Uploaded by Prakash Yadayu

CARPENTER et al-2008-Haemophilia

Haemophilia (2008), 14, 1250–1254
DOI: 10.1111/j.1365-2516.2008.01766.x
a2-Antiplasmin and its deficiency: fibrinolysis out of balance
*Department of Pediatrics, University of Texas Health Science Center, San Antonio, TX, USA; and Department of
Pediatrics, Ted R. Montoya Hemophilia Program, University of New Mexico, Albuquerque, New Mexico
Summary. Fibrinolysis serves an important role in
the process of coagulation, ensuring that clots that
are formed in response to injury resolve after the
injured tissue is repaired. Fibrinolysis occurs because
the protein plasminogen is converted to the active
serine protease plasmin by its activating molecules
(primarily tissue plasminogen activator). One of the
inhibitors of fibrinolysis is a2-antiplasmin, which
acts as the primary inhibitor of plasmin(ogen).
Congenital deficiency of a2-antiplasmin causes a
rare bleeding disorder because of increased fibrinolysis. Despite the rare nature of this disorder,
understanding of the actions of a2-antiplasmin and
Plasmin is generated from its progenitor protein
plasminogen to initiate fibrinolysis. To prevent excess
bleeding and tissue damage, it is important that the
process of fibrinolysis be precisely coordinated. Inhibitors of fibrinolysis include plasminogen activator
inhibitor (PAI-1), which is the main inhibitor of tissue
plasminogen activator (tPA), thrombin activated fibrinolysis inhibitor (TAFI) and a2-antiplasmin (a2-AP),
which acts as the primary inhibitor of plasmin(ogen).
This natural inhibitor of plasmin found in humans has
been known variously as a2-AP, a2-plasmin inhibitor
and primary plasmin inhibitor.
The congenital deficiency was initially described in
1969 by Masateru Kohakura in a 16-year-old boy
with repeated bleeding. Unable to establish haemophilia from laboratory testing, it was noted that the
Correspondence: Shannon L. Carpenter, MD, 333 North Santa
Rosa St., 8th Floor, CHRISTUS Santa Rosa Hospital, Division of
Pediatric Hematology/Oncology/Immunology, Department of
Pediatrics, University of Texas Health Science Center, San Antonio, TX 78207, USA.
Tel.: +1 210 704 3454; fax: +1 210 704 2396;
e-mail: [email protected]
Accepted after revision 7 April 2008
the results of its deficiency has provided the opportunity for better understanding of the fibrinolytic
system in both how it affects the risk of bleeding and
its impact on other bodily systems. Here, we review
the history of the discovery of a2-antiplasmin, our
understanding of its genetics and function, and our
current knowledge of its congenital deficiency. We
also discuss some of the current avenues of investigation into its impact on other diseases and physiological states.
Keywords: antiplasmin, bleeding, deficiency, fibrinolysis
blood clot formed during the test for whole blood
clotting time lysed in 12 h at room temperature, a
shorter time than expected. This phenomenon led to
the consideration of abnormalities in the process of
fibrinolysis as the cause of this young manÕs bleeding
complaints. Subsequent study after many years of
evaluation eventually identified, in 1977, the specific
abnormality in this patient as being a deficiency of
a2-AP. This inhibitor had been isolated from other
inhibitors of fibrinolysis such as a2-macroglobulin
and a2-antitrypsin, by three independent groups in
1976 [1,2].
Although congenital deficiency of a2-AP (once
called Miyasato disease [3]) is a rare disorder, it is
important to rule out this disease in the evaluation of
patients with an unknown bleeding disorder because
of the absence of abnormalities on typical screening
laboratory test results. In addition, investigation of
the disease state of a2-AP deficiency has improved
our understanding of fibrinolysis as a whole. Here,
we seek to review the current status of the knowledge
related to a2-AP deficiency.
Materials and methods
An OVID search was conducted for the term
antiplasmin, which yielded 1423 citations. From
2008 The Authors
Journal compilation 2008 Blackwell Publishing Ltd
these citations, those related to bleeding and fibrinolysis were selected and then visually scanned for
the most recent and appropriate articles. References
from these articles were also evaluated to ensure
that the most recent and pertinent articles were
In conjunction with thrombin activatable fibrinolysis
inhibitor (TAFI) and plasminogen activator inhibitor
(PAI-1), a2-AP acts as the principal regulator of
fibrinolysis [4]. Fibrinolysis is regulated by a2-AP in
three ways: (i) by forming a complex with plasmin;
(ii) by inhibiting adsorption of plasminogen to fibrin;
and (iii) by making fibrin more resistant to local
plasmin through cross-linking via factor XIIIa
(FXIIIa) [5]. Both thrombus-associated and plasma
a2-AP act in regulating fibrinolysis. a2-AP rapidly
inactivates plasmin resulting in the formation of a
stable inactive complex, plasmin-a2-AP. Two forms
of a2-AP circulate in human plasma: a 464-residue
protein with methionine as the amino-terminus
(Met-a2-AP) and an N-terminally shortened 452residue form with asparagine as the amino-terminus
(Asn-a2-AP). Human plasma-a2-AP consists of
approximately 30% Met-a2-AP and 70% Asn-a2AP [6]. In regulating fibrinolysis, the C-terminal end
of a2-AP binds with strong affinity to the lysinebinding site of plasminogen, where fibrin is also noncovalently bound. In this way, a2-AP competitively
inhibits the binding of fibrin to plasminogen. After
binding to the lysine-binding site, a2-AP is rapidly
cleaved by plasmin at the reactive site of the
molecule, resulting in release of a peptide and
formation of a covalent plasmin-a2-AP complex.
The reaction time between plasminogen and a2-AP is
very fast, in the order of 2 · 10)7 mol)1 s)1. Fibrinbound plasmin is protected from this rapid interaction with a2-AP, with a rate of inhibition 10 times
slower than that of free plasmin [6]. In addition,
during the process of blood clotting, a portion of
circulating plasma a2-AP is rapidly covalently bound
to fibrin by FXIIIa, resulting in increased resistance
of the fibrin to fibrinolysis (Fig. 1). It is in this
mechanism of action that the Asn form gains
increased inhibitory capacity when compared with
the Met form, in that the site where cross-linking
takes place is at the second residue from the amino
terminus of the Asn form (Gln 14 in the Met form),
and this form is cross-linked 3–13 times more
quickly than the Met form [7]. Plasminogen activators tPA and urokinase are also inhibited by a2-AP.
Recently, it has also been shown that lysine residues
2008 The Authors
Journal compilation 2008 Blackwell Publishing Ltd
Fig. 1. Schematic representation of the assembly of fibrinolytic
proteins. (a) In the absence of fibrin clot formation, the principal
fibrinolytic proteins are free in plasma. (b) Activation of coagulation results in the fibrin clot formation. Antiplasmin is crosslinked by its N-terminus to fibrin. Tissue plasminogen activator
(tPA) and plasminogen assemble on fibrin leading to the generation
of plasmin. tPA can be inhibited by plasminogen activator inhibitor (PAI-1) either in solution or at the fibrin surface. Kringle
domains on plasmin allow binding to lysine residues on fibrin or
alternatively in the C-terminus of antiplasmin. (c) While plasmin
remains bound to fibrin, it is relatively protected from antiplasmin
and fibrinolysis occurs. Antiplasmin is cross linked to the fibrin
surface and is well placed to inactivate free plasmin in this
in the C-terminus of a2-AP interact with the endothelial cells, although the details of this interaction
are still being elucidated [8].
The bleeding associated with a deficiency in a2-AP
is because of the premature dissolution of haemostatic plugs before tissue and vessel repair. Bleeding
may be delayed after trauma or invasive procedures.
Acquired deficiency of a2-AP may be seen in patients
with severe liver disease with plasma levels falling as
low as 8% [9]. Decreased levels have also been
reported in patients with renal disease and disseminated intravascular coagulation, as well as in
patients undergoing thrombolytic therapy [10]. In
addition, low levels of a2-AP have been found in a
patient with a congenital disorder of glycosylation
Haemophilia (2008), 14, 1250–1254
and intracranial haemorrhage. In this case, the level
of antiplasmin was found to be 41% and was
thought to contribute to the occurrence of the
intracranial haemorrhage but cannot be attributed
as the primary cause. This is of interest because a2AP is a glycosylated molecule [11].
A member of the serpin family of enzyme inhibitors,
a2-AP is synthesized in the liver as a single-chain
glycoprotein with a molecular weight of 51 000 Da. It
circulates in the body either bound to plasminogen or
in an unbound free form. The typical plasma concentration is 0.7 mg mL)1, with a half-life of 2–6 days.
Paediatric reference ranges have been established and
remain fairly consistent with adult values [12].
The gene for a2-AP has been mapped to chromosome 17 (17pter-p12) and contains 10 exons and nine
introns [13]. Polymorphisms of the gene have been
described. The mature protein has been found to have
464 amino acids with three functional regions. The
amino acid structure of the serpin family of proteins
is highly conserved among species. The human a2-AP
molecule shares 80% homology with the bovine
protein and 74% with the murine molecule [14].
After the protein is formed, antiplasmin-cleaving
enzyme shortens the N-terminal to convert the 464
amino acid form (Met form) to the 452 amino acid
form (Asn form), which comprises 60–70% of
circulating antiplasmin and is more physiologically
active than the Met form [7,15].
Clinical manifestations
Congenital deficiency of a2-AP is a rare disorder that
is inherited in an autosomal recessive manner. The
real prevalence of the disease is unknown; approximately 40 cases have been reported to date [16–33].
In the few patients for whom the molecular defect
has been defined, mutations have variously resulted
in impaired intracellular transport, decreased activity
because of an abnormal protein, and absence of the
protein [6,34]. Consanguinity is common in families
with homozygous deficiency.
Patients with homozygous a2-AP deficiency may
exhibit severe bleeding symptoms, often presenting
in childhood and appearing similar to those patients
with congenital haemophilia. Umbilical bleeding
may be the first presentation of the disease. The
delayed bleeding associated with the disorder may be
reminiscent of bleeding that occurs with congenital
deficiency of FXIII. The unusual symptom of intramedullary haemorrhage into the diaphyses of long
Haemophilia (2008), 14, 1250–1254
bones has been described, presenting as pain in the
affected limb visualized as homogeneous hyperlucent
lesions without marginal sclerosis on radiographs and
homogeneous hyperintense signal in the medulla with
surrounding hypointense signal on magnetic resonance imaging. These lesions have been reported to
occur spontaneously and in response to trauma [16].
Heterozygous individuals, in contrast, may have
milder bleeding or may be asymptomatic [31,32].
Bleeding tends to occur in response to trauma,
surgery or dental procedures. Intramedullary haematomas have not been described. Symptoms may
increase with age as a result of falling plasma levels,
sometimes erupting a new in elderly patients with
heterozygous deficiency [34].
Often the results of screening tests such as the
prothrombin time, activated partial thromboplastin
time, thrombin time, and PFA-100 are normal;
therefore, a high index of suspicion is necessary. The
euglobulin lysis time may be shortened in patients
with a2-AP deficiency [5]. In patients with a history
of bleeding in whom all screening test results are
normal, a specific assay for a2-AP deficiency should
be performed. Patients may either have type I
deficiency, wherein the antigen and activity level
are equally decreased, or type II deficiency, with
lower activity compared with antigen level [9].
Intramedullary haematomas have been successfully
treated with surgical evacuation and instillation of a
combination of tranexamic acid and fibrin glue [16].
Treatment of bleeding episodes is typically successful
with fibrinolytic inhibitors, such as -aminocaproic
acid or tranexamic acid. These drugs can be used in
response to bleeding complications or as prophylaxis
before invasive procedures. The oral dose of tranexamic acid that has been recommended is 7.5–
10 mg kg)1 every 6 h or i.v. 20 mg kg)1 before an
invasive procedure [24]. No specific dose of aminocaproic acid has been suggested in the literature.
Antifibrinolytic agents primarily act by preventing
the binding of plasminogen to fibrin.
Fresh frozen plasma (FFP) may be used as an
alternative to antifibrinolytic agents [17]. Depending
on the preparative method of FFP, the a2-AP
contained therein may have variable activity. For
this reason, and in the interest of reducing the risk of
viral transmission that is inherent in FFP administration, antifibrinolytics are the treatment of choice.
2008 The Authors
Journal compilation 2008 Blackwell Publishing Ltd
Desmopressin acetate, which is commonly used to
treat bleeding by enhancing platelet activity, should
be avoided in a2-AP deficiency because it may induce
secretion of plasminogen activator [17].
Future directions
Mice with a2-AP gene deficiency have been developed. These mice are particularly interesting in that
although the deficiency leads to increased fibrinolytic
activity, no overt bleeding has been observed [35].
The fibrinolytic pathway has also been linked to roles
in many different physiological processes, such as
ovulation, embryogenesis, atherosclerosis, metastasis
and even obesity [36]. However, recent evidence in
mice with inactivation of the a2-AP gene shows no
increase in adipose tissue development when compared with that of normal mice [36].
Some studies have shown that the levels of
plasmin-a2-AP complex in plasma are elevated in
acute stroke, myocardial infarction, unstable angina,
and atrial fibrillation suggesting a self-defense system
against complications of ischemic events [37], but the
physiological roles of a2-AP in these conditions are
still not understood. However, in a recent study,
Matsuno et al. showed in an experimental model
that lack of a2-AP promotes acute cor-pulmonale via
over-release of vascular endothelial growth factor
(VEGF) after acute myocardial infarction [38].
Another recent experimental study showed that lack
of a2-AP improved cutaneous wound healing via
fibroblast VEGF-induced angiogenesis in wound
lesions [39].
Because of its efficacy in preventing fibrinolysis,
a2-AP has been used to prevent bleeding complications secondary to thrombolytic therapy without
major effect on thrombolysis and to stabilize clots in
patients after coiling of patent ductus arteriosi [6].
Efforts are currently being taken to investigate
methods of altering the normal a2-AP molecule for
therapeutic benefit [40]. More studies will need to be
performed in these areas to elucidate the true impact
of the fibrinolytic pathway and its components on
these phenomena.
Experts in the field
Professor Michael Gallimore, Kent Haemophilia
Centre, Kent and Canterbury Hospital, Ethelbert
Road, Canterbury, Kent UK CT1 3NG Institute for
Surgical Research, Rikshospitalet, Sognvansveien 20,
Oslo, Norway.
Tel.: 0044 1227 783 168; fax: 0044 1227 783 157
Dr Nobuo Aoki: e-mail [email protected]
2008 The Authors
Journal compilation 2008 Blackwell Publishing Ltd
Dr Paul Coughlin: e-mail [email protected]
Links to organizations
National Hemophilia Foundation: http://www.
Hemostasis and Thrombosis Research Society:
International Society of Thrombosis and Hemostasis: http://www.med.unc.edu/isth
The authors stated that they had no interests which
might be perceived as posing a conflict or bias.
1 Aoki N. Discovery of a2-plasmin inhibitor and its
congenital deficiency. J Thromb Haemost 2005; 3:
2 Gallimore MJ. More on: discovery of a2-plasmin
inhibitor and its congenital deficiency. J Thromb
Haemost 2006; 4: 284–5.
3 Koie K, Kamiya T, Ogata K, Takamatsu J. Alpha2plasmin-inhibitor deficiency (Miyasato disease). Lancet
1978; 2: 1334–6.
4 Mutch NJ, Thomas L, Moore NR, Lisiak KM, Booth
NA. TAFIa, PAI-1 and a2-antiplasmin: complementary
roles in regulating lysis of thrombi and plasma clots.
J Thromb Haemost 2007; 5: 812–7.
5 Goodnight SH, Hathaway WE. Disorders of Hemostasis and Thrombosis: a Clinical Guide, 2nd edn. New
York, NY: McGraw-Hill, 2001: 187–9.
6 Lee KN, Jackson KW, Christiansen VJ, Chung KH,
McKee PA. a2-Antiplasmin: potential therapeutic roles
in fibrin survival and removal. Curr Med Chem 2004;
2: 303–10.
7 Sumi Y, Ichikawa Y, Nakamura Y, Miura O, Aoki N.
Expression and characterization of pro alpha-2-antiplasmin inhibitor. J Biochem (Tokyo) 1989; 106: 703–7.
8 Thomas L, Moore NR, Miller S, Booth NA. The
C-terminus of a2-antiplasmin interacts with endothelial
cells. Br J Haematol 2006; 136: 472–9.
9 Favier R, Aoki N, de Moerloose P. Congenital
alpha(2)-plasmin inhibitor deficiencies: a review. Br J
Haematol 2001; 114: 4–10.
10 Juhan-Vague I, Alessi MC, Declerck PJ. Pathophysiology of fibrinolysis. Balliè€res Clin Haem 1995; 8: 329–
11 Cohn RD, Eklund E, Bergner AL et al. Intracranial
hemorrhage as the initial manifestation of a congenital
disorder of glycosylation. Pediatrics 2006; 118: e514–
12 Flanders MM, Phansalkar MS, Crist RA, Roberts WL,
Rodgers GM. Pediatric reference intervals for uncom-
Haemophilia (2008), 14, 1250–1254
mon bleeding and thrombotic disorders. J Pediatr
2006; 149: 275–7.
Hirosawa S, Nakamura Y, Miura O, Sumi Y, Aoki N.
Organization of the human a2-plasmin inhibitor gene.
Proc Natl Acad Sci USA 1988; 85: 6836–40.
Coughlin PB. Antiplasmin: the forgotten serpin? FEBS
J 2005; 272: 4852–7.
Bangert K, Johnsen AH, Christensen U, Thorsen S.
Different N-terminal forms of a2-plasmin inhibitor in
human plasma. Biochem J 1993; 291: 623–5.
Miyauchi Y, Mii y, Aoki M, Tamai S, Takahashi Y,
Yoshioka A. Operative treatment of intramedullary
hematoma associated with congenital deficiency of
a2-plasmin inhibitor: a report of three cases. J Bone
Joint Surg Am 1996; 78: 1409–14.
Shahian DM, Levine JD. Open-heart surgery in a
patient with heterozygous a2-antiplasmin deficiency.
Chest 1990; 97: 1488–90.
Griffin GC, Mammen EF, Sokol RJ, Augustine LP,
Stoyanovich A, Abildgaard CF. a2-Antiplasmin deficiency: an overlooked cause of hemorrhage. Am
J Pediatr Hemotol Oncol 1993; 15: 328–30.
Kordich L, Feldman L, Porterie P, Lago O. Severe
hemorrhagic tendency in heterozygous a2-antiplasmin
deficiency. Thromb Res 1985; 40: 645–51.
Hanss MML, Farcis M, French PO, de Mazancourt P,
Dechavanne M. A splicing donor site point mutation in
intron 6 of the plasmin inhibitor (a2 antiplasmin) gene
with heterozygous deficiency and a bleeding tendency.
Blood Coagul Fibrinolysis 2003; 14: 107–11.
Hayward CPM, Cina CS, Staunton M, Jurriaans E.
Bleeding and thrombotic problems in a patients with
alpha2 plasmin inhibitor deficiency. J Thromb Haemost 2005; 3: 399–401.
Paqueron X, Favier R, Richard P, Maillet J, Muirat I.
Severe postadenoidectomy bleeding revealing congenital a2 antiplasmin deficiency in a child. Anesth Analg
1997; 84: 1147–9.
Yoshioka A, Kanitsuji H, Takase T, Iida Y, Mikami S,
Fukui H. Congenital deficiency of a2-plasmin inhibitor
in three sisters. Haemostasis 1982; 11: 176–84.
Morimoto Y, Yoshioka A, Imai Y, Takahashi Y,
Minowa H, Kirita T. Haemostatic management of intraoral bleeding in patients with congenital deficiency
of a2-plasmin inhibitor or plasminogen activator
inhibitor-1. Haemophilia 2004; 10: 669–74.
Miles LA, Plow EF, Donnelly KJ, Hougie C, Griffin JH.
A bleeding disorder due to deficiency of a2-antiplasmin. Blood 1982; 59: 1246–50.
Devaussuzenet VMP, Ducon-le-Pointe HA, Doco AM,
Mary PM, Montagne JPR, Favier R. A case of intramedullary haematoma associated with congenital
a2-plasmin inhibitor deficiency. Pediatr Radiol 1998;
28: 978–80.
Haemophilia (2008), 14, 1250–1254
27 Yoshinaga H, Hirosawa S, Chung DH, Miyasaka N,
Aoki N, Favier R. A novel point mutation of the
splicing donor site in the intron 2 of the plasmin
inhibitor gene. Thromb Haemost 2000; 84: 307–11.
28 Miura O, Sugahara Y, Aoki N. Hereditary a2-plasmin
inhibitor deficiency caused by a transport-deficient
mutation (a2-PI-Okinawa). J Biol Chem 1989; 264:
29 Kluft C, Vallenga E, Brommer EJP, Wingaards G.
A familial hemorrhagic diathesis in a Dutch family: an
inherited deficiency of a2-antiplasmin. Blood 1982; 59:
30 Kettle P, Mayne EE. A bleeding disorder due to deficiency of alpha 2-antiplasmin. J Clin Pathol 1985; 38:
31 Leebeek FW, Stibbe J, Knot EA, Kluft C, Gomes MJ,
Beudeker M. Mild haemostatic problems associated
with congenital heterozygous a2-antiplasmin deficiency. Thromb Haemost 1988; 59: 96–100.
32 Yalcun S, Yalcun B, Demiroglu H. Dialysis for a
patient who had congenital deficiency of a2-plasmin
inhibitor. Clin Nephrol 1998; 49: 335.
33 Kluft C, Nieuwenhuis HK, Rijken DC et al. a2-antiplasmin Enschede: dysfunctional a2-antiplasmin molecule associated with an autosomal recessive
hemorrhagic disorder. J Clin Invest 1987; 80: 1391–
34 Harish VC, Zhang L, Huff JD, Lawson H, Owen J.
Isolated antiplasmin deficiency presenting as a spontaneous bleeding disorder in a 63-year-old man. Blood
Coagul Fibrinolysis 2006; 17: 673–5.
35 Lijnen HR, Okada K, Matsuo O, Collen D, Dewerchin
M. a2-Antiplasmin gene deficiency in mice is associated
with enhanced fibrinolytic potential without overt
bleeding. Blood 1999; 93: 2274–81.
36 Lijnen HR. Deficiency of a2-antiplasmin does not affect
murine adipose tissue development. J Thromb Haemost 2007; 5: 420–1.
37 Matsuno H, Kozawa O, Yoshimi N et al. Lack of
a2-antiplasmin promotes heart failure via overrelease
of VEGF after acute myocardial infarction. Blood
2002; 100: 2487–93.
38 Matsuno H. a2-Antiplasmin on cardiovascular diseases. Curr Pharm Des 2006; 12: 841–7.
39 Kanno Y, Hirade K, Ishisaki A et al. Lack of alpha2antiplasmin improves cutaneous wound healing via
over-released vascular endothelial growth factor-induced angiogenesis in wound lesions. J Throm Haemost 2006; 4: 1602–10.
40 Lee KN, Jackson KW, Christiansen VJ, Chung KH,
McKee PA. A novel plasma proteinase potentiates
a2-antiplasmin inhibition of fibrin digestion. Blood
2004; 103: 3783–8.
2008 The Authors
Journal compilation 2008 Blackwell Publishing Ltd