RESUMO
Tissue factor (TF), the main activator of the blood coagulation cascade, has been shown to be expressed by platelets. Despite the evidence that both megakaryocytes and platelets express TF mRNA, and that platelets can make de novo protein synthesis, the main mechanism thought to be responsible for the presence of TF within platelets is through the uptake of TF positive microparticles. In this study we assessed 1) whether human megakaryocytes synthesise TF and transfer it to platelets and 2) the contribution of platelet-TF to the platelet hemostatic capacity. In order to avoid the cross-talk with circulating microparticles, we took advantage from an in vitro cultured megakaryoblastic cell line (Meg-01) able to differentiate into megakaryocytes releasing platelet-like particles. We show that functionally active TF is expressed in human megakaryoblasts, increased in megakaryocytes, and is transferred to a subset of shed platelets where it contributes to clot formation. These data were all confirmed in human CD34pos-derived megakaryocytes and in their released platelets. The effect of TF silencing in Meg-megakaryoblasts resulted in a significant reduction of TF expression in these cells and also in Meg-megakaryocytes and Meg-platelets. Moreover, the contribution of platelet-TF to the platelet hemostatic capacity was highlighted by the significant delay in the kinetic of thrombin formation observed in platelets released by TF-silenced megakaryocytes. These findings provide evidences that TF is an endogenously synthesised protein that characterises megakaryocyte maturation and that it is transferred to a subset of newly-released platelets where it is functionally active and able to trigger thrombin generation.
Assuntos
Plaquetas/metabolismo , Megacariócitos/metabolismo , Comunicação Parácrina , Trombina/metabolismo , Tromboplastina/biossíntese , Antígenos CD34/metabolismo , Biomarcadores/metabolismo , Linhagem Celular , Humanos , Cinética , Interferência de RNA , RNA Mensageiro/biossíntese , Transdução de Sinais , Tromboelastografia , Tromboplastina/genética , TransfecçãoRESUMO
AIMS: The discovery of a specific prorenin receptor (PRR) suggests a biological function of prorenin that is independent of angiotensin I production. In the present study, we investigated the role of PRR on smooth muscle cell (SMC) migration. METHODS AND RESULTS: PRR was expressed in human mammary arteries and in cultured human aortic SMCs. Prorenin induced SMC migration in a dose-dependent manner, as assessed by Boyden chamber chemotaxis assay, and increased SMC random motility, as determined by video microscopy. The prorenin decoy peptide inhibited SMC migration in response to prorenin, and knockdown of PRR by small interfering RNA completely inhibited the migratory response to prorenin, demonstrating that the chemotactic action of prorenin is mediated by the PRR. Prorenin induced cytoskeleton reorganization and lamellipodia formation and increased the intracellular levels of both RhoA-GTP and Rac1-GTP through PRR. These effects were required for SMC migration, because the suppression by small interfering RNA of either Rac1 or RhoA GTP-bound forms completely blocked the PRR-mediated chemotactic effect. Prorenin also induced the formation of larger focal adhesions and cleavage of the focal adhesion kinase (pp125(FAK)) into two main fragments with molecular weights of 50 and 90 kDa. The generation of these two fragments of pp125(FAK) was reduced by the calpain inhibitor ALLN, which also inhibited SMC migration in response to prorenin. CONCLUSIONS: These results demonstrate that prorenin is a chemotactic factor for human aortic SMCs expressing PRR. This effect is elicited through reorganization of the cytoskeleton and focal adhesion, activation of RhoA and Rac1, and calpain-mediated cleavage of pp125(FAK).
Assuntos
Quimiotaxia , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Precursores de Proteínas/metabolismo , Receptores de Superfície Celular/metabolismo , Renina/metabolismo , Aorta/metabolismo , Calpaína/antagonistas & inibidores , Calpaína/metabolismo , Células Cultivadas , Quimiotaxia/efeitos dos fármacos , Inibidores de Cisteína Proteinase/farmacologia , Citoesqueleto/metabolismo , Quinase 1 de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Humanos , Microscopia de Vídeo , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Fragmentos de Peptídeos/metabolismo , Interferência de RNA , Receptores de Superfície Celular/genética , Transdução de Sinais , Fatores de Tempo , Transfecção , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Receptor de Pró-ReninaRESUMO
In the last ten years the contribution of both vessel wall-derived tissue factor (TF) and platelets to atherosclerosis has been revisited. At the beginning of the 2000 a circulating blood-borne TF has been proposed to sustain coagulation activation and propagation on the edge of a growing thrombus. Concomitantly with the observation that platelets not only contribute to thrombus formation, but also take part to the onset of the atherosclerotic lesion, evidences have been provided that they express functionally active TF, making them able to trigger the coagulation cascade.
