• The procoagulant nature of HIT can be simulated in a microfluidic model using human blood and its components.• PF4/glycosaminoglycans/ immunoglobulin G complexes activate monocytes through FcgRIIA to generate TF and thrombin, leading to coated platelets in HIT.Heparin-induced thrombocytopenia (HIT) is characterized by a high incidence of thrombosis, unlike other antibody-mediated causes of thrombocytopenia. We have shown that monocytes complexed with surface-bound platelet factor 4 (PF4) activated by HIT antibodies contribute to the prothrombotic state in vivo, but the mechanism by which this occurs and the relationship to the requirement for platelet activation via fragment crystallizable (Fc)gRIIA is uncertain. Using a microfluidic model and human or murine blood, we confirmed that activation of monocytes contributes to the prothrombotic state in HIT and showed that HIT antibodies bind to monocyte FcgRIIA, which activates spleen tyrosine kinase and leads to the generation of tissue factor (TF) and thrombin. The combination of direct platelet activation by HIT immune complexes through FcgRIIA and transactivation by monocyte-derived thrombin markedly increases Annexin V and factor Xa binding to platelets, consistent with the formation of procoagulant coated platelets. These data provide a model of HIT wherein a combination of direct FcgRIIA-mediated platelet activation and monocyte-derived thrombin contributes to thrombosis in HIT and identifies potential new targets for lessening this risk. (Blood. 2016;127(4):464-472) IntroductionHeparin-induced thrombocytopenia (HIT) is an iatrogenic, immunemediated disorder characterized by antibodies that recognize complexes between the platelet chemokine platelet factor 4 (PF4, CXCL4) and heparin or cell surface glycosaminoglycans (GAGs). 1,2 It is estimated that up to 50% of patients with HIT develop thrombosis that might be limb-and/or life-threatening. [3][4][5] Even with early recognition, cessation of heparin, and institution of alternative forms of anticoagulation, recurrent thromboembolic complications may occur and 10% to 20% of patients go on to amputation and/or death. 6 Thus, there is a need for a better understanding of the pathogenesis of HIT and to determine how this information can be used to mitigate the risk of thrombosis.Thrombocytopenia and thrombosis in HIT have been attributed to binding of PF4/heparin/immunoglobulin G (IgG) immune-complexes to the platelets through the IgG fragment crystallizable (Fc) region, which activates platelets through their immunoreceptor tyrosine-based activation motif (ITAM) receptor, FcgRIIA. 7,8 However, monocytes, endothelial cells, and other cell types might also be activated by these immune complexes and contribute to the underlying pathology, 9 but their contribution to the process is less well characterized. Indeed, recent evidence suggests that thrombosis in HIT is initiated by binding of pathogenic antibodies to antigenic complexes of PF4 and GAGs expressed by the endothelium as well as circulating cells, includi...

SummaryHeparin-induced thrombocytopenia (HIT) is an autoimmune disorder characterised by thrombocytopenia and thrombosis. The mechanisms leading to platelet destruction are complex and the thrombotic complications of HIT appear to be due to multiple different intravascular targets. The dual binding of HIT antibodies to platelet surface PF4/GAG complexes and to FcγRIIA likely leads to both platelet clearance and to their direct activation. Monocytes and endothelial cells bind PF4 with higher avidity than platelets and are more resistant to competitive removal of surface-bound PF4 in the presence of heparin. Binding of HIT antibodies to PF4/glycosaminoglycan complexes on the surface on these cells leads to their activation and increased procoagulant activity. Binding of higher levels of PF4 released from activated platelets to the endothelium may lead to changes of the anticoagulant properties of the glycocalyx and target the endothelial cells for HIT antibodies. Pathogenic antibodies bound to endothelial cells further promote prothrombotic conditions by a mechanism that is independent of FcγR activation, yet not completely understood. A more detailed understanding of the role of monocytes and endothelium may identify new targets for intervention to mitigate the risk of thrombosis with less impact on systemic haemostasis than current approaches to treatment for this serious disorder.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by antibodies to platelet factor 4 (PF4)/heparin complexes. PF4 released from platelets binds to surface glycosaminoglycans on hematopoietic and vascular cells that are heterogenous in composition and differ in affinity for PF4. PF4 binds to monocytes with higher affinity than to platelets, and depletion of monocytes exacerbates thrombocytopenia in a murine HIT model. Here we show that the expression of PF4 on platelets and development of thrombocytopenia are modulated by the (re)distribution of PF4 among hematopoietic and endothelial cell surfaces. Binding of PF4 to platelets in whole blood in vitro varies inversely with the white cell count, likely because of the greater affinity of monocytes for PF4. In mice, monocyte depletion increased binding of PF4 to platelets by two- to three-fold. Induction of HIT in mice caused a transient >80-fold increase in binding of HIT antibody to monocytes vs 3.5-fold increase to platelets and rapid transient monocytopenia. Normalization of monocyte counts preceded the return in platelet counts. Exposure of blood to endothelial cells also depletes PF4 from platelet surfaces. These studies demonstrate a dynamic interchange of surface-bound PF4 among hematopoetic and vascular cells that may limit thrombocytopenia at the expense of promoting prothrombotic processes in HIT.

BACKGROUND. COVID-19 is a prothrombotic disease, characterized by endotheliopathy, hypercoagulability, and thromboembolic complications. We hypothesized that the pathogenesis of thromboembolism associated with COVID-19 might differ from thromboembolism in patients without COVID-19. In this study, we sought to evaluate the proteomic signatures of plasma from patients with venous thromboembolism with and without COVID-19. METHODS. Between December 17, 2020 and February 25, 2021 blood was collected from 48 hospitalized patients. Of these 24 had a confirmed diagnosis of COVID-19 infection (COVID+) and radiologic confirmation of arterial or venous thromboembolism (TE+); 17 had COVID-19 infection with absence of arterial thrombosis clinically and absence of venous thromboembolism on lower extremity Doppler ultrasound or chest CT angiography (COVID+/TE-), while 7 were arterial or venous thromboembolism in the absence of COVID-19 (COVID-/TE+). Blood was collected in sodium citrate tubes and centrifuged at 4000 rpm for 20 minutes, with resulting plasma supernatant used for protein profiling performed at Eve Technologies (Calgary, Alberta, Canada). Institutional Review Board approval was obtained for this study. Statistical analysis was performed using GraphPad Prism (v9.1, GraphPad Software, San Diego, CA) and R (v4, R Core Team). P values <0.05 were considered statistically significant. A heatmap was generated using Heatmapper (heatmapper.ca) to represent the concentrations of proteins. RESULTS. The median age was 63 years; overall 25 (52%) were men (13 [54%] among COVID+/TE+, 11 [65%] among COVID+/TE-, and 1 [14%] among COVID-/TE+). In COVID-19 patients who developed thromboembolic events, several proteins associated with inflammation, complement activation, and hemostasis were present at higher levels than in non-COVID-19 patients who developed thromboembolic events (Fig. 1). These included complement factors C2 and C5a, pentraxin-3 (PTX-3), lipocalin-2 (LCN2), resistin (RETN), platelet endothelial cell adhesion molecule-1 (Pecam1), serum amyloid A (SAA), and tissue factor (TF). The heatmap indicates relative protein levels detected in each subject (columns) for proteins (rows) that had statistically significant differences between groups (Fig. 2). Heatmap revealed relatively lower levels of all proteins in patients with thromboembolism without COVID-19 and relatively higher levels of proteins in patients with COVID-19, and especially in ICU patients with COVID-19 and thromboembolism. CONCLUSIONS. Thromboembolic complications in patients with COVID-19 are associated with increased levels of various proteins involved in complement activation and immunothrombotic cascades, compared to thrombotic events in the absence of COVID-19. Activation of the classical complement pathway as evidenced by a relative increase in complement factor C2 may lead to increased TF activation, reflecting more substantial endothelial damage in COVID-19 patients. Higher levels of Pecam1, SAA, LCN2, and RETN all point to increased endotheliopathy, inflammation, and tissue damage in COVID-19 compared to non-COVID-19 thrombosis. These findings may offer insights into novel therapeutic strategies to treat immunothrombotic complications of COVID-19. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

BACKGROUND. COVID-19 is a prothrombotic disease, characterized by both arterial and venous thrombosis. The pathogenesis of coagulopathy in COVID-19 is multifactorial, with activation of endothelial cells, platelets, neutrophils, and other immune system effectors playing crucial roles. High levels of von Willebrand factor (VWF) and reduced levels of ADAMTS13 have been observed in COVID-19, sepsis, and immunothrombotic states, and an elevated VWF/ADAMTS13 ratio has been previously described in ischemic stroke1. In this study, we sought to evaluate potential associations of VWF activity and antigen ratios to ADAMTS13 with clinical outcomes in hospitalized patients with COVID-19 and to compare these ratios with other cytokines and markers of inflammation in critical illness. METHODS. Institutional Review Board approval was obtained for this study. Blood was collected from 49 hospitalized patients with a confirmed diagnosis of COVID-19 between April 13 and April 24, 2020, which included 40 patients in an intensive care unit and 9 on a non-ICU unit. Medical records were reviewed, and patient characteristics including age, sex, comorbidities, pertinent medications, imaging studies, history of arterial or venous thrombosis, and other pertinent patient information were compiled. Blood was collected in sodium citrate tubes and centrifuged at 4000 rpm for 20 minutes, with the resulting plasma supernatant used for further testing. Measurements of VWF antigen and VWF activity were performed on an ACL TOP machine using our institution's standard clinical laboratory protocol. Biomarker profiling analyses were performed at Eve Technologies (Calgary, Alberta, Canada). Statistical analysis was performed using GraphPad Prism (v8.4.3, GraphPad Software, San Diego, CA) and R (v4, R Core Team, 2020). P values <0.05 were considered statistically significant. RESULTS. The median age was 64 years; 36 (73%) were male, 11 (22.4%), 29 (59.2%), and 9 (18.4%) had BMI<25, between 25 and 40, and >40 kg/m2, respectively. The ratio of VWF activity/ADAMTS13 was significantly higher in patients with higher BMI (median [interquartile range, IQR] VWF activity/ADAMTS13 ratios: 0.66 [0.39-0.88] for BMI<25 kg/m2, 0.865 [0.769-1.102] for BMI 25-40 kg/m2, and 1.041 [0.747-2.032] for BMI>40 kg/m2; p = 0.0408, Kruskal-Wallis test). In patients who required mechanical ventilation, median [IQR] VWF activity/ADAMTS13 was 0.876 [0.75-1.769] vs 0.792 [0.566-0.932] for those who did not (p = 0.0497, Mann-Whitney U-test). The difference for the VWF antigen/ADAMTS13 ratio was more pronounced, 1.32 [0.991-2.456] vs 0.981 [0.628-1.162] (p = 0.0045, Mann-Whitney U-test). There was also a significant correlation between both VWF activity/ADAMTS13 and VWF antigen/ADAMTS13 ratios and neutrophil indices such as absolute neutrophil count (R = 0.57, p <0.0001 and R = 0.54, p = 0.0001, respectively) and neutrophil to lymphocyte count (R = 0.6, p <0.0001 and R = 0.57, p <0.0001, respectively). We also found significant correlations of these ratios with markers of neutrophils activation (HGF, resistin, and lipocalin-2; Figure 1). CONCLUSIONS. Ratios VWF activity/ADAMTS13 and VWF antigen/ADAMTS13 are elevated in patients with more severe COVID-19 disease and correlate with markers of neutrophils activation. These findings and that azurophillic granules in neutrophils contain α-defensins implicated in thrombosis2 suggest possible mechanistic links among VWF, ADAMTS13, contact pathway, and neutrophil function in mediating immunothrombotic complications in COVID-19, which might highlight potential therapeutic targets to attenuate the severity of coagulopathy and merit further investigation with a larger cohort of patients. REFERENCES 1. Taylor A, et al. Blood Adv. 2020;4(2):398-407 2. Abu-Fanne R, et al. Blood. 2019;133(5):481-493 Disclosures No relevant conflicts of interest to declare.