Abstract

Abstract 270

HIT is an immune thrombocytopenic disorder associated with a high risk of thrombosis. Mechanistic studies have focused on circulating platelet factor 4 (PF4)/heparin complexes and on the subsequent activation of platelets. We and others have shown that binding of PF4 to cell surface glycosaminoglycans (GAGs) not only makes platelets critical targets in the pathogenesis of HIT, but monocytes and neutrophils as well. Our previous observations that monocyte depletion using clodronate-laden liposomes prior to induction of HIT in a passive immunization model mitigated the prothrombotic state, but paradoxically exacerbated initial thrombocytopenia, suggest that our understanding of the relationship between monocyte activation, platelet activation, thrombocytopenia and thrombosis is unsettled. Chondroitin sulfate is the predominant GAG expressed by platelets; therefore, they bind PF4 less avidly than monocytes, which have a cell surface rich in heparan and dermatan sulfates. Based on this cell-type difference in surface affinity for PF4, we hypothesized that: 1) monocytes and perhaps other vascular cells bind PF4 and form surface PF4/HIT antigenic complexes preferentially when compared with platelets, and 2) depletion of this high-affinity (monocyte) “sink” shifts PF4 binding to platelets making them more targeted. Flow cytometric analysis of whole mouse and human blood support these hypotheses as monocytes bind ∼100-fold more FITC-labeled human (h) PF4 than platelets or red blood cells and ∼10-fold more than neutrophils or lymphocytes. When isolated platelets and white blood cells are admixed, the amount of exogenously added hPF4 bound to platelets is inversely related to the leukocyte:platelet ratio. In vitro, exposure of whole blood to the HIT-like monoclonal antibody KKO plus recombinant hPF4 generated an intense platelet activation characterized by binding of annexin V and factor Xa, consistent with formation of coated platelets. Importantly, formation of coated platelets was attenuated by monocyte depletion from whole blood samples or by inhibition of thrombin by PPACK. We then infused KKO into transgenic mice expressing hPF4 and hFcgRIIA to induce HIT and followed the temporal profile of antibody binding to various cell types. Binding of KKO to monocytes was detected within 30 min of injection. These monocytes remained the predominant target over the first 4 hrs, after which binding decreased as the circulating monocytes were depleted. The mechanism and implications of this relatively late monocytopenia during HIT is under study. However, these data provide an explanation of how clodronate-laden monocyte depletion prior to inducing HIT exacerbated thrombocytopenia in the murine model of HIT, while decreasing the prothrombotic state: Early in the disease when PF4 is limited, monocytes selectively bind the PF4 and are targeted by HIT antibodies, which induces tissue factor and generates thrombin, but limits initial thrombocytopenia. Later, after induction of large amounts of TF, activated monocytes are cleared, shifting the target of antigen-antibody interactions to the surface of platelets, enhancing their response to available thrombin, thereby establishing a feed-forward prothrombotic cycle and more platelet clearance. In conclusion, we propose that HIT evolves from a monocyte-focused to a platelet-focused disease and that early intervention to prevent monocyte activation provides a new important potential therapeutic target for intervention at the earliest stages of disease recognition that may become less effective as time passes.

Disclosures:

Cines:Amgen Inc.: Consultancy; GlaxoSmithKline: Consultancy; Eisai: Consultancy.

Author notes

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Asterisk with author names denotes non-ASH members.