Bleeding disorders Regulation of coagulation by the fibrinolytic system: expecting the unexpected

K.A. Hajjar ABSTRACT

Department of Cell and Recent investigations into fibrinolysis and coagulation have yielded exciting and unexpected findings. Developmental Biology, Of note, deficiencies of the major components of the classical fibrinolytic system, namely plasminogen, Department of Pediatrics tissue plasminogen activator, and urokinase, are now recognized to be rare causes of macrovascular Department of Medicine, thrombosis. At the same time, both plasminogen activator excess and fibrinolytic inhibitor deficiencies Weill Cornell Medical College, are associated with clinically significant bleeding, while elevated inhibitor levels, particularly thrombin New York, New York, USA activatable fibrinolysis inhibitor, appear to confer clinically significant risk for thrombosis. Receptor- mediated cell surface fibrinolysis adds a new dimension to the regulation of fibrin balance. Exaggerated expression of the annexin A2 complex, for example, is associated with hemorrhage in acute promyelo- Hematology Education: cytic leukemia, whereas autoantibodies directed against A2 correlate with thrombotic disease in the education program for the patients with antiphospholipid syndrome. The contributions of the urokinase receptor and a diverse annual congress of the European array of plasminogen receptors to fibrin homeostasis remain to be defined. Future studies are likely to Hematology Association reveal novel mechanisms that co-regulate coagulation and fibrinolysis. 2012;6:49-56

Introduction involving compartmentalization, functional redundancies, and multiple points of cross Fibrinolysis is a finely tuned process in regulation with the coagulation system. which cross-linked fibrin is proteolyzed, giv - ing rise to a defined series of degradation products 1-3 (Figure 1). In the major pathway, Classical fibrinolysis either of two serine proteases, tissue plas - minogen activation (tPA) or urokinase (uPA), Plasminogen deficiency cleaves a single peptide bond within the inac - In both humans and rodents, plasminogen- tive zymogen plasminogen to yield active deficiency is more likely to be associated with plasmin. Serine protease inhibitors, primarily ligneous mucositis, especially conjunctivitis, 12-14 a2antiplasmin ( a2AP), serve to dampen plas - rather than thrombosis. (Table 1) Ligneous min activity, while plasminogen activator mucositis in humans, which usually presents inhibitor-1 (PAI-1) blocks plasmin formation in infancy (mean age 9.5 months), is charac - by inhibiting tPA- or uPA-mediated plasmino - terized by the formation of fibrin-containing gen activation. Other key regulators include pseudomembranes usually of the conjunctiva, fibrin, the most potent known cofactor for but occasionally involving other mucous tPA-dependent plasminogen activation, and membranes, such as the gingiva, upper and thrombin activatable fibrinolysis inhibitor lower respiratory tract, middle ear, gastroin - (TAFI), a plasma carboxypeptidase. TAFI testinal tract, female genital tract, and kid - cleaves C-terminal lysine residues on plas - neys. Fibrin deposition in this disorder can be min-modified fibrin, eliminating binding sites prevented with the use of anticoagulants, and, for plasminogen and tPA, and impeding fur - thus, the defect in ligneous conjunctivitis ther plasmin generation. A number of cell sur - appears to represent unopposed procoagulant face receptors, including the urokinase recep - activity within the microvasculature, possibly tor (uPAR), the annexin A2/p11 complex, and in association with a localized vascular leak. a variety of plasminogen-binding proteins, Thus, while plasmin may be uniquely impor - may contribute to fibrinolytic balance by tant for clearing fibrin associated with mucos - stimulating the catalytic efficiency of plasmin al microvessels, other proteases, such as generation on the surface of blood vessels. membrane type 1 matrix metalloproteinase, Understanding the precise contributions of kallikrein, factor XIa, and factor XIIa may the fibrinolytic system to hemostatic balance replace its function, at least partially, within has proven challenging. For example, while larger vessels. 15-18 fibrinolytic deficiency rarely seems to cause Mice deficient in plasminogen have surpris - clinical thrombosis, 4-9 loss of fibrinolytic ingly normal embryonic development and fer - inhibitors is a major cause of clinical bleed - tility, but display significant runting, and ing. 10,11 This apparent paradox invites intra- and extravascular fibrin accumulation reassessment of the intricacies of fibrinolysis, in multiple tissues. 19,20 Depending upon back - and suggests a far more complex system ground strain, they also may exhibit ligneous

Hematology Education: the education programme for the annual congress of the European Hematology Association | 2012; 6(1) | 49 | 17 th Congress of the European Hematology Association conjunctivitis. 12 Plg –/– mice clear injury-induced thrombi ficially induced pulmonary thrombi. 29 Doubly deficient much less readily than wild-type mice, 21 a defect that can (tPA –/– ; uPA –/– ) mice, on the other hand, exhibit extensive be reversed by bolus intravenous injection of exogenous spontaneous fibrin accumulation in multiple organs, rec - plasminogen. 22 tal prolapse, runting, and cachexia, all reminiscent of the Plg –/– phenotype. Thus, while tPA and uPA are not essen - Plasminogen activator deficiency tial for normal embryologic development in the mouse, Clinically significant gene mutations in tPA and uPA they do seem to play overlapping roles in fibrinolytic sur - have not been well-documented in humans, and deficien - veillance and in the lysis of spontaneous and induced cies in these factors are not associated with thrombosis. 23 thrombi. Although the endothelium is the principal source of tPA in blood, tPA expression appears to be highly restricted to Plasminogen activator excess smaller vessels in specific anatomic locations. In the Occasionally, excessive uPA activity leads to hyperfib - baboon, for example, neither tPA antigen nor tPA mRNA rinolytic hemorrhage. In metastatic prostate cancer, for was detected in endothelial cells of femoral artery or vein, example, hyperfibrinolysis with hemorrhagic sequelae is carotid artery, or aorta, but both were clearly present in thought to reflect overproduction of urokinase. 30 precapillary arterioles, postcapillary venules, and the vasa Similarly, in Factor V Quebec, a rare, moderately severe, vasora. 24 In the mouse lung, similarly, pulmonary blood autosomal dominant bleeding disorder, 31-33 platelets harbor vessels were uniformly negative for tPA antigen while increased quantities of uPA due to a duplication of the bronchial blood vessels displayed strong signals, 25 espe - uPA gene. Excessive activation of plasmin leads to degra - cially at branch points. 26 dation of factor V, fibrinogen, and other procoagulant fac - In humans, deficiency of functional tPA has been tors stored within platelet granules. The resultant bleeding reported in association with livedoid vasculopathy of the usually occurs at 12-24 hours after surgery, dental extrac - lower extremities and ulceration-induced atrophic scars tion, or trauma. (atrophie blanche). Livedoid vasculopathy, an occlusive Exaggerated release of tPA from the endothelium, its microvascular disorder, can be due to hyperproduction of site of synthesis, may also initiate hyperfibrinolytic plasminogen activator inhibitor-1 (PAI-1) as a result of bleeding under extreme circumstances. In patients under - the 4G/4G promoter polymorphism, or due to a marked going cardiopulmonary bypass, blood contact with non- defect in post-venous occlusion release of tPA. 27,28 To date, endothelial cell surfaces may lead to generation of throm - there are no reported hemostatic disorders related to uPA bin, and subsequent endothelial cell release of tPA, lead - deficiency in humans. ing to excessive activation of plasmin. 34 Hyperfibrinolysis Mice deficient in uPA or tPA exhibit little spontaneous is also a contributor to the coagulopathy of heat stroke 35,36 thrombosis. uPA –/– mice exhibit occasional fibrin deposi - and to trauma-induced coagulopathy, which arises most tion, and tPA-deficient mice display impaired lysis of arti - likely from the combined effects of tPA release from

Figure 1. Schematic model for classical and cell surface fibrinolysis. In classical, fibrin-based fibrinolysis, fibrin forms in response to vascular injury, and tPA and plasminogen (Plg) bind to lysine (K) residues on plasmin-modified fibrin. Fibrin increases the catalytic efficiency of tPA-dependent plasminogen activation by approximately 500-fold. uPA-mediated acti - vation of Plg is fibrin-independent. Assembly of tPA and Plg on fibrin is reduced in the presence of TAFIa, which eliminates their binding sites by cleaving off C-terminal K residues. The action of tPA and uPA on Plg is further dampened by the action of PAI-1, the principal plasminogen activator inhibitor. Once formed, plasmin can reduce fibrin to its fibrin degra - dation products (FDPs). The action of plasmin is inhibited by a2-antiplasmin ( a2AP). Cell surface fibrinolysis is mediated by cell surface receptors that bind plasminogen and/or its activators. The annexin (A2-p11) 2 heterotetrameric complex binds both tPA and Plg, thereby increasing the efficiency of plasmin generation, and promoting fibrin clearance. A series of plasminogen receptors (PlgR), as well as urokinase (uPA) bound to the urokinase receptor (uPAR), may also regulate fibrin balance.

| 50 | Hematology Education: the education programme for the annual congress of the European Hematology Association | 2012; 6(1) Amsterdam, The Netherlands, June 14-17, 2012

Table 1. Human fibrinolytic disorders.

Protein Deficiency Excess References

Plasminogen Ligneous mucositis in congenital deficiency Not reported 12-14

Tissue plasminogen activator Livedoid vasculopathy and atrophie blanche Possible cause of bleeding in heat stroke, in disorders of tPA release cardiopulmonary bypass, trauma 27, 28; 34-37

Urokinase Not reported Cause of bleeding in Factor V Quebec and possibly prostate cancer 30-33 a2-antiplasmin Bleeding in congenital deficiency, hepatic failure, Not reported 10 nephritic syndrome, and amyloidosis

Plasminogen activator inhibitor-1 Bleeding in congenital deficiency and hepatic failure Conflicting data 11; 44

Thrombin activatable fibrinolysis inhibitor Possible bleeding in hepatic failure Increased risk of thrombosis 38, 39; 44-46

Annexin A2 complex Autoantibodies associated with thrombosis in Contributor to bleeding in acute 99-102; 105; antiphospholipid syndrome and with cerebral vascular promyelocytic leukemia 96-98 thrombosis; polymorphisms associated with risk of stroke and osteonecrosis in sickle cell disease

injured endothelium together with shock-related inhibi - plasma at a concentration of about 75 nM and undergoes tion of PAI-1. 37 proteolytic activation in the presence of thrombin. 41,42 Active TAFI removes carboxyl-terminal lysine or argi - Fibrinolytic inhibitor deficiency nine residues from fibrin, thereby reducing plasminogen 43 Primary deficiencies of a2AP and PAI-1 are rare, but binding and activation. Half-maximal efficiency for this well-documented causes of hyperfibrinolytic hemor - reaction is reached at TAFI concentrations of approxi - 10,11 rhage. a2AP deficiency is a rare autosomal recessive mately 1 nM, which are well below the usual plasma disorder that usually presents in childhood, often with level. The potentially antifibrinolytic effect of thrombin umbilical bleeding that may be delayed, or later with appears to be at least partially mediated through its ability intraoral bleeding or intramedullary hemorrhage of bone. to activate TAFI in the presence of thrombomodulin. Patients with congenital PAI-1 deficiency usually exhibit Elevated levels of TAFI are associated with global fib - a mild to moderate delayed bleeding disorder that is man - rinolytic deficiency (GFD), as defined by a tissue factor- ifest in the setting of injury, trauma, or surgery. initiated, tPA-induced clot lysis time (CLT). GFD has Congenital deficiency of TAFI has not been reported. recently emerged as a risk factor for thrombosis, 44-46 as a Acquired loss of fibrinolytic inhibitors occurs in the CLT prolonged beyond the 90th percentile roughly dou - setting of an underlying, systemic process that leads to bles the risk of deep vein thrombosis (DVT). 45 To date, reduction in circulating levels of a fibrinolytic inhibitor. GFD has been associated mainly with elevated levels of Chronic liver disease or the anhepatic phase of orthotopic TAFI, with little evidence to implicate abnormalities in liver transplantation, for example, may be associated with plasminogen, tPA, a2AP, or PAI-1. The association reduced synthesis of a2AP, PAI-1, or TAFI, but the clini - between GFD and venous thrombosis is substantially cal significance of inhibitor loss is uncertain, mainly due increased in the presence of additional prothrombotic risk to the lack of valid assessments of overall fibrinolytic factors, such as the use of oral contraceptives, the pres - function. 38,39 A recent study suggests that TAFI deficiency ence of Factor V Leiden, or abnormalities in natural anti - in hepatic cirrhosis is associated with accelerated plasma coagulants, such as protein S, protein C, and antithrombin fibrinolysis and may be a valid predictor of survival in III. The connection between elevated PAI-1 and arterial 40 this setting. Similarly, urinary loss of a2AP may occur in thrombosis is less strong. While some studies report con - the nephrotic syndrome, whereas tissue deposition of flicting results, 44 low plasma fibrinolytic potential dou - a2AP in light chain (LC)-amyloidosis may exhaust circu - bled the relative risk of arterial thrombosis in a cohort of lating levels and lead to hemorrhage. young survivors of coronary artery disease, ischemic stroke, or peripheral artery disease. 47 Fibrinolytic inhibitor excess There is increasing evidence that elevations in fibri - nolytic inhibitor levels, unlike plasminogen activator Cell surface fibrinolysis deficiencies, can lead to thrombotic diatheses. The major fibrinolytic inhibitors include thrombin-activatable fibri - Although fibrinolysis was originally described as a nolysis inhibitor (TAFI), plasminogen activator inhibitor- process confined to the surface of a thrombus, 1 the pres -

1 (PAI-1), and a2-antiplasmin ( a2AP). Recent studies ence of small amounts of endothelial cell-dependent fib - have identified TAFI as a common culprit in global fibri - rinolytic activity associated with blood vessels was first nolytic deficiency states. reported by Todd et al. in the 1950s and 1960s. 48,49 TAFI is a plasma carboxypeptidase that circulates in Decades later, several groups identified endothelial cell

Hematology Education: the education programme for the annual congress of the European Hematology Association | 2012; 6(1) | 51 | 17 th Congress of the European Hematology Association receptors that promote fibrinolytic activity on the vessel pp60 src , an event that stimulates the transit of A2 to the 84 surface by binding plasminogen and/or its activators, pro - cell surface. Formation of the (A2•p11) 2 complex is viding a catalytic boost to protease activity, or protecting largely mediated by hydrophobic contacts between the C- active proteases from their inhibitors, thus fulfilling terminal region of p11 and the amino terminal tail (S 1-G 14 ) Todd’s prediction. 50 of the A2 monomer. 85-88 Phosphorylation of S 11 within the p11 binding domain, has been observed to disrupt het - Plasminogen receptors erotetramer formation, 89 thereby constituting a molecular An interesting array of plasminogen binding proteins “switch” that might regulate the equilibrium between has been identified on many cell types, including membrane-associated and cytosolic A2. Recent data sug - endothelial cells, monocytes, macrophages, neuronal gest that serine phosphorylation of A2 may be regulated cells, and tumor cells. 51 Reported receptors that bear C- by plasmin in a negative feedback loop that limits cell terminal lysine or arginine residues and interact with the surface plasmin generation. 90 Interestingly, loss of kringle structures of plasminogen include a-enolase, 52 endothelial cell A2 leads to dramatic reduction in p11, TIP49, 53 histone H2B, 54 and protein p11. 55 Proposed recep - due to exposure of a polyubiquitination site on p11 that is tors that lack C-terminal basic residues in their synthe - normally masked by A2. 91 In addition, endothelial cell 56 57 58 sized form include annexin A2, aMβ2, and amphoterin. synthesis of A2 mRNA and protein are upregulated 2-3 On endothelial cells, retention of tPA, possibly via a rele - fold under hypoxic conditions through the direct tran - vant receptor, may promote generation of plasmin, which scriptional action of hypoxia-inducible factor (HIF-1) on may then modify surface receptors generating new and the A2 promoter. 92 A2 translocation to the cell surface, additional C-terminal lysine residues for plasminogen moreover, is strongly stimulated by heat stress or activa - binding. 59,60 Recently, the first C-terminal lysine-bearing tion of the thrombin receptor. 84,93 transmembrane plasminogen receptor, Plg-RKT, was Although Anxa2 -/- mice undergo normal development, identified, 61 and found to regulate macrophage and mono - are fertile, and have a full lifespan, their tissues display a cyte invasion, migration, and recruitment. 62 To date, how - general increase in microvascular fibrin. 94 Plasma clotting ever, with the exception of the annexin A2/p11 complex and clot lysis times are normal in Anxa2 -/- mice, but iso - (see below), no studies implicate a specific plasminogen lated microvascular endothelial cells lack normal “cofac - receptor in any clinical disorder of hemostasis. tor activity,” rendering them unable to support tPA-

dependent plasminogen activation. FeCl 3-induced carotid The urokinase receptor artery injury, moreover, leads to complete vascular occlu - The urokinase receptor (uPAR) is anchored to the cell sion in Anxa2 -/- mice much more frequently than in membrane of monocytes, macrophages, tumor cells and Anxa2 +/+ littermates. Data derived from multiple in vivo activated, migrating endothelial cells through a glyco - models involving growth factor stimulation (Matrigel sylphosphatidylinositol linkage. 63-66 While uPA bound to implant and corneal pocket) or oxygen priming (oxygen- its receptor maintains its activity and its susceptibility to induced retinopathy) reveal that the A2 system also plays PAI-1, formation of uPA-PAI-1 complexes appears to has - a central role in postnatal angiogenesis, 94 and is regulated ten clearance of uPA by hepatic or monocytoid cells. 65,67,68 by hypoxia as mentioned above. 92 In mice with hyperho - Interestingly, complete deficiency of uPAR in mice is mocysteinemia, moreover, A2 is derivatized by homocys - associated with a normal phenotype and unimpaired lysis teine through a disulfide linkage within the N-terminal, of intravenously injected plasma clots, suggesting that its tPA-binding tail domain. These animals display fibrin role in fibrinolysis may be minimal. 69 Indeed, uPAR accumulation and defective corneal angiogenesis, which appears to play a novel role in cellular signaling and can be rescued by infusion of non-homocysteinylated, adhesion events by regulating the signaling function of recombinant A2. 95 Thus, the hyperhomocysteinemic integrins and caveolins in migrating tumor and/or mouse represents a phenocopy of the A2 knockout. endothelial cells. 70-75 Thus, integrin function may be regu - Together, these data reveal a defect in fibrin clearance or lated by uPAR, signifying an integrated relationship “surveillance”, especially in smaller blood vessels, when between cellular adhesion and cell surface proteolysis. annexin A2 is deficient or dysfunctional. Although congenital deficiencies of A2 or p11 have not The Annexin A2/p11 complex been identified in humans, several pieces of evidence sug - Annexin A2 (A2) belongs to the annexin family of Ca 2+ - gest an actual role for the annexin A2 system in human dependent phospholipid-binding proteins, twelve mem - health and disease. First, high-level cell surface expres - bers of which are found in humans. 76 Like all annexins, sion of the annexin A2/p11 complex on circulating blast A2 consists of a core domain comprised of four Ca 2+ - cells has been associated with hyperfibrinolytic bleeding binding “annexin” repeats, and a smaller N-terminal and disseminated intravascular coagulopathy in acute “interaction” or “tail” domain that is essentially promyelocytic leukemia (APL). 96,97 Induction of remission unique. 77,78 The annexin A2 complex binds both Plg and with differentiation therapy in APL patients led to a tPA on resting and activated endothelial cells. 79-81 marked decline in A2 expression and a reduction in cell In the endothelial cell, A2 can exist either as a soluble surface plasmin generation. 98 Second, polymorphisms in monomer within the cytoplasm, or in a membrane-associ - A2 are associated with increased risk for vaso-occlusive ated, stable heterotetrameric complex with protein p11 stroke and osteonecrosis in sickle cell disease. 99-101 Third, (S100A10), a member of the S100 protein family. 82,83 The recent evidence identifies A2 as a target for autoantibod - A2 complex acts as a highly regulated co-receptor for ies arising in patients with anti-phospholipid syndrome. plasminogen and tPA, accelerating the catalytic efficiency Patient-derived anti-annexin A2 antibodies blocked of plasmin formation by 1-2 log orders. In response to endothelial surface tPA-dependent plasmin generation, endothelial cell activation, A2 is phosphorylated at Y 23 by and also “activated” cultured endothelial cells, inducing

| 52 | Hematology Education: the education programme for the annual congress of the European Hematology Association | 2012; 6(1) Amsterdam, The Netherlands, June 14-17, 2012 them to express elevated levels of prothrombotic tissue factor. 102 Other groups have noted that A2 can serve as a binding site for β(2)- glycoprotein I, a common antigenic target for autoantibodies on APS on cultured endothelial cells. 103,104 Finally, in a recent study of a Mexican Mestizo cohort with cerebral vascular thrombosis, 12.5% exhibit - ed anti-A2 antibody titers that were greater than three standard deviations above the mean value for healthy controls (2.1%; P <0.01), suggesting a possible etiologic role in thrombosis for inhibition of A2 activity by autoan - tibodies. 105 Cross-regulation of coagulation and fibrinolysis Several possible points of cross-regulation between the fibrinolytic and coagulation systems are evident (Figure 2). Among its many actions, thrombin can inhibit fibri - nolytic activity by virtue of its ability to activate TAFI, which can then eliminate C-terminal lysine residues on fibrin, hindering further clot-associated plasminogen acti - vation. Procoagulant thrombin activates factors Va and VIIIa (not shown), while overproduction of plasmin deac - tivates these factors, as in the Factor V Quebec syndrome Figure 2. Points of cross-regulation between the coagula - or in prostate cancer. Thrombin can be profibrinolytic tion and fibrinolytic systems. Thrombin cleaves fibrinogen through its ability to stimulate the release of tPA from (FGN), permitting its polymerization to form insoluble fibrin. endothelial cells, and through its ability to initiate translo - When no longer needed, fibrin is further processed by plas - min, giving rise to fibrin degradation products (FDPs). When cation of annexin A2 to the cell surface. It is likely that thrombin is associated with thrombomodulin, it can exert additional points of interaction between coagulation and two additional activities. Thrombin can generate activated fibrinolysis will emerge in coming years. protein C (aPC) from its parent compound (PC), and aPC can then attenuate thrombin generation by inactivating coagulation cofactors Va and VIIIa (not shown). In addition, thrombomodulin-associated thrombin can activate throm - Conclusions bin-activatable fibrinolysis inhibitor (TAFI), which then mod - ifies fibrin based binding sites for tPA and plasminogen (Plg), attenuating plasmin formation. Finally, thrombin Because of the delicate and dynamic balance between accelerates trafficking of the annexin A2 complex (A2/p11) thrombosis and hemorrhage, pathways leading to optimal to the endothelial cell surface, promoting cell surface plas - min generation (dotted line • • •). Plasmin, however, can fibrin formation and fibrin dissolution must be highly reg - attenuate its own formation at the cell surface by signaling ulated and coordinated. These pathways involve several the activation of protein kinase C, which phosphorylates dozen different proteins acting at multiple levels as A2, thereby disrupting the annexin (A2-p11) 2 heterote - enzymes, cofactors, inhibitors, and receptors. In some tramer and preventing further translocation to the cell sur - face (solid line ____ ). In addition, plasmin can impair cases, a single agent, such as thrombin or plasmin, may thrombin generation by degrading Va and VIIIa (dashed have seemingly contradictory activities. Within this com - line –––). Thus, both thrombin and plasmin have dual pro - plex system, however, multiple points of interaction coagulant and profibrinolytic actions. 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