Pathogenesis Of Heparin-induced Thrombocytopenia Research Articles

New perspectives on the induction and acceleration of immune-associated thrombosis by PF4 and VWF.

Platelet factor 4 (PF4), also known as chemokine (C-X-C motif) ligand 4 (CXCL4), is a specific protein synthesized from platelet α particles. The combination of PF4 and heparin to form antigenic complexes is an important mechanism in the pathogenesis of heparin-induced thrombocytopenia (HIT), but vaccine-induced immune thrombotic thrombocytopenia (VITT) related to the COVID-19 vaccine makes PF4 a research hotspot again. Similar to HIT, vaccines, bacteria, and other non-heparin exposure, PF4 can interact with negatively charged polyanions to form immune complexes and participate in thrombosis. These anions include cell surface mucopolysaccharides, platelet polyphosphates, DNA from endothelial cells, or von Willebrand factor (VWF). Among them, PF4-VWF, as a new immune complex, may induce and promote the formation of immune-associated thrombosis and is expected to become a new target and therapeutic direction. For both HIT and VITT, there is no effective and targeted treatment except discontinuation of suspected drugs. The research and development of targeted drugs based on the mechanism of action have become an unmet clinical need. Here, this study systematically reviewed the characteristics and pathophysiological mechanisms of PF4 and VWF, elaborated the potential mechanism of action of PF4-VWF complex in immune-associated thrombosis, summarized the current status of new drug research and development for PF4 and VWF, and discussed the possibility of this complex as a potential biomarker for early immune-associated thrombosis events. Moreover, the key points of basic research and clinical evaluation are put forward in the study.

Elucidation of Cellular Contributions to Heparin-Induced Thrombocytopenia Using Omic Approaches.

Heparin-induced thrombocytopenia (HIT) is an unpredictable, complex, immune-mediated adverse drug reaction associated with a high mortality. Despite decades of research into HIT, fundamental knowledge gaps persist regarding HIT likely due to the complex and unusual nature of the HIT immune response. Such knowledge gaps include the identity of a HIT immunogen, the intrinsic roles of various cell types and their interactions, and the molecular basis that distinguishes pathogenic and non-pathogenic PF4/heparin antibodies. While a key feature of HIT, thrombocytopenia, implicates platelets as a seminal cell fragment in HIT pathogenesis, strong evidence exists for critical roles of multiple cell types. The rise in omic technologies over the last decade has resulted in a number of agnostic, whole system approaches for biological research that may be especially informative for complex phenotypes. Applying multi-omics techniques to HIT has the potential to bring new insights into HIT pathophysiology and identify biomarkers with clinical utility. In this review, we review the clinical, immunological, and molecular features of HIT with emphasis on key cell types and their roles. We then address the applicability of several omic techniques underutilized in HIT, which have the potential to fill knowledge gaps related to HIT biology.

ChAdOx1 interacts with CAR and PF4 with implications for thrombosis with thrombocytopenia syndrome.

Vaccines derived from chimpanzee adenovirus Y25 (ChAdOx1), human adenovirus type 26 (HAdV-D26), and human adenovirus type 5 (HAdV-C5) are critical in combatting the severe acute respiratory coronavirus 2 (SARS-CoV-2) pandemic. As part of the largest vaccination campaign in history, ultrarare side effects not seen in phase 3 trials, including thrombosis with thrombocytopenia syndrome (TTS), a rare condition resembling heparin-induced thrombocytopenia (HIT), have been observed. This study demonstrates that all three adenoviruses deployed as vaccination vectors versus SARS-CoV-2 bind to platelet factor 4 (PF4), a protein implicated in the pathogenesis of HIT. We have determined the structure of the ChAdOx1 viral vector and used it in state-of-the-art computational simulations to demonstrate an electrostatic interaction mechanism with PF4, which was confirmed experimentally by surface plasmon resonance. These data confirm that PF4 is capable of forming stable complexes with clinically relevant adenoviruses, an important step in unraveling the mechanisms underlying TTS.

Platelet Factor 4 Binds to Released Von Willebrand Factor Strands from Injured Endothelium and Forms a Prothrombotic HIT Antigenic Complex

Heparin-induced thrombocytopenia (HIT) is driven by the binding of pathogenic HIT antibodies to platelet factor 4 (PF4) complexed to heparin. We, and others, have shown that PF4 bound to a variety of other polyanionic molecules, like surface glycosaminoglycans, also form HIT antigenic complexes. This is likely due to the formation of high molecular weight aggregates by the PF4. While studying the pathogenesis of HIT in a microfluidic system lined with human umbilical endothelial cells (HUVEC), we noted that PF4 did not bind diffusely to the HUVECs after a hematoporphyrin-induced photochemical injury as one would expect if binding were to the injured surface GAGs, but rather in a linear pattern described previously for von Willebrand Factor (vWF) under flow. We confirmed this by concurrent staining using PF4 and vWF-specific labeled antibodies. We also demonstrated that the HIT-like monoclonal antibody KKO, as well as IgG isolated from patients with likely HIT as determined by clinical assessment and ELISA positivity and positive serotonin release assay, bound to these PF4-vWF complexes. The HIT-like complexes were not uniformly distributed along the vWF, but occurred in a periodic fashion along thicker vWF strands. This suggested that the complexes were composed of multiple molecules of PF4 and vWF forming cross-chained high molecular weight aggregates that induce or enhance binding of HIT antibodies as previously reported for PF4/heparin in solution. We then wanted to identify the potential for therapeutics to intervene with PF4-vWF-KKO complex formation or stability. We tested this by introducing heparin; 2-0,3-0 desulfated heparin (ODSH); ADAMTS13 or n-acetylcysteine into channels post-complex formation. All interventions at therapeutically relevant concentrations were able to significantly remove or disrupt complex formation. When whole human blood + PF4 + KKO were flowed through channels lined with injured endothelium, large platelet aggregates developed within 10 mins along the extended strands of vWF. Significantly fewer platelet aggregates formed in the absence of PF4 or with KKO alone or PF4 plus an isotype control TRA. After 1 hour of flow, aggregates in the PF4 + KKO channels had grown significantly, but had largely disappeared in the other settings. Furthermore, by adding the FcgammaRIIA blocking antibody, IV.3, platelet binding could be reduced to background levels. These studies identify large vWF polymeric strands as a binding site for PF4 and a HIT antigen target. The Fc portions of the bound HIT antibody then contribute to platelet binding to the vWF. We are presently examining whether PF4-vWF-KKO aggregates contribute to thrombotic complications in HIT and defining the vWF domain(s) involved as these targets offer a potential novel intervention target in HIT. [Display omitted] DisclosuresCines:Amgen: Consultancy; Ionic: Consultancy; Juno: Consultancy; Astellas: Consultancy; T2 Biosystems: Research Funding; Rigel: Consultancy; Syntimmune: Research Funding.

Genome-Wide Association Study Identifies Variation in ABO As Risk Factor for Platelet Reactivity in Heparin-Induced Thrombocytopenia

Background: Heparin-induced thrombocytopenia (HIT) is an unpredictable, potentially catastrophic adverse effect of heparin treatment resulting from an immune response to platelet factor 4 (PF4)/heparin complexes. Genome-wide association studies (GWAS) have the potential to identify genetic risk factors and generate insights into biological mechanisms of adverse drug reactions. However, published HIT GWAS are underpowered and do not include confirmation of HIT using functional assays. We aimed to identify genetic associations with HIT using a GWAS approach in two European ancestry populations tested for functional assay and PF4/heparin antibody levels. Methods: We performed a GWAS with positive functional assay as the outcome in a large discovery cohort of patients, including heparin-induced platelet aggregation (HIPA) positive cases (n=1243), antibody positive but functional assay negative controls (n=1091), and antibody negative controls (n=1718). Logistic regressions were adjusted for age, gender, and first two principal components. Significant associations from the discovery cohort (alpha=5.00x10-8) were then investigated in a replication cohort of serotonin-release assay (SRA)-confirmed HIT cases (n=173), antibody positive controls (n=125), and antibody negative controls (n=488). All patients were genotyped with the HumanOmniExpressExome BeadChip, including 9,065,510 variants after imputation and quality control. Genes near significantly associated loci were resequenced. Results: In the discovery cohort, a genome-wide significant association was observed between common variation in the ABO gene and HIPA positive status (rs505922 odds ratio 0.738 [0.668-0.816], p=2.813x10-9). This variant was also significantly associated with SRA-confirmed HIT in the replication cohort (odds ratio 0.392 [0.172-0.891], p=0.025). Meta-analysis indicated strong association of ABO variation with positive functional assay (Figure 1). Sequencing and fine-mapping of the ABO locus indicated this effect was driven by ABO blood groups, with the O blood group being a risk factor for HIT (odds ratio 1.42 [1.25-1.61], p=6.11x10-8). Conclusions: We identify common variation in ABO as a risk factor for platelet reactivity and HIT. Strong association of ABO variation may have important implications for prediction and understanding of HIT pathogenesis. Figure 1 Disclosures No relevant conflicts of interest to declare.

The role of fluid-phase immune complexes in the pathogenesis of heparin-induced thrombocytopenia

Immune complexes assemble on the platelet surface and cause Fc-mediated platelet activation in heparin-induced thrombocytopenia (HIT); however, it is not known if fluid-phase immune complexes contribute to HIT. The objective of this study was to understand the role of fluid-phase immune complexes in platelet activation and HIT. Binding of wild-type and 15 platelet factor 4 (PF4) mutants to platelets was measured using flow cytometry. Platelet activation was measured using the PF4-dependent 14C-serotonin release assay (PF4-SRA) with KKO and a HIT-patient plasma in the presence of wild-type or PF4 mutants. To activate platelets, we found that a minimal level of wild-type PF4 is required to bind the platelet surface in the presence of KKO (2.67 relative MFI) or HIT-patient plasma (1.71 relative MFI). Only a subset of PF4 mutants was able to support platelet activation, despite having lower surface binding than the minimum binding required of wild-type PF4 (9 mutants with KKO and 2 mutants with HIT-patient plasma). Using individual PF4 mutants, we identified that HIT immune complexes can be formed in fluid-phase and induce platelet activation. Further studies are required to investigate the role of fluid-phase HIT immune complexes in the development of thrombocytopenia and thrombosis associated with clinical HIT.

Heparin-induced thrombocytopenia (HIT): Review of incidence, diagnosis, and management.

Heparin-induced thrombocytopenia (HIT) is a life and limb-threatening complication of heparin exposure. Here, we review the pathogenesis, incidence, diagnosis, and management of HIT. The first step in thwarting devastating complications from this entity is to maintain a high index of clinical suspicion, followed by an accurate clinical scoring assessment using the 4Ts. Next, appropriate stepwise laboratory testing must be undertaken in order to rule out HIT or establish the diagnosis. In the interim, all heparin must be stopped immediately, and the patient administered alternative anticoagulation. Here we review alternative anticoagulation choice, therapy alternatives in the difficult-to-manage patient with HIT, and the problem of overdiagnosis.

Physicochemical Characteristics of Platelet Factor 4 under Various Conditions are Relevant for Heparin-Induced Thrombocytopenia Testing

Heparin-induced thrombocytopenia (HIT), an adverse drug effect, has gained much attention. Affected patients have a high risk of new thrombotic complications. In addition, HIT is also a model to study mechanisms of immune-mediated disorders. Platelet factor 4 (PF4) is the key protein involved. It is the basis for many diagnostic tests for HIT and is used for in vitro studies and in mouse models on the pathogenesis of HIT. Purified PF4 is known to easily form aggregates, which can cause artifacts in experiments. The impact of storage buffer, storage period, lyophilization, and temperature on the size of PF4 and PF4/heparin (H) complexes was assessed by dynamic light scattering (DLS), while enzyme immunoassay (EIA) was used to test the binding of anti-PF4/H antibodies (aPF4/H Abs) to PF4/H complexes. PF4 size was more stable in Hank's balanced salt solution (HBSS) compared to phosphate-buffered saline (PBS), especially during storage. Lyophilization further facilitated the formation of PF4 aggregates, while incubation of reconstituted lyophilized PF4 in PBS at 37 °C reduced PF4 aggregates. Complexes formed between lyophilized PF4 and heparin were larger, and they enhanced the binding of aPF4/H Abs in EIA compared to complexes between nonlyophilized PF4 and heparin, both in HBSS and PBS, possibly influencing in vitro test results. Our results may be helpful for mechanistic studies on the biological function of PF4 and for the improvement of assays for detecting aPF4/H Abs.

Heparin induces neutrophil elastase-dependent vital and lytic NET formation.

Heparin is used extensively as an anticoagulant in a broad range of diseases and procedures; however, its biological effects are not limited to coagulation and remain incompletely understood. Heparin usage can lead to the life-threatening complication known as heparin-induced thrombocytopenia (HIT), caused by the development of antibodies against heparin/PF4 complexes. Here, we demonstrate the ability of heparin to induce neutrophil extracellular traps (NETs). NETs occurred with cell lysis and death, but live neutrophils releasing extracellular DNA strands, known as vital NETs, also occurred abundantly. Formation of NETs was time and dose dependent, and required reactive oxygen species and neutrophil elastase. Other compounds related to heparin such as low molecular weight heparin, fondaparinux and heparan sulfate either failed to induce NETs, or did so to a much lesser extent. Our findings suggest the ability of heparin to directly induce NET formation should be considered in the context of heparin treatment and HIT pathogenesis.

New Monoclonal Antibodies Reveal Distinct Specificities for Platelet Factor 4 Bound to Heparin Vs. Platelets: Implications for HIT Pathogenesis

Heparin induced thrombocytopenia (HIT) is a severe adverse reaction to heparin treatment characterized by antibodies to PF4/polyanion complexes. However, HIT antibodies are heterogeneous. Some bind to heparin/polyanion complexes in solid-phase enzyme-linked immunoassay (EIA) but do not activate platelets; other EIA-positive antibodies also activate platelets in assays such as the serotonin release assay (SRA) and in the recently described PF4-dependent p-selectin expression assay (PEA). It is generally thought that only platelet-activating antibodies are pathogenic. On the basis of studies which showed that platelet-activating antibodies, but not those positive only in EIA recognize PF4 bound to platelets (Blood. 2015;125(1):155-61 and others), we hypothesized that pathogenic HIT antibodies may recognize epitopes exposed on PF4 when it binds to a target, possibly chondroitin sulfate, on the platelet surface that are not created when PF4 forms a complex with heparin or other polyanions. Here, we describe studies utilizing newly developed monoclonal antibodies (mAbs) that demonstrate the feasibility of this concept and suggest that HIT antibodies having this specificity could be important in HIT pathogenesis. mAbs were produced in mice immunized with PF4/heparin complexes. We employed a novel dual screening strategy for candidate clones utilizing the PF4/polyanion EIA as well as the PEA. mAbs selected for study were designated 431.3, 431.4 and 447.1 and were compared with KKO, a widely studied HIT-like mAb (generous gift from G Arepally) and an isotype control (IC; Fig 1A). In the EIA, KKO strongly recognized PF4/polyvinylsulfonate (PF4/PVS) but not PF4 alone and was inhibited by high-dose heparin (100U/mL). mAb 447.1 had an EIA profile similar to KKO but with significantly lower optical densities (0.56 vs. 2.43 OD) and 431.3 gave reactions indicating it recognizes PF4 alone but demonstrated much stronger reactivity to PF4/PVS complexes (1.96 OD). However, mAb 431.4, similar to isotype control, was completely non-binding in the EIA (Fig 1A). In contrast, all 4 mAbs strongly recognized PF4-treated platelets (Fig 1B). In addition, all four mAbs produced strong positive reactions in the PEA in which mAbs interact with PF4-primed platelets to induce p-selectin expression (Fig 1C). The EIA requires multiple washing steps, whereas studies performed to assess IgG binding to PF4-treated platelets utilized only a single wash and the PEA is a "no-wash" assay. Thus, failure of 431.4 to react in EIA could be explained by susceptibility of the mAb to being removed by washing, possibly due to low avidity for its target. We therefore studied mAb binding to PF4/heparin complexes in real time using Bio-layer interferometry (Octet system, Pall ForteBio). In this system, mAbs 431.4 and KKO produced a steadily increasing signal, reaching saturation, whereas 431.4 demonstrated no binding to PF4/heparin consistent with results seen in EIA (Fig 1D). These findings indicate that mAb 431.4 binds to PF4 bound to the platelet surface and induces platelet activation as measured by p-selectin expression but fails to react with PF4 in a complex with heparin or PVS. This behavior contrasts strikingly with that of the "HIT-like" mAb KKO that reacts in both assays. Although antibodies from patients with HIT frequently give positive reactions in both platelet-activating assays and in EIA, there are clear examples of antibodies that are positive in one assay and not the other (e.g. EIA-positive, SRA-negative antibodies are common in clinical practice). Thus in HIT patients, multiple antibodies with different specificities are undoubtedly present. Our data support the growing body of evidence that while strength of the reaction in EIA (optical density) is often correlated with platelet-activating antibodies, there are notable exceptions (such as mAb 447.1). These results suggest that studies to isolate and characterize human antibodies according to specificity and reaction strength are needed to define the extent to which antibodies like mAb 431.4 and 447.1 contribute to the pathogenesis of HIT. Disclosures Padmanabhan: Versiti Wisconsin: Patents & Royalties: Related to HIT patents; Retham Technologies: Equity Ownership; Terumo BCT: Consultancy; Veralox Therapeutics: Membership on an entity's Board of Directors or advisory committees; Janssen R&D: Consultancy. Jones:Retham Technologies: Equity Ownership; Versiti Wisconsin: Patents & Royalties.

Diagnosis and management of heparin-induced thrombocytopenia: a consensus statement from the Thrombosis and Haemostasis Society of Australia and New Zealand HIT Writing Group.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder that occurs following the administration of heparin and is caused by antibodies to platelet factor 4 and heparin. Diagnosis of HIT is essential to guide treatment strategies using non-heparin anticoagulants and to avoid unwanted and potential fatal thromboembolic complications. This consensus statement, formulated by members of the Thrombosis and Haemostasis Society of Australia and New Zealand, provides an update on HIT pathogenesis and guidance on the diagnosis and management of patients with suspected or confirmed HIT. A 4Ts score is recommended for all patients with suspected HIT prior to laboratory testing. Further laboratory testing with a screening immunoassay or confirmatory functional assay is not recommended in individuals with a low 4Ts score. However, if there are missing or unreliable clinical data, then laboratory testing should be performed. A positive functional assay result confirms the diagnosis of HIT and should be performed to confirm a positive immunoassay result. Heparin exposure must be ceased in patients with suspected or confirmed HIT and initial treatment with a non-heparin alternative instituted. Non-heparin anticoagulants (danaparoid, argatroban, fondaparinux and bivalirudin) used to treat HIT should be given in therapeutic rather than prophylactic doses. Direct oral anticoagulants may be used in place of warfarin after patients with HIT have responded to alternative parenteral anticoagulants with platelet count recovery. These are the first Australasian recommendations for diagnosis and management of HIT, with a focus on locally available diagnostic assays and therapeutic options. The importance of examining both clinical and laboratory data in considering a diagnosis of HIT cannot be overstated.

Regulatory T Cells Control PF4/Heparin Antibody Production in Mice

Heparin-induced thrombocytopenia (HIT) is an immune-mediated disorder that can cause fatal arterial or venous thrombosis/thromboembolism. Immune complexes consisting of heparin, platelet factor 4 (PF4) and PF4/heparin-reactive antibodies are central to the pathogenesis of HIT. Regulatory T (Treg) cells are a subpopulation of CD4 T cells that modulate immune system by suppressing or down-regulating immune response and play an important role in immune homeostasis. However, the role of Treg cells in controlling PF4/heparin-specific antibody production is not known. Here we found that FoxP3-deficient mice, which have no functional Treg cells, spontaneously generated PF4/heparin-specific antibodies as early as three weeks after birth. Similarly, following transplantation with bone marrow cells from FoxP3-deficient mice, Rag1-deficient mice that have no endogenous B cells and T cells spontaneously produced PF4/heparin-specific antibodies. In contrast, Rag1-deficient mice that received wild-type bone marrow cells failed to produce PF4/heparin-specific antibodies. In addition, adoptively transferred Treg cells prevented spontaneous production of PF4/heparin-specific antibodies in FoxP3-deficient mice. Adoptively transferred Treg cells, not conventional CD4 T cells, also suppressed PF4/heparin complex-induced production of PF4/heparin-specific IgGs in wild-type mice. Treg cells suppress immune response mainly through releasing anti-inflammatory cytokines, such as interleukin-10 (IL-10). Deficiency of IL-10 led to spontaneous production of PF4/heparin-specific antibodies in mice. Moreover, BM chimeric mice with CD4 T cell-specific deletion of IL-10 increased PF4/heparin-specific IgG production following PF4/heparin complex challenge. Taken together, these findings demonstrate that Treg cells play an important role in suppressing PF4/heparin-specific antibody production in mice. DisclosuresHaribhai:AbbVie Inc.: Employment. Aster:BloodCenter of Wisconsin: Patents & Royalties.

A CASE OF SPONTANEOUS HEPARIN-INDUCED THROMBOCYTOPENIA AND THROMBOSIS (HITT)

SESSION TITLE: Critical Care 2 SESSION TYPE: Med Student/Res Case Rep Postr PRESENTED ON: 10/09/2018 01:15 PM - 02:15 PM INTRODUCTION: Spontaneous heparin-induced thrombocytopenia and thrombosis (HITT) is established with positive serum platelet factor 4 (PF4) antibody, thrombosis, and without prior heparin exposure. Only a handful of spontaneous HITT cases have been reported. Due to history of no exposure to heparin products, diagnosis is often challenging and treatment delayed leading to increased patient morbidity and mortality. Here we present a case of spontaneous HIT and thrombosis after a left total knee arthroplasty (TKA). CASE PRESENTATION: A 71 year old male presented on postoperative day (POD) 7 from left TKA with acute onset abdominal pain, bright red blood per rectum, and coffee ground emesis. Prior to his TKA, he was noted to have a platelet count of 185 x 103/uL. Post-operatively he was discharged on Clindamycin 600mg and Aspirin 325mg twice daily for deep venous thrombosis (DVT) prophylaxis. On admission, he was found to have a platelet count of 47 x 103/uL and further work-up revealed splenic vein and bilateral lower extremity deep venous thrombosis. Due to acute bleeding patient was not started on anticoagulation and an inferior vena cava filter was placed. On hospital day 3, patient became acutely short of breath and computed tomography angiogram of the chest revealed bilateral pulmonary artery emboli. Serum PF4 [define PF4 here] antibody resulted strongly positive at 3.004 optical density (OD) (Range: 0.0 – 0.4 OD) indicating HITT associated thrombosis. Once the serum PF4 antibody titer was resulted positive, patient was started on Argatroban drip with eventual increase in platelet count past 150 x 103/uL. DISCUSSION: HIT is a complication of exposure to heparin that could be life threatening. It is caused by autoantibodies that recognize and bind to highly antigenic multi-molecular complexes formed of heparin binding to endogenous protein PF4 and causing platelet activation.1 Multiple theories have been proposed but the exact mechanism behind spontaneous HIT remains unclear. In vitro studies have shown that heparin antibodies are not specific to heparin and can also recognize PF4 bound to other polyanions and have shown to be elicited by reaction with lipopolysaccharides from gram negative bacteria.2 This may explain why cases of spontaneous HIT are seen in patient following total knee or hip arthroplasty where significant limb ischemia and tissue injury occur. CONCLUSIONS: Although exceedingly rare, spontaneous HITT is important to be recognized early to reduce patient morbidity and mortality. Patients without prior exposure to heparin who have undergone orthopedic procedures and present with worsening platelet count or new arterial/venous thrombosis should be considered to have spontaneous HITT and treated accordingly. Reference #1: Rauova L, Zhai L, Kowalska MA, et al. Role of platelet surface PF4 antigenic complexes in heparin-induced thrombocytopenia pathogenesis: diagnostic and therapeutic implications. Blood 2006; 107:2346. Reference #2: G. Pongas, S. K. Dasgupta, and P. Thiagarajan, “Antiplatelet factor 4/heparin antibodies in patients with gram negative bacteremia,” Thrombosis Research, vol. 132, no. 2, pp. 217–220, 2013. Reference #3: Warkentin T.E., Cook R.J., Marder V.J., Greinacher A. Anti-PF4/heparin antibody formation postorthopedic surgery thromboprophylaxis: the role of nondrug risk factors and evidence for a stoichiometry-based model of immunization. J Thromb Haemost. 2010; 8:504. DISCLOSURES: No relevant relationships by Bryan Anderson, source=Web Response No relevant relationships by Chinthaka Bulathsinghala, source=Web Response No relevant relationships by Palla De Silva, source=Web Response No relevant relationships by Ahmed Hassan, source=Web Response No relevant relationships by aftab mahmood, source=Web Response No relevant relationships by Chirag Patel, source=Web Response Speaker/Speaker's Bureau relationship with Sunovian Please note: $5001 - $20000 Added 11/20/2017 by Salim Surani, source=Web Response, value=Honoraria Advisory Committee Member relationship with Astra Zeneca Please note: $5001 - $20000 Added 11/20/2017 by Salim Surani, source=Web Response, value=Consulting fee No relevant relationships by Deanna Yamamura, source=Web Response

Influence of Human Leukocyte Antigen (HLA) Alleles and Killer Cell Immunoglobulin-Like Receptors (KIR) Types on Heparin-Induced Thrombocytopenia (HIT).

Heparin-induced thrombocytopenia (HIT) is an unpredictable, life-threatening, immune-mediated reaction to heparin. Variation in human leukocyte antigen (HLA) genes is now used to prevent immune-mediated adverse drug reactions. Combinations of HLA alleles and killer cell immunoglobulin-like receptors (KIR) are associated with multiple autoimmune diseases and infections. The objective of this study is to evaluate the association of HLA alleles and KIR types, alone or in the presence of different HLA ligands, with HIT. HIT cases and heparin-exposed controls were identified in BioVU, an electronic health record coupled to a DNA biobank. HLA sequencing and KIR type imputation using Illumina OMNI-Quad data were performed. Odds ratios for HLA alleles and KIR types and HLA*KIR interactions using conditional logistic regressions were determined in the overall population and by race/ethnicity. Analysis was restricted to KIR types and HLA alleles with a frequency greater than 0.01. The p values for HLA and KIR association were corrected by using a false discovery rate q<0.05 and HLA*KIR interactions were considered significant at p<0.05. Sixty-five HIT cases and 350 matched controls were identified. No statistical differences in baseline characteristics were observed between cases and controls. The HLA-DRB3*01:01 allele was significantly associated with HIT in the overall population (odds ratio 2.81 [1.57-5.02], p=2.1×10-4 , q=0.02) and in individuals with European ancestry, independent of other alleles. No KIR types were associated with HIT, although a significant interaction was observed between KIR2DS5 and the HLA-C1 KIR binding group (p=0.03). The HLA-DRB3*01:01 allele was identified as a potential risk factor for HIT. This class II HLA gene and allele represent biologically plausible candidates for influencing HIT pathogenesis. We found limited evidence of the role of KIR types in HIT pathogenesis. Replication and further study of the HLA-DRB3*01:01 association is necessary.

Endothelial antigen assembly leads to thrombotic complications in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder initiated by antibodies against complexes between human platelet factor 4 (hPF4) and heparin. A better understanding of the events that initiate the prothrombotic state may improve approaches to antithrombotic management. Here, we visualized thrombus formation in an in vivo murine model and an endothelialized microfluidic system that simulate the pathogenesis of HIT. hPF4 released from platelets predominantly bound to peri-injury endothelium and formed HIT antigenic complexes that were dissociated by heparin. In mice expressing both hPF4+ and human platelet IgG Fc receptor IIA (FcγRIIA), infusion of the HIT-like monoclonal antibody KKO increased fibrin and platelet deposition at sites of injury, followed immediately by antigen formation on proximate endothelial cells. After a few minutes, HIT antigen was detected within the thrombus itself at the interface between the platelet core and the surrounding shell. We observed similar results in the humanized, endothelialized microfluidic system. hPF4 and KKO selectively bound to photochemically injured endothelium at sites where surface glycocalyx was reduced. These studies support the concept that the perithrombus endothelium is the predominant site of HIT antigen assembly. This suggests that disrupting antigen formation along the endothelium or protecting the endothelium may provide a therapeutic opportunity to prevent thrombotic complications of HIT, while sparing systemic hemostatic pathways.

TULA-2 Inhibits the Platelet FcγRIIA-Syk Pathway and Heparin-Induced Thrombocytopenia in Mice

Heparin-induced thrombocytopenia (HIT) is a life-threatening disease in which IgG antibodies against the heparin-PF4 complex activate platelets via FcγRIIA. We previously reported that TULA-2 serves as a negative regulator of FcγRIIA pathway by dephosphorylating Syk in HEL cells. To further investigate the effect of TULA-2 on the FcγRIIA pathway and HIT, we crossed TULA-2-/- with FcγRIIA+/+ mice. Ablation of TULA-2 resulted in hyperphosphorylation of Syk, LAT, and PLCγ2 in platelets after FcγRIIA activation. Integrin activation, calcium mobilization, and P-selectin exposure were also enhanced in TULA-2-/- murine platelets compared to TULA-2+/+. Further, anti-GPIX antibody-induced HIT-like thrombocytopenia and thrombin generation were also augmented in TULA-2-/- mice (Figure A). We also found that decreased TULA-2 level shortened tail-bleeding time in mice (Figure B), suggesting a role of TULA-2 in physiological hemostasis.Additionally, TULA-2 KO and WT platelets did not show significant differences in platelet spreading and clot retraction, indicating that outside-in signaling is not affected by the absence of TULA-2. At the protein level, TULA-2 heterozygous knockout (TULA-2+/-) platelets express 50% as much protein as their wildtype counterparts. Interestingly, TULA-2+/- mice showed significantly increased platelet reactivity and more severe thrombocytopenia in vivo compared with TULA-2+/+ mice. Together the data indicate that not only the absence of TULA-2, but also the relative level of TULA-2 expression modulate FcγRIIA-mediated platelet reactivity, HIT pathogenesis, and hemostasis. Considering that TULA-2 is also a key regulator in platelet GPVI pathway, measuring TULA-2 expression may be a valuable predictor for HIT susceptibility and platelet reactivity in general. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

A case of heparin-induced thrombocytopenia (HIT) triggered by methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia

Heparin-induced thrombocytopenia (HIT) is a potentially life-threatening complication of heparin anticoagulation. Recently, infection has been implicated in the development of HIT. We herein present a case of HIT diagnosed by a repeated functional assay. A 67-year-old female with diabetic nephropathy was admitted to our hospital with hypoxemia caused by volume overload. Due to her diuretic-resistant condition, dialysis therapy anticoagulated with low-molecular-weight heparin (LMWH) was initiated. The coagulation of the whole extracorporeal blood circuit occurred on day 13. Methicillin-susceptible Staphylococcus aureus (MSSA) bacteremia derived from a catheter-related bloodstream infection (CRBSI) was detected on day 15. Based on the hypercoagulable state due to infection, we increased the heparin dose during dialysis therapy; however, coagulation of the dialysis circuit persisted. We suspected HIT on day 17 in spite of a normal platelet count and performed a functional assay, which was negative. Since thrombocytopenia subsequently developed, the functional assay was repeated and a positive result was obtained on day 27, which definitely established a diagnosis of HIT. Based on the present clinical course, MSSA bacteremia appears to have contributed to the pathogenesis of HIT.

Single-molecule force spectroscopy applied to heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT), occurring up to approximately 1% to 5% of patients receiving the antithrombotic drug heparins, has a complex pathogenesis involving multiple partners ranging from small molecules to cells/platelets. Recently, insights into the mechanism of HIT have been achieved by using single-molecule force spectroscopy (SMFS), a methodology that allows direct measurements of interactions among HIT partners. Here, the potential of SMFS in unraveling the mechanism of the initial steps in the pathogenesis of HIT at single-molecule resolution is highlighted. The new findings ranging from the molecular binding strengths and kinetics to the determination of the boundary between risk and non-risk heparin drugs or platelet-surface and platelet-platelet interactions will be reviewed. These novel results together have contributed to elucidate the mechanisms underlying HIT and demonstrate how SMFS can be applied to develop safer drugs with a reduced risk profile.

TULA-2 (T-Cell Ubiquitin Ligand-2) Inhibits the Platelet Fc Receptor for IgG IIA (FcγRIIA) Signaling Pathway and Heparin-Induced Thrombocytopenia in Mice.

The objective of this study is to investigate the role of T-cell ubiquitin ligand-2 (TULA-2) in the platelet Fc receptor for IgG IIA (FcγRIIA) pathway and in the pathogenesis of heparin-induced thrombocytopenia (HIT). HIT is a life-threatening thrombotic disease in which IgG antibodies against the heparin-platelet factor 4 complex activate platelets via FcγRIIA. We reported previously differential expression of TULA-2 in human population was linked to FcγRIIA responsiveness. In this study, we investigated the role of TULA-2, a protein phosphatase, in the FcγRIIA pathway and HIT pathogenesis by crossing TULA-2-/- mice with transgenic FcγRIIA +/+ mice. Ablation of TULA-2 resulted in hyperphosphorylation of spleen tyrosine kinase, linker for the activation of T cells, and phospholipase Cγ2 in platelets via FcγRIIA activation. Platelet integrin activation, granule secretion, phosphatidylserine exposure, and aggregation were also enhanced in TULA-2-/- murine platelets. Compared with wild-type mice, TULA-2-/- mice showed aggravated antibody-mediated thrombocytopenia, augmented thrombin generation, and shortened tail bleeding time. In contrast, there was no significant difference between TULA-2-/- and TULA-2+/+ platelets in platelet spreading and clot retraction. Of note, heterozygous TULA-2+/- mice, whose platelets contained 50% as much protein as the TULA-2+/+ platelets, showed significantly increased platelet reactivity and more severe thrombocytopenia in vivo compared with TULA-2+/+ mice. Together, the data demonstrate that not only the absence of TULA-2 but also the relative level of TULA-2 expression modulates FcγRIIA-mediated platelet reactivity and HIT in vivo. TULA-2 expression could be a valuable marker for HIT and inhibiting TULA-2 may serve as a potential therapy to reverse the bleeding adverse effect of anticoagulants.

Diagnosis and treatment of heparin-induced thrombocytopenia (HIT) based on its atypical immunological features

Heparin-induced thrombocytopenia (HIT) is a prothrombotic side effect of heparin therapy caused by HIT antibodies, i.e., anti-platelet factor 4 (PF4)/heparin IgG with platelet-activating properties. For serological diagnosis, antigen immunoassays are commonly used worldwide. However, such assays do not indicate their platelet-activating properties, leading to low specificity for the HIT diagnosis. Therefore, over-diagnosis is currently the most serious problem associated with HIT. The detection of platelet-activating antibodies using a washed platelet activation assay is crucial for appropriate HIT diagnosis. Recent advances in our understanding of the pathogenesis of HIT include it having several clinical features atypical for an immune-mediated disease. Heparin-naïve patients can develop IgG antibodies as early as day 4, as in a secondary immune response. Evidence for an anamnestic response on heparin re-exposure is lacking. In addition, HIT antibodies are relatively short-lived, unlike those in a secondary immune response. These lines of evidence suggest that the mechanisms underlying HIT antibody formation may be compatible with a non-T cell-dependent immune reaction. These atypical clinical and serological features should be carefully considered while endeavoring to accurately diagnose HIT, which leads to appropriate therapies such as immediate administration of an alternative anticoagulant to prevent thromboembolic events and re-administration of heparin during surgery involving cardiopulmonary bypass when HIT antibodies are no longer detectable.