Heparin-induced thrombocytopenia (HIT) is an iatrogenic severe prothrombotic disease caused by an immune response to complexes of platelet factor 4 (PF4) and polyanions, such as heparin, accompanied by mild- moderate thrombocytopenia. We have studied the biological basis of this prothrombotic state in vitro using a novel human endothelial-lined microfluidic system, and in vivo, using a HIT model focused on mice that are transgenic for both human (h) PF4 and FcgRIIA. We found that in both, PF4 released from platelets followed by HIT-associated antibodies (HIT Abs) bound predominantly to the perithrombus endothelium. These endothelial-bound complexes propel thrombus growth and extension. Additionally, we found that PF4 enhanced neutrophil binding to injured endothelium and caused activated neutrophils to extrude its chromatin termed neutrophil extracellular traps (NETs). NETs were compacted by PF4, enhancing their resistance to digestion by DNase. HIT Abs bound to these compacted NETs, further enhancing their DNase resistance. We propose that this sequence of events contributes to the intense prothrombotic sequelae of HIT and resistance to anti-thrombotic therapy. We will now further pursue these observations:
Specific Aim (SA) #1: Understanding the role of the endothelium in HIT. We showed that PF4 released from platelets binds preferentially to injured perithrombus endothelium concurrent with loss of its heparan-sulfate-rich glycocalyx. It is unclear how endothelial injury with loss of glycocalyx increases PF4 binding. This unexpected increase will be pursued in the microfluidic system and in mice with genetic defects in glycocalyx formation. Strategies to attenuate the prothrombotic nature of HIT directed at the endothelium will also be pursued. SA#2: Understanding the role of neutrophils in HIT. PF4/HIT Abs complexes augment neutrophil adhesion to injured endothelium in vitro. In addition, in situ studies in HIT mice show enhanced neutrophil adhesion to thrombi in HIT and formation of neutrophil/platelet hetero-aggregates in hepatic vessels. These observations suggest important role(s) for neutrophils in HIT. We will extend the in situ studies of neutrophils in HIT to other target organs and we will study neutropenic HIT mice to confirm the importance of neutrophils to HIT- associated thrombosis and thrombocytopenia. We will test whether adhesion of neutrophils via P-selectin and its ligands mediate these observations and whether blocking this pathway ameliorates the thrombosis and/or thrombocytopenia. SA#3: Understanding the role of NET-release (NETosis) in HIT. We posit that PF4/HIT- Ab-stabilized NETs are highly prothrombotic. We have begun to test this concept in vitro and in vivo using HIT mice that cannot undergo NETosis, and will also measure soluble markers released from NETs in HIT. Thus, this proposal focuses on the roles of the endothelium and neutrophils in the development of HIT, and may lead to novel therapeutic approaches to complement current anticoagulant therapies. We also believe that insights gained from these studies may be applicable to other proinflammatory, prothrombotic disorders.

Public Health Relevance

This grant will develop a better understanding of how the blood thinner heparin can sometimes lead to a serious, life-threatening complication, termed heparin-induced thrombocytopenia (HIT), where the patients are at risk of developing life-threatening clots. We will focus on the roles that the blood vessel lining (called the endothelium) and white cells (called neutrophils) contribute to this process. These studies should lead to new insights into why clots develop in HIT, anticipating that such new knowledge can be translated to the care of these patients and patients with related clotting disorders due to inflammation.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
1R01HL139448-01
Application #
9414507
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Kindzelski, Andrei L
Project Start
2017-12-19
Project End
2021-11-30
Budget Start
2017-12-19
Budget End
2018-11-30
Support Year
1
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
073757627
City
Philadelphia
State
PA
Country
United States
Zip Code
19104