Flow-Cytometry Evaluation of Cellular Surface FVIII in Blood Cells and Its Correlation with FVIII Activity and Coagulation
Authors: Al-Mohannadi Anjud , Elnaggar Muhammad , Bibawi Hani , Lachica Che-Ann , Pavlovski Igor , Gentilcore Giusy , Kizhakayil Dhanya , Saleh Ayman , Elgaali Elkhansa E , Elanbari Mohammed , Cugno Chiara , Deola Sara 
Published: November 2021

Abstract
FVIII deficiency or inactivity leads to coagulation malfunction known as Hemophilia A (HA). FVIII overactivity on the contrary is commonly linked to a hypercoagulability state. In steady state, FVIII remains quiescent by forming a non-covalent complex with vWF, and upon coagulation cascade activation, thrombin liberates active FVIII (FVIIIa) from vWF. FVIIIa binds to the plasma membrane of the platelets and other blood cells via its C2 domain phospholipids binding motif to exert its coagulative role.
Recently, we developed a reliable Flow-Cytometry (FC) platform for FVIII protein detection (Elnaggar M. et al, 2020). The assay is suitable to measure both intracellular and extracellular FVIII. When applied to human blood it showed that by overexpressing FVIII through a lentiviral-vector-based transduction, a high proportion of circulating FVIII is bound on leukocytes membranes. Leukocyte abundance is reported to be highly associated with thrombotic events but with no clear mechanistic explanation. Therefore, we sought to evaluate the percentage of FVIIIa bound on different leukocyte subsets (T cells, B cells, monocytes and granulocytes) in different clinical scenarios, including HA and other abnormal coagulation states and to correlate it to the plasmatic levels of FVIII.
The analysis was conducted on 31 pediatric/young-adult subjects (median age 9y), including patients diagnosed with HA (n=8), patients with coagulopathies (von Willebrand Disease, menorrhagia and unexplained bleedings (n=11) and 1 patient with known high levels of FVIII), and patients without coagulopathies/healthy controls (n=11) referred to Hematology/Oncology clinic at Sidra Medicine-Qatar.
Fresh blood samples were analyzed with a Sysmex XN1000 Hematologic Counter for complete blood count and with a BD-Symphony FC for the following surface markers: CD3/CD4/CD8/CD14/CD16/CD19/CD33/Live/Dead and for FVIII protein A2 domain (Ab clone GMA8024), after light-chain Zenon-labeling, with proper IgG controls, as described in the publication above. In parallel, plasma FVIII measurements were performed for clinical diagnostic purposes, and results were correlated.
Expectedly, HA patients showed the lowest FVIII percentage in plasma (p&lt;0.0001), while leukocytes' surface FVIII was generally low (on average &lt;1%), trended to a lower rate in patients with coagulopathies and HA compared to healthy-controls and was significantly higher in the patient with hypercoagulability (p=0.0007). Surprisingly, among leukocytes' subpopulations, screened on patients with bleeding diathesis and HA subjects vs healthy-controls, only CD8-surface bound FVIIIa was significantly lower compared to healthy-controls (p=0.015 healthy-controls vs HA, and p=0,0093 healthy-controls vs bleeding diathesis). These results raise the possibility of a CD8-cell mediated controlled binding mechanism regulating FVIII availability in case of biological need of the protein, as bleeding.
We did not find any significant correlation between leukocyte-surface bound FVIII and plasma FVIII level, and this further indicates that the differences in FVIIIa leukocyte surface binding are not directly proportional to FVIII availability in the circulation, rather actively regulated by a biological still unknown mechanism.
While the study is still ongoing and recruiting other subjects with hypercoagulation state, we showed in this preliminary analysis that our in-house FC platform for FVIII protein detection screens in depth FVIII protein adherence to blood cells, may shed light on coagulation novel mechanisms, and potentially serve as a diagnostic and prognostic tool.
Figure 1 Figure 1.

Disclosures
No relevant conflicts of interest to declare.