Evidence for Two Populations of Disulphide Bonds on Blood Platelets
Evidence for Two Populations of Disulphide Bonds on Blood Platelets
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血小板上两个二硫键群的证据
DOI：
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发表时间：
1974
期刊：
影响因子：
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通讯作者：
J. L. Gordon
中科院分区：
文献类型：
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作者：
D. Macintyre;J. L. Gordon
当膜二硫键被还原时，人血小板的聚集被抑制（Born，1967）。将血小板暴露于紫外光诱导血小板聚集（Dickson等人，1971）也可能通过二硫键的还原（Doery等人，1973年）。通过使用二硫苏糖醇作为还原剂，我们已经证实，低浓度抑制血小板聚集，但在较高浓度的二硫苏糖醇本身诱导聚集。通过差速离心从人和大鼠血液制备富血小板血浆，并在0.1 ml样品中用光度法测量血小板聚集（Gordon & Drummond，1974）。使用白蛋白密度梯度，从2 ml体积的富血小板血浆中制备洗涤的人血小板混悬液（沃尔什，1972）。在血小板聚集之前和之后，通过荧光法测量血小板和血浆或悬浮介质中的总腺嘌呤核苷酸和5-羟色胺（Gordon & Drummond，1974; Drummond & Gordon，1974），以确定血小板释放反应的程度。通过Wroblewski & La Due（1955）的方法测量乳酸脱氢酶以检查血小板的可能溶解。当胶原蛋白或ADP加入到搅拌的富血小板血浆中时，血小板聚集。对胶原蛋白的反应的特征在于几秒钟的延迟，随后是由血小板释放胺和核苷酸引起的不可逆聚集。相反，对低浓度ADP的聚集反应是快速的、可逆的，并且与血小板成分的释放无关。在富含血小板的血浆样品中加入二硫苏糖醇可抑制ADP和胶原蛋白诱导的聚集，但二硫苏糖醇对ADP的作用较弱。对胶原和ADP的I.C.50值（抑制50%聚集的二硫苏糖醇浓度）分别为0.45m ~ 2和2.15m ~ 2。在每种情况下，使用半最大聚集刺激，并在加入聚集剂之前，将二硫苏糖醇在富含血小板的血浆中于37°C预孵育2分钟。加入1 m-二硫苏糖醇可消除人富血小板血浆中胶原诱导的聚集，并完全抑制伴随的血小板释放反应：对照样品中胶原释放34%的血小板5-羟色胺，但在含有1 m-二硫苏糖醇的样品中未引起释放。二硫苏糖醇在人和大鼠富血小板血浆中的抑制效力相似。当富含血小板的血浆样品在加入聚集剂之前与二硫苏糖醇在37°C下预孵育时，抑制随时间增加，在10- 20分钟后达到最大值。当将二硫苏糖醇以3 μ M及以上的浓度加入人富血小板血浆中时，它在延长的滞后期（层粘连蛋白）后引起血小板聚集。滞后的持续时间减少，聚集率随着二硫苏糖醇浓度的增加而增加。在洗涤过的人血小板的悬浮液中，二硫苏糖醇不产生聚集，除非还加入纤维蛋白原。即使存在纤维蛋白原，在加入二硫苏糖醇和开始聚集之间也总是有约2分钟的滞后。当二硫苏糖醇与血小板悬液在无纤维蛋白原存在下孵育2 min或更长时间时，随后加入纤维蛋白原引起立即聚集。二硫苏糖醇诱导聚集前的滞后是由于二硫苏糖醇对血小板的作用而不是对纤维蛋白原的作用，因为二硫苏糖醇与纤维蛋白原的预孵育不会改变滞后的持续时间（图1）。二硫苏糖醇引起的人血小板聚集与10- 15%的血小板5-羟色胺和腺嘌呤核苷酸的释放有关。没有释放的乳酸脱氢酶从血小板聚集过程中检测到二硫苏糖醇，这表明释放的胺和核苷酸是选择性的，而不是由血小板裂解。当二硫键被二硫苏糖醇还原时，
The aggregation of human blood platelets is inhibited when membrane disulphide bonds are reduced (Born, 1967). Exposure of blood platelets to U.V. light induces platelet aggregation (Dickson et al., 1971) possibly also by the reduction of disulphide bonds (Doery et al., 1973). By using dithiothreitol as a reducing agent, we have confirmed that low concentrations inhibit platelet aggregation but at higher concentrations dithiothreitol itself induces aggregation. Platelet-rich plasma was prepared from human and rat blood by differential centrifugation, and platelet aggregation was measured photometrically in 0.1 ml samples (Gordon & Drummond, 1974). Suspensions of washed human platelets were prepared from 2ml volumes of platelet-rich plasma by using an albumin density gradient (Walsh, 1972). Total adenine nucleotides and 5-hydroxytryptamine in platelets and in plasma or suspending medium were measured fluorimetrically (Gordon & Drummond, 1974; Drummond & Gordon, 1974) before and after platelet aggregation to determine the extent of the platelet-release reaction. Lactate dehydrogenase was measured by the method of Wroblewski & La Due (1955) to check for possible lysis of platelets. Platelets aggregate when collagen or ADP is added to stirred platelet-rich plasma. The response to collagen is characterized by a lag of several seconds duration, followed by irreversible aggregation caused by release of amines and nucleotides from the platelets. In contrast, the aggregation response to low concentrations of ADP is rapid, reversible and not associated with the release of platelet constituents. The addition of dithiothreitol to platelet-rich plasma samples inhibited aggregation induced by both ADP and collagen, but dithiothreitol was less effective against ADP. The I.C.50 value (concentration of dithiothreitol inhibiting aggregation by 50%) was 0 .45m~ for collagen and 2 .15m~ for ADP. In each case, a half-maximal aggregating stimulus was used and dithiothreitol was preincubated in platelet-rich plasma for 2min at 37°C before adding the aggregating agent. The addition of 1 m-dithiothreitol abolished collagen-induced aggregation in human platelet-rich plasma and also completely inhibited the accompanying plateletrelease reaction : collagen released 34 % of platelet 5-hydroxytryptamine in control samples, but caused no release in samples containing 1 m-dithiothreitol. The inhibitory potency of dithiothreitol was similar in human and in rat platelet-rich plasma. When samples of platelet-rich plasma were preincubated with dithiothreitol at 37°C before addition of the aggregating agent, inhibition increased with time, reaching a maximum after 10-2Omin. When dithiothreitol was added to human platelet-rich plasma in concentrations of 3 m ~ and above, it caused platelet aggregation after a prolonged lag phase (lamin). The duration of the lag decreased and the rate of aggregation increased with increasing dithiothreitol concentrations. In suspensions of washed human platelets dithiothreitol did not produce aggregation unless fibrinogen was also added. Even when fibrinogen was present there was always a lag of about 2min between the addition of dithiothreitol and the start of aggregation. When dithiothreitol had been incubated with the platelet suspension for 2min or longer in the absence of fibrinogen, the subsequent addition of fibrinogen caused immediate aggregation. The lag before dithiothreitol-induced aggregation was due to an action of dithiothreitol on the platelets and not on the fibrinogen, since preincubation of dithiothreitol with fibrinogen did not alter the duration of the lag (Fig. 1). Aggregation of human platelets by dithiothreitol was associated with the release of 10-15 % of platelet 5-hydroxytryptamine and adenine nucleotides. No release of lactate dehydrogenase from the platelets could be detected during aggregation by dithiothreitol, suggesting that the release of amines and nucleotides was selective and not caused by platelet lysis. When disulphide bonds are reduced by dithiothreitol, there is concomitant formation