Platelet phosphatidylserine is the critical mediator of thrombosis in heparin-induced thrombocytopenia

Abstract

Heparin-induced thrombocytopenia (HIT) is a severe immune-mediated prothrombotic disorder caused by antibodies (Ab) reactive to complexes of platelet factor 4 and heparin. Platelets (PLT) and their interaction with different immune cells contribute to prothrombotic conditions in HIT. However, the exact mechanisms and the role of different PLT subpopulations in this prothrombotic environment remain poorly understood. In this study, we observed that HIT patient Ab induce a new PLT population that is characterized by increased P-selectin expression and phosphatidylserine (PS) externalization. Formation of this procoagulant PLT subpopulation was dependent on engagement of PLT Fc-γ-RIIA by HIT Ab and resulted in a significant increase of thrombin generation on the PLT surface. Using an ex vivo thrombosis model and multi-parameter assessment of thrombus formation, we observed that HIT Ab-induced procoagulant PLT propagated formation of large PLT aggregates, leukocyte recruitment and most importantly, fibrin network generation. These prothrombotic conditions were prevented via the upregulation of PLT intracellular cAMP with Iloprost, a clinically approved prostacyclin analogue. Additionally, the functional relevance of P-selectin and PS was dissected. While inhibition of P-selectin did not affect thrombus formation, the specific blockade of PS prevented HIT Ab-mediated thrombin generation and most importantly procoagulant PLT-mediated thrombus formation ex vivo. Taken together, our findings indicate that procoagulant PLT are critical mediators of prothrombotic conditions in HIT. Specific PS targeting could be a promising therapeutic approach to prevent thromboembolic events in HIT patients.

Figure 1.

Heparin-induced thrombocytopenia antibodies induce procoagulant platelets and thrombin generation in a heparin-dependent manner. (A) Platelets (PLT) were incubated with sera from patients with suspected heparin (Hep)-induced thrombocytopenia (HIT, N=37), control sera (healthy controls [HC], N=6) or (B) with corresponding immunoglobulin G (IgG) isolates and tested for changes in the expression levels of P-selectin (CD62p) and phosphatidylserine (PS) via double staining in flow cytometry. Where indicated, PLT were co-incubated with low- (0.2 IU/mL) or high-dose (100 IU/mL) heparin. (C) Representative thrombin generation curve induced on PLT after incubation with IgG from HC (black lines) or HIT patients (red lines) in the presence of buffer or heparin (0.2 IU/mL). Each curve represents the amounts of generated thrombin over time in the presence of buffer (dashed lines) or heparin (0.2 IU/mL, [solid lines]). (D) Data were quantified as peak thrombin generated (nM) using Thrombinoscope software and Graphpad prism. The number of patients/IgG tested is reported in each graph. Violin plots showing the distribution of the values were generated using Graphpad Prism 8. *P<0.05, **P<0.01, ***P<0.001, and ****P< 0.0001. ns: non-significant; CAT: calibrated automated thrombogram; pos.: positive; neg.: negative.

Figure 2.

Heparin dependency of the ex vivo heparin-induced thrombocytopenia thrombosis model. Platelet-rich plasma from healthy individuals was incubated with Immunoglobulin G (IgG) from heparin (Hep)-induced thrombocytopenia (HIT) patients in the presence of buffer (upper panel) or heparin (lower panel) prior to labeling of platelets (PLT) with DiOC₆ (green), procoagulant PLT with AF647 Annexin V (red), AF546 Fibrinogen (magenta) and leukocytes with Hoechst 33342 (blue). After labeling, samples were reconstituted into autologous whole blood. Samples were then recalcified and perfused through microfluidic channels at a venous shear rate of 250s^-1 (10 dyne) for 10 minutes. Images were acquired at x40 magnification in different fluorescence channels using a Zeiss Axio Observer 7 microscope. Scale bar 20μm. Images were processed identically using adjusted threshold settings and exclusion of image artefacts using Fiji image processing software. Violin plots showing the percentage of total surface area coverage (% SAC) by DiOC₆, phosphatidylserine (PS), Fibrin (-ogen), number of Hoechst-positive labeled cells and cumulative area with DiOC₆, PS and Fibrin (-ogen) labeled thrombus captured in the microfluidic channel. *P<0.05, **P<0.01 and ***P<0.001. ns: non-significant.

Figure 3.

Fc-γ-RIIA inhibition prevents thrombus formation by heparin-induced thrombocytopenia immunoglobulin G. Platelets (PLT) from healthy individuals were incubated with immunoglobulin G (IgG) from heparin (Hep)-induced thrombocytopenia (HIT) patients in the presence of heparin (0.2 IU/mL), and monoclonal antibody IV.3 (lower panel) or isotype control (upper panel) before reconstitution into whole blood and perfusion through microfluidic channels at a venous shear rate of 250s^-1 (10 dyne) for 10 minutes. After perfusion, images were acquired at x40 magnification. Scale bar 20μm. Violin plots showing the percentage of total surface area coverage (% SAC) by DiOC₆, phosphatidylserine (PS), Fibrin (-ogen), count of Hoechst-positive labeled cells and cumulative total % SAC with DiOC₆, PS and Fibrin(-ogen) labeled thrombus captured in the microfluidic channel. *P<0.05, **P<0.01 and ***P<0.001.

Figure 4.

Upregulation of platelet cAMP protects from heparin-induced thrombocytopenia antibody-induced thrombus formation. Platelets (PLT) from healthy individuals were incubated with immunoglobulin G (IgG) from heparin-induced thrombocytopenia (HIT) patients in the presence of vehicle (upper panel) or Iloprost (20 nM, [lower panel]) and heparin (Hep) (0.2 IU/mL). After reconstitution into autologous whole blood and recalcification, samples were perfused through microfluidic channels at a venous shear rate of 250s (10 dyne) for 10 minutes. After perfusion, images were acquired at x40 magnification. Scale bar 20µm. Violin plots showing the percentage of total surface area coverage (% SAC) by DiOC₆, phosphatidylserine (PS), Fibrin (-ogen), count of Hoechst-positive labeled cells and cumulative % SAC with DiOC₆, PS and Fibrin (-ogen) labeled thrombus captured in the microfluidic channel. *P<0.05, **P<0.01 and ***P<0.001.

Figure 5.

Increased platelet phosphatidylserine and not CD62p causes higher thrombin generation in heparin-induced thrombocytopenia. Panel (A) shows thrombin generation potential on platelets (PLT) after incubation with wit immunoglobulin g (IgG) from heparin-induced thrombocytopenia (HIT) patients (red line) or healthy controls (HC) (black line) in the presence of vehicle or anti-CD62p blocking antibody (Ab). (C) Thrombin generation on PLT that were incubated with different HIT patient IgG (red line) or HC IgG (black line) and treated with Lactadherin (Lact.) or vehicle, before calibrated automated thrombogram (CAT) analysis was performed. Each curve represents the amounts of generated thrombin over time induced by HIT IgG in the presence of vehicle (solid lines) or anti-CD62p blocking Ab or Lactadherin (dashed lines), respectively. (B and D) Data were quantified as peak thrombin generated (nM) using Thrombinoscope software and Graphpad prism. *P<0.05, **P<0.01 and ***P<0.001. ns: nonsignificant; CD62p: P-selectin.

Figure 6.

Heparin-induced thrombocytopenia antibodies can induce thrombus independent of CD62p. Platelets (PLT) from healthy individuals were incubated with heparin-induced thrombocytopenia (HIT) patient immunoglobulin G (IgG) and treated with vehicle (upper panel) or anti-CD62p blocking antibody (lower panel). After reconstitution into autologous whole blood and recalcification, samples were perfused through microfluidic channels at a venous shear rate of 250s^-1 (10 dyne) for 10 minutes. After perfusion, images were acquired at x40 magnification. Scale bar 20 µm. Violin plots showing the percentage of total surface area coverage (% SAC) by DiOC₆, phosphatidylserine, Fibrin (-ogen), count of Hoechst-positive labeled cells and cumulative area of DiOC₆, PS and Fibrin (-ogen) labeled thrombus in the microfluidic channel. *P<0.05, **P<0.01 and ***P<0.001. ns: non-significant. Hep: heparin.

Figure 7.

Platelet phosphatidylserine externalization is essential for heparin-induced thrombocytopenia antibody-induced thrombus formation. Platelets (PLT) from healthy individuals were incubated with heparin-induced thrombocytopenia (HIT) patient immunoglobulin G (IgG) and treated with vehicle (upper panel) or Lactadherin (lower panel). After reconstitution into auto-logous whole blood and recalcification, samples were perfused through microfluidic channels at a venous shear rate of 250s^-1 (10 dyne) for 10 minutes. Images were acquired at x40 magnification. Scale bar 20μm. Violin plots showing the percentage of total surface area coverage (% SAC) by DiOC₆, phosphatidylserine (PS), Fibrin (-ogen), count of Hoechst-positive labeled cells and cumulative area with DiOC₆, PS and Fibrin (-ogen) positive labeled thrombus in the microfluidic channel. *P<0.05, **P<0.01 and ***P<0.001. ns: non-significant. Hep: heparin.