Plasmin, the major clot-dissolving protease in humans, is formed upon the cleavage of a single peptide bond within the zymogen plasminogen (Chap. 135). This tightly regulated reaction is strongly influenced by cells of the blood vessel wall, including endothelial cells, smooth muscles cells, and macrophages, which express plasminogen activators, plasminogen activator inhibitors, and fibrinolytic receptors.
ENDOTHELIAL CELL PRODUCTION OF FIBRINOLYTIC PROTEINS
In 1958, Todd demonstrated that human blood vessels possess fibrinolytic activity that is dependent upon an intact endothelium.102,103 We now know that the endothelium is the principal source of t-PA in vivo where it appears to be highly restricted to small blood vessels in specific anatomic locations, a pattern that likely reflects the heterogeneity of endothelial cells as they respond to a myriad of tissue-specific cues.104,105 In the baboon, for example, sites of t-PA production include 7 to 30 μm precapillary arterioles and postcapillary venules, but not large arteries and veins.106 In the mouse lung, similarly, bronchial, but not pulmonary, endothelial cells express t-PA.107 Moreover, enhanced expression of t-PA at branch points of pulmonary blood vessels may reflect stimulation by laminar shear stress.108 In addition, peripheral sympathetic neurons that invest the walls of small arteries may represent a significant source of circulating t-PA.109
Although in vitro studies suggest that t-PA expression in cultured endothelial cells is regulated by a wide array of factors, only a few of these pathways have been confirmed in vivo. Thrombin,110 histamine,111,112 oxygen radicals,113 phorbol myristate acetate,114 DDAVP (deamino D-arginine vasopressin),115 and butyric acid liberated from dibutyryl cAMP116 all increase t-PA mRNA in cultured endothelial cells. Both thrombin and histamine appear to act via receptor-mediated activation of the protein kinase C pathway.105 Laminar shear stress stimulates both t-PA secretion117 and steady-state mRNA levels.118 Hyperosmotic stress and repetitive stretch also enhance t-PA expression.119,120 In addition, differentiating agents, such as retinoids,121,122 stimulate transcription of t-PA in endothelial cells in vitro.
In vivo, the circulating half-life of t-PA is approximately 5 minutes. Infusion of DDAVP, bradykinin, platelet-activating factor (PAF), endothelin, or thrombin is associated with an acute release of t-PA, and a burst of fibrinolytic activity can be detected within minutes.123 In the mouse lung, exposure to hyperoxia leads to 4.5-fold upregulation of t-PA mRNA in small-vessel endothelial cells.107 In humans, infusion of TNF into patients with malignancy is associated with an increase in plasma t-PA.123 Deficient release of t-PA in response to venous occlusion in humans is associated with deep venous thrombosis,124 as well as atrophie blanche and other cutaneous vasculitides.125
In vivo, urokinase plasminogen activator (u-PA) is not a product of resting endothelium,126 but is produced primarily by renal tubular epithelium.127 Expression of u-PA mRNA in endothelium, however, is strongly stimulated during wound repair and physiologic angiogenesis within ovarian follicles, corpus luteum, and maternal decidua.128 Endothelial cells passaged in culture do synthesize u-PA,129 and expression of its mRNA is stimulated by TNF-α by 5- to 30-fold.130 Small increases in u-PA have also been observed in vitro in response to IL-1 and LPS.131,132,133
The association of u-PA with the blood vessel wall appears to reflect its association with the u-PA receptor (uPAR) which may fulfill a variety of nonproteolytic functions ranging from directed cell migration to cellular adhesion, differentiation, and proliferation (Fig. 115–6).134 In the adult mouse, uPAR mRNA is not normally detected by in situ hybridization in the endothelium of either large or small blood vessels.135 However, upon stimulation with endotoxin, expression is detected in endothelium lining aorta, as well as arteries, veins, and capillaries in heart, kidney, brain, and liver,135 and in renal tubular epithelial cells.127
Schematic of principal endothelial cell fibrinolytic receptors. A. The annexin A2/S100A10 heterotetrameric complex. Annexin A2 consists of a hydrophilic aminoterminal tail domain (A-Tail, approximately 3 kDa), and a membrane-oriented carboxyl terminal core domain (approximately 33 kDa).311,312 The tail domain contains residues required for tissue-type plasminogen activator (t-PA) binding. The core domain is composed of four homologous annexin repeats (A1, A2, A3, and A4), each consisting of five α-helical regions that contribute to calcium-dependent phospholipid binding sites. Repeat 2 appears to be most important for the interaction of annexin A2 with the endothelial cell surface. Plasminogen (PLG) binding requires lysine residue 307 within helix C of repeat 4. B. Urokinase plasminogen activator receptor (uPAR) is a 55- to 60-kDa, glycosylphosphatidylinositol-linked protein that consists of three disulfide-linked domains (U1, U2, U3).314 Domain 1 contains sequences required for urokinase plasminogen activator (u-PA) binding, while domains 2 and 3 mediate the receptor’s interaction with matrix proteins such as vitronectin. Domain 3 contains glycosylphosphatidylinositol-linked membrane anchor. (A, adapted with permission from Gerke V, Creutz CE, Moss SE: Annexins: linking Ca2+ signalling to membrane dynamics. Nat Rev Mol Cell Biol 6(6):449–461.)
Plasminogen activator inhibitor (PAI)-1 is likely to function as a major regulator of plasmin generation in the vicinity of the endothelial cell. Thrombin, IL-1, transforming growth factor β, TNF, lipoprotein(a) (Lp[a]), and LPS all induce dramatic increases in steady state PAI-1 message levels.110,131,132,136,137 Heparin-binding growth factor 1 reduces PAI-1 mRNA production by cultured endothelial cells, but has no effect on t-PA.138 Thus, synthesis and secretion of PAI-1 by the endothelial cell in vitro appears to be regulated independently of t-PA.
In vivo, elevated levels of circulating PAI-1 have been linked epidemiologically to risk for myocardial infarction.124 Although the liver is the major source of plasma PAI-1, endothelial expression of PAI-1 is detected near neovascular sprouts during decidual neovascularization in the ovary.128 In addition, inflammatory cytokines are powerful stimuli for induction of PAI-1 in a variety of tissues including liver, as injection of TNF in both rats and humans with active malignancy results in a striking increase plasma concentrations of PAI-1.105,123
The endothelial cell coreceptor for t-PA and plasminogen, the annexin A2/S100A10 complex (see Fig. 115–6), appears to be expressed constitutively in vivo by endothelial cells in a wide variety of tissues in the chicken,139 mouse,140 rat,141 and human.142 Annexin A2 is upregulated transcriptionally by hypoxia both in vivo and in endothelial cells in vitro,143 and by nerve growth factor in neuronal-like PC12 cells.144 In addition, the in vitro transition of human monocyte to macrophage is associated with a severalfold increase in both annexin A2 protein and steady state mRNA expression.145
The evidence that the annexin A2 system plays a role in maintaining vascular patency includes the findings that (1) overexpression of annexin A2 in blast cells in acute promyelocytic leukemia blast cells increases plasmin production and contributes to hyperfibrinolytic bleeding,146,147,148,149 (2) systemic injection of annexin A2 diminishes thrombotic vascular occlusion resulting from vascular injury in experimental animals,150 (3) annexin A2–deficient mice display fibrin deposition on microvessels and impaired clearance of arterial thrombi following vascular injury,151 (4) high titer antibodies directed against annexin A2 are associated with thrombosis in antiphospholipid syndrome and in individuals with cerebral venous thrombosis,152,153 and (5) that polymorphisms in the ANXA2 gene are associated with cerebral vascular occlusion and osteonecrosis of bone in patients with sickle cell disease.154,155,156 Whether defects in S100A10, which could serve either as a chaperone for annexin A2 or as a direct binding site for plasminogen,157 might also be associated with these clinical entities remains to be determined.
NONFIBRINOLYTIC VASCULAR FUNCTIONS OFPLASMIN
Although not yet demonstrated in vivo, plasmin may inactivate factor Va in vitro by cleaving both the heavy and light chains of this 168-kDa protein, in a manner that is distinct from the action of activated protein C.158,159 Plasmin can also inactivate factor VIIIa, a procoagulant cofactor that is structurally related to factor Va.160 In addition, platelet GPIIb/IIIa and GPIb, the cell surface receptors for fibrinogen and VWF, respectively, are both plasmin substrates.161,162 Thus, plasmin formation in the vicinity of a hemostatic plug could lead to impaired adhesion and poor aggregation in response to agonists. In vivo, prolonged bleeding times were found in patients 90 minutes after t-PA infusion for thrombolysis, suggesting early impairment of platelet function upon plasmin generation.163 However, there is also evidence that platelets may promote thrombotic reocclusion following successful thrombolytic therapy.164
FIBRINOLYTIC FUNCTION IN VASCULARINJURY
Transgenic mouse models of vascular disease have helped to elucidate the complex role of the fibrinolytic system in atherosclerosis (Table 115–3).165,166 In mice, the general effects of plasminogen deficiency include runting, fibrin deposition in intra- and extravascular locations, and premature death.167,168 In addition, the mice display impaired healing of cutaneous wounds,169 a response that appears to depend largely on the fibrinolytic action of plasmin as loss of fibrinogen eliminates these defects.170 Mice doubly deficient in plasminogen and apolipoprotein E (ApoE) showed an increased predisposition to atherosclerosis compared to animals deficient in ApoE alone (Fig. 115–7A).171 Mice with ApoE deficiency combined with deficiency of either u-PA or t-PA showed the same predilection for early fatty streaks and advanced plaques as was observed in mice with isolated ApoE deficiency, suggesting that complete elimination of plasmin generating activity is required to exacerbate the proatherogenic state.172 Finally, mice doubly deficient in ApoE and PAI-1 exhibit no change in early plaque size at the aortic root,173,174 decreased early plaque size at the carotid bifurcation,173,174 but increased advanced plaque size with accelerated deposition of matrix.175
Table 115–3.The Fibrinolytic System in Cardiovascular Disease—Transgenic Mouse Models ||Download (.pdf) Table 115–3. The Fibrinolytic System in Cardiovascular Disease—Transgenic Mouse Models
|Genotype ||Result ||Reference(s) |
|PLG–/– ApoE–/– ||Increased atherogenesis ||178 |
|t-PA–/– ApoE–/– ||Unchanged atherogenesis ||179 |
|u-PA–/– ApoE–/– ||Unchanged atherogenesis ||179 |
|PAI-1–/– ApoE–/– ||Decrease in early plaque size; increase in advanced plaque size ||180,181,182 |
|Transplant arteriosclerosis: |
|PLG–/– ||Reduced leukocyte invasion in transplantation model; reduced extent of disease ||185 |
|Coronary ligation: |
|u-PA–/– ||Protection from ventricular rupture; but poor revascularization and late death from heart failure ||186 |
|t-PA–/– ||No protection ||186 |
|uPAR–/– ||No protection ||186 |
|Aortic aneurysm: |
|u-PA–/– ApoE–/– ||Protected ||179 |
|t-PA–/– ApoE–/– ||Not protected ||179 |
|Early oxidative injury: |
|PAI-1–/– ||Attenuated thrombotic occlusion (Rose Bengal) ||194 |
|PAI-1–/– ||Attenuated thrombotic occlusion (FeCl3) ||195 |
|u-PA–/– ||Increased thrombosis (FeCl3) ||196 |
|t-PA–/– ||Increased thrombosis (FeCl3) ||196 |
|A2–/– ||Increased thrombosis (FeCl3) ||155 |
|Restenosis with prominent thrombosis: |
|PAI-1–/– ||No neointima (Cu cuff) ||199 |
|PAI-1–/– ||Reduced neointima (ligation) ||317 |
|PAI-1–/– ||Reduced neointima (FeCl3) ||317 |
|PAI-1–/– ApoE–/– ||Reduced neointima (FeCl3) ||198 |
|Restenosis without prominent thrombosis: |
|PLG–/– ||Reduced neointima (electrical) ||187,188 |
|t-PA–/– ||No change (electrical or mechanical) ||187,189 |
|u-PA–/– ||Reduced neointima (electrical or mechanical) ||187,189 |
|u-PA–/– t-PA–/– ||Reduced neointima (electrical or mechanical) ||187,189 |
|uPAR–/– ||No change (electrical) ||190 |
|PAI-1–/– ||Increased neointima (ligation) ||318 |
|PAI-1–/– ||Increased neointima (electrical or mechanical) ||191 |
Working model for the actions of the fibrinolytic system in vascular disease. A. Plaque formation. Atheromatous plaque is thought to form in response to endothelial cell (EC) (orange) injury or perturbation. Following the initial injury, perturbed endothelial cells may fail to clear fibrin on the blood vessel surface, and may also promote adhesion and invasion of leukocytes (blue). In addition, smooth muscle cells arising in the tunica media invade the developing plaque within the intima (green). Endothelial cells may utilize cell-surface receptors for focal activation of plasmin to maintain a thromboresistant vascular surface. Leukocytes, macrophages, and smooth muscle cells may use plasmin to migrate into the evolving plaque (cells outlined in red). B. Aneurysm. Fragmentation and dissolution of the elastic laminae of the arterial wall may occur may occur upon matrix metalloproteinase activation via plasmin-dependent pathways, possibly mediated by smooth muscle cells. Cells migrating outward toward the adventitial surface of the vessel induce further matrix degradation, and the potential for rupture. C. Restenosis. In response to vascular injury, smooth muscle cells proliferate and, together with leukocytes, invade the subendothelial space establishing a thickened neointima that compromises vascular patency. In all three scenarios, cell migration is thought to require plasmin activity, possibly in association with cell surfaces. EEL, external elastic lamina; IEL, internal elastic lamina.
Once the atherosclerotic plaque is established, plasmin may affect its evolution by mediating invasion of leukocytes (see Table 115–3).176 In the peritoneal cavity, recruitment of inflammatory cells is profoundly influenced by the presence or absence of plasminogen.177 In transplant-associated arteriosclerosis, the extent of disease is significantly reduced in plasminogen-deficient mice, reflecting, at least in part, reduced influx of macrophages, with an associated reduction in medial necrosis, fragmentation of elastic laminae, and remodeling of the adventitia.178 Thus, the role of plasmin in degrading fibrin and other matrix constituents in the early lesion limits atherosclerosis, whereas its ability to promote cellular invasion later on appears to promote atherogenesis.
During aortic aneurysm formation in mice, deficiency of u-PA, but not t-PA, was associated with reduced medial destruction and impaired activation of downstream plasmin-dependent matrix metalloproteinases (Fig. 115–7B and Table 115–3).172 Similarly, u-PA–, but not t-PA–, deficient mice were protected from cardiac rupture secondary to ventricular aneurysm. In this study, temporary administration of PAI-1 or the general matrix metalloproteinase inhibitor, tissue inhibitor of metalloproteinase (TIMP)-1, completely protected wild-type mice from aortic rupture, reinforcing the concept that plasmin-based protease activity promotes aneurysm progression.179
Vascular remodeling may occur following acute arterial injury induced by interventions for vascular compromise, leading to vascular restenosis (Fig. 115–7C and Table 115–3). This process reflects leukocyte invasion, proliferation and migration of smooth muscle cells, deposition of extracellular matrix, and reendothelialization. Electrical or mechanical injury studies in gene-targeted mice indicate that neointima formation, an initial step in restenosis, requires intact expression of plasminogen and u-PA, but not t-PA.180,181,182 Interestingly, loss of uPAR has no effect on neointima formation,183 whereas loss of PAI-1 is associated with increased neointimal stenosis.184,185 In these injury models, which do not induce severe thrombosis, it is thought that vascular occlusion, reflecting migration of smooth muscle cells and leukocytes, is impaired when fibrinolytic potential is attenuated.186
In the ferric chloride, Rose Bengal, and copper cuff models, on the other hand, thrombosis is observed within minutes of arterial injury (see Fig. 115–7 and Table 115–3). In these systems, deficiency of PAI-1 is associated with later and less-extensive thrombotic occlusion of the injured artery,187,188 while loss of u-PA is associated with more rapid and more significant thrombotic occlusion.189 At the same time, the absence of PAI-1 led to reduced vascular stenosis, regardless of whether ApoE was absent190,191 or present.192,193 In balloon-injured rat carotid arteries, finally, transduction of a PAI-1–expressing gene led to increased restenosis of the vessel, again suggesting that clearance of the initial thrombus may have longterm effects on vessel patency and neointima formation.194 In these models, the predominant effect of the fibrinolytic system may be to clear the initial thrombus, which may provide a provisional scaffolding for later restenosis.