Blood clotting in minimally altered whole blood
Blood clotting in minimally altered whole blood
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DOI：
10.1182/blood.v88.9.3432.bloodjournal8893432
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发表时间：
1996-11-01
期刊：
影响因子：
20.3
通讯作者：
Mann, KG
中科院分区：
文献类型：
--
作者：
Rand, MD;Lock, JB;Mann, KG
使用因子V、凝血酶原、血小板因子4、抗凝血酶III和纤维蛋白原的定量Western印迹法研究了在37 ℃下组织因子引发的全血凝血过程中调节凝血酶生成的事件序列，其中接触途径被玉米胰蛋白酶抑制剂抑制。此外，纤维蛋白肽A（FPA），凝血酶-抗凝血酶III（达特）复合物的形成，和凝血酶原片段1.2（F1.2）通过市售的酶联免疫吸附试验（ELISA）进行测量。在用40 pmol/l重组组织因子启动的典型实验中，在凝血时间（4.5分钟）可见之前是因子V的显著裂解，导致65%的因子Va重链生成，但仅10%的轻链形成。此时，50%的血小板因子4被释放，表明已经产生了一半（约700 pmol/L）的可用血小板凝血酶原酶位点。在凝血时间，存在约15 nmol/L凝血酶B链;然而，FPA释放分析表明，只有15%的凝血酶作用于纤维蛋白原。这种凝血酶由7 pmol/L凝血酶原酶作用产生。凝血酶产生的最大速率在凝血时间后很久达到，并且与约150 pmol/L凝血酶原酶的存在一致（约7分钟）。这些结果表明因子Xa是凝血酶生成的限制因子。60分钟后，75%的初始凝血酶原（1.24 μ mol/L）被消耗，产生440 nmol/L凝血酶原2和360 nmol/L凝血酶（B链）产物。这些值的总和（800 nmol/L）与ELISA法测定的（校正）F1.2浓度相似。凝血酶原的不完全裂解表明凝血酶原酶复合物和凝血酶原酶的形成在反应中均受到抑制。通过ELISA测量达特复合物几乎等于B链浓度，但十二烷基硫酸钠稳定的凝血酶-抗凝血酶III复合物直到凝块形成后很久才观察到，并且从不等于ELISA-TAT值。在凝块形成时，80%的纤维蛋白原从流体相中耗尽，而仅35%至45%的FPA被释放，这表明未裂解的纤维蛋白原显著掺入到形成的初始凝块中。(C)1996年，美国血液学会。
The sequences of events regulating thrombin generation during tissue factor-initiated clotting in whole blood at 37 degrees C in which the contact pathway was suppressed with corn trypsin inhibitor are studied using quantitative Western blotting of factor V, prothrombin, platelet factor 4, antithrombin III, and fibrinogen. In addition, fibrinopeptide A (FPA), thrombin-antithrombin III (TAT) complex formation, and prothrombin fragment 1.2 (F1.2) were measured via commercially available enzyme-linked immunosorbent assays (ELISAs). In a typical experiment initiated with 40 pmol/l recombinant tissue factor, visual clot time (4.5 minutes), was preceded by significant cleavage of factor V resulting in 65% factor Va heavy-chain generation but only 10% light-chain formation. At this point, 50% of the platelet factor 4 is released, suggesting that half (approximately 700 pmol/L) of the platelet prothrombinase sites available have been generated. At clot time, approximately 15 nmol/L thrombin B-chain is present; however, analyses of FPA release demonstrate that only 15% of the thrombin is acting on fibrinogen. This thrombin is produced by the action of 7 pmol/L prothrombinase. The maximum rate of thrombin production is reached well after clot time and is consistent with the presence of approximately 150 pmol/L prothrombinase (at about 7 minutes). These results suggest that factor Xa is the limiting factor for thrombin generation. After 60 minutes, 75% of the initial prothrombin (1.24 mu mol/L) is consumed yielding 440 nmol/L prethrombin 2 and 360 nmol/L thrombin (B-chain) products. The sum of these values (800 nmol/L) is similar to the (corrected) F1.2 concentration determined by ELISA. The incomplete cleavage of prothrombin indicates both the prothrombinase complex and the formation of prothrombinase are inhibited in the reaction. TAT complex measured by ELISA is almost equivalent to B-chain concentration, but sodium dodecyl sulfate stable thrombin-antithrombin III complexes are not observed until well after clot formation and are never equivalent to ELISA-TAT values. At the point of clot formation, 80% of the fibrinogen is depleted from the fluid phase, whereas only 35% to 45% of the FPA is released, suggesting a significant incorporation of uncleaved fibrinogen into the initial clot formed. (C) 1996 by The American Society of Hematology.