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2308 Part XII: Hemostasis and Thrombosis Chapter 135: Fibrinolysis and Thrombolysis 2309

uPAR is anchored to the plasma membrane through glycosylphosphat- (HC), a thiol-containing amino acid that accumulates in association
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idylinositol linkages. u-PA bound to its receptor maintains its activity with nutritional deficiencies of vitamin B , vitamin B , or folic acid, or in
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and susceptibility to the physiologic inhibitor, PAI–1. Formation of inherited abnormalities of cystathionine β-synthase, methylenetetrahy-
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u-PA–PAI-1 complexes hastens clearance of u-PA by hepatic or mono- drofolate reductase, or methionine synthase, and is associated with
cytoid cells. 158–161 atherothrombotic disease. 195,196 In vitro, HC impairs t-PA–dependent
Although originally thought to function only as a means of localiz- plasmin generation at the endothelial cell surface by approximately 50
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ing Plg activation to the cell surface, uPAR now appears to play a central percent by forming a covalent derivative with Cys, and mice with diet-
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role in cellular signaling and adhesion events. 152,162 The uPAR-deficient induced hyperhomocysteinemia have deficient annexin A2 function
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mouse has normal development and fertility, and unimpaired fibrin clot The half-maximal dose of HC for inhibition of t-PA binding to annexin
lysis (see Table 135–2). 163,164 uPAR binds the adhesive glycoprotein vit- A2 is approximately 11 μM HC, a value close to the upper limit of nor-
ronectin at a site distinct from the u-PA binding domain 165,166 and u-PA mal for HC in plasma (12 μM).
transfected renal epithelial cells acquire enhanced adhesion to vitronec- The important role of S100A10 in fibrin balance has recently been
tin while they lose their adhesion to fibronectin. uPAR, furthermore, underscored. S100A10–/– mice display increased deposition of fibrin
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colocalizes with integrins in focal contacts and at the leading edge of in the vasculature and reduced clearance of batroxobin-induced vas-
migrating cells, and also associates with caveolin, a major component cular thrombi, and S100A10-deficient endothelial cells demonstrate
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of caveolae, structures abundant in endothelial cells and thought to par- a 40 percent reduction in Plg binding and plasmin generation in vitro
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ticipate in signaling events. 169–171 In addition, cleaved and soluble forms (see Table 135–2). S100A10 also appears to contribute to Plg-dependent
of uPAR have recently been detected in the sera of patients with cancer, macrophage invasion in vitro by enhancing plasmin-dependent activa-
and these modified forms are thought to regulate the activity of several tion of matrix metalloporetinase-9. 200
receptors involved in inflammatory and angiogenic responses. 153 Several studies suggest a physiologic role for the annexin A2 sys-
The Annexin A2-S100a10 System Annexin A2, a Mr 36,000, tem in fibrin homeostasis. First, blast cells from human patients with
339-amino-acid member of the annexin superfamily of calcium- acute promyelocytic leukemia overexpress annexin A2 in proportion to
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dependent, phospholipid-binding proteins, forms a heterotetramer with their degree of hyperfibrinolytic coagulopathy ; S100A10 also appears
the S100 family protein, S100A10 (see Table 135–1). 172–174 It is highly con- to be upregulated by the PML-RAR-α oncoprotein, and both annexin
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served, and abundantly expressed on endothelial cells, 175–178 monocyte/ A2 and S100A10 are downregulated by treatment with all-trans-retinoic
macrophages, 179,180 early myeloid cells, developing neuronal cells, and acid. Second, in rats, arterial thrombosis can be significantly attenuated
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some tumor cells. 183–185 All of the more than 60 annexin family members by pretreatment with intravenous annexin A2. Third, the prevalence
have in common a conserved membrane-binding C-terminal “core” of high-titer anti–annexin A2 antibodies correlates with a history of
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region and a more variable N-terminal “tail.” The human annexin A2 severe thrombosis in humans with antiphospholipid syndrome and in
gene consists of 13 exons distributed over 40 kb of genomic DNA on a cohort of individuals with cerebral venous thrombosis. 203,204 Finally,
chromosome 15 (15q21). 187 mice with total deficiency of annexin A2 display impaired clearance of
Annexin A2 is unique among fibrinolytic receptors in that it artificial arterial thrombi, fibrin deposition in the microvasculature, and
possesses binding affinity for both Plg (Kd 114 nM) and t-PA (Kd angiogenic defects in a variety of tissues (see Table 135–2). 205
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30 nM), but not u-PA. In a fluid phase system of purified proteins,
native human annexin A2 stimulates the catalytic efficiency of t-PA– Clearance Receptors
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dependent Plg activation by 60-fold. This effect is completely inhib- Clearance of serpin-enzyme complexes, such as t-PA–PAI–1 and u-PA–
ited in the presence of lysine analogues or upon treatment of annexin PAI-1, occurs mainly in the liver, and is mediated by a large two–chain
A2 with carboxypeptidase B, an agent that removes basic carboxyl- receptor called the LDL receptor–related protein 1 (LRP1). 206,207 LRP1
terminal amino acids. Although it lacks a classical signal peptide, binds a large number of serpin-protease complexes and other ligands,
annexin A2 is constitutively translocated to the endothelial cell surface indicating a multifunctional role in mammalian physiology. An addi-
within 16 hours of its biosynthesis. This translocation event can be stimu- tional Mr 39,000 “receptor associated protein” copurifies with LRP1 and
lated either by thrombin or by heat stress, in a process that requires phos- appears to regulate the binding and uptake of LRP1 ligands. Inter-
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phorylation of annexin A2 at Tyr23, the action of a Src family kinase, and estingly, LRP1 “knockout” embryos undergo developmental arrest by
the presence of the annexin A2 binding protein p11 (S100A10). 189 13.5 days after conception, suggesting that regulation of serine pro-
At the cell surface, A2 binds phospholipid via core repeat 2, which tease activity may be crucial for early embryogenesis. 209,210 Although
contains the linear amino acid sequence KGLGT and downstream PAI–1–independent clearance pathways for t-PA have been proposed
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aspartate residue (Asp 161); together these moieties constitute a clas- involving the mannose receptor, or an α–fucose–specific receptor,
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sical “annexin” motif. The annexin A2 heterotetramer, which consists in vivo studies in mice suggest that LRP1 and the mannose receptor play
of two A2 monomers and two protein p11 subunits and constitutes the a dominant role in t-PA clearance. 213
cell surface form of A2, appears to have even greater stimulatory effects
on t-PA–dependent plasmin generation. Interestingly, A2 regulates THE FIBRINOLYTIC ACTIONS
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endogenous levels of protein p11 in the endothelial cell by masking
a polyubiquitination site on p11, which otherwise directs p11 to the OF PLASMIN
proteasome where it is rapidly degraded. 191
Plg and t-PA appear to bind to distinct domains. Lys307 appears DEGRADATION OF FIBRINOGEN AND FIBRIN
to be crucial for the effective interaction of Plg with annexin A2, and Fibrinogen
may be revealed upon limited proteolysis of the parent protein. The Plasmin releases carboxyl–terminal Aα and N–terminal fibrinopep-
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atherogenic low-density lipoprotein (LDL)-like particle, lipoprotein(a), tide B moieties from fibrinogen (Fig. 135–3). This reaction is distinct
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competes with Plg for binding to annexin A2 in vitro, thereby reduc- from the proteolytic cleavage of fibrinogen by thrombin, which releases
ing cell surface plasmin generation. t-PA binding to annexin A2 requires fibrinopeptide A, exposing the Gly–Pro–Arg tripeptide sequence
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a domain consisting of residues 8 to 13 (LCKLSL) within the receptor’s and allowing fibrinogen to polymerize and form insoluble fibrin.
amino terminal “tail” domain. This region is a target for homocysteine Plasmin cleavage of fibrinogen (Mr 340,000) initially produces
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Kaushansky_chapter 135_p2303-2326.indd 2309 9/18/15 5:13 PM