Coagulation Factor IXa Binding to Activated Platelets and Platelet-Derived Microparticles: A Flow Cytometric Study

Summary

Factor IX plays a central role in blood coagulation, since it can be activated by either XIa (intrinsic pathway) or tissue factor-VIIa (extrinsic pathway). Activated factor IX (IXa), in a surface-bound complex with factor Villa, then activates factor X. Platelets provide the catalytic surface upon which this Xase complex is assembled in vivo. We have used flow cytometry to examine binding of factor IXa to thrombin-activated platelets in the absence of added Villa. Platelet-bound IXa and platelet protein GPIb were detected by indirect immunofluorescence staining followed by two-color flow cytometric analysis. Microparticles were identified by their light scattering characteristics. Two binding sites for factor IXa were detected. The high affinity binding site saturated at about 10 nM, with a K _d of 1.6 nM. A second binding curve, with a K _d of about 100 nM, was observed at higher concentrations of IXa. The high affinity factor IXa binding sites comprise about 7% of the total factor IXa binding. Binding to both sites was dependent on the presence of calcium. Thus, we conclude that factor IXa, in addition to its high affinity binding, has a calcium-dependent low affinity association with activated platelets and microparticles. Sims et al. have shown that binding sites for a different coagulation factor, factor Va, are concentrated on microparticles relative to platelet membrane proteins, such as GPIb. GPIb is distributed on platelets and microparticle in proportion to plasma membrane surface. At 100 nM factor IXa, 1–3% of the total bound factor IXa was associated with microparticles, as was a similar proportion of the total GPIb. However, at 10 nM IXa, factor IXa binding to microparticles was concentrated about 2-fold relative to GPIb staining. Thus, the high affinity factor IXa binding site is concentrated on microparticles to a small degree, while the low affinity binding site is not.

• References

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