Miraglia C C, Greenberg C S
Anal Biochem. 1985 Jan;144(1):165-71. doi: 10.1016/0003-2697(85)90099-5.
A method to directly measure the formation of blood coagulation Factor XIIIa in platelet-poor plasma unmodified by heat is described. The synthetic peptide glycyl-L-prolyl-L-arginyl-L-proline, a fibrin-polymerization inhibitor, was used to prevent clotting of platelet-poor plasma. Plasma was diluted to a final concentration of 2.5% (v/v) in 0.1 M Tris-HCl, pH 8.5, buffer containing 25% glycerol, 5 mM calcium chloride, and 0.25 mM glycyl-L-prolyl-L-arginyl-L-proline and then activated by thrombin (20 U/ml) for 15 min. The Factor XIIIa-catalyzed incorporation of [3H]putrescine into Hammersten casein was used to measure Factor XIIIa formation. The assay detected Factor XIIIa in 2.5 to 50 microliter of thrombin-treated plasma. When purified Factor XIII was added to Factor XIII-deficient plasma, there was complete recovery of the Factor XIII added. Glycyl-L-prolyl-L-arginyl-L-proline did not inhibit Factor XIIIa activity in thrombin-treated plasma or purified platelet Factor XIIIa. Glycerol stabilized Factor XIIIa activity in thrombin-treated plasma and buffer for 60 min. The presence of fibrinogen in plasma did not modify the assay results. The time course of thrombin-catalyzed Factor XIIIa formation in platelet-poor plasma containing glycyl-L-prolyl-L-arginyl-L-proline was directly measured using the assay.
描述了一种直接测量未受热修饰的乏血小板血浆中凝血因子XIIIa形成的方法。合成肽甘氨酰-L-脯氨酰-L-精氨酰-L-脯氨酸，一种纤维蛋白聚合抑制剂，用于防止乏血小板血浆凝固。血浆在含有25%甘油、5 mM氯化钙和0.25 mM甘氨酰-L-脯氨酰-L-精氨酰-L-脯氨酸的0.1 M Tris-HCl，pH 8.5缓冲液中稀释至最终浓度2.5%（v/v），然后用凝血酶（20 U/ml）激活15分钟。因子XIIIa催化的[3H]腐胺掺入哈默斯坦酪蛋白用于测量因子XIIIa的形成。该测定法可检测2.5至50微升凝血酶处理血浆中的因子XIIIa。当将纯化的因子XIII添加到因子XIII缺乏的血浆中时，添加的因子XIII可完全回收。甘氨酰-L-脯氨酰-L-精氨酰-L-脯氨酸不抑制凝血酶处理血浆或纯化的血小板因子XIIIa中的因子XIIIa活性。甘油在凝血酶处理血浆和缓冲液中稳定因子XIIIa活性60分钟。血浆中纤维蛋白原的存在不改变测定结果。使用该测定法直接测量了在含有甘氨酰-L-脯氨酰-L-精氨酰-L-脯氨酸的乏血小板血浆中凝血酶催化因子XIIIa形成的时间进程。
