抗血栓蛇毒蛋白對嗜中性白血球附著、嗜中性
白血球-血小板交互作用與其作用機轉之研究

Disintegrins是一群具有特定胺基酸序列Arg(或Lys)-Gly-Asp之蛇毒蛋白的總稱。它們能藉由拮抗ΖIbβ3 integrin，進而抑制血小板的凝集作用。Rhodostomin是一種由馬來亞腹蛇(Calloselasma rhodostoma)純化出的disintegrin，我們首先利用它去研究disintegrin對嗜中性白血球弁鄋獐v響。將rhodostomin接合上FITC以作為探針工具，並利用流式細胞儀分析全血樣本，我們發現rhodostomin除了作用在血小板上之外，它也能作用於嗜中性白血球與單核球上。然而，rhodostomin並不作用於淋巴球上。Rhodostomin與嗜中性白血球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或fMLP的存在下，rhodostomin與嗜中性白血球之結合會明顯增加。我們也證實rhodostomin不會被嗜中性白血球所吞噬，這說明了rhodostomin與嗜中性白血球的結合應具有特異性。EDTA並無法抑制rhodostomin與嗜中性白血球的結合。CD11b/CD18 (Mac-1)的天然結合物─纖維蛋白原，其能抑制rhodostomin與被PMA活化之嗜中性白血球的結合；GRGDS亦能抑制這種結合。然而，包括單株抗體7E3在內，對抗Κ與β2 integrin的抗體卻無法抑制上述的結合，暗示rhodostomin能與嗜中性白血球上其他的受體結合。Rhodostomin能部分抑制經由Mac-1受體附著於固定相纖維蛋白原上的嗜中性白血球，進而減少超氧化物的產生。我們認為超氧化物產量的減少乃歸因於附著之嗜中性白血球減少之故。上述的發現或陳酮郢hodostomin是否具抗發炎的性質提出部分的佐證。除了disintegrins以外，腹蛇科蛇毒中已有多種具生物活性的成分被研究，這些成分能專一性的影響細胞與細胞外間質的作用。根據抗附著(anti-adhesive)的理論，某些蛇毒成分已成戊Q用來發展成抗血栓或抗血管新生藥物。利用血小板凝集器與流式細胞儀，我們首先篩選出日本龜殼花(Trimeresurus flavoviridis)蛇毒中能抑制稀釋之全血樣本中嗜中性白血球與血小板間彼此附著的成分。該蛋白成分之分子量約為28 kDa，其蛋白質N端序列類似其他的蛇毒金屬蛋白酶。利用azocasein作為受質，我們發現該蛋白具有酵素活性，其活性能被EDTA或phenanthroline所抑制，但無法被PMSF所抑制，故該蛋白屬於蛇毒金屬蛋白酶。我們將之命名為triflamp (a metalloproteinase from Trimeresurus flavoviridis)。此外，triflamp為一種?纖維蛋白原酶。從人體血小板與嗜中性白血球的實驗發現，triflamp頗具選擇性的抑制與醣蛋白Ib?(GPIb?有關之血小板凝集作用。此外，triflamp能抑制經由PSGL-1與selectin所造成之嗜中性白血球彼此間的黏附作用。經由細胞混合實驗發現，triflamp作用於嗜中性白血球即已足夠抑制嗜中性白血球與血小板之間的黏附作用。Triflamp的這種抗附著作用乃是隨著劑量或作用時間的增加而加強。經流式細胞儀的分析發現，triflamp會明顯減少嗜中性白血球上PSGL-1以及血小板上醣蛋白Ibㄙ漯穛{。西方點墨法證實PSGL-1與醣蛋白Ibㄖ′配riflamp的受質。Triflamp可藉由切除PSGL-1來抑制嗜中性白血球對P-selectin的附著。Triflamp分解PSGL-1的方式與cathepsin G不同。除了PSGL-1的N端外，triflamp應有其他的分解作用點。Phenanthroline能抑制triflamp對PSGL-1之分解。此外，triflamp不會造成嗜中性白血球釋放cathepsin G。值得注意的是，當血小板先以triflamp處理過後，它們仍能與被活化劑如PAF或fMLP刺激的嗜中性白血球黏附，這顯示出triflamp分解血小板上醣蛋白Ibㄙ漣@用並不能影響嗜中性白血球與血小板的黏附。換言之，分解PSGL-1才是其作用機轉。 Triflamp的發現為腹蛇科的蛇毒金屬蛋白酶在抑制血球細胞間的作用上提供一種新的角色，亦即它或釵頃蝷O能應用為抗發炎藥物。然而在人類全血樣本中，即使高達6微克/毫升的triflamp並無法分解嗜中性白血球上的PSGL-1與血小板的醣蛋白IbㄐC人類血液中的?-巨分子球蛋白(?-macroglobulin)能有效中和 triflamp分解PSGL-1、醣蛋白Ib˙P纖維蛋白原的能力。根據分解azocasein的實驗去定量triflamp之酵素活性顯示出，從化學計量學的角度而言，大約一莫耳的人類?-巨分子球蛋白能中和一莫耳triflamp的酵素活性。SDS-膠體電泳實驗顯示，triflamp能在所謂的誘餌區(bait-region)將?-巨分子球蛋白切開。西方點墨法證實，triflamp會與被切下的?-巨分子球蛋白之C端片段形成數種高分子量的複合物。在大量的親核性化合物methylamine的競爭下，triflamp的酵素活性就不被?-巨分子球蛋白所抑制。活體實驗發現，小鼠嗜中性白血球上PSGL-1的表現量不被triflamp所影響。然而，由小鼠血液分離的嗜中性白血球上的PSGL-1與血小板的醣蛋白Ibㄓ敞鉧Dtriflamp所分解。該結果顯示出，一如發生於人類血液樣本上，小鼠血清中的成分應能中和triflamp的酵素活性。?-巨分子球蛋白-triflamp複合物的形成能合理解釋為何triflamp在人類全血樣本與動物模式下會失去酵素活性。在探討抗血栓蛇毒蛋白對嗜中性白血球之細胞附著因子的影響之模式中，期能開發以抗附著理論為基礎的抗發炎藥物提供一個全新且具潛力的研究方向。適當的藥物篩選模式能尋找出具有高度專一性的產物。雖然蛇毒金屬蛋白酶能應用成為一藥理學研究之工具，但由於動物活體之血液中存在有內生性蛋白酶抑制劑(如?-巨分子球蛋白)，使此類蛇毒金屬蛋白酶在藥物開發上難以應用。釐清各類蛇毒金屬蛋白酶與內生性蛋白酶抑制劑的作用，將有助於這類酵素蛋白的應用。此外，研究具有抗附著弁鄋漣C分子量蛇毒蛋白，如disintegrin，或蛇毒中其他非酵素的成分在抗發

關鍵字

血栓；蛇毒蛋白；嗜中性白血球；血小板

並列摘要

Disintegrins are a group of Arg(or Lys)-Gly-Asp-containing snake venom proteins which inhibit platelet aggregation via the blockade of ΖIbβ3 integrin. We first studied the effect of rhodostomin, a disintegrin purified from the venom of Calloselasma rhodostoma, on the functions of neutrophils. By flow cytometric analysis of whole blood, we found that rhodostomin interacted with leukocytes of the myeloid and monocytic lineage as well as with platelets. The binding of rhodostomin to neutrophils was dose-dependent and saturable, and its binding was increased in PMA- and fMLP-stimulated neutrophils. EDTA did not inhibit the binding of rhodostomin. In addition, bound rhodostomin was not internalized. Soluble fibrinogen, a natural ligand of Mac-1 (CD11b/CD18, Κβ2), and the peptide, GRGDS, inhibited the binding of rhodostomin to PMA-activated neutrophils, while 7E3, a monoclonal antibody (mAb) raised against β3 integrin, or mAbs raised against Κ and β2 integrin did not. Rhodostomin blocked the Mac-1-dependent adhesion of neutrophils to immobilized fibrinogen, in parallel with decreasing the production of superoxide from adherent neutrophils. These data indicate that rhodostomin binds to activated neutrophils in an RGD-dependent manner, blocks the adhesion of activated neutrophils to fibrinogen and attenuates superoxide production, suggesting that rhodostomin may have anti-inflammatory activity. Besides disintegrins, other biologically active components from Viperidae venoms specifically affect cell-matrix interactions and have been utilized for developing anti-adhesive therapy as the anti-thrombotic and anti-angiogenic agents. Utilizing platelet aggregometry coupled with flow cytometry, we first found that a metalloproteinase isolated from the venom of Trimeresurus flavoviridis, termed triflamp, inhibited heterotypic adhesion between platelets and neutrophils in whole blood samples. Triflamp is a monomeric glycoprotein with a molecular weight of ~28 kDa. Triflamp has a N-terminal amino acid sequence homologous to other venom metalloproteinases isolated from T. flavoviridis. The enzymatic activity of triflamp towards azocasein was inhibited by EDTA and phenanthroline but not by PMSF. Moreover, triflamp is a pure ?fibrinogenase. Studies aimed at determining the nature of triflamp in affecting platelets or neutrophils revealed a selective inhibitory activity to glycoprotein (GP) Ib?dependent platelet aggregation and P-selectin glycoprotein ligand-1 (PSGL-1)- dependent neutrophil homotypic aggregation, indicating that its effects are rather specific. Coincubation studies demonstrate that direct interaction of triflamp with neutrophils is sufficient to inhibit the formation of neutrophil-platelet complexes. Its anti-adhesive effect is in a concentration- and incubation time-dependent manner. Triflamp reduces the expression of PSGL-1 on neutrophils and GPIb?on platelets as probed by flow cytometry. As judged by Western blotting, GPIb?on platelets and PSGL-1 on neutrophils are the substrates of triflamp. Moreover, triflamp disrupts P-selectin-mediated adhesion by cleaving PSGL-1 from the neutrophil surface. There are obvious differences regarding PSGL-1 proteolysis by triflamp and cathepsin G. Besides the NH2-terminus of PSGL-1, other sites are truncated by triflamp. The inhibitory effect of triflamp on PSGL-1 expression was blocked by pretreatment with a metalloproteinase inhibitor, phenanthroline. However, triflamp-treated platelets fully kept the ability for binding to PAF- or fMLP-stimulated neutrophils. Degradation of platelet GPIb?by triflamp did not interfere with neutrophil-platelet adhesion. Its effect on neutrophil PSGL-1 appears to be a critical factor for its inhibition on neutrophil-platelet interaction. This study on triflamp revealed the novel role of venom metalloproteinase from Viperidae in affecting the blood cell-cell interactions, thus offering a potential approach for further exploration of anti-inflammatory agents. In human whole blood preparation, however, triflamp (6 μg/ml) failed to cleave neutrophil PSGL-1 and platelet GPIb? Human ?-macroglobulin (?M) was mainly responsible for the neutralization of the proteolytic effects of triflamp on PSGL-1, GPIb?and fibrinogen. Human ?M neutralized triflamp at a stoichiometry about 1:1 (molar basis) as determined by azocaseinolysis. SDS-PAGE analysis revealed that triflamp cleaved the bait-region of ?M. Western blot demonstrated that triflamp interacted with the C-terminal half-subunits of truncated ?M resulting in the formation of high-molecular-weight species of ?M-triflamp complexes. In the presence of a competing nucleophile, methylamine, the proteolytic activity of triflamp was conserved. In vivo we found that mice neutrophils were resistant to the cleavage of PSGL-1 by triflamp. However, mouse PSGL-1 and GPIb?were susceptible to be cleaved by triflamp in washed mouse neutrophil and platelet preparation, respectively. Similarly, mouse serum was also responsible for the inactivation of the proteolytic activity of triflamp. This study provides direct evidences for the reasonable explanation regarding the reduced proteolytic activity of triflamp toward its substrates in whole blood preparation and in vivo model. Taken together, our data clearly show that the anti-adhesive effects of anti-thrombotic snake venom proteins on neutrophils may become a potentially pharmacological approach for the development of anti-inflammatory agents. A suitable bioassay is helpful for screening specific components from venom mixture. The application of triflamp in vivo is restricted because of being inhibited by endogenous proteinase inhibitors. Dissecting the interactons between snake venom metalloproteinases and endogenous proteinase inhibitors is attempted. Understanding the effect and binding targets of disintegrins on neutrophils and searching other potential anti-adhesive components from snake venom are further tasks.

並列關鍵字

triflamp ； disintegrins ； neutrophils ； platelets ； PSGL-1 ； GPIb ； sanke venom metalloproteinases

參考文獻

Chapter 1

1. Ouyang C, Huang TF. Potent platelet aggregation inhibitor from Trimeresurus gramineus snake venom. Biochim Biophys Acta 1983; 757: 332-41.

2. Huang TF, Holt JC, Kirby EP, Niewiarowski S. Trigramin: primary structure and its inhibition of von Willebrand factor binding to glycoprotein IIb/IIIa complex on human platelets. Biochemistry 1989; 28: 661-6.

3. Ouyang C, Teng CM, Huang TF. Characterization of snake venom components acting on blood coagulation and platelet function. Toxicon 1992; 30: 945-66.

4. Huang TF. What have snakes taught us about integrins? Cell Mol Life Sci 1998; 54: 527-40.

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抗血栓蛇毒蛋白對嗜中性白血球附著、嗜中性白血球-血小板交互作用與其作用機轉之研究

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