“Endothelialized” Hemostasis-on-a-Chip Reveals Rapid Platelet Recruitment By Locally Concentrated Ultra-Large Von Willebrand Factor (VWF) Release from Endothelial Cells Surpasses Plasma and Platelet VWF in Initiating Hemostasis

von Willebrand Factor (vWF) has a critical role to play in primary hemostasis. Although it is widely known that circulating plasma vWF recruits platelets on exposed subendothelium at the site of bleeding, the importance of ultra-large vWF (ULVWF) from local injured endothelial cells (ECs) in hemostasis has not been well-defined. Our endothelialized microfluidic bleeding device directly reveals how locally damaged ECs release ULVWF immediately after injury and recruit platelets to initiate hemostasis. The response is faster than both circulating vWF and vWF released from platelets. To identify newly released ULVWF from the injury site, we perfused washed platelets through the device and noted their tethering/binding location in the vasculature and bleeding area.This showed that phosphatidylserine (PS)-positive damaged ECs released long string structures immediately after injury, and at the same time, platelets were trapped along the strings in the vasculature and bleeding area. The string-platelet interactions were disrupted by a GP1b blocking antibody, confirming that the strings were unfolded A1 domain-exposing ULVWF under flow. Our system reveals that locally concentrated vWF release from ECs at injury is the frontrunner for initiating primary hemostasis at the bleeding site.

The polydimethylsiloxane-based microfluidic bleeding device (Sakurai et al., 2018) is coated with collagen and seeded with human umbilical vein endothelial cells, which are grown to confluence under flow. As the ECs grow, the vascular channel and valve membrane of the device remain closed. Healthy human blood is collected in anticoagulants and used as whole blood or processed further for platelet isolation. A vascular injury is created by positive pressure into the vascular channel, which partially disrupts the endothelium and creates an opening into the “bleeding” channel. Whole blood or washed platelets are re-calcified and flown into the channels, and time lapse images of the process of hemostasis in bright field and fluorescence (labeled ECs, platelets, and PS) are collected by a confocal microscope for a 600 second time course.

When healthy whole blood treated with a GP1b blocking antibody, AK2, was perfused at a shear rate of 1000 s^-1, hemostasis was impaired, and bleeding did not stop within 600 seconds compared to the control, which stopped bleeding at an median bleeding time of 415 seconds. When whole blood was perfused into the vascular channel without the presence of ECs (i.e. collagen coating only), hemostasis was also delayed, and bleeding did not stop within 600 seconds, indicating that EC-derived factors play a role in facilitating prompt initiation of hemostasis.

To detect only newly released vWF from ECs, we used washed platelets without the presence of circulating plasma vWF and ADAMTS13, at a concentration of 250 x 10³ µL^-1, and flowed them at a shear rate of 1500 s^-1. Upon injury, we saw affected ECs in the injury area started expressing PS on their surface via annexin V binding. At the same time, we observed platelets tethering on string-like structures originating from affected ECs. The strings with platelets appeared both in vasculature and the bleeding area at a high frequency, ranging from 4 to 15 strings (10.75 in average) per experiment, and had tethering lengths of up to 136 µm. With GP1b blocking, the strings and platelet tethering were significantly reduced in frequency, ranging from 0 to 6 strings (2.67 in average), and length, up to 53 µm. GP1b directly binds to vWF's A1 domain. This suggests that those strings are A1 domain-exposing ULVWF. When washed platelets were perfused without ECs on a collagen coated channel, we did not observe strings, which suggests that ULVWF is released from ECs, not from platelets. The amount of platelets present directly correlated to the amount and length of strings. In this study, we microscopically confirmed, in real time, that the incorporation of ULVWF released from EC mechanical injury facilitates rapid platelet recruitment in healthy localized hemostasis. Without EC-initiated prompt response to injury, hemostasis delays and excess bleeding occurs. Bleeding disorders have previously only been characterized as “blood” disorders, but now we cannot emphasize enough the importance of endothelial function. A more holistic approach to vasculature/blood assessment for bleeding disorders will be critical for future treatments.

Lam:Sanguina, Inc.: Current equity holder in private company.