Activation of platelets in whole blood by recombinant factor VIIa by a thrombin-dependent mechanism.
Using a diluted whole blood method of flow cytometric analysis, we have shown that platelets could be activated in vitro in the presence of high concentrations (100 nmol/l) of recombinant factor (F) VIIa (rFVIIa; NovoSeven(R)) and 2.5 mmol/l calcium chloride. This was demonstrated by a significant increase in the mean percentage of platelets expressing CD62P and their mean fluorescent intensity (MFI) after 30 min versus platelets incubated with calcium or rFVIIa alone or diluted blood alone. The presence of rFVIIa and calcium increased the exposure of the PAC-1 activation epitope of glycoprotein (Gp) IIb/IIIa. This effect was equally influenced by the presence of calcium alone but not by rFVIIa. The effect of rFVIIa was time and concentration dependent. Thrombin generation was also necessary, as the effect of rFVIIa was completely abrogated by the additional presence of hirudin. Furthermore, soy bean trypsin inhibitor (SBTI) but not corn trypsin inhibitor (CTI) abrogated CD62P exposure, suggesting that thrombin was derived via FX but not FXII activation. Exposure of CD62P demonstrated a significant lag phase, sometimes of the order of > 30 min, as well as large intersubject variation. Significant platelet activation was observed at a concentration as low as 25 nmol/l rFVIIa. Platelet-leucocyte aggregation was also increased in the presence of 25 nmol/l rFVIIa and calcium. No significant difference was observed between levels of CD62P in diluted whole blood and platelet-rich plasma adjusted to an identical platelet count after their exposure to rFVIIa and calcium for 30 min.
Wilbourn
Barry
B
Haemostasis Research Unit, Department of Haematology, University College London, 3rd Floor Jules Thorn Building, 48 Riding House Street, London W1W 7EY, UK. b.wilbourn@ucl.ac.uk
Harrison
Paul
P
Mackie
Ian J
IJ
Liesner
Ri
R
Machin
Samuel J
SJ
D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology; D002118:Calcium / Q000494:pharmacology; D002449:Cell Aggregation / Q000187:drug effects; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D015942:Factor VIIa / Q000494:pharmacology*; D005434:Flow Cytometry; D006801:Humans; D007962:Leukocytes / Q000187:drug effects / Q000502:physiology; D015539:Platelet Activation / Q000187:drug effects*; D011480:Protease Inhibitors / Q000494:pharmacology; D011994:Recombinant Proteins / Q000494:pharmacology; D013917:Thrombin / Q000502:physiology*
D011480:Protease Inhibitors; D011994:Recombinant Proteins; D015942:Factor VIIa; D013917:Thrombin; D002118:Calcium