RESUMO
BACKGROUND: Protease nexin-1 (PN-1) is an important physiological regulator of thrombin in the brain. PN-1 is also present in aortic smooth muscle cells and may thus participate in vascular biology. However, little is known about its function in the vessel wall. OBJECTIVES: In this study, we investigated the effect of PN-1 overexpression in smooth muscle cells (SMCs), on their sensitivity to thrombin, and their capacity for adhesion, spreading and migration. RESULTS: Two clones exhibiting a two- to threefold increase in PN-1 expression were selected and compared with untransfected and mock-transfected cells. Overexpression of PN-1 was observed to inhibit thrombin-induced cell responses as indicated by a twofold decrease in induction of PAI-1 expression, a decreased calcium mobilization in response to low thrombin concentrations and a twofold increase in the capacity to inhibit thrombin catalytic activity. Overexpression of PN-1 did not modify adhesion, spreading, and migration of SMCs on type I collagen. In contrast, SMCs overexpressing PN-1 exhibited a 40% reduction in adhesion, a 50% reduction in spreading and a complete absence of migration on vitronectin when compared with control SMCs. CONCLUSIONS: Our studies thus reveal that PN-1 is likely to play a critical role in regulating essential cell functions such as (i) thrombin-induced responses, which are dependent on its antiprotease activity, and (ii) adhesion, spreading, and migration, which are independent of its antiprotease activity and may be related to its interaction with other partners, such as vitronectin in the present case.
Assuntos
Precursor de Proteína beta-Amiloide/metabolismo , Músculo Liso Vascular/metabolismo , Receptores de Superfície Celular/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Sinalização do Cálcio/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/fisiologia , Células Cultivadas , DNA Complementar/genética , Expressão Gênica , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Nexinas de Proteases , Ratos , Receptores de Superfície Celular/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Trombina/farmacologia , Transfecção , Vitronectina/metabolismoRESUMO
We have recently identified (Akhavan S et al., Thromb Haemost 2000; 84: 989-97) a patient with a mild bleeding diathesis associated to an homozygous mutation in the thrombin B chain (Gly25Ser, chymotrypsinogen numbering, i.e. position 330 in human prothrombin numbering). Transient transfection of wild-type prothrombin (FII-WT) and mutant prothrombin (designated FII-G25(330)S) cDNA in COS-7 cells showed a mild reduction (50%) in FII-G25(330)S production. Recombinant proteins, stably expressed in Chinese hamster ovary cells, were isolated and activated by Taipan snake or Echis carinatus venoms. We show that the G25(330)S mutation results in a decrease in the rate of prothrombin proteolytic activation. The mutation also significantly decreases (i) the catalytic activity of thrombin with a 9-fold reduction in catalytic efficiency of the mutant toward S-2238; (ii) the interaction with benzamidine; (iii) the rate of inhibition by TLCK and antithrombin; and (iv) the rate of hydrolysis of macromolecular substrates (fibrinogen, protein C). In contrast, exosite I does not appear to be affected by the molecular defect. These results, together with molecular modeling and dynamics, indicate that Gly25(330) is important for proper expression and probably proper folding of prothrombin, and also plays a critical role in both the alignment of the catalytic triad and the flexibility of one of the activation segments of prothrombin.
Assuntos
Glicina/química , Trombina/química , Animais , Sítios de Ligação , Células CHO , Células COS , Catálise , Cricetinae , Análise Mutacional de DNA , DNA Complementar/metabolismo , Fibrina/química , Fibrinogênio/química , Homozigoto , Humanos , Hidrólise , Cinética , Modelos Moleculares , Mutação , Conformação Proteica , Estrutura Terciária de Proteína , Protrombina/química , Protrombina/genética , Proteínas Recombinantes/química , Venenos de Serpentes , Serpentes , Trombina/antagonistas & inibidores , Fatores de Tempo , TransfecçãoRESUMO
Hereditary protein S deficiency was detected in three subjects belonging to three generations of one single family. The deficiency was heterozygous and was associated with recurrent venous thromboembolism in two of the subjects affected. Plasma analysis by two-dimensional immunoelectrophoresis showed that the protein S fraction bound to C4b-binding protein was quantitatively normal, whereas the free protein S fraction was much reduced. Since it has been well established that only free protein S intervenes in the regulation of blood coagulation, any quantitative deficiency, even moderate, in protein S resulting in redistribution of its free and bound fractions will have major functional repercussions.
