Anti-platelet factor 4 (PF4)/heparin antibodies (Abs) and activation of platelet Fc?Rlla are involved in the pathogenesis of heparin-induced thrombocytopenia (HIT). However, the actual basis of the observed thrombocytopenia and thrombosis are not well understood, and markers of risk or diagnosis and targeted therapies remain limited. This Project focuses on furthering our understanding of the cellular events underlying the thrombocytopenia and thrombosis in HIT. We believe that platelets, leukocytes and endothelium, and derived microparticles (MP) contribute to both the thrombocytopenia and thrombosis. In this Project, we propose three novel interrelated cellular mechanisms that underlie the thrombocytopenia and prothrombotic nature of HIT:
Specific Aim (SA) 1. Leukocyte:platelet surface PF4 levels and platelet clearance in HIT. We observed that monocyte depletion paradoxically exacerbates thrombocytopenia in a murine model of HIT. We hypothesize that this observation is due to the higher affinity of monocyte surface glycosaminoglycans (GAGs) for PF4 than platelets, which normally limits platelet surface PF4 levels and subsequent targeting by HIT Abs. In the absence of monocytes (or other leukocytes), platelets are targeted more and undergo more hepatosplenic clearance than microthrombi consumption. In vitro and in vivo murine and human studies will test this hypothesis, and along with the second aim, provide new insights into the relationship between thrombocytopenia and thrombosis in HIT. SA 2. Contribution of leukocytes to platelet activation and thrombosis in Hit. We propose that HIT is an immune thrombocytopenia associated with a high risk of thrombosis because monocytes (and perhaps other leukocytes) bind PF4 well and are selectively activated by HIT Abs. These activated monocytes express tissue factor, leading to thrombin generation and dual platelet activation via thrombin and via pathogenic HIT Ab Fc?Rlla activation, leading to prothrombotic "coated platelets". These studies will be done in close collaboration with Projects 2 and 4, and will utilize both human and murine in vitro and in vivo systems. SA 3. "Rolling recruitment" model of endothelial cell (EC) contribution to thrombosis in HIT. We show that perithrombus ECs are persistently and vigorously activated in HIT. We hypothesize that this observation is secondary to a rolling recruitment of the perithrombus ECs, wherein activated platelets release PF4 that forms activating antigenic complexes on surrounding ECs, amplifying and extending the injury and thrombus. We will test this hypothesis both in vitro in a microfluidic system using both human and murine sample sources and in vivo in the murine HIT model. The studies in this Project should expand our understanding of how targeting of PF4 to surface GAGs on vascular cells contribute to the thrombocytopenia and thrombosis in HIT, should lead to development of new strategies to assess risk of developing HIT complementary to efforts in Projects 2 and 3, and offer opportunities to develop and test novel therapeutics, some of which are the focus of studies in Project 4 of this application.

Public Health Relevance

The proposed studies on the cellular events underlying the development of heparin-induced thrombocytopenia will enhance our understanding of how this devastating complication of heparin anticoagulation results in patients developing life-threatening clots. In the context of this Program Project, these studies may lead to better risk assessment, diagnostic tests, and more effective therapies.

Agency
National Institute of Health (NIH)
Type
Research Program Projects (P01)
Project #
5P01HL110860-03
Application #
8695470
Study Section
Heart, Lung, and Blood Program Project Review Committee (HLBP)
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Children's Hospital of Philadelphia
Department
Type
DUNS #
City
Philadelphia
State
PA
Country
United States
Zip Code
19104
McKenzie, Steven E; Sachais, Bruce S (2014) Advances in the pathophysiology and treatment of heparin-induced thrombocytopenia. Curr Opin Hematol 21:380-7
Cines, Douglas B; Lebedeva, Tatiana; Nagaswami, Chandrasekaran et al. (2014) Clot contraction: compression of erythrocytes into tightly packed polyhedra and redistribution of platelets and fibrin. Blood 123:1596-603
Yeung, Jennifer; Tourdot, Benjamin E; Fernandez-Perez, Pilar et al. (2014) Platelet 12-LOX is essential for Fc?RIIa-mediated platelet activation. Blood 124:2271-9
Cines, Douglas B; Cuker, Adam; Semple, John W (2014) Pathogenesis of immune thrombocytopenia. Presse Med 43:e49-59
Kowalska, M Anna; Zhao, Guohua; Zhai, Li et al. (2014) Modulation of protein C activation by histones, platelet factor 4, and heparinoids: new insights into activated protein C formation. Arterioscler Thromb Vasc Biol 34:120-6
Litvinov, Rustem I; Yarovoi, Serge V; Rauova, Lubica et al. (2013) Distinct specificity and single-molecule kinetics characterize the interaction of pathogenic and non-pathogenic antibodies against platelet factor 4-heparin complexes with platelet factor 4. J Biol Chem 288:33060-70
Zhi, Huiying; Rauova, Lubica; Hayes, Vincent et al. (2013) Cooperative integrin/ITAM signaling in platelets enhances thrombus formation in vitro and in vivo. Blood 121:1858-67
Lee, Grace M; Arepally, Gowthami M (2013) Diagnosis and management of heparin-induced thrombocytopenia. Hematol Oncol Clin North Am 27:541-63
Lee, Grace M; Welsby, Ian J; Phillips-Bute, Barbara et al. (2013) High incidence of antibodies to protamine and protamine/heparin complexes in patients undergoing cardiopulmonary bypass. Blood 121:2828-35
Cuker, Adam; Rux, Ann H; Hinds, Jillian L et al. (2013) Novel diagnostic assays for heparin-induced thrombocytopenia. Blood 121:3727-32

Showing the most recent 10 out of 11 publications