PB0814 - Endothelial cells, not blood, dictate where, when, and how hemostasis initiates under flow conditions: mechanistic studies using a vascularized hemostasis-on-a-chip system

Background: Canonically, hemostasis initiates when platelets adhere to exposed subendothelium at the vascular injury. However, little is known about how this process changes under flow conditions in which von Willebrand factor (vWF)-dependent platelet adhesion is the critical component in primary hemostasis. As measuring plasma vWF levels and function neglects vWF secreted from endothelium at the injury, we leveraged our “endothelialized” microengineered mechanical injury bleeding model under flow regimes.

Aims: To investigate the role of endothelium in initiating hemostasis by direct observation of platelet adhesion by basolateral vWF and injury-inflicted ultralarge vWF (ULVWF) release, and spatially-regulated fibrin formation.

Methods: Microfluidic devices were constructed with a pneumatic valve, creating a mechanical disruption in endothelialized channel, leading to “bleeding”. Channel with injured endothelial cell (EC)s were stained for vWF. Washed platelets were tracked on damaged EC under flow realtime. We also perfused whole blood, and recorded bleeding time, phosphatidylserine exposure, platelet/fibrin accumulation with/without vWF inhibitors.

Results: Subendothelial vWF was detected where ECs were damaged (Figure1A). Washed platelets tethered on ULVWF strings released from phosphatidylserine-exposed ECs immediately after the injury (Figure1B). When whole blood was perfused at shear rate of 1500 s-1, presence of ECs increased initial platelet adhesion on exposed collagen-coated surface (Figure1C). Blocking A1 domain of vWF attenuated platelet adhesion on exposed subendothelium. The results suggest endothelial-dependent platelet recruitment at injury site in early hemostasis. Fibrin formation occurs at phosphatidylserine-exposed ECs over time (Figure2), thus, ECs regulate local thrombin generation as well.

Conclusion(s): Our hemostasis-on-a-chip demonstrates temporo-spatially-regulated endothelial response to the injury during hemostasis, especially at higher shear rates. The level contribution of blood vs endothelial-secreted vWF to hemostasis will be quantitatively assessed. We offer more holistic bleeding model for congenital and acquired endothelial dysfunction. Ongoing work will incorporate Endothelial Colony Forming Cells (ECFCs) from von Willebrand disease patients to assess the contribution of patient ECs.