Thrombin generation on vascular cells in the presence of factor VIII and/or emicizumab.

BACKGROUND: The effect of factor VIII (FVIII) or emicizumab on thrombin generation is usually assessed in assays using synthetic phospholipids. Here, we assessed thrombin generation at the surface of human arterial cells (aortic endothelial cells [hAECs] and aortic vascular smooth muscle cells [hVSMCs]). OBJECTIVES: To explore the capacity of hAECs (resting or stimulated) and hVSMCs to support thrombin generation by FVIII or emicizumab. METHODS: Primary hVSMCs and hAECs were analyzed for tissue factor (TF)-activity and antigen, phosphatidylserine (PS)-exposure, tissue factor pathway inhibitor (TFPI)-content and thrombomodulin expression. Cells were incubated with FVIII-deficient plasma spiked with FVIII, emicizumab, activated prothrombin complex concentrate (APCC) or combinations thereof. RESULTS: TF activity and PS-exposure were present on both hVSMCs and hAECs. In contrast, thrombomodulin and TFPI were expressed on hAECs, while virtually lacking on hVSMCs, confirming the procoagulant nature of hVSMCs. Tumor necrosis factor α-mediated stimulation of hAECs increased not only TF antigen, TF activity, and PS-exposure but also TFPI and thrombomodulin expression. As expected, FVIII and emicizumab promoted thrombin generation on nonstimulated hAECs and hVSMCs, with more thrombin being generated on hVSMCs. Unexpectedly, FVIII and emicizumab increased thrombin generation to a lesser extent on stimulated hAECs compared with nonstimulated hAECs. Finally, adding emicizumab to FVIII did not further increase thrombin generation, whereas the addition of emicizumab to APCC resulted in exaggerated thrombin generation. CONCLUSION: Tumor necrosis factor stimulation of hAECs increases both pro- and anticoagulant activity. Unexpectedly, the increased anticoagulant activity is sufficient to limit both FVIII- and emicizumab-induced thrombin generation. This protective effect disappears when emicizumab is combined with APCC.

Pharmacodynamics of eftrenonacog-alfa (rFIX-Fc) in severe hemophilia B patients: A real-life study.

Eftrenonacog-alfa is a recombinant factor IX-Fc fusion protein increasingly prescribed in hemophilia B patients. We aimed to assess its pharmacodynamics (PD) in real-life setting via FIX activity measurement and thrombin generation assay (TGA). Sixty samples from 15 severe hemophilia B treated patients were collected at different time points. FIX activity was measured using product-specific one-stage clotting assay (reference method) and two chromogenic assays (CSA) (Biophen FIX and Rox FIX). TGA was triggered with 1 pM tissue factor. Five parameters were analyzed: lag time (LT), time to peak (TTP), peak height (PH), endogenous thrombin potential (ETP), and velocity. PD models were built to characterize their relationships with FIX activity, using mixed effects models. Mean trough FIX level was estimated at 4.64 (±1.50) IU/dl with a recovery at 0.78 (±0.16) IU/dl per 1 IU/kg injected dose. FIX activity ranged between 1 and 86 IU/dl with 21.5 IU/dl median value. Biophen FIX and Rox FIX allowed reliable measurements except in samples with FIX <20 IU/dl in which values were underestimated (delta >30%). PD models revealed that velocity was the most sensitive TGA parameter to FIX activity followed by PH, ETP, TTP and finally LT. Following FIX activity peak after eftrenonacog-alfa injection, velocity decreased first, followed by PH then ETP. Both CSA failed to accurately measure FIX in severe hemophilia B patients receiving eftrenonacog-alfa throughout the measuring range. TGA could be an additional valuable tool to evaluate hemostasis balance in treated patients.

Camelid-derived single-chain antibodies in hemostasis: Mechanistic, diagnostic, and therapeutic applications.

Hemostasis is a complex process involving the concerted action of molecular and vascular components. Its basic understanding as well as diagnostic and therapeutic aspects have greatly benefited from the use of monoclonal antibodies. Interestingly, camelid-derived single-domain antibodies (sdAbs), also known as VHH or nanobodies, have become available during the previous 2 decades as alternative tools in this regard. Compared to classic antibodies, sdAbs are easier to produce and their small size facilitates their engineering and functionalization. It is not surprising, therefore, that sdAbs are increasingly used in hemostasis-related research. In addition, they have the capacity to recognize unique epitopes unavailable to full monoclonal antibodies. This property can be used to develop novel diagnostic tests identifying conformational variants of hemostatic proteins. Examples include sdAbs that bind active but not globular von Willebrand factor or free factor VIIa but not tissue factor-bound factor VIIa. Finally, sdAbs have a high therapeutic potential, exemplified by caplacizumab, a homodimeric sdAb targeting von Willebrand factor that is approved for the treatment of thrombotic thrombocytopenic purpura. In this review, the various applications of sdAbs in thrombosis and hemostasis-related research, diagnostics, and therapeutic strategies will be discussed.