Thrombosis In Heparin-induced Thrombocytopenia Research Articles

Heparin-induced thrombocytopenia thrombosis after pancreaticoduodenectomy without definitive prophylactic or therapeutic use of heparin: a case report

Heparin-induced thrombocytopenia thrombosis (HITT) is a rare and potentially life-threatening complication after abdominal surgery, and it always occurs after the prophylactic or therapeutic use of heparin. HITT after pancreaticoduodenectomy (PD) has not been reported before. Herein, we reported a case of HITT after PD without prophylactic or therapeutic use of heparin. A 74-year-old female patient who suffered resectable pancreatic head cancer was transferred to our center for surgery. An open PD procedure was performed, and the operation was smooth. No heparin was used after surgery. Nine days after surgery, the platelet sharply declined to 48 × 109/L (100–350), and the D-dimer soared up to 33.56 mg/L (0–0.55). Ultrasound examination showed vein thrombosis in both the lower limb and the right upper limb. HIT-antibody was 6.3 U/mL (0–0.6). The diagnosis of HITT was confirmed. Fondaparinux was used. On postoperative day (POD) 23, the platelet recovered to the normal range. On POD 27, she was discharged without thromboembolism or active bleeding, and oral rivaroxaban was prescribed. One month after discharge, the platelet remained normal, and she did not complain of discomfort.

PB0342 Platelet Phosphatidylserine is the Critical Mediator of Thrombosis in Heparin-Induced Thrombocytopenia

PB0342 Platelet Phosphatidylserine is the Critical Mediator of Thrombosis in Heparin-Induced Thrombocytopenia

PB1359 Fc Modified KKO Blocks Thrombosis in Heparin-Induced Thrombocytopenia (HIT), But not in Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT)

PB1359 Fc Modified KKO Blocks Thrombosis in Heparin-Induced Thrombocytopenia (HIT), But not in Vaccine-Induced Immune Thrombotic Thrombocytopenia (VITT)

Treatment of thrombocytopenia and thrombosis in HIT in mice using deglycosylated KKO: a novel therapeutic?

AbstractHeparin-induced thrombocytopenia (HIT) is characterized by thrombocytopenia associated with a highly prothrombotic state due to the development of pathogenic antibodies that recognize human platelet factor 4 (hPF4) complexed with various polyanions. Although nonheparin anticoagulants are the mainstay of care in HIT, subsequent bleeding may develop, and the risk of developing new thromboembolic events remain. We previously described a mouse immunoglobulin G2bκ (IgG2bκ) antibody KKO that mimics the sentinel features of pathogenic HIT antibodies, including binding to the same neoepitope on hPF4–polyanion complexes. KKO, like HIT IgGs, activates platelets through FcγRIIA and induces complement activation. We then questioned whether Fc-modified KKO could be used as a novel therapeutic to prevent or treat HIT. Using the endoglycosidase EndoS, we created deglycosylated KKO (DGKKO). Although DGKKO retained binding to PF4–polyanion complexes, it inhibited FcγRIIA-dependent activation of PF4-treated platelets triggered by unmodified KKO, 5B9 (another HIT-like monoclonal antibody), and IgGs isolated from patients with HIT. DGKKO also decreased complement activation and deposition of C3c on platelets. Unlike the anticoagulant fondaparinux, injection of DGKKO into HIT mice lacking mouse PF4, but transgenic for hPF4 and FcγRIIA, prevented and reversed thrombocytopenia when injected before or after unmodified KKO, 5B9, or HIT IgG. DGKKO also reversed antibody-induced thrombus growth in HIT mice. In contrast, DGKKO was ineffective in preventing thrombosis induced by IgG from patients with the HIT-related anti-PF4 prothrombotic disorder, vaccine-induced immune thrombotic thrombocytopenia. Thus, DGKKO may represent a new class of therapeutics for targeted treatment of patients with HIT.

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

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.

Monoclonal gammopathy of thrombotic/thrombocytopenic significance

Letter to Blood| April 6, 2023 Monoclonal gammopathy of thrombotic/thrombocytopenic significance Adam J. Kanack, Adam J. Kanack Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN Search for other works by this author on: This Site PubMed Google Scholar Jordan K. Schaefer, Jordan K. Schaefer Department of Medicine, University of Michigan, Ann Arbor, MI https://orcid.org/0000-0002-7166-386X Search for other works by this author on: This Site PubMed Google Scholar Meera Sridharan, Meera Sridharan Department of Medicine, Mayo Clinic, Rochester, MN https://orcid.org/0000-0001-7225-8228 Search for other works by this author on: This Site PubMed Google Scholar Noah P. Splinter, Noah P. Splinter Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN Search for other works by this author on: This Site PubMed Google Scholar Mindy C. Kohlhagen, Mindy C. Kohlhagen Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN Search for other works by this author on: This Site PubMed Google Scholar Bandana Singh, Bandana Singh Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN Search for other works by this author on: This Site PubMed Google Scholar Silvana B. De Lorenzo, Silvana B. De Lorenzo Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN Search for other works by this author on: This Site PubMed Google Scholar Emily E. Mauch, Emily E. Mauch Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN Search for other works by this author on: This Site PubMed Google Scholar Maen A. Hussein, Maen A. Hussein Florida Cancer Specialists, Orlando, FL Search for other works by this author on: This Site PubMed Google Scholar Marwan Shaikh, Marwan Shaikh Department of Medicine, Mayo Clinic, Jacksonville, FL Search for other works by this author on: This Site PubMed Google Scholar Shaji Kumar, Shaji Kumar Department of Medicine, Mayo Clinic, Rochester, MN https://orcid.org/0000-0001-5392-9284 Search for other works by this author on: This Site PubMed Google Scholar Renren Wen, Renren Wen Department of Microbiology and Immunology, Versiti Blood Research Institute and Medical College of Wisconsin, Milwaukee, WI https://orcid.org/0000-0003-2420-8212 Search for other works by this author on: This Site PubMed Google Scholar Demin Wang, Demin Wang Department of Microbiology and Immunology, Versiti Blood Research Institute and Medical College of Wisconsin, Milwaukee, WI https://orcid.org/0000-0001-5549-3795 Search for other works by this author on: This Site PubMed Google Scholar David Murray, David Murray Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN Search for other works by this author on: This Site PubMed Google Scholar Anand Padmanabhan Anand Padmanabhan Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, MN https://orcid.org/0000-0003-2519-4377 Search for other works by this author on: This Site PubMed Google Scholar Blood (2023) 141 (14): 1772–1776. https://doi.org/10.1182/blood.2022018797 Article history Submitted: October 19, 2022 Accepted: December 27, 2022 Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Request Permissions Cite Icon Cite Search Site Citation Adam J. Kanack, Jordan K. Schaefer, Meera Sridharan, Noah P. Splinter, Mindy C. Kohlhagen, Bandana Singh, Silvana B. De Lorenzo, Emily E. Mauch, Maen A. Hussein, Marwan Shaikh, Shaji Kumar, Renren Wen, Demin Wang, David Murray, Anand Padmanabhan; Monoclonal gammopathy of thrombotic/thrombocytopenic significance. Blood 2023; 141 (14): 1772–1776. doi: https://doi.org/10.1182/blood.2022018797 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBlood Search Subjects: Thrombosis and Hemostasis TO THE EDITOR: Anti–platelet factor 4 (PF4) antibodies have a central role in the pathophysiology of thrombosis and thrombocytopenia in heparin-induced thrombocytopenia (HIT)1,2 and vaccine-induced immune thrombotic thrombocytopenia (VITT).3-7 Here, we present the case of a patient with recurrent thrombosis and thrombocytopenia, secondary to a persistent anti-PF4/polyanion monoclonal antibody due to an underlying neoplastic condition, which we refer to as monoclonal gammopathy of thrombotic/thrombocytopenic significance (MGTS). The implications of these findings for the investigation of unexplained thrombophilia are discussed. A 64-year-old man presented for evaluation of recurrent thrombotic episodes. His medical history included segmental and subsegmental pulmonary emboli, portal vein thrombosis, non-ST elevation myocardial infarction, splenic infarction, and ST elevation myocardial infarction (Figure 1A). Extensive investigations performed did not support a thrombophilia diagnosis (supplemental Table 1 and supplemental Materials; available on the Blood website). His...

Variable serotonin release assay pattern and specificity of PF4‐specific antibodies in HIT, and clinical relevance

BackgroundThe diagnosis of heparin‐induced thrombocytopenia (HIT) requires functional assays to demonstrate that platelet factor 4 (PF4)‐specific antibodies activate platelets, typically when therapeutic heparin (H) concentrations are tested (“classical” pattern). Some HIT samples also activate platelets without heparin (“atypical” pattern), but with unclear clinical significance. ObjectivesWe aimed to assess whether platelet activation pattern and some characteristics of PF4‐specific antibodies were associated with the severity of HIT. Patients/MethodsSerotonin release assay (SRA) pattern of 81 HIT patients were analyzed and compared with their clinical and biological data, including levels of anti‐PF4/H immunoglobulin G (IgG) and anti‐PF4 IgG in 47 of them. ResultsHigher anti‐PF4/H IgG titers were measured in patients with an “atypical” SRA (optical density 2.52 vs. 1.94 in those with a “classical” pattern, p < .001). Patients of both groups had similar platelet count (PC) nadir and time to recovery, but those with an “atypical” SRA more frequently developed thrombotic events (69% vs. 34%, p = .037). Significant levels of anti‐PF4 IgG were detected in both groups (38% and 61%, respectively). Whatever the SRA pattern, a lower PC nadir (35 vs. 53 G/L, p = .006) and a longer PC recovery time (6 vs. 3 days, p = .015) were evidenced in patients with anti‐PF4 antibodies, compared with those with anti‐PF4/H IgG only. ConclusionsAn atypical SRA pattern with elevated anti‐PF4/H IgG titers seems associated with an increased risk of thrombosis in HIT. IgG antibodies to native PF4 may contribute to more severe and persistent thrombocytopenia, and their detection could be useful in clinical practice.

Incidence and features of heparin-induced thrombocytopenia in mechanical heart valves and immunologic cardiac diseases

Background: Heparin-induced thrombocytopenia/thrombosis (HIT/T) is characterized by a fall in platelet count 5-10days after starting heparin therapy and is diagnosed with specific 4-T clinical features and laboratory tests. This complication is relatively common in Cardiothoracic surgery patients. Objective: To evaluate the positive and negative predictive value of various HIT laboratory tests and assess any correlation between HIT, the underlying diagnosis, underlying procedure, and mechanical cardiac devices. Patients and methods: The patient’s medical records were correlated with two laboratories HIT diagnostic tests, the pan-specific screening test with IgG, IgA, and IgM antibodies, followed by HIT specific IgG ELISA. Results: Total n = 80 patients were assessed, 48% (n = 38) were HIT screen pan-specific negative and 50% (n = 40) were HIT pan-specific positive and 2 cases were inconclusive. 17% (n = 14) were both pan-specific and specific HIT IgG ELISA positive. There were 5 atypical cases. One patient had Eosinophilic myocarditis and was HIT ELISA IgG neg. Argatroban was given on clinical grounds with successful recovery. One patient with Sarcoidosis had an aggressive course and received IV Immunoglobulin (IVIG) but succumbed secondary to liver failure. One patient progressed to gut ischemia and had surgical intervention but succumbed. Two patients with mechanical heart valves were on Argatroban but relapsed and responded to IVIG therapy. Conclusion: Our study indicates that 9/16 (&gt; 50%) HIT-positive patients had valve replacement or cardiac devices suggesting that like knee arthroplasty there is a high incidence of HIT in patients with mechanical heart valves and cardiac devices and this warrants further prospective study.

Fc-Modified Kko: A Novel Therapeutic for Heparin-Induced Thrombocytopenia (HIT), Reversing Both the Thrombocytopenia and Thrombosis

Heparin induced thrombocytopenia (HIT) is an immunogenic prothrombotic disorder caused by antibodies that recognize human platelet factor 4 (PF4) complexed to polyanions. We had previously shown using chimeric constructs of hPF4 and mouse (m) PF4 and chimeras with the related chemokine, neutrophil-activating peptide-2 that there is a single antigenic locus on hPF4 in these complexes to which most HIT antibodies bind. KKO is a mouse monoclonal IgG2b k anti-hPF4/polyanion monoclonal antibody that mimics pathogenic antibodies that induce HIT and provokes thrombosis in a murine model of HIT. We previously established that specific hydrolysis of N-linked glycans in the Fc-region of KKO by endoglycosidase from Streptococcus pyogenes EndoS (Genovis) yields >97% deglycosylation on LC-MS/MS generating DGKKO. This modification has no significant effect on binding to PF4-heparin complexes as shown by ELISA and by dynamic light scattering, but abrogates FcgRIIA-mediated binding and platelet activation, and decreases complement activation as evaluated by flow cytometry. To examine if DGKKO reduces prothrombotic effects, we compared DGKKO with KKO in human microfluidic system lined with human umbilical vein cells (HUVECs) that are then photochemically injured and a murine model involving “HIT mice” (mice that express FcgRIIA and human PF4 and lack mouse PF4). Using the microfluidic system described above and infusing blood from healthy donors with added human PF4 (25 µg/ml) and KKO (50 µg/ml) or HIT IgG from three individuals with SRA-positive HIT (1mg/ml) resulted in increased platelet adherence to the injured endothelium (Figure 1). Addition of DGKKO (50 µg/ml) 15 minutes after addition of HIT antibodies eliminated platelet accumulation (Figure 1). In the HIT murine model, we found that intraperitoneal (IP, 200 µg/mice) or intravenous (IV, 20 µg/mice) DGKKO did not induce thrombocytopenia in HIT mice, but reversed the thrombocytopenia induced by either IP KKO (200 µg/mice) or HIT IgG (1 mg/mice) even when the DGKKO is given 6 hrs after HIT induction (Figure 2A). We used an intravital cremaster laser arteriole injury model in HIT mice to study the efficacy of DGKKO as an antithrombotic agent. We found that unlike KKO that enhances growth of established thrombi in these mice, DGKKO significantly reversed the size of the observed thrombi (Figure 2B). These studies suggest that DGKKO obstructs the HIT antigenic site recognized by HIT antibodies and leads to a reversal of thrombocytopenia and thrombus size. Additional studies are underway to examine if DGKKO can be used as a monotherapy or adjunctive therapy in the murine model of HIT thrombosis. [Display omitted] DisclosuresCines: Rigel: Consultancy; Dova: Consultancy; Treeline: Consultancy; Arch Oncol: Consultancy; Jannsen: Consultancy; Taventa: Consultancy; Principia: Other: Data Safety Monitoring Board.

Inhibition of NADPH oxidase blocks NETosis and reduces thrombosis in heparin-induced thrombocytopenia

AbstractHeparin-induced thrombocytopenia (HIT) is associated with severe and potentially lethal thrombotic complications. NETosis was recently shown to be an important driver of thrombosis in HIT. We investigated the role of reactive oxygen species (ROS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) and their contributions to thrombus development in HIT. We showed that neutrophil activation by HIT immune complexes induced ROS-dependent NETosis. Analysis of thrombi formed in a microfluidics system showed ROS production in both platelets and neutrophils, and abundant neutrophil extracellular traps (NETs) and ROS distributed throughout the clot. Neutrophil-targeted ROS inhibition was sufficient to block HIT-induced NETosis and thrombosis using human blood. Inhibition of NOX2 with diphenyleneiodonium chloride or GSK2795039 abrogated HIT-induced thrombi in vivo using FcγRIIa+/hPF4+-transgenic mice. Thrombocytopenia in mice remained unaffected by ROS inhibition. Increased ROS production in activated neutrophils was also confirmed using fresh blood from patients with active HIT. Our findings show that ROS and NOX2 play a crucial role in NETosis and thrombosis in HIT. This enhances our understanding of the processes driving thrombosis in HIT and identifies NOX2 as a potential new therapeutic target for antithrombotic treatment of HIT.

HIT in the head: a systematic review of cerebral venous sinus thrombosis in classical and autoimmune heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) causes thrombosis and thrombocytopenia, usually due to prior heparin exposure, so-called classical HIT. However, in the autoimmune form, the signs and symptoms of HIT occur without prior heparin exposure. Development of cerebral venous sinus thrombosis (CVST) secondary to HIT is a rare occurrence, with relatively few reports in the literature. There is a need to better understand the clinical presentation and treatment paradigms in these rare cases. Therefore, we present the first systematic review of CVST occurring in classical and autoimmune HIT. Cases of HIT-induced CVST were identified through a systematic search of Pubmed from the date of inception to March 2021. Literature search revealed 21 cases of HIT and associated CVST with six cases (28.6%) of autoimmune HIT. Patients presented with signs and symptoms consistent with increased intracranial pressure, intracerebral hemorrhage (ICH), and/or focal neurologic deficits. Headache was the most common symptom with 12 patients (60.0%) presenting as such. 10 patients (47.6%) included in the study developed ICH. Non-heparin anticoagulants, especially direct thrombin inhibitors, were the first-line treatment for the majority of patients (55.6%). Intravenous immunoglobulin (IVIG) was used as treatment for select patients (16.7%) with autoimmune HIT. Few patients received surgical intervention for CVST (14.3%) or ICH (30.0%). Four patients had a full recovery, four patients had residual deficits, and seven patients ultimately expired. Symptoms of HIT-induced CVST are often related to CNS dysfunction. Non-heparin anticoagulants are important to treat CVST, even when patients have concomitant ICH, and may be supplemented with IVIG if treating autoimmune HIT. Rapid identification and treatment of HIT-induced CVST is imperative in order to prevent morbidity and mortality.

Heparin-Induced Thrombocytopenia: A Focus on Thrombosis.

Heparin-induced thrombocytopenia is an immune-mediated disorder caused by antibodies that recognize complexes of platelet factor 4 and heparin. Thrombosis is a central and unpredictable feature of this syndrome. Despite optimal management, disease morbidity and mortality from thrombosis remain high. The hypercoagulable state in heparin-induced thrombocytopenia is biologically distinct from other thrombophilic disorders in that clinical complications are directly attributable to circulating ultra-large immune complexes. In some individuals, ultra-large immune complexes elicit unchecked cellular procoagulant responses that culminate in thrombosis. To date, the clinical and biologic risk factors associated with thrombotic risk in heparin-induced thrombocytopenia remain elusive. This review will summarize our current understanding of thrombosis in heparin-induced thrombocytopenia with attention to its clinical features, cellular mechanisms, and its management.

Evolving concepts of pathogenesis of heparin‐induced thrombocytopenia: Diagnostic and therapeutic implications

Heparin-induced thrombocytopenia (HIT) is an immune reaction to heparin. It often causes severe thrombosis which may lead to limb gangrene and thrombosis-associated death. The concept of its pathogenesis has been evolving during the past five decades. Initially, HIT was thought to be caused by disseminated intravascular coagulation. Later it became clear that HIT was mediated by an immune mechanism whereby an IgG antibody induced platelet aggregation, release of procoagulant materials and consequently thrombus formation. The antigen comprises Platelet Factor 4 (PF4) and heparin which have a tendency to form ultralarge complexes. The HIT immune response has atypical features. IgG antibody appears early without IgM precedence and lasts transiently. One explanation is that there is prior priming by bacterial infection. Another unique characteristic is that it is processed as if it is a particulate antigen involving complement activation and B cells. Antigen-presenting cells/monocytes are also involved but the role of T cells is controversial. Recent advances have provided new insights into the underlying mechanisms of HIT-related thrombosis. Previously, platelets were believed to play a central role; their activation and consequently the induction of blood coagulation was the basis of the hypercoagulability in HIT. More recently, several studies have provided clear evidence that neutrophil and NETosis, monocytes and endothelial cells contribute significantly to the thrombosis in HIT. These new insights may result in development of better diagnostic laboratory assays and more effective treatments for HIT.

Predictive value of HIT-antibodies detection for new thrombosis in heparin-induced thrombocytopenia

Objective To investigate the predictive value of HIT-antibodies(HIT-Ab) detection for new thrombus in suspected Heparin-Induced thrombocytopenia (HIT). Methods Retrospective cohort study. 472 suspected HIT patients were collected from July 2016 to November 2018, and all subjects underwent a 4Ts score and were sent for HIT-Ab tests. According to the results of HIT-Ab, there were four groups: 412 cases of negative HIT-Ab (0-0.9 U/ml), 45 cases of weak-positive HIT-Ab (1.0-4.9 U/ml), 12 cases of moderate-positive HIT-Ab (5.0-15.9 U/ml), and 3 cases of strong-positive HIT-Ab (≥16.0 U/ml) respectively. Ultrasound or CT examination was used to confirm new thrombosis as a standard to evaluate the value of HIT-Ab for predicting new thrombus. The diagnostic efficacy of HIT-Ab for HIT was evaluated in clinically confirmed HIT. Results The incidence rates of new thrombus in each group were: 15.8% in Negative HIT-Ab group (62/412), 48.9% in Weak-positiveHIT-Ab group (22/45), 75.0% in Moderate-positive HIT-Ab group (9/12), and100% in Strong-positive HIT-Ab group (3/3) (P<0.00) . When HIT-Ab≥1.0 U/ml, the specificity for diagnosing new thrombus was 93.0%, the sensitivity was 34.2%, the negative predictive value (NPV) was 84.2%, and the positive predictive value (PPV) was 56.5%. The diagnostic rates of HIT in each group were: negative 0% (0/412), weak-positive 62.2% (28/45), moderate-positive (12/12) and strong-positive (3/3) were 100%. When HIT-Ab ≥ 1.0 U/ml, the specificity for HIT diagnosis was 96.0%, the sensitivity was 100%, NPV was 100%, and PPV was 71.5%. Conclusions In suspected HIT patients, the incidence of new thrombosis increases with the elevated HIT-Ab level. HIT-Ab detection can be used as a crucial tool for new thrombosis prediction and HIT diagnosis in suspected HIT patients. Clinicians can develop treatment strategies based on HIT-Ab levels. Key words: Thrombocytopenia; Autoantibodies; Heparin; Venous thrombosis; Forecasting

Neutrophil activation and NETosis are the major drivers of thrombosis in heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia/thrombosis (HIT) is a serious immune reaction to heparins, characterized by thrombocytopenia and often severe thrombosis with high morbidity and mortality. HIT is mediated by IgG antibodies against heparin/platelet factor 4 antigenic complexes. These complexes are thought to activate platelets leading to thrombocytopenia and thrombosis. Here we show that HIT immune complexes induce NETosis via interaction with FcγRIIa on neutrophils and through neutrophil-platelet association. HIT immune complexes induce formation of thrombi containing neutrophils, extracellular DNA, citrullinated histone H3 and platelets in a microfluidics system and in vivo, while neutrophil depletion abolishes thrombus formation. Absence of PAD4 or PAD4 inhibition with GSK484 abrogates thrombus formation but not thrombocytopenia, suggesting they are induced by separate mechanisms. NETs markers and neutrophils undergoing NETosis are present in HIT patients. Our findings demonstrating the involvement of NETosis in thrombosis will modify the current concept of HIT pathogenesis and may lead to new therapeutic strategies.

Neutrophil Activation and Netosis Are the Key Drivers of Thrombosis in Heparin-Induced Thrombocytopenia

Adverse drug effects are common in clinical practice and often have a negative impact on patient safety. Heparin and heparin-derived drugs may induce an immune reaction, termed heparin-induced thrombocytopenia (HIT). HIT is mediated by IgG antibodies with specificity for heparin/platelet factor 4 (PF4) antigenic complexes. HIT is a hypercoagulable state which often causes severe and extensive thrombosis that results in high morbidity and mortality. The prevailing view is that these immune complexes activate platelets via FcγRIIa receptors leading to thrombocytopenia and thrombosis.Neutrophil extracellular traps (NETs) are DNA-containing structures released by neutrophils that are increasingly being reported in patients with infection and thrombosis associated with various autoimmune and non-immune disorders. Here we show that HIT immune complexes directly activate neutrophils via FcγRIIa and induce NETs formation. In addition, NETosis is also induced by activated platelets/neutrophil interactions mediated by P-selecting and PSGL-1. Ex vivo reconstitution of the HIT condition in a microfluidics system demonstrated the formation of thrombi rich in platelets, neutrophils, extracellular DNA and citrullinated histone 3 (Figure 1A, left panels). Neutrophil depletion abolished thrombus formation. Conversely neutrophil reconstitution restored thrombus deposition (Figure 1A, middle panels and right panels). Moreover, neutrophils alone treated with HIT IgG plus heparin formed thrombi containing extracellular DNA networks and citrullinated histone 3 on P-selectin coated channels (Figure 1B).Establishment of HIT in hFcγRIIa+/hPF4+ transgenic mice (HIT mice) using HIT patient's IgG or the HIT-like monoclonal antibody KKO recapitulated the hallmarks of NETosis: citrullinated histone 3, cell free DNA and MPO were detected in plasma and the presence of neutrophils, extracellular DNA and citrullinated histone 3 was found in lung thrombi. Low density neutrophils were also present in HIT mice treated with HIT IgG plus heparin but not in animals treated with control IgG. Treatment of HIT mice with DNase I or NETs formation inhibition with the PAD4 inhibitor GSK484 led to a dramatic decrease in thrombosis. This was corroborated by deletion of PAD4 in HIT mice. No thrombi were detected in hFcγRIIa+/hPF4+/PAD4-/- mice treated with HIT IgG and heparin (Figure 1C), indicating that NETs formation is required for thrombosis. Unlike thrombosis, thrombocytopenia was not affected by the absence of NETs formation, suggesting that these are separable processes. However, they are FcγRIIa-mediated mechanisms as anti-FcγRIIa antibodies abolished both processes. Analyses of sera from HIT patients revealed the presence of NETs markers and a significant proportion of neutrophils from patients with active HIT were undergoing NETosis. Our observations demonstrate that NETs formation is present in HIT and that it is essential for the development of thrombosis. Thrombocytopenia is not affected by the absence of NETosis. These findings suggest a new concept of the pathogenesis of thrombosis in HIT and as such are of clinical significance. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Neutrophil accumulation and NET release contribute to thrombosis in HIT.

Heparin-induced thrombocytopenia (HIT) is an immune-mediated thrombocytopenic disorder associated with a severe prothrombotic state. We investigated whether neutrophils and neutrophil extracellular traps (NETs) contribute to the development of thrombosis in HIT. Using an endothelialized microfluidic system and a murine passive immunization model, we show that HIT induction leads to increased neutrophil adherence to venous endothelium. In HIT mice, endothelial adherence is enhanced immediately downstream of nascent venous thrombi, after which neutrophils undergo retrograde migration via a CXCR2-dependent mechanism to accumulate into the thrombi. Using a microfluidic system, we found that PF4 binds to NETs, leading them to become compact and DNase resistant. PF4-NET complexes selectively bind HIT antibodies, which further protect them from nuclease digestion. In HIT mice, inhibition of NET formation through Padi4 gene disruption or DNase treatment limited venous thrombus size. PAD4 inactivation did affect arterial thrombi or severity of thrombocytopenia in HIT. Thus, neutrophil activation contributes to the development of venous thrombosis in HIT by enhancing neutrophil-endothelial adhesion and neutrophil clot infiltration, where incorporated PF4-NET-HIT antibody complexes lead to thrombosis propagation. Inhibition of neutrophil endothelial adhesion, prevention of neutrophil chemokine-dependent recruitment of neutrophils to thrombi, or suppression of NET release should be explored as strategies to prevent venous thrombosis in HIT.

TROMBOSITOPENIA PADA PENGOBATAN DENGAN HEPARIN

Heparin induced thrombocytopenia (HIT), a well known side effect of heparin therapy, occurs in 1–5% of adults exposed to heparin.Unlike other drug induced thrombocytopenia, HIT does not usually cause bleeding, but instead cause thrombosis about 50% of HIT.The thrombosis in HIT can lead to limb gangrene or even death. The Importance to know the HIT is the wide use of heparin led tothe increasing recognition of untoward complications including HIT, relatively uncommon but severe side effect of heparin therapy,unpredictable, and difficulty in diagnosing and treating HIT. HIT is mediated by an antibody that recognizes an epitope on the plateletfactor (PF4)-heparin complex. The platelet factor (PF4)-heparin complex binds to FcgRII receptor on the platelet surface and crosslinksthereceptors.Thisinducesintenseplateletactivationandaggregationandsimultaneouslyactivatesbloodcoagulationpathways,thesechangesareprobablythebasisofthethrombosiseventsinHIT.HITwasclassifiedintotype1and2baseonthepathogenesisandtheseverityofHIT.RegularplateletcountmonitoringisbestsuitedforearlydiagnosisofHIT.Functional(serotoninrelease,plateletaggregationtest)andantigenassays(solidphaseenzymeimmunoassay,fluidphase,andparticlegelimmunoassay)areavailabletoconfirmHIT.HITwasmadebaseontheclinicalfindingandlaboratoryexamination.OnceHITisclinicallysuspected,heparinshouldbestoppedimmediatelyandtreatmentwithanalternativeanticoagulant,waitingforlaboratoryconfirmationmaybecatastrophic.Earlydiagnosisof HIT will decrease the morbidity and mortality.

Is low serum albumin level a significant predictor for the development of heparin-induced thrombocytopenia-thrombosis?

The present study investigates the effect of albumin levels in patients who have developed heparin-induced thrombocytopenia (HIT) and heparin-induced thrombocytopenia-thrombosis (HITT). A retrospective observational cohort study was conducted at King Abudlaziz Medical City (KAMC), a university teaching hospital, on patients diagnosed with HIT between June 2013 and December 2014. Clinical and laboratory findings were used to confirm HIT. Albumin levels were reported on admission as baseline and during HIT occurrence. Twenty-eight patients were identified as HIT positive by enzyme-linked immunosorbent assay (ELISA), with a cutoff value of ≥1 optical density units and pretest probability "4Ts" score of ≥4. Of the 28 patients, nine (32%) developed HITT. Demographic characteristics of the patients who developed HIT and HITT were similar. The mean albumin level for patients who developed HITT was significantly lower than that for patients who developed HIT (p<0.001). Our findings suggest that patients with low serum albumin levels are at greater risk of developing HITT. This finding awaits confirmation in larger prospective clinical trials.

Activated Platelet-Derived and Leukocyte-Derived Circulating Microparticles and the Risk of Thrombosis in Heparin-Induced Thrombocytopenia: A Role for PF4-Bearing Microparticles?

Though the presence of platelets-derived microparticles (MPs) have previously been described in heparin-induced thrombocytopenia (HIT), the mechanism of thrombosis in HIT remains poorly understood. We aimed to assess the presence and origin of MPs in patients with HIT and their possible contribution to HIT with thrombosis (HITT). Forty-five patients with HIT and 45 matched hospitalized patients with not confirmed HIT (HIT-negative) were enrolled. Twelve HIT patients (27%) developed HITT. MPs expressing phosphatidylserine (Annexin V-MP), activated platelet-derived (P-Selectin+), activated leukocyte-derived (L-Selectin+), PF4-bearing and tissue factor-bearing (TF+) MPs were measured by flow-cytometry. HIT patients showed significantly higher median levels of P-Selectin+, L-Selectin+, PF4-bearing, L-Selectin+/TF + MPs than HIT-negative; PF4-bearing MP showed the highest statistical difference. As compared to HIT patients, HITT patients showed a trend of higher median levels of all MP subtypes considered but the differences were not statistically significant. Only levels of activated-leukocyte/TF + MPs (L-Selectin + CD142+) were significantly higher (P = 0.015). Sensitive analyses showed that HIT patients with activated-leukocyte/TF + MPs above the cut-off (52 MP/µL) had an odds ratio (OR) for thrombosis of 3.78 (95%CI, 0.98-14.5, P = 0.045). The combination of activated-leukocyte/TF + MPs and PF4-bearing-MPs above the cut-off (1416 MP/uL) resulted in a higher risk of HITT (OR 4.49 (95% CI, 1.17-8.05, P = 0.014). We showed for the first time the presence of circulating PF4-bearing MPs derived from activated platelets in patients with HIT; activated leukocyte/TF + MPs are associated with an increased thrombotic risk. Our findings confirm that HIT antibodies complexes may determine a TF-driven prothrombotic state through the activation of platelets and leukocytes. © 2017 International Clinical Cytometry Society.