Diagnostic and genetic studies on fibrin-stabilizing factor with a new assay based on amine incorporation.
Diagnostic and genetic studies on fibrin-stabilizing factor with a new assay based on amine incorporation.
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使用基于胺掺入的新测定法对纤维蛋白稳定因子进行诊断和遗传学研究。
DOI：
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发表时间：
1969
影响因子：
15.9
通讯作者：
H. Nossel
中科院分区：
文献类型：
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作者：
L. Lorand;T. Urayama;J. W. De Kiewiet;H. Nossel
纤维蛋白连接酶是一种纤维蛋白交联酶，在正常血液凝固过程中短暂出现，可催化荧光胺单丹酰尸胺[或N-（5-氨基戊基）-5-二甲氨基-1-萘磺酰胺]掺入酪蛋白。该反应提供了灵敏的荧光方法的基础，用于测量酶的活性（以及类似的其他转肽酶，如转氨酶）。在涉及血浆的试验中，必须克服某些困难，这主要是由于酶本身不存在于柠檬酸盐血浆中。只有它的前体（纤维蛋白稳定因子或因子XIII）存在，仍然需要有限的凝血酶蛋白水解激活。因此，为了测量以血浆作为因子来源的胺掺入，必须加入凝血酶。这就需要对内源性纤维蛋白原进行差异脱敏，使后者不能凝结，从而不能干扰胺的掺入。此外，血浆的凝血酶灭活能力必须达到饱和，才能使该因子完全转化为转肽酶。酪蛋白，monodansyl尸胺，钙，和氢离子的浓度进行选择，以允许几乎最大速度的胺掺入。只有在这种条件下，才能获得与血浆浓度的线性关系。目前还没有类似的检测方法可用于定量评价血浆中的纤维蛋白稳定因子水平。胺掺入试验应用于遗传性总纤维蛋白稳定因子缺乏症的临床病例。研究了输血治疗的效果，并对患者的一些亲属进行了检查。而父亲的阿姨和叔叔给出的值在正常范围内，兄弟和母亲被证明是部分缺陷，可以被认为是杂合子携带者。父亲的纤维蛋白稳定因子水平似乎降低了，但不像其他两个亲戚那么低。两次输注（每次1升）新鲜的正常血浆，间隔约26小时给药，使患者血浆中的水平接近母亲和兄弟中发现的水平。然而，输血的矫正能力在5-6天内迅速下降。最后一次输血无效可归因于针对前体稳定因子的中和抗体的出现，这是一种严重的并发症。一般诊断的通用性和潜力的定量胺掺入试验与血浆进行了讨论。
Fibrinoligase, the fibrin cross-linking enzyme, transiently appearing during the course of coagulation in normal blood, was shown to catalyze the incorporation of a fluorescent amine, monodansylcadaverine [or N-(5-aminopentyl)-5-dimethylamino-1-naphthalene-sulfonamide] into casein. The reaction provided the basis of a sensitive fluorimetric method for measuring the activity of the enzyme (and also of similar other transpeptidases, such as transglutaminase). In tests involving plasma, certain difficulties had to be overcome which were mainly due to the fact that the enzyme itself does not occur in citrated plasma. Only its precursor (fibrin-stabilizing factor or factor XIII) is present, still requiring limited proteolytic activation by thrombin. Thus, in order to measure amine incorporation with plasma as a source of the factor, thrombin must be added. This necessitated a differential desensitization of the intrinsic fibrinogen so that the latter could not clot and could not thereby interfere with amine incorporation. Also, the thrombin-inactivating capacity of plasma had to be saturated to enable full conversion of the factor to the transpeptidase. Concentrations of casein, monodansylcadaverine, calcium, and hydrogen ions were chosen to permit almost maximal velocity of amine incorporation. A linear relationship with regard to plasma concentration could be obtained only under such conditions. No similar assay is presently available for quantitatively evaluating fibrin-stabilizing factor levels in plasma.The amine incorporation test was applied to a clinical case of hereditary total fibrin-stabilizing factor deficiency. The effect of transfusion therapy was studied, and some of the patient's relatives were examined. Whereas a paternal aunt and uncle gave values well within the normal range, a brother and the mother proved to be partially deficient and could be considered as heterozygous carriers. The father appeared to have a reduced level of fibrin-stabilizing factor, though not quite as low as the other two relatives. Two infusions (1 liter each) of fresh normal plasma, administered about 26 hr apart, brought levels in the patient's plasma close to those found in the mother and brother. The corrective power of the transfusions, however, rapidly declined within 5-6 days. Futility of the last transfusion could be ascribed to the appearance of a neutralizing antibody directed against the precursor stabilizing factor, a serious complication. General diagnostic versatility and potential of the quantitative amine incorporation assay with plasma is discussed.