Binding Of Recombinant Activated Factor VII (rFVIIa) To Human Platelets Identifies Donor Variation and Platelet Subpopulations With Increased Binding Of rFVIIa In Platelet Concentrates From Healthy Donors

Inhibitor bypassing therapies including recombinant activated factor VII (rFVIIa) are used to treat hemophilia patients with inhibitors. However, response to treatment varies, and there is no standardized laboratory method to monitor and predict treatment response (Bentorp, Haemophilia 2009, 15:3; Hedner, Haemophilia 2010, 16:372).

TF-independent factor X (FX) activation on the surface of platelets, which by-passes the lack of factor VIII or IX in hemophilia, is suggested to be the main contributor to rFVIIa’s procoagulant effect (Monroe, 1997, Br J Haematol, 99(3):542). Binding of rFVIIa to platelets is inefficient; therefore, high concentrations of rFVIIa are required. These concentrations correlate well with therapeutic doses. Platelet characteristics and functions vary between individuals and might influence rFVIIa platelet binding and treatment response (Monroe, 2002, Arterioscler Thromb Vasc Biol 22:1381). rFVIIa has been shown to preferentially bind “coated” platelets, characterized by the exposure of fibrinogen and other procoagulant proteins (Kjalke, 2007, JTH, 5:774). Variation in hemophilia patients’ ability to form this platelet subpopulation influences bleeding in this population (Saxena, 2010 JTH 8:1140).

Fresh platelet concentrates were incubated with rFVIIa at concentrations of 50 to 2000 nM for 7 min at 37°C with or without platelet activators thrombin and convulxin, a collagen receptor GPVI agonist. Platelets were detected using flow cytometry by staining for CD61 and P-selectin (CD62P) or fibrinogen and forward/side scatter gating to determine their activation status. rFVIIa bound to activated or non-activated platelets was quantified using a fluorescent-labeled anti human FVIIa antibody. Median fluorescence intensities of rFVIIa bound to all platelets and to subpopulations varying in rFVIIa binding were measured. Platelets stained for FVIIa without addition of rFVIIa served as controls.

Using FACS technology, we quantified binding of rFVIIa to platelets concentrates of 21 healthy donors. rFVIIa binding to non-activated and dual agonist activated platelets was concentration dependent and was saturated at rFVIIa concentrations of 200 to 1200 nM. Median fluorescence intensity increase at 100 nM rFVIIa ranged 4- to 33-fold for non-activated and 5- to 37-fold for activated platelets compared with controls without rFVIIa. Assessing the total platelet population, binding constants for activated platelets ranged from 29 to 1025 nM (mean ± standard deviation 392 ± 355 nM; n=6), and from 56 to 357 nM (mean ± standard deviation 184 ± 88; n=10) for non-activated platelets.

A platelet subpopulation of more than 2% was detected in 7/21 non-activated platelet donor concentrates, and in 15/21 after dual activation. At 100 nM, in non-activated platelets, this subpopulation bound 32x more rFVIIa than the main platelet population, and 6x more in activated platelets. Two to 24% of total platelets were characterized by high capacity of rFVIIa binding in platelet concentrates from donors positive for this subpopulation.

The rFVIIa high binding capacity subpopulation formation did not correlate with platelet activation or “coated” platelet formation, as all dual agonist activated platelets had similar distribution of CD62P and fibrinogen expression after activation.

We observed considerable interindividual variation in binding of rFVIIa to resting and activated platelets, and identified platelet subpopulations with substantially higher rFVIIa binding capacity.

Whether the occurrence of these platelet subpopulations correlates with treatment response requires further population-based functional and phenotypic characterization.

Koehn:Baxter Innovations GmbH: Employment. Boehm:Baxter Innovations GmbH: Employment. Dockal:Baxter Innovations GmbH, Vienna, Austria: Employment. Sedivy:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH, Vienna, Austria: Employment.