Haemophilia (2008), 14, 1250–1254 DOI: 10.1111/j.1365-2516.2008.01766.x ORIGINAL ARTICLE a2-Antiplasmin and its deficiency: fibrinolysis out of balance S. L. CARPENTER* and P. MATHEW *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 Introduction 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: carpenters3@uthscsa.edu Accepted after revision 7 April 2008 1250 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 REVIEW OF a 2 -ANTIPLASMIN DEFICIENCY 1251 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 obtained. Pathophysiology 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 microenvironment. 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 1252 S. L. CARPENTER and P. MATHEW 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]. Genetics 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]. Diagnosis 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]. Management 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 REVIEW OF a 2 -ANTIPLASMIN DEFICIENCY 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 aokinobi@kvd.biglobe.ne.jp 2008 The Authors Journal compilation 2008 Blackwell Publishing Ltd 1253 Dr Paul Coughlin: e-mail paulcoughlin@med. monash.edu.au Links to organizations National Hemophilia Foundation: http://www. hemophilia.org Hemostasis and Thrombosis Research Society: http://www.htrs.org International Society of Thrombosis and Hemostasis: http://www.med.unc.edu/isth Disclosures The authors stated that they had no interests which might be perceived as posing a conflict or bias. References 1 Aoki N. Discovery of a2-plasmin inhibitor and its congenital deficiency. J Thromb Haemost 2005; 3: 623–31. 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– 43. 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– 21. 12 Flanders MM, Phansalkar MS, Crist RA, Roberts WL, Rodgers GM. Pediatric reference intervals for uncom- Haemophilia (2008), 14, 1250–1254 1254 13 14 15 16 17 18 19 20 21 22 23 24 25 26 S. L. CARPENTER and P. MATHEW 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: 18213–9. 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: 1169–79. 30 Kettle P, Mayne EE. A bleeding disorder due to deficiency of alpha 2-antiplasmin. J Clin Pathol 1985; 38: 428–9. 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– 400. 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