Billy D, Briedé J, Heemskerk J W, Hemker H C, Lindhout T
Department of Biochemistry, Maastricht University, The Netherlands.
Blood Coagul Fibrinolysis. 1997 Apr;8(3):168-74. doi: 10.1097/00001721-199704000-00003.
Prothrombin activation by prothrombinase was investigated on platelets adhered onto a fibrinogen-coated coverslip mounted in a flow chamber. Once bound to the fibrinogen, platelets gradually changed their morphology: they developed pseudopods, spread over the surface and finally transformed into balloon-shaped cells. This last morphologic change required the presence of calcium and was accompanied by the exposure of procoagulant phospholipid at the outer membrane as detected by the capability of the platelets to bind fluorescein-labelled annexin V. Prothrombinase complexes were allowed to assemble on these adhered platelets by perfusion with factor Xa and varying concentrations of factor Va and prothrombin. The steady-state rate of thrombin formation during continuous flow increased with the prothrombin concentration but not with the factor Va between 0.05 and 0.5 nM. Once prothrombinase was assembled, factor Xa could be omitted from the perfusion mixture without affecting the steady state rate of thrombin production. Our study demonstrates the efficient ability of the procoagulant surface of adherent platelet to support the assembly of stable prothrombinase complexes. Thrombin production was limited by the rate of supply of prothrombin towards the catalytic surface.
在流动腔中安装的纤维蛋白原包被盖玻片上黏附的血小板上，研究了凝血酶原酶对凝血酶原的激活作用。一旦与纤维蛋白原结合，血小板会逐渐改变其形态：它们形成伪足，在表面铺展，最终转变为气球状细胞。这最后的形态学变化需要钙离子的存在，并且伴随着促凝磷脂在外膜的暴露，这可通过血小板结合荧光素标记的膜联蛋白V的能力检测到。通过用因子Xa和不同浓度的因子Va及凝血酶原灌注，使凝血酶原酶复合物在这些黏附的血小板上组装。在连续流动过程中，凝血酶形成的稳态速率随凝血酶原浓度增加而增加，但在0.05至0.5 nM之间不随因子Va浓度增加。一旦凝血酶原酶组装完成，灌注混合物中可省略因子Xa而不影响凝血酶产生的稳态速率。我们的研究证明了黏附血小板的促凝表面支持稳定凝血酶原酶复合物组装的高效能力。凝血酶的产生受凝血酶原向催化表面供应速率的限制。
