Gene targeting in hemostasis. alpha2-antiplasmin

Abstract

Apha2-antiplasmin (AP), the main physiological plasmin inhibitor in mammalian plasma, is a 70 kDa single chain serpin (serine proteinase inhibitor) with reactive site peptide bond Arg-Met. It inhibits plasmin very rapidly (second-order inhibition rate constant of = 2 x 107 M-1.s-1) following formation of an inactive 1:1 stoichiometric complex. The high reaction rate requires the presence of a free active site and free lysine-binding site(s) in plasmin. The pathophysiologic relevance of AP is suggested by the finding that homozygous deficient patients show a bleeding tendency; heterozygotes, in contrast, frequently have no or only mild bleeding complications. Inactivation of the AP gene in mice was achieved by replacing, via homologous recombination in embryonic stem cells, a 7 kb genomic sequence encoding the entire murine protein with the neomycin resistance expression cassette. Homozygous AP deficient mice display normal fertility, viability and development. They have an enhanced endogenous fibrinolytic capacity without overt bleeding; this is reflected by a higher spontaneous lysis rate of experimental pulmonary emboli, by a reduced fibrin deposition in the kidneys following challenge with endotoxin, by more limited photochemically induced arterial thrombosis, and by reduced infarct size following induction of focal cerebral ischemia by ligation of the left middle cerebral artery. In a vascular injury restenosis model, AP deficiency has no significant effect on smooth muscle cell migration and neointima formation. These data suggest that, at least in the murine system, the main role of alpha2-antiplasmin is in regulating plasmin activity in the circulating blood and in controlling intravascular fibrinolysis.

Vascular Functions of the Plasminogen Activation System

“…21,22,27,29 -31 Homozygous ␣ 2 -antiplasmin-deficient mice do not exhibit abnormal bleeding, but do demonstrate diminished thrombosis after vascular injury and accelerated lysis of experimental pulmonary emboli. 32,33 These results suggest that the main in vivo function of ␣ 2 -antiplasmin is to regulate circulating plasmin activity and intravascular fibrinolysis. Dewerchin et al studied mice with combined deficiency of PAI-1 and ␣ 2 -antiplasmin in several bleeding and thrombosis models and compared doubledeficient mice to mice with isolated deficiency of each factor to study the relative roles of these inhibitors in regulating fibrinolysis in vivo.…”

α2-Antiplasmin is involved in bone loss induced by ovariectomy in mice

“…Discussion α 2 -AP is a serine protease inhibitor that rapidly sequesters plasmin into a stable inactive complex and thereby regulates fibrinolysis [17,18]. However, Kawashita et al reported that α 2 -AP also enhances dendritic growth in hippocampal neurons independently of the fibrinolytic system [24].…”

“…Significant bleeding tendency is observed in patients with homozygous α 2 -AP deficiency, highlighting its physiological significance [17,18]. Heterozygous patients have mild or no bleeding tendency [17,18]. The role of α 2 -AP in bone metabolism and in the pathogenesis of osteoporosis remains unknown, although several studies suggest various activities besides inhibition of fibrinolysis [19,20].…”

“…Plasmin inhibition by α 2 -AP is almost instantaneous, and leads to the formation of a stable inactive complex [17]. Significant bleeding tendency is observed in patients with homozygous α 2 -AP deficiency, highlighting its physiological significance [17,18]. Heterozygous patients have mild or no bleeding tendency [17,18].…”

“…α 2 -AP is mainly produced in the liver but circulated in the plasma [15,16]. Plasmin inhibition by α 2 -AP is almost instantaneous, and leads to the formation of a stable inactive complex [17]. Significant bleeding tendency is observed in patients with homozygous α 2 -AP deficiency, highlighting its physiological significance [17,18].…”