화학물질 유도 적혈구의 혈액응고 활성화 및 용혈에 의한 혈전생성촉진

Abstract

Silver nanoparticles (AgNP) are widely used in medical practices owing to their distinct antibacterial, antiviral and anticancer activities. However, with increasing use of AgNP, concerns over its potential toxicity are also escalating. Here, we demonstrated the potential thrombotic effect of AgNP which was mediated by the procoagulant activity of red blood cells (RBCs). In freshly isolated human RBCs, AgNP, but not silver microparticles (AgMP), elicited morphological changes, phosphatidylserine (PS) exposure and microvesicles (MV) generation, the key indicators of procoagulant activity in RBCs at relatively low concentration ranges (≦ 100 μg/mL) that were free of significant hemolysis. In line with this, AgNP potentiated thrombin generation and adherence of RBCs to endothelial cells, while AgMP did not. Oxidative stress, intracellular calcium increase, and ATP depletion were found to underlie the procoagulant activity of AgNP, which led to altered activity of membrane aminophospholipid translocases. These in vitro findings were well reproduced in rat in vivo, where intravenously exposure to AgNP promoted venous thrombosis significantly. Of note, RBCs isolated from cancer patients, who inherently convey the risk of thrombosis, were more sensitive to the procoagulant effects of AgNP. In addition, AgNP significantly potentiated the procoagulant effects of a chemotherapeutic drug, paclitaxel. Collectively, these results suggest that AgNP may have prothrombotic risks by promoting procoagulant activity of RBCs and caution shall be taken for its use in the population sensitive to thrombosis like cancer patients. Thrombotic complications often concur with hemolytic anemia as adverse effects of some xenobiotics. However, little is known about the cross-talk between these two events. Here, employing ginsenoside Rg3, one main active component of ginseng with hemolytic activity, we investigated whether the hemolysis may provoke thrombosis and elucidated the mechanism underlying. Rg3 induced hemolysis of freshly isolated human erythrocytes in time- and concentration-dependent manners. Along with hemolysis, Rg3 led to the formation of holes on the cell membrane of erythrocytes and irregular fragmentation. Resulting erythrocyte debris was observed to expose phosphatidylserine (PS) while residual erythrocytes did not. This phenomenon did not accompany calcium increase or caspase-3 activation, microparticles (MP) generation that are the typical mechanisms underlying PS exposure and erythrocyte-elicited thrombosis as observed with lead acetate (Pb2+). Nevertheless, erythrocytes exposed to Rg3 and Pb2+, both promoted thrombin generation and exhibited increased adherence to endothelial cells, reflecting that hemolysis, itself can induce procoagulant activation of erythrocytes. These results were further confirmed in vivo where Rg3 administration led to PS-bearing erythrocyte debris and increased thrombosis. Pb2+ also increased thrombosis but did not produce fragmented erythrocytes. Collectively, these results demonstrated that hemolysis can provoke thrombosis through generating PS-bearing debris, providing an important clue to explain the thrombotic complications appearing as a co-morbidity of hemolysis.

본 연구는 화학물질에 의한 적혈구 형태변화와 세포독성은 혈전을 일으킨다는 문헌보고를 참고하여 두가지 연구목적으로 수행되었다. 첫 번째 연구주제는 최근 사용이 증가되고 있는 은 나노 (Silver nanoparticle; AgNP)의 잠재적 혈액독성을 검토하기 위하여 적혈구 (RBCs)의 응고 촉진 활성에 의해 매개 된 AgNP에 의한 혈전 유발 가능성을 연구하였다. AgNP 는 적혈구의 혈액응고 촉진의 주요 지표인 PS 노출, MV 생성, 형태 변화를 유도했다. 그 결과 AgNP는 thrombin 생성 및 적혈구의 내피세포 부착을 증가시켰으며 이러한 변화는 ROS 생성 및 세포내 칼슘증가등 기전을 매개한다. 또한, 이러한 in vitro 결과는 정맥혈전 동물모델에서 혈전을 유발됨이 확인됐다. 또한, AgNP는 항암제 paclitaxel의 응고 촉진 효과를 크게 강화시켰으며 혈전증의 위험이 큰 암 환자로부터 분리 된 적혈구에서도 관찰되었다. 종합적으로, AgNP는 적혈구의 응고 촉진 작용을 촉진하여 혈전 형성 위험을 갖고 있으며, 암 환자와 같은 혈전증에 민감한 인구 집단에서는 더욱 위험할 수 있음을 제시하는 결과이다. 두 번째 연구주제는 용혈을 유발하는 여러 화학물질은 혈전위험이 증가됨이 알려져 있지만 그 원인에 관하여는 연구된 바 없다. 따라서 용혈성 활성이 있는 ginsenoside Rg3를 사용하여 용혈이 어떤 과정을 거쳐 혈전생성이 증가하는지를 규명하였다. Rg3는 인간 적혈구의 용혈을 유도하며 세포막에 pore가 생겨 불규칙적인 debris가 생성되었다. 생성된 debris는 표면에 PS를 노출하고 있으며, 그 결과 thromin 생성을 증가시켰다. In vivo 실험에서도 Rg3에 의한 PS-bearing debris는 혈전생성을 촉진시키며, 이는 Pb2+에 의해 유도된 적혈구 형태변화, PS 노출에 의한 혈전생성과는 차이가 있음을 밝혔다. 결론적으로 AgNP, Pb2+, Rg3와 같은 화학물질이 적혈구에 노출되면 서로 다른 경로의 형태변화, 용혈, PS 노출을 매개하여 procoagulant 활성과 혈관내피세포에 부착능의 증가를 통하여 in vivo에서 혈전생성이 증가됨을 밝힘으로써 화학물질에 의한 적혈구를 매개한 심혈관계 질환 위험성의 새로운 지견을 제시한다.