and rarely by detrimental mutations in the ADAM-

TS13 gene. The presence of inhibitors in plasmas from

acquired TTP patients is, however, not always obvious [3–5].

This can be explained, in part, by the poor methods currently

available to resolve low-affinity inhibitors. On the other hand,

auxiliary factors controlling enzymatic activity could contrib-

ute significantly to deficiency, either alone or in synergy with

these weak (undetectable) inhibitors.

The most evident of these auxiliary factors are polymor-

phisms in the ADAMTS13 coding sequence that influence

plasma levels and/or enzyme activity [6]. In addition,

non-immunoglobulin inhibitors such as free hemoglobin or

Correspondence: Karen Vanhoorelbeke, Laboratory for Thrombosis

Research, Katholieke Universiteit Leuven Campus Kortrijk, E.

Sabbelaan 53, 8500 Kortrijk, Belgium.

Tel.: +32 56 246019; fax: +32 56 246997.

E-mail: [email protected]

Received 27 May 2010, accepted 1 June 2010

Journal of Thrombosis and Haemostasis, 8: 2053–2062 DOI: 10.1111/j.1538-7836.2010.03942.x

� 2010 International Society on Thrombosis and Haemostasis

unidentified molecules may influence ADAMTS13 activity [7–

9]. Enzymatic activity may also be (in)directly modulated by

plasma proteins such as thrombospondin-1 and factor (F)VIII

[10,11]. Finally, ADAMTS13 is a substrate for serine proteases,

including granulocyte elastase in sepsis-induced disseminated

intravascular coagulation, and also plasmin and thrombin, at

least in vitro [12,13]. The physiologic significance of serine

proteases controlling ADAMTS13 levels is, however, poorly

understood [14].

In the subset of idiopathic TTP patients without ADAM-

TS13 mutations, diagnosed primary illness or identifiable

(inhibitory) autoantibodies, one or more of these auxiliary

factors may significantly contribute to or even determine the

observed ADAMTS13 deficiency. However, as the underlying

causes of ADAMTS13 deficiency are generally not required for

diagnosis and treatment [15], further investigation is usually

considered to be unnecessary, and interesting phenomena may

be overlooked.

Here, we report such a case of acquired TTP and demon-

strate that excessive proteolysis of ADAMTS13 took place

during the acute bout. Our data indicate an acquired but

transient deficiency of a2-antiplasmin activity which may

underlie the cleavage of ADAMTS13.

Patient, materials and methods

Patient course and treatment

In April 2006, a 42-year-old man noted hematuria and

spontaneous ecchymoses while on a skiing holiday. The patient

had no medical history of bleeding events following tooth

extraction, trauma or surgery. A platelet count was performed

in his holiday resort, and a thrombocytopenia of

35 · 109 platelets L–1 was found. The patient started taking

methylprednisolone 32 mg daily. He presented at our outpa-

tient clinic after 15 days. Hematuria and ecchymoses were

completely resolved. Blood tests showed hemolytic anemia

[hemoglobin (Hb) level of 12.6 g dL)1, elevated lactate dehy-

drogenase (LDH) level of 694 U L)1, haptoglobin level of

< 0.20 g L)1, Coombs-negative], mild thrombocytopenia

(116 · 109 platelets L)1), excess fragmented red cells (> 30/

1000 red blood cells), and a normal coagulation profile

(prothrombin time of 10.4 s, activated partial thromboplastin

time of 22.8 s, D-dimers normal). After 3 weeks, all blood tests

had normalized, but ADAMTS13 activity was < 10% of

normal, as measured by a method published previously [16].

Corticosteroids were rapidly tapered. In September 2006, the

patient returned to the outpatient department complaining of

dark urine and reappearance of ecchymoses. The patient had

no kidney or neurologic abnormalities, but blood tests showed

anemia (Hb level of10.3 g dL)1) and profound thrombocyto-

penia (16 · 109 platelets L)1), a total bilirubin level of

3.31 mg dL)1, an LDH level of 1890 U L)1, a haptoglobin

level of < 0.20 g L)1, > 30 fragmented red cells/1000 red

blood cells, andADAMTS13 activity< 5%.At that point, the

diagnosis of TTP was made, and the patient was treated with

daily infusions of 30 mL kg)1 fresh frozen plasma, with

complete recovery of platelet count and normalization of

LDHafter 6 days. Prior to this treatment, a plasma sample was

taken, which is referred to as �acute-phase� plasma throughout

this article. Rapid relapse occurred when plasma infusions were

tapered, and plasma exchange was started mid-October 2006.

Remissions were not sustained when the frequency of plasma

exchange was decreased, and mid-January 2007 treatment with

rituximab 375 mg m)2 weekly for 4 weeks was added. Com-

plete remission was achieved following a final plasma exchange

on 9 February 2007. One sample taken during remission in

May 2007 will be indicated throughout the article as �remission

phase�. The patient has remained in complete remission for

almost 3 years. All studies were approved by the Institutional

Review Board of the Katholieke Universiteit Leuven, Belgium,

and informed consent was given by the patient(s) involved.

Materials

Human a2-antiplasmin was purified from plasma (Fig. S1),

and its activity was determined by titration with plasmin [17].

Plasmin was obtained by activation of native plasminogen with

urokinase plasminogen activator (u-PA), and its activity was

determined by titration with p-nitrophenyl-p¢-guanidinobenzo-ate [18]. Two-chain u-PA (100 000 IU mg)1) was obtained by

activation of recombinant single-chain u-PA (Grunenthal

GmbH, Aachen, Germany) by Sepharose-immobilized plas-

min. 4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride

(AEBSF) was from Roche Applied Science (Basel, Switzer-

land). Aprotinin, a1-antitrypsin, amiloride and e-aminocaproic

acid were from Sigma-Aldrich (St Louis, MO, USA). Phe-Pro-

Arg-chloromethylketone (PPACK) was from Haematologic

Technologies (Essex Junction, VT, USA) and chromogenic

plasmin substrate S-2403 (Pyro-Glu-Phe-Lys-pNA) was from

Chromogenix (Antwerp, Belgium). Purified VWF was recon-

stituted lyophilized VWF–FVIII concentrate (Haemate-P)

from CSL Behring (Marburg, Germany). Peroxidase-labeled

anti-VWF was from Dako (Glostrup, Denmark). In-house-

developed monoclonal antibodies (mAbs) against ADAM-

TS13 were mapped in binding assays using truncated ADAM-

TS13 variants (kind gift from J. E. Sadler and P. J. Anderson,

Washington University, St Louis, MO, USA), and mAbs

20A5, 17B10, 5C11, 12H6 and 3H9 bind to the eighth

thrombospondin type 1 repeat, thrombospondin type 3 repeat,

thrombospondin type 2 repeat (TSR2), CUB1-2 and metallo-

protease domains, respectively.

ADAMTS13 antigen and activity, autoantibody and genotype

determination

ADAMTS13 antigen was measured in diluted citrated plasma

using an enzyme-linked immunosorbent assay (ELISA),

essentially as previously described, using mAb 20A5 for

coating and biotinylated 5C11 for detection [19]. Enzyme

activity was measured using the fluorogenic FRETS-VWF73

substrate (Peptides International, Louisville, KY, USA) in the

2054 H. B. Feys et al


presence of 5 mM AEBSF, as described previously [20]. To

assess inhibitors in the patient�s plasma, a 1 : 1 mix of the

patient�s heat-inactivated plasma with normal human pooled

plasma (NHP) (n = 20) was prepared [21], and analyzed using

the FRETS-VWF73 substrate. As a negative control, NHP

was mixed with heat-inactivated NHP instead of the patient�splasma. Because the final dilution required for this assay (� 70-

fold) could cause dissociation of weak inhibitors, an additional

method requiring a � 20-fold dilution was performed in

parallel [22]. The patient sample and NHP were mixed and

dialyzed against a buffer containing 1.5 M urea for 16 h at

37 �C. The residual uncleaved VWF was detected with a

collagen-binding assay [16]. Both antigen and activity data are

expressed relative to the amount of ADAMTS13 in NHP,

arbitrarily set to 100%.

To detect (non-inhibitory) autoantibodies [23] to ADAM-

TS13, the Imubind ADAMTS13 autoantibody kit was used,

following the instructions of the provider (American Diagnos-

tica, Greenwich, CT, USA). Anti-ADAMTS13 immunoglob-

ulins were bound to immobilized recombinant ADAMTS13

(rADAMTS13) and detected with a peroxidase-labeled anti-

human serum. Another assay for the detection of anti-

ADAMTS13 autoantibodies used immunoprecipitation of

rADAMTS13 as described by Luken et al. [24]. Briefly,

magnetic protein G beads (Invitrogen, Carlsbad, CA, USA)

were incubated with plasma samples to bind the immunoglob-

ulin fraction. NHP was used as a negative control, and

unlabeled anti-V5 was added to NHP as a positive control

(final concentration of 5 lg mL)1); the sample was then treated

in the same way as patient samples. Following a washing step,

the immunoglobulin-containing beads were added to 10 nM

rADAMTS13 and tested for binding by immunoprecipitation

and sodium dodecylsulfate polyacrylamide gel electrophoresis

(SDS-PAGE) western blotting of the precipitate. ADAMTS13

was then detected with the use of horseradish peroxidase

(HRP)-labeled anti-V5.

ADAMTS13 exons with exon–intron boundaries were

amplified from genomic DNA by polymerase chain reaction,

with primers described elsewhere [25]. Sequencing reactions

were outsourced (GATC Biotech AG, Konstanz, Germany).

Western blotting of plasma ADAMTS13 and VWF

SDS-PAGE (7.5%) was performed in a Tris–glycine-buffered

system [25 mM Tris, pH 8.8, with 200 mM glycine and 1% (m/

v) sodium dodecylsulfate (SDS)] at a constant current of

20 mA. Plasmas were diluted five-fold in 50 mM Tris (pH 7.4)

with 10% (v/v) glycerol, 4% SDS and 0.04% Bromophenol

blue, of which 30 lL was loaded. Transfer was onto a

nitrocellulose membrane (Hybond C Extra; GE Healthcare,

Waukesha, WI, USA) in transfer buffer [50 mM Tris, pH 8.6,

with 40 mM glycine and 20% (v/v) methanol] for 1.5 h at a

constant 16 V. The membrane was blocked with 3% skimmed

milk in Tris-buffered saline (TBS). The anti-ADAMTS13mAb

17B10 was incubated at 10 nM in TBS with 0.3% skimmed

milk. In all cases, samples were run in non-reducing conditions,

as 17B10 binds a conformational epitope that is destroyed

upon b-mercaptoethanol treatment of ADAMTS13. Bound

antibody was detected with HRP-labeled goat anti-mouse

secondary antiserum (Jackson Immunoresearch, West Grove,

PA,USA) and chemiluminescence (ECLPlus; GEHealthcare),

according to the manufacturer�s instructions. The Precision

Plus precolored standard (Bio-Rad, Hercules, CA, USA) was

used in all electrophoreses. All experiments were performed in

duplicate, including a background control in which the primary

mAb was omitted.

Detection of VWF was performed with a commercial

polyclonal anti-VWF antibody and in the presence of 1% (v/

v) b-mercaptoethanol as reducing agent.

Recombinant ADAMTS13 and its cleavage in acute-phase

plasma samples

rADAMTS13 was produced in 293 T-REx cells (Invitrogen),

as published elsewhere [20]. Conditioned media were diluted

four-fold with ultrapure water, and buffered with 1 M 2-(N-

morpholino)-ethanesulfonic acid (MES) to give a final MES

concentration of 50 mM and pH 6.6. The sample was applied

to a Heparin Sepharose column (GEHealthcare), and this was

followed by washing with 50 mM MES (pH 6.6) containing

25 mM NaCl. Then, rADAMTS13 was eluted with 50 mM

MES (pH 6.6) containing 1 M NaCl. Positive fractions were

pooled, concentrated with Amicon concentrators (Millipore,

Billerica, MA, USA), and dialyzed against 50 mM HEPES

(pH 7.4), 5 mMCaCl2, 1 lMZnCl2 and 150 mMNaCl. Purified

protein was stored at – 80 �C in aliquots. The ADAMTS13

antigen concentration was determined by ELISA [19], and

specific activity was tested by FRETS-VWF73. Purity was

estimated by SDS-PAGE with subsequent Simply Blue Safe

Stain staining (Invitrogen; Fig. S1).

Plasma samples were generally spiked by addition of purified

rADAMTS13 to a final concentration of 15 nM (unless stated

otherwise). Dilution as a consequence of rADAMTS13 addi-

tion was negligible (typically one volume of rADAMTS13

solution per 40 volumes of plasma). Then, the mixture was

incubated at 37 �C for the indicated times with gentle mixing

on a planar rotator. rADAMTS13 proteolysis was quenched

by addition of electrophoresis sample buffer (containing SDS),

and this was followed by analysis by SDS-PAGE western blot.

a2-Antiplasmin antigen, plasmin–a2-antiplasmin (PAP)

complex and plasmin activity determination

a2-Antiplasmin antigen levels were determined by ELISA,

using the home-made mAbs 33B1 and 34F7 (polycondensed)

for coating and mAb 39A1A3 for HRP conjugation. The

production and application of these mAbs has been described

previously [26,27]. Plasmin-a2–antiplasmin complex levels were

also measured by ELISA using mAb 34D3D10 directed

against human plasmin for HRP conjugation, and mAbs

34F7 and 7B9 (polycondensed) against human a2-antiplasmin

for coating. The production and application of these mAbs has

TTP and a2-antiplasmin deficiency 2055


been described previously [28,29]. a2-Antiplasmin activity was

determined by addition of plasmin and quantification of

residual plasmin activity after 10 s of incubation with S-2403,

as described elsewhere [30].

Recombinant ADAMTS13 proteolysis in a2-antiplasmin-

deficient plasma

To investigate ADAMTS13 cleavage in a2-antiplasmin-defi-

cient plasma, 5 nM active plasmin was added and incubated for

6 h at 37 �C. Alternatively, 250 IU mL)1 u-PA was added and

quenched by 100 lM amiloride following 30 min of incubation

at 37 �C to initiate plasmin generation. All indicated concen-

trations are final, and stock concentrations were high enough

for sample dilution to be negligible. These samples were then

also incubated for another 6 h at 37 �C. In both cases, active

plasmin was neutralized by adding electrophoresis sample

buffer (containing SDS), and this was followed by analysis of

endogenous ADAMTS13 proteolysis by SDS-PAGE western

blot as described above.

Results

ADAMTS13 deficiency is not caused by mutations or

identifiable autoantibodies

The acute-phase TTP sample contained no measurable

ADAMTS13 antigen or activity (Fig. 1A). In remission-phase

plasma samples, the ADAMTS13 activity and antigen values

were still low, at 14% ± 1% (mean ± standard deviation,

n = 3) and 55% ± 14% (n = 5; Fig. 1A), respectively.

Consequently, the activity/antigen ratio was significantly

skewed at 0.3 (normal range: 0.9–1.3 [19]). Mixing of normal

plasma with acute-phase patient plasma did not enable the

detection of an inhibitor of ADAMTS13 by FRETS-VWF73

(Fig. 1B) or by an urea-based activity assay (not shown). In

addition, non-inhibitory autoantibodies binding to rADA-

MTS13 [23] were not detected in the commercially available

Imubind ELISA (not shown) or with immunoprecipitation of

purified rADAMTS13 using the patient�s immunoglobulin

fraction [24] (Fig. S2). Furthermore, genotyping of the AD-

AMTS13 exons and exon–intron boundaries revealed a

combination of known [2] single-nucleotide polymorphisms

(SNPs) (Table 1) but no mutations.

ADAMTS13 is truncated at the C-terminus during the acute

phase

Because the reasons for complete deficiency of ADAMTS13

activity are unclear, plasmas were further investigated by SDS-

PAGE and western blotting, which showed that full-length

ADAMTS13 (175 kDa) was absent during the acute phase.

However, this sample contained an immunoreactive band at

100 kDa and a faint one at 130 kDa, both of which resolved

during remission (Fig. 2A). Detection of ADAMTS13 was

specific, as a sample taken from a non-related congenital TTP

patient [31] was negative.

Following this, the combination of mAbs used to measure

ADAMTS13 antigen in Fig. 1 might not have detected the

smaller ADAMTS13 variant from acute-phase plasma. If this

was the case, then the dominant 100-kDa fragment in Fig. 2A

no longer contained either the 20A5 (anti-thrombospondin

80A

B

60

40%

20

0

80

60

40

% a

ctiv

ity

20

0CTR AC

AC RE

Fig. 1. ADAMTS13 antigen and activity, and ADAMTS13 inhibitors, in

acute-phase and remission-phase plasma. (A) Endogenous ADAMTS13

antigen (open bars, n = 5) and activity (filled bars, n = 3) measured by

sandwich enzyme-linked immunosorbent assay and FRETS-VWF73,

respectively in acute-phase plasma (AC) and remission-phase plasma (RE)

samples shows complete absence during acute disease. The dashed line

indicates the lower limit of the assay. (B) The presence of an anti-AD-

AMTS13 inhibitor was tested by mixing heat-inactivated normal human

pooled plasma (NHP) [control (CTR)] or acute-phase TTP plasma (AC)

with NHP in a 1 : 1 ratio, andmeasuring residual ADAMTS13 activity by

FRETS-VWF73 (n = 3). No inhibition of normal ADAMTS13 could be

observed in the presence of patient plasma. Data are expressed relative to

the activity or antigen arbitrarily set as 100% in NHP.

Table 1 Identification of single-nucleotide polymorphisms (SNPs)

Patient�s SNPs Zygosity Amino acid change Allele frequency Genotype frequency NCBI SNP ID

19C>T C/T R7W T: 10.0% (n = 120)* 17.4% (n = 46)� rs34024143

1342C>G G/G Q448E G: 29.3% (n = 454)� 1.8% (n = 454)� rs2301612

1852C>G C/G P618A G: 9.2% (n = 120)* 4.3% (n = 46)� rs28647808

Nucleotide numbering starts at the initiating ATG codon of the coding sequence of ADAMTS13. *Antoine et al. [46]. �Kokame et al. [25].

�Retrieved from the National Center for Biotechnology Information (http://ncbi.nlm.nih.gov).



type 8 repeat, C-terminal) or the 5C11 (anti-TSR2, middle)

epitope. To discriminate between these two options, an ELISA

was set up with different mAb couples that have known

domain specificity: the anti-metalloprotease domain mAb 3H9

(N-terminal) was used for immobilization, and either 5C11 or

the anti-CUB1-2 mAb 12H6 (C-terminal) for detection

(Fig. 3C). This experiment showed antigen in acute-phase

plasma with 5C11 but not with 12H6 (Fig. 3A). This is in

contrast to measurements of ADAMTS13 antigen in NHP,

which was detected equally by both combinations (Fig. 3B).

These data indicate that the missing fragment in the 100-kDa

ADAMTS13 protein of the patient is C-terminal.

Proteolytic degradation of ADAMTS13 in the acute phase but

not the remission phase

We hypothesized that the small ADAMTS13 variant is

acquired and results from the action of a protease. Therefore,

wild-type full-length purified rADAMTS13 was added to both

acute-phase and remission-phase plasma samples, and incu-

bated for 16 h. Western blot analysis revealed that the

recombinant enzyme was indeed (partially) proteolysed in

acute-phase but not remission-phase samples (Fig. 2B). The

signal of the 100-kDa band increased considerably, indicating

that more of this fragment was generated following incubation.

The additional band of higher molecular mass (130 kDa) as

well as the uncleaved rADAMTS13 antigen of 175 kDa were

also detected, indicating that proteolysis was incomplete under

these conditions.

Proteolytic degradation of ADAMTS13 is dose-dependently

inhibited by a2-antiplasmin

In the search for the protease responsible for cleaving

ADAMTS13, multiple inhibitors were added to acute-phase

plasma prior to spiking with rADAMTS13 and incubation for

24 h. Heat inactivation (1 h at 50 �C) (Fig. 4A), or addition of

ACA

B

250

150

100

75

50

250

150100

75

50

AC AC RE RE++ ––

RE cTTP NHP

Fig. 2. ADAMTS13 degradation in the acute phase but not in the

remission phase. (A) Western blotting of endogenous ADAMTS13 anti-

gen shows that the acute-phase plasma (AC) sample contains aberrant

forms of ADAMTS13 that are not present in remission-phase plasma

(RE) phase or in normal human pooled plasma (NHP). An unrelated

congenital thrombotic thrombocytopenic purpura TTP (cTPP) sample

was included as a specificity control. (B) Acute-phase (AC) and remission

(RE) phase plasma samples were spiked (+) with rADAMTS13 or with

buffer ()) and incubated for 16 h. Western blotting revealed substantial

but partial proteolysis of rADAMTS13 in acute but not remission plasma.

In both panels, blotted ADAMTS13 was detected by monoclonal anti-

body 17B10 and horseradish peroxidase-labeled goat anti-mouse second-

ary antiserum. Molecular masses (kDa) are on the left.

0.3A

B

C

Acute phase

0.2

OD

490

nmO

D49

0 nm

0.1

00 20 40 60

% plasma (v/v)

0

3H9

MDTCS 2 3 4

17B10

72 kDa

119 kDa79 kDa

5 6 7 8 CUB1-2

5C11 20A5 12H6

5 10 15 20% plasma (v/v)

NHP0.8

0.6

0.4

0.2

0

Fig. 3. ADAMTS13 is cleaved at its C-terminus. Enzyme-linked immu-

nosorbent assays with domain-specific monoclonal antibodies (mAbs)

were used to delineate what portion is missing from the low molecular

mass fragments detected in Fig. 2 by mAb 17B10. (A) A dilution series of

acute-phase plasma (top panel) was applied to immobilizedmAb 3H9, and

bound ADAMTS13 was detected either with mAb 5C11 (¤) or with mAb

12H6 ( ) and horseradish peroxidase-labeled goat anti-mouse secondary

antiserum. mAb 12H6 does not recognize 3H9-immobilized ADAMTS13,

whereas (B) a similar experiment with normal human pooled plasma

(NHP) (middle panel) shows binding for both 12H6 and 5C11. (C) A

schematic representation of ADAMTS13, depicting the domains to which

the mAbs bind (bottom panel). The arrows below the drawing indicate

that a putative N-terminally truncated fragment detected by 17B10 and

20A5 but not 5C11 (dashed line) is too small to correspond to the domi-

nant 100-kDa band in Fig. 2. Fragments lacking the C-terminus (solid

lines) therefore corroborate better with the experimental data.



700 lM e-aminocaproic acid (Fig. 4A), 10 mM EDTA or

50 lM PPACK (not shown) could not prevent rADAMTS13

proteolysis. However, the broad-range serine protease inhibitor

AEBSF (5 mM) (Fig. 4A) and aprotinin (40 lM) (Fig. 4A)

inhibited rADAMTS13 cleavage. Several serine proteases are

inhibited by this concentration of aprotinin, including kallik-

rein, plasmin, FXIa and leukocyte elastase [32–34]. Elastase is

however an unlikely candidate, because no inhibition was

observed in the presence of 1 lM a1-antitrypsin (not shown), a

potent elastase inhibitor. On the other hand, the natural

plasmin antagonist a2-antiplasmin (Fig. 4A) [35] also effec-

tively prevented rADAMTS13 breakdown in a dose-dependent

manner (Fig. 4B). Note that we opted to reduce the illumina-

tion times of the X-ray film in Fig. 4 as compared with Fig. 2,

allowing us to �select� for the more abundant rADAMTS13,

avoiding the signal of the endogenously cleaved ADAMTS13

fragment in acute-phase plasma.

a2-Antiplasmin activity is deficient in the acute-phase TTP

sample

The above experiments indirectly demonstrate that plasmin

may be responsible for cleaving ADAMTS13. However, in

normal plasma, active plasmin is rapidly inhibited by circulat-

ing a2-antiplasmin [35]. Consequently, in an attempt to detect

putative defects in the PAP axis, concentrations of a2-antiplasmin and PAP complex were determined in the absence

or presence of exogenously added active purified plasmin

(Fig. 5A). This experiment demonstrated both normal a2-antiplasmin antigen and normal endogenous PAP complex

levels. However, formation of de novo PAP complex in

response to addition of exogenous plasmin was significantly

impaired in acute-phase but not remission-phase samples.

Furthermore, addition of 1% (v/v) acute-phase plasma to

5 nM purified plasmin and 0.3 mM chromogenic substrate

pyro-Glu-Phe-Lys-pNA did not inhibit plasmin, in contrast to

NHP, demonstrating a defect in a2-antiplasmin inhibitory

activity (Fig. 5B).

Plasmin will selectively cleave arginyl-X and lysyl-X peptide

bonds in many target proteins, so if active plasmin was indeed

present in the patient�s circulation, other potential substratescould be subjected to (partial) proteolysis. VWF is one of those

proteins targeted by plasmin, thereby coincidently generating

proteolytic bands of largely similar size to those produced by

ADAMTS13, despite different scissile bond locations [36,37].

Therefore, available plasma samples were analyzed for VWF

proteolysis by western blotting in reducing conditions

ACA

B

250

150

100

75

50

250

150

100

75

PF– + 1.5 1.0 0.5 0.2 0.1

α2-antiplasmin (µM)

RE PF CA TR HI AP

Fig. 4. Recombinant ADAMTS13 (rADAMTS13) cleavage is inhibited

by a2-antiplasmin in a dose-dependent manner. (A) A series of inhibitors

was tested for their potency in blocking proteolysis of rADAMTS13 in

acute-phase plasma. Plasma samples were pretreated with 4-(2-amino-

ethyl)benzenesulfonyl fluoride hydrochloride (AEBSF) (PF), e-amino-

caproic acid (CA), aprotinin (TR), heat inactivation (HI) or a2-antiplasmin (AP), and then spiked with rADAMTS13 as in Fig. 2B. The

experiment shows that a serine protease cleaves rADAMTS13. Acute-

phase plasma (AC) and remission-phase plasma (RE) controls contained

no inhibitors. (B) a2-Antiplasmin inhibition of rADAMTS13 proteolysis

demonstrates dose-dependency. Varying concentrations (lM) are indicatedabove the panel. Samples inhibited by AEBSF (+) or not ()) are includedas a reference. In both panels, ADAMTS13 was detected using mono-

clonal antibody 17B10 and horseradish peroxidase-labeled goat anti-

mouse secondary antiserum. Molecular masses (kDa) are on the left.

200A

B

150

µg m

L–1

100

50

0

706050

dA/d

t (O

D40

5/m

in)

40302010

01.0 2.0

Plasma (µL)

NHP AC RE

Fig. 5. De novo plasmin–a2-antiplasmin (PAP) complex formation is

deficient in acute-phase but not remission-phase plasma. (A) Concentra-

tions of PAP complexes before (open bars) and after (gray bars) addition

of exogenous active plasmin determined in normal human pooled plasma

(NHP), acute-phase plasma (AC) and remission-phase plasma (RE)

samples show that de novo formation of PAP complexes is impaired

during the acute phase of thrombotic thrombocytopenic purpura. In

parallel, a2-antiplasmin antigen concentrations (black bars) were found to

be normal. (B) The activity of endogenous a2-antiplasmin was determined

indirectly by monitoring inhibition of plasmin by available plasma sam-

ples. Residual plasmin activity was measured by cleavage of a chromo-

genic substrate as a function of time (dA/dt) in the presence of increasing

concentrations of NHP (s) or acute-phase plasma ( ). All experiments

were performed in duplicate.



optimized not to resolve endogenous fragments generated by

ADAMTS13 (Fig. S3). As anticipated, VWF in acute-phase

samples was partially proteolysed, in contrast to that in NHP

or remission-phase samples (Fig. S3).

ADAMTS13 proteolysis in an unrelated congenitally a2-

antiplasmin-deficient patient

Plasmin generated at sites of coagulation is inhibited slowly by

inhibitors in plasma, such as a2-macroglobulin, substituting for

a2-antiplasmin when it is deficient [35]. Moreover, it remains

unclear if and to what extent a2-antiplasmin deficiency causes

vulnerability of ADAMTS13 to proteolysis. Therefore, a

plasma sample from an unrelated (previously reported [38])

patient with congenital a2-antiplasmin deficiency was used to

show that plasmin is able to cleave ADAMTS13 in plasma.

Western blotting showed that endogenous cleavage of

ADAMTS13 was insignificant (Fig. 6A) in a2-antiplasmin-

deficient plasma. However, upon addition of 10 nM plasmin,

significant proteolysis of ADAMTS13 was observed in a2-antiplasmin-deficient plasma but not in a2-antiplasmin hetero-

zygous plasma, NHP or remission-phase plasma (Fig. 6B).

Moreover, following generation of endogenous plasmin by

preincubation with u-PA, endogenous ADAMTS13 was

cleaved in all samples, but to completion only in a2-antiplas-min-deficient plasma (Fig. 6C). Purified rADAMTS13 itself is

not a substrate for u-PA in these conditions, as it was not

cleaved in the absence of plasma (Fig. 6C). Furthermore, in the

absence of plasma proteins, purified rADAMTS13 was readily

cleaved by purified plasmin in the presence of 1 mg mL)1

purified a2-macroglobulin (Fig. S4). Taken together, the data

show that a2-antiplasmin deficiency increases the susceptibility

to proteolysis of ADAMTS13 by plasmin, and that alternative

inhibitors in plasma cannot prevent this. In addition, u-PA

causes slow activation of plasminogen, indicating that kinetic

defects in the inhibition of plasmin by a2-antiplasmin probably

do not underly the deficiency.

ADAMTS13 proteolysis in autoimmune TTP plasma samples

It is currently not known whether and to what extent

ADAMTS13 is subjected to proteolysis in other cases of

TTP. To investigate this, a series of 32 randomly selected acute-

phase TTP samples with detectable ADAMTS13 inhibitors

were analyzed. Interestingly, all cases had identifiable ADAM-

TS13 immunoreactive bands, but none presented with sub-

stantially proteolysed ADAMTS13 (Fig. 7A).

In addition, nine of these TTP samples were also investi-

gated for PAP complex concentration in the presence or

absence of exogenous plasmin (Fig. 7B). As expected, none of

the samples had high endogenous PAP complex concentra-

tions, although some samples (1, 8 and 9) had PAP levels

> 3 lg mL)1 (vs. 0.8 lg mL)1 in NHP), indicating that some

plasmin was formed at some point prior to sampling. Upon

addition of exogenous plasmin, the PAP complex concentra-

tion increased to various degrees in all nine samples,

demonstrating that active a2-antiplasmin was present in these

TTP plasma samples.

Discussion

We present a peculiar case of mild TTP that initially responded

fairly well to both immunosuppressive therapy and single

plasma infusions, suggesting the presence of a weak ADAM-

A

B

C

250AC

AC

AP–/–

AP–/–

AP+/–

AP+/–

NHP

NHPRE

ACAP–/– AP+/– NHP rA

150

10075

50

37

250150

100

75

50

37

250150

100

75

50

37

Fig. 6. ADAMTS13 cleavage by plasmin in unrelated congenitally a2-antiplasmin-deficient plasma. (A) a2-Antiplasmin-deficient plasma (AP)/)), a2-antiplasmin-semideficient plasma (heterozygous donor, AP+/)) ornormal human pooled plasma (NHP) were analyzed for endogenous

ADAMTS13 by western blot. As a positive control for endogenously

cleaved ADAMTS13, acute-phase plasma (AC) was included. Traces of

cleaved ADAMTS13 can be seen, but are insignificant. (B) AP)/), AP+/

), NHP and remission-phase plasma (RE) samples were spiked with

exogenous active plasmin and incubated, and this was followed by analysis

of endogenous ADAMTS13 by western blot. Cleavage of ADAMTS13 is

seen only in the AP)/) sample. AC was included as a positive control for

cleaved ADAMTS13. (C) AP)/), AP+/) and NHP samples were �acti-vated� with urokinase plasminogen activator (u-PA) for a short time, and

this was followed by incubation. Untreated AC was included as a

reference, and rADAMTS13 (rA) was incubated with u-PA alone to assess

whether the enzyme presents a substrate for u-PA. All samples show

ADAMTS13 cleavage, but the AP)/) sample is cleaved more substan-

tially. In all panels, ADAMTS13 was detected using monoclonal antibody

17B10 and horseradish peroxidase-labeled goat anti-mouse secondary

antiserum. Molecular masses (kDa) are on the left.



TS13 immunoglobulin inhibitor or a gene defect. The mixing

assay could not pick up an inhibitor, indicating that there was

none or that the assaywas insufficient. It has been reported that

non-inhibiting immunoglobulins can also cause ADAMTS13

deficiency [23], but these were not found in this patient, with the

use of two different assays. In that regard, the seemingly good

response to both methylprednisolone and rituximab is puz-

zling. Perhaps low-affinity autoantibodies to ADAMTS13 are

present, and are dissociated by mixing and dilution. Alterna-

tively, the activity assay in vitromight not reflect the inhibitory

potential of certain binders in vivo.

An inherited ADAMTS13 deficiency was not found,

although an interesting combination of SNPs may explain

the fairly low levels of activity in this patient�s remission-phase

samples. The heterozygous SNP P618A decreases both antigen

and activity, either alone (i.e. P618A/Q448E) or in combination

with the other heterozygous SNP (i.e. P618A/Q448E/R7W), to

levels below 70% and 35%, respectively [6]. Nonetheless, one

�normal� allele should be present, as the other two SNPs (R7W

andQ448E) do notmodify ADAMTS13 levels, at least in vitro.

Therefore, the skewed activity/antigen ratio in the remission-

phase sample is not unexpected but is rather extreme, and the

SNPs cannot explain the complete deficiency observed during

the acute phase.

Western blotting confirmed the absence of full-length

ADAMTS13 during the acute-phase episode, but resolved

smaller fragments of 100 kDa and 130 kDa, which apparently

cannot cleave FRETS-VWF73. Consequently, ADAMTS13

antigen is not actually depleted but, rather, is functionally

abolished. This is unexpected, as it is well known that C-

terminally truncated ADAMTS13 forms retain normal func-

tion towards FRETS-VWF73 [39,40]. It should be noted,

however, that our experimental setup can only pick up bond

cleavages in interdomain connecting sequences, as treatment

with reducing agents destroys the 17B10 epitope. Therefore,

auxiliary cuts localized within the disulfide-linked secondary

structure of critical domains (metalloprotease and/or spacer)

cannot be uncovered. Indeed, Crawley et al. [13] have demon-

strated that plasmin targets numerous peptide bonds within

these crucial domains, preventing proteolysis of multimeric

VWF.

It is unusual to find an active protease in the presence of

plasma proteins, which include many protease inhibitors that

prevent enzymes from acting non-specifically, especially plas-

min, which is known to be quickly inhibited by a2-antiplasmin

through the formation of an equimolar inhibitor complex.

Deficiency of a2-antiplasmin may nevertheless cause active

plasmin to circulate and cleave other targets prior to being

inhibited by substitute inhibitors [35]. a2-Antiplasmin defi-

ciency can be congenital, but this is very rare [41] and is mainly

caused by secretion defects. However, the deficiency in our

patient is not congenital but acquired, as it resolved during

remission and the patient never presented with bleeding

problems [41] prior to this. Acquired a2-antiplasmin deficiency

is usually associated with primary illnesses such as nephrotic

syndrome [42], acute promyelocytic leukemia [43] and amyloi-

dosis [44]. These diseases mainly cause a2-antiplasmin antigen

depletion, and were not diagnosed in our patient. Alternatively,

this acquired deficiency could be explained by a circulating

inhibitor blocking a2-antiplasmin function. Indeed, inhibition

of rADAMTS13 proteolysis in the acute-phase sample

required far more exogenous a2-antiplasmin (0.5 lM, Fig. 5)than expected from the estimated concentration of active

plasmin/protease present in the acute-phase sample [£ 0.5 nM

(not shown)]. Moreover, if the putative a2-antiplasmin inhib-

itor is an immunoglobulin, an explanation for the seemingly

good response to immunosuppressive treatment is provided.

However, attempts to detect immunoglobulins that would

recognize linear epitopes by binding to blotted a2-antiplasmin

and conformational epitopes by binding to mAb-immobilized

purified a2-antiplasmin were unsuccessful (not shown).

Our data leave much room for speculation on the contribu-

tion of the a2-antiplasmin deficiency and the consequent

ADAMTS13 degradation to this patient�s TTP. We propose

two hypotheses, either including or excluding a2-antiplasmin

deficiency as a contributing factor to TTP etiology. On the one

hand, if a2-antiplasmin activity was deficient prior to micro-

angiopathy, active plasmin might have been generated at sites

of (minor) trauma, degrading ADAMTS13. In that case, the

slow replenishment of ADAMTS13 [45] may have instigated a

NHPA

B

AC

250

150

100

75

50

200

150

PAP

(µg

mL–

1 )

100

50

0NHP 1 2 3 4 5 6 7 8 9 AC

1 2 3 4 5 6

Fig. 7. Cleaved ADAMTS13 is not found in randomly selected samples

from acquired thrombotic thrombocytopenic purpura (TTP) patients. (A)

ADAMTS13 antigen was determined by western blotting in acute-phase

plasma samples from 32 patients with acquired TTP, six of which are

depicted. Normal human pooled plasma (NHP) and acute-phase plasma

(AC) were included as negative and positive controls, respectively. AD-

AMTS13 was detected using monoclonal antibody 17B10 and horseradish

peroxidase-labeled goat anti-mouse secondary antiserum. (B) Concentra-

tions of plasmin–a2-antiplasmin (PAP) complexes before (open bars) and

after (gray bars) addition of exogenous active plasmin as determined in

NHP, AC and nine of the above-mentioned TTP samples [numbers are

not necessarily paired to those in (A)].



period with insufficient systemic VWF proteolysis, eventually

underlying TTP. Another scenario is that the patient acquired

acute TTP for a different, hitherto unknown, reason, thereby

initiating fibrinolysis in the unfortunate moment of unavailable

active a2-antiplasmin. The remaining ADAMTS13 was then

exposed to active plasmin and degraded, putatively aggravating

or accelerating the disease. Indeed, plasmin generation during

acute-phase TTP does not seem odd, as other TTP patients

seemingly have small amounts of circulating PAP complex.

Nonetheless, the innately low levels of ADAMTS13 activity

as a consequence of the SNPs as well as the good response to

immunosuppressives should not be ignored. Therefore, the

most reasonable explanation for this case of TTP is a series of

unfortunate events, including the hereditary factor, a putative

autoimmune factor, and the acquired activation of fibrinolysis.

A role for the PAP axis in controlling ADAMTS13 activity in a

physiologic setting is therefore not proven, but may need to be

(re)investigated now that it is obvious that, unlike thrombin

[14], plasmin is able to cleave ADAMTS13 in plasma even at

low concentrations.

Addendum

H. B. Feys designed and performed research, coordinated the

study, analyzed and interpreted data, provided essential

reagents and tools, and wrote the paper. K. Vanhoorelbeke

designed research, provided essential reagents, interpreted

data, and wrote the paper. H. R. Lijnen designed research,

analyzed and interpreted data, and provided essential reagents

and tools. N. Vandeputte performed research, and analyzed

and interpreted data. H. Deckmyn, F. Peyvandi, R. Palla and

K. Peerlinck provided essential reagents and tools and/or

patient material. All authors critically reviewed themanuscript.

Acknowledgements

We thank A. Vandenbulcke (KU Leuven Campus Kortrijk,

Belgium), E. Majerus, P. J. Anderson, J. E. Sadler and L.

Westfield (Washington University Medical School in St Louis,

MO, USA) for help with mapping mAbs. We thank B. Van

Hoef for technical help with PAP experiments. H. B. Feys is a

fellow of the Research Foundation Flanders [Fonds voor

Wetenschappelijk Onderzoek Vlaanderen (FWO)]. This work

was supported by grants of the FWO (G.0299.06) and the K.

U. Leuven (GOA/2004/09). The Centre for Molecular and

Vascular Biology is supported by the �ExcellentiefinancieringK. U. Leuven� (Project EF/05/013).

Disclosure of Conflict of Interests

The authors state that they have no conflict of interest.

Supporting Information

Additional Supporting Informationmay be found in the online

version of this article:

Fig. S1. Analysis of purified proteins a2-antiplasmin and

ADAMTS13 by SDS-PAGE with Coomassie Brilliant Blue

staining.

Fig. S2. Autoantibodies to ADAMTS13 are not detected.

Fig. S3. Cleavage of endogenous VWF in acute-phase but not

remission-phase plasma.

Fig. S4. Cleavage of rADAMTS13 by plasmin in the presence

of a2-macroglobulin.

Please note: Wiley-Blackwell are not responsible for the

content or functionality of any supporting materials supplied

by the authors. Any queries (other than missing material)

should be directed to the corresponding author for the article.

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Inactivation of ADAMTS13 by plasmin as a potential cause of thrombotic thrombocytopenic purpura

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