探討抗血栓蛇毒蛋白Rhodostomin 對於單核性白血球之結合及其效應

Abstract

蛇毒成份中,有一類低分子量,結構中均富含cysteine且含特定氨基酸序列RGD (Arg-Gly-Asp) loop， 並以適當的雙硫結構構成一個具彈性的髮夾環，進而凸顯RGD序列，使其能跟黏著分子(integrin)產生緊密的結合，此類蛇毒蛋白總稱為抗黏著蛇毒蛋白disintegrins.此類蛇毒蛋白能藉由它們對未受活化或受到刺激劑活化之血小板上的αllb β3 integrin的高度親合力而去拮抗αllbβ3 integrin之作用，進而抑制血小板的凝集作用。 Rhodostomin是一種由馬來亞腹蛇(Calloselasma rhodostoma) 蛇毒中純化而來的一種中鏈的disintegrin，主要作用可以選擇性地去阻斷αllbβ3及 αvβ3這兩個黏著分子，具有抗血栓和血管新生之活性；但對於白血球方面的研究仍在起步階段，因此我們想利用rhodostomin去研究disintegrin對於人類單核性白血球功能性的影響。將rhodostomin接合上FITC來作為探針工具，並利用流式細胞儀分析人類單核性白血球與它的反應。我們發現rhodostomin除了能與單核性白血球產生結合外，也發現它們之間的結合具有呈濃度相關的性質併呈現飽和的趨勢。在活化劑PMA的存在下，rhodostomin跟單核性白血球之結合現象會明顯增加。我們也證實rhodostomin不會被單核性白血球所吞噬，這說明了rhodostomin與單核性白血球之結合應具有特異性。EDTA並無法抑制rhodostomin與單核性白血球的結合。CD11b/CD18(Mac-1)的天然結合物-纖維蛋白原(2 mg/ml)，其能部份抑制rhodostomin與被PMA活化之單核性白血球的結合(35%的抑制)；GRGDS (300 μM)也有同樣的抑制作用。我們同時也利用附著試驗觀察rhodostomin跟單核性白血球之間的附著現象，發現它們之間有濃度相關性的附著現象；LDL-receptor-related protein是一個多功能的受體，它可調控白血球上 β2 integrin 的表現和 β2 integrin附著到內皮細胞的現象，在共軛焦顯微鏡也發現它與β2 integrin也有colocalization的現象，因此我們接著去探討rhodostomin與LRP的關係。我們利用流式細胞儀分析，可發現rhodostomin會抑制α4,αM,αX以及LRP單源抗體結合至受刺激劑刺激的人類單核性白血球上；也發現單核球上確實有大量LRP的表現，而LRP單源抗體明顯競爭性地抑制β2黏著分子在單核球上的表現。且rhodostomin不但會競爭性地抑制LRP單源抗體結合上單核球，且隨著rhodostomin濃度的增加，抑制的程度也有增加的傾向。另外在附著試驗我們也發現α4,αM,αX以及LRP單源抗體皆可抑制rhodostomin跟單核性白血球之間的附著現象 Rhodostomin 能部份抑制經由Mac-1受體附著到固定相纖維蛋白原上的單核性白血球，也能抑制經由受α4β1體附著到固定相fibronectin上的單核性白血球，也間接地印證在流式細胞儀發現rhodostomin會作用在α4,αM,αX integrin的現象。另外我們也發現rhodostomin可以濃度相關性地去抑制經由LPS刺激下單核球上組織因子的表現及促凝血活性。 綜合上述結果可知rhodostomin可與單核性白血球結合，可能經由和單核性白血球上的 β2 integrin或是LRP作用，進而影響單核性白血球的功能，例如跟基質蛋白的附著、組織因子的表現及活性。因此， rhodostomin可能具有抗發炎療效的潛力，研究disintegrin或類似蛇毒蛋白成份在抗發炎的活體效應應是另一重要探討之方向。

Disintegrin , a group of low molecular weight , cysteine-rich snake venom polypeptides which contain an Arg-Gly-Asp (RGD) loop maintained by specific disulfide bridge. They exhibit high binding affinities to both resting and activated αIIbβ3 on platelets in a divalent cation-dependent manner , interfering with fibrinogen binding to αIIbβ3 integrin and consequently blocking platelet aggregation. Rhodostomin , a medium chain disintegrin isolated from the venom of Calloselasma rhodostoma , blocks platelet aggregation and angiogenesis through selective binding to platelet αIIbβ3 and endothelial αVβ3 , respectively. The research regarding the effect of rhodostomin on leukocytes is just in the beginning , so we studied the effect of rhodostomin on the function of monocytes in this study. By flow cytometric analysis of human monocytic THP-1 cells , we found that rhodostomin interacted with monocytes, and the binding was dose-dependent and saturable. EDTA did not inhibit the binding of rhodostomin. In addition, bound rhodostomin was not internalized. Soluble fibrinogen (2mg/ml), a natured ligand of Mac-1 (CD11b/CD18), and the peptides, GRGDS(300 μM), inhibited the binding of the rhodostomin to PMA-activated THP-1 cells, indicating the RGD motif in rhodostomin is essential for its binding activity. We also found that THP-1 cells bound to immobilized rhodostomin in a dose-dependent manner. PMA also increased their binding to immobilized rhodostomin. β-integrin clustering on activation is a key event in leukocyte adhesion to the endothelium during the inflammatory response. LDL-receptor-related protein (LRP), a multifunctional receptor, can regulate the expression of β2 integrin, and affects the adhesion between monocytes and endothelial cells. Confocal microscopy also revealed that endogenously expressed LRP and αLβ2 colocalized in monocytes. In this study, using flow cytometry, we found that rhodostomin significantly inhibited the binding of mAb raised against α4、αM、αX and LRP to PMA-activated THP-1 cells. In addition , we found that LRP was highly expressed on THP-1 cells and LRP mAb inhibited the binding of LRP mAb to PMA-activated THP-1 cell, and rhodostomin inhibited the binding of LRP mAb to PMA-activated THP-1 cells in a dose-dependent manner. Rhodostomin blocked the Mac-1 and VLA-4-dependent adhesion of activated THP-1 cells to immobilized fibrinogen and fibronectin, respectively. On the other hand, we also found that rhodostomin blocked the expression and procoagulant activity of tissue factor on THP-1 cells stimulated by LPS in a dose-dependent manner. Take together ,these data suggest that rhodostomin may affect the adhesion and tissue factor expression of THP-1 cells through its binding to LRP or α4、αM、αX integrin subunit. Whether rhodostomin and its derivatives exhibit anti-inflammatory activity in vivo needs to be investigated .