McPherson J, Brownlea S, Zucker M B
Blood. 1987 Aug;70(2):546-50.
The platelet retention test provides a measure of the number of platelets retained in a column of glass beads and is one of the few in vitro platelet function tests that is abnormal in von Willebrand's disease (vWd). In a two-stage test, 1 mL of blood (designated A) was passed through the column, followed by 5 mL of isotonic saline and then 5 mL of blood (B) in which platelet retention was measured. With normal blood as A and B, retention is very high in all 5 mL of blood B. In the first stage, platelets adhere to the glass beads; this requires fibrinogen but not von Willebrand factor (vWf). The platelet-platelet adhesion in the second stage requires vWf, is dependent on release of ADP, and fails to occur if thrombasthenic platelets are tested. Retention was normal when blood from a patient with afibrinogenemia was used as blood B. We have now used monoclonal antibodies to elucidate further the mechanism of platelet retention. Five antibodies to different epitopes on vWf essentially abolished retention in the one-stage test and in the second stage of the two-stage test, but had no effect on the first stage. Thus, the entire vWf molecule must be free of antibody to function in the platelet-platelet adhesion of the second stage of this test. Binding of the antigen-antibody complex to the platelet Fc receptor was not responsible, as Fab and F(ab')2 fragments of one of the antibodies were as effective as intact antibody, and as neither heat-aggregated IgG nor a polyclonal antibody to plasma factor IX inhibited retention. F(ab')2 fragments of 6D1, an antibody to platelet GP Ib that prevents binding of vWf to platelets, also inhibited the second phase of retention. An antibody that inhibits binding of fibrinogen and vWf to GP IIb/IIIa (LJ-CP8) inhibited both the first and second stages of retention, whereas LJ-P5, an antibody that inhibits only the binding of vWf to GP IIb/IIIa, caused slight inhibition of retention when normal or afibrinogenemic blood was used as blood B and was reported to cause only partial inhibition of ADP-induced platelet aggregation in this afibrinogenemic patient. The results suggest that vWf is altered during rapid passage of blood through the glass-bead column so that it attaches to GP Ib, exposing GP IIb/IIIa, which then binds the altered vWf or fibrinogen, either of which can induce platelet aggregation (platelet-platelet adhesion) and thus retention in the column.
血小板滞留试验可测定保留在玻璃珠柱中的血小板数量，是少数几种在血管性血友病（vWd）中异常的体外血小板功能试验之一。在两阶段试验中，将1 mL血液（标记为A）通过柱，随后是5 mL等渗盐水，然后是5 mL血液（B），并测定其中的血小板滞留情况。以正常血液作为A和B时，所有5 mL血液B中的滞留率都很高。在第一阶段，血小板黏附于玻璃珠；这需要纤维蛋白原，但不需要血管性血友病因子（vWf）。第二阶段的血小板-血小板黏附需要vWf，依赖于ADP的释放，如果检测血小板无力症的血小板则不会发生。当将无纤维蛋白原血症患者的血液用作血液B时，滞留正常。我们现在使用单克隆抗体进一步阐明血小板滞留的机制。针对vWf上不同表位的五种抗体在单阶段试验和两阶段试验的第二阶段基本消除了滞留，但对第一阶段没有影响。因此，整个vWf分子必须没有抗体才能在该试验第二阶段的血小板-血小板黏附中发挥作用。抗原-抗体复合物与血小板Fc受体的结合不是原因，因为其中一种抗体的Fab和F(ab')2片段与完整抗体一样有效，而且热聚集的IgG和针对血浆因子IX的多克隆抗体均未抑制滞留。6D1的F(ab')2片段是一种针对血小板糖蛋白Ib的抗体，可阻止vWf与血小板结合，也抑制了第二阶段的滞留。一种抑制纤维蛋白原和vWf与糖蛋白IIb/IIIa结合的抗体（LJ-CP8）抑制了滞留的第一和第二阶段，而LJ-P5是一种仅抑制vWf与糖蛋白IIb/IIIa结合的抗体，当以正常或无纤维蛋白原血症的血液作为血液B时，对滞留产生轻微抑制，并且据报道在该无纤维蛋白原血症患者中仅对ADP诱导的血小板聚集产生部分抑制。结果表明，血液快速通过玻璃珠柱时vWf发生改变，使其附着于糖蛋白Ib，暴露糖蛋白IIb/IIIa，然后糖蛋白IIb/IIIa结合改变后的vWf或纤维蛋白原，其中任何一种都可诱导血小板聚集（血小板-血小板黏附），从而导致在柱中的滞留。
