Deciphering and disrupting PIEZO1-TMEM16F interplay in hereditary xerocytosis.

ABSTRACT: Cell-surface exposure of phosphatidylserine (PS) is essential for phagocytic clearance and blood clotting. Although a calcium-activated phospholipid scramblase (CaPLSase) has long been proposed to mediate PS exposure in red blood cells (RBCs), its identity, activation mechanism, and role in RBC biology and disease remain elusive. Here, we demonstrate that TMEM16F, the long-sought-after RBC CaPLSase, is activated by calcium influx through the mechanosensitive channel PIEZO1 in RBCs. PIEZO1-TMEM16F functional coupling is enhanced in RBCs from individuals with hereditary xerocytosis (HX), an RBC disorder caused by PIEZO1 gain-of-function channelopathy. Enhanced PIEZO1-TMEM16F coupling leads to an increased propensity to expose PS, which may serve as a key risk factor for HX clinical manifestations including anemia, splenomegaly, and postsplenectomy thrombosis. Spider toxin GsMTx-4 and antigout medication benzbromarone inhibit PIEZO1, preventing force-induced echinocytosis, hemolysis, and PS exposure in HX RBCs. Our study thus reveals an activation mechanism of TMEM16F CaPLSase and its pathophysiological function in HX, providing insights into potential treatment.

Inhibition of the C1s Protease and the Classical Complement Pathway by 6-(4-Phenylpiperazin-1-yl)Pyridine-3-Carboximidamide and Chemical Analogs.

The classical pathway (CP) is a potent mechanism for initiating complement activity and is a driver of pathology in many complement-mediated diseases. The CP is initiated via activation of complement component C1, which consists of the pattern recognition molecule C1q bound to a tetrameric assembly of proteases C1r and C1s. Enzymatically active C1s provides the catalytic basis for cleavage of the downstream CP components, C4 and C2, and is therefore an attractive target for therapeutic intervention in CP-driven diseases. Although an anti-C1s mAb has been Food and Drug Administration approved, identifying small-molecule C1s inhibitors remains a priority. In this study, we describe 6-(4-phenylpiperazin-1-yl)pyridine-3-carboximidamide (A1) as a selective, competitive inhibitor of C1s. A1 was identified through a virtual screen for small molecules that interact with the C1s substrate recognition site. Subsequent functional studies revealed that A1 dose-dependently inhibits CP activation by heparin-induced immune complexes, CP-driven lysis of Ab-sensitized sheep erythrocytes, CP activation in a pathway-specific ELISA, and cleavage of C2 by C1s. Biochemical experiments demonstrated that A1 binds directly to C1s with a Kd of ∼9.8 µM and competitively inhibits its activity with an inhibition constant (Ki) of ∼5.8 µM. A 1.8-Å-resolution crystal structure revealed the physical basis for C1s inhibition by A1 and provided information on the structure-activity relationship of the A1 scaffold, which was supported by evaluating a panel of A1 analogs. Taken together, our work identifies A1 as a new class of small-molecule C1s inhibitor and lays the foundation for development of increasingly potent and selective A1 analogs for both research and therapeutic purposes.

Red cell exchange modulates neutrophil degranulation responses in sickle cell disease.

BACKGROUND: Neutrophils in sickle cell disease (SCD) are activated, contributing to disease. Red cell exchange (RCE), with the goal of lowering hemoglobin S (HbS), is an important part of therapy for many SCD patients. Whether RCE impacts neutrophil reactivity is unknown. STUDY DESIGN AND METHODS: To determine the effect of RCE on neutrophil activation, SCD patients undergoing RCE in steady-state were enrolled. Neutrophil degranulation responses were examined before/after RCE. Kinetic studies were completed to determine the duration of the effect of RCE on neutrophil function. Degranulation results were examined in relation to white blood cell count, neutrophil count, and HbS levels. The effect of RCE on RBC phosphatidylserine (PS) exposure was examined as a possible contributor to modulation of neutrophil function by RCE. RESULTS: Twenty-two patients with SCD, genotype SS, who underwent RCE (average pre-RCE HbS 33 ± 14%) were included for the study. RCE significantly decreased neutrophil degranulation responses. The effect of RCE on neutrophil activation was unrelated to cell count and instead directly correlated with HbS. The effect of RCE on neutrophil activation was sustained over several days post-apheresis. Furthermore, while increased RBC PS exposure results in increased neutrophil degranulation, RCE decreases RBC PS exposure. DISCUSSION: To our knowledge, this is the first study demonstrating that RCE significantly decreases neutrophil activation in a sustained HbS-dependent manner. Modulation of PS exposure by RCE may be a contributing mechanism by which RCE modulates neutrophil activation. These studies raise the possibility that modulation of neutrophil activation contributes significantly to the therapeutic effect of RCE.

Vaccine-induced immune thrombotic thrombocytopenia: what we know and do not know.

The development of vaccines to fight COVID-19 has been a remarkable medical achievement. However, this global immunization effort has been complicated by a rare vaccine-related outcome characterized by thrombocytopenia and thrombosis in association with platelet-activating anti-platelet factor 4 antibodies. In this Spotlight, we will discuss the recently described complication of vaccine-induced immune thrombotic thrombocytopenia (VITT) occurring in response to certain COVID-19 vaccines. Although information about this clinical condition is rapidly evolving, we will summarize our current understanding of VITT.

Complement mediates binding and procoagulant effects of ultralarge HIT immune complexes.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by ultra-large immune complexes (ULICs) containing immunoglobulin G (IgG) antibodies to a multivalent antigen composed of platelet factor 4 and heparin. The limitations of current antithrombotic therapy in HIT supports the need to identify additional pathways that may be targets for therapy. Activation of FcγRIIA by HIT ULICs initiates diverse procoagulant cellular effector functions. HIT ULICs are also known to activate complement, but the contribution of this pathway to the pathogenesis of HIT has not been studied in detail. We observed that HIT ULICs physically interact with C1q in buffer and plasma, activate complement via the classical pathway, promote codeposition of IgG and C3 complement fragments (C3c) on neutrophil and monocyte cell surfaces. Complement activation by ULICs, in turn, facilitates FcγR-independent monocyte tissue factor expression, enhances IgG binding to the cell surface FcγRs, and promotes platelet adhesion to injured endothelium. Inhibition of the proximal, but not terminal, steps in the complement pathway abrogates monocyte tissue factor expression by HIT ULICs. Together, these studies suggest a major role for complement activation in regulating Fc-dependent effector functions of HIT ULICs, identify potential non-anticoagulant targets for therapy, and provide insights into the broader roles of complement in immune complex-mediated thrombotic disorders.

Fc-modified HIT-like monoclonal antibody as a novel treatment for sepsis.

Sepsis is characterized by multiorgan system dysfunction that occurs because of infection. It is associated with high morbidity and mortality and is in need of improved therapeutic interventions. Neutrophils play a crucial role in sepsis, releasing neutrophil extracellular traps (NETs) composed of DNA complexed with histones and toxic antimicrobial proteins that ensnare pathogens, but also damage host tissues. At presentation, patients often have a significant NET burden contributing to the multiorgan damage. Therefore, interventions that inhibit NET release would likely be ineffective at preventing NET-based injury. Treatments that enhance NET degradation may liberate captured bacteria and toxic NET degradation products (NDPs) and likely be of limited therapeutic benefit as well. We propose that interventions that stabilize NETs and sequester NDPs may be protective in sepsis. We showed that platelet factor 4 (PF4), a platelet-associated chemokine, binds and compacts NETs, increasing their resistance to DNase I. We now show that PF4 increases NET-mediated bacterial capture, reduces the release of NDPs, and improves outcome in murine models of sepsis. A monoclonal antibody KKO which binds to PF4-NET complexes, further enhances DNase resistance. However, the Fc portion of this antibody activates the immune response and increases thrombotic risk, negating any protective effects in sepsis. Therefore, we developed an Fc-modified KKO that does not induce these negative outcomes. Treatment with this antibody augmented the effects of PF4, decreasing NDP release and bacterial dissemination and increasing survival in murine sepsis models, supporting a novel NET-targeting approach to improve outcomes in sepsis.

Recognition of PF4-VWF complexes by heparin-induced thrombocytopenia antibodies contributes to thrombus propagation.

Heparin-induced thrombocytopenia (HIT) is a prothrombotic disorder mediated by complexes between platelet factor 4 (PF4) and heparin or other polyanions, but the risk of thrombosis extends beyond exposure to heparin implicating other PF4 partners. We recently reported that peri-thrombus endothelium is targeted by HIT antibodies, but the binding site(s) has not been identified. We now show that PF4 binds at multiple discrete sites along the surface of extended strings of von Willebrand factor (VWF) released from the endothelium following photochemical injury in an endothelialized microfluidic system under flow. The HIT-like monoclonal antibody KKO and HIT patient antibodies recognize PF4-VWF complexes, promoting platelet adhesion and enlargement of thrombi within the microfluidic channels. Platelet adhesion to the PF4-VWF-HIT antibody complexes is inhibited by antibodies that block FcγRIIA or the glycoprotein Ib-IX complex on platelets. Disruption of PF4-VWF-HIT antibody complexes by drugs that prevent or block VWF oligomerization attenuate thrombus formation in a murine model of HIT. Together, these studies demonstrate assembly of HIT immune complexes along VWF strings released by injured endothelium that might propagate the risk of thrombosis in HIT. Disruption of PF4-VWF complex formation may provide a new therapeutic approach to HIT.

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.

Bad weed: synthetic cannabinoid-associated coagulopathy.

Recent multistate outbreaks of coagulopathy caused by brodifacoum-tainted synthetic cannabinoids or "fake weed" highlight the public health impact of long-acting anticoagulant rodenticides (LAARs). Patients presenting with this syndrome have had recent exposure to synthetic cannabinoids, evidence of isolated vitamin K antagonism with or without bleeding, and detectable levels of brodifacoum and other LAARs in circulation. This article will provide information on synthetic cannabinoids, LAARs, and coagulopathic manifestations arising from use of adulterated synthetic cannabinoids and their management.

Dynamic intercellular redistribution of HIT antigen modulates heparin-induced thrombocytopenia.

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.

Polyreactive IgM initiates complement activation by PF4/heparin complexes through the classical pathway.

The mechanisms by which exposure to heparin initiates antibody responses in many, if not most, recipients are poorly understood. We recently demonstrated that antigenic platelet factor 4 (PF4)/heparin complexes activate complement in plasma and bind to B cells. Here, we describe how this process is initiated. We observed wide stable variation in complement activation when PF4/heparin was added to plasma of healthy donors, indicating a responder "phenotype" (high, intermediate, or low). Proteomic analysis of plasma from these healthy donors showed a strong correlation between complement activation and plasma immunoglobulin M (IgM) levels (r = 0.898; P < .005), but not other Ig isotypes. Complement activation response to PF4/heparin in plasma displaying the low donor phenotype was enhanced by adding pooled IgM from healthy donors, but not monoclonal IgM. Depletion of IgM from plasma abrogated C3c generation by PF4/heparin. The complement-activating features of IgM are likely mediated by nonimmune, or natural, IgM, as cord blood and a monoclonal polyreactive IgM generate C3c in the presence of PF4/heparin. IgM facilitates complement and antigen deposition on B cells in vitro and in patients receiving heparin. Anti-C1q antibody prevents IgM-mediated complement activation by PF4/heparin complexes, indicating classical pathway involvement. These studies demonstrate that variability in plasma IgM levels correlates with functional complement responses to PF4/heparin. Polyreactive IgM binds PF4/heparin, triggers activation of the classical complement pathway, and promotes antigen and complement deposition on B cells. These studies provide new insights into the evolution of the heparin-induced thrombocytopenia immune response and may provide a biomarker of risk.

Heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) is an immune complication of heparin therapy caused by antibodies to complexes of platelet factor 4 (PF4) and heparin. Pathogenic antibodies to PF4/heparin bind and activate cellular FcγRIIA on platelets and monocytes to propagate a hypercoagulable state culminating in life-threatening thrombosis. It is now recognized that anti-PF4/heparin antibodies develop commonly after heparin exposure, but only a subset of sensitized patients progress to life-threatening complications of thrombocytopenia and thrombosis. Recent scientific developments have clarified mechanisms underlying PF4/heparin immunogenicity, disease susceptibility, and clinical manifestations of disease. Insights from clinical and laboratory findings have also been recently harnessed for disease prevention. This review will summarize our current understanding of HIT by reviewing pathogenesis, essential clinical and laboratory features, and management.

The antigenic complex in HIT binds to B cells via complement and complement receptor 2 (CD21).

Heparin-induced thrombocytopenia is a prothrombotic disorder caused by antibodies to platelet factor 4 (PF4)/heparin complexes. The mechanism that incites such prevalent anti-PF4/heparin antibody production in more than 50% of patients exposed to heparin in some clinical settings is poorly understood. To investigate early events associated with antigen exposure, we first examined the interaction of PF4/heparin complexes with cells circulating in whole blood. In healthy donors, PF4/heparin complexes bind preferentially to B cells (>90% of B cells bind to PF4/heparin in vitro) relative to neutrophils, monocytes, or T cells. Binding of PF4 to B cells is heparin dependent, and PF4/heparin complexes are found on circulating B cells from some, but not all, patients receiving heparin. Given the high proportion of B cells that bind PF4/heparin, we investigated complement as a mechanism for noncognate antigen recognition. Complement is activated by PF4/heparin complexes, co-localizes with antigen on B cells from healthy donors, and is present on antigen-positive B cells in patients receiving heparin. Binding of PF4/heparin complexes to B cells is mediated through the interaction between complement and complement receptor 2 (CR2 [CD21]). To the best of our knowledge, these are the first studies to demonstrate complement activation by PF4/heparin complexes, opsonization of PF4/heparin to B cells via CD21, and the presence of complement activation fragments on circulating B cells in some patients receiving heparin. Given the critical contribution of complement to humoral immunity, our observations provide new mechanistic insights into the immunogenicity of PF4/heparin complexes.

Indications for and outcomes of therapeutic plasma exchange after cardiac transplantation: A single center retrospective study.

INTRODUCTION: Limited data are available describing indications for and outcomes of therapeutic plasma exchange (TPE) in cardiac transplantation. METHODS: In a retrospective study of patients who underwent cardiac transplantation at Duke University Medical Center from 2010 to 2014, we reviewed 3 TPE treatment patterns: a Single TPE procedure within 24 h of transplant; Multiple TPE procedures initiated within 24 h of transplant; and 1 or more TPE procedures beginning >24 h post-transplant. Primary and secondary outcomes were overall survival (OS) and TPE survival (TS), respectively. RESULTS: Of 313 patients meeting study criteria, 109 (35%) underwent TPE. TPE was initiated in 82 patients within 24 h, 40 (37%) receiving a single procedure (Single TPE), and 42 (38%) multiple procedures (Multiple TPE). Twenty-seven (25%) began TPE >24 h after transplant (Delayed TPE). The most common TPE indication was elevated/positive panel reactive or human leukocyte antigen antibodies (32%). With a median follow-up of 49 months, the non-TPE treated and Single TPE cohorts had similar OS (HR 1.08 [CI, 0.54, 2.14], P = .84), while the Multiple and Delayed TPE cohorts had worse OS (HR 2.62 [CI, 1.53, 4.49] and HR 1.98 [CI, 1.02, 3.83], respectively). The Multiple and Delayed TPE cohorts also had worse TS (HR 2.59 [CI, 1.31, 5.14] and HR 3.18 [CI, 1.56, 6.50], respectively). Infection rates did not differ between groups but was independently associated with OS (HR 2.31 [CI, 1.50, 3.54]). CONCLUSIONS: TPE is an important therapeutic modality in cardiac transplant patients. Prospective studies are needed to better define TPE's different roles in this patient population.

Changing practice of anticoagulation: will target-specific anticoagulants replace warfarin?

The target-specific oral anticoagulants are a class of agents that inhibit factor Xa or thrombin. They are effective and safe compared to warfarin for the prevention of stroke and systemic embolism in patients with atrial fibrillation and for the treatment of venous thromboembolism, and they are comparable to low-molecular-weight heparin for thromboprophylaxis after hip or knee arthroplasty. For other indications, however, such as the prevention of stroke in patients with mechanical heart valves, initial studies have been unfavorable for the newer agents, leaving warfarin the anticoagulant of choice. Further studies are needed before the target-specific anticoagulants can be recommended for patients with cancer-associated thrombosis or heparin-induced thrombocytopenia. Concerns also persist about difficulties with the laboratory assessment of anticoagulant effect and the lack of a specific reversal agent. For these reasons, we anticipate that the vitamin K antagonists will continue to be important anticoagulants for years to come.

Etiology and complications of thrombocytopenia in hospitalized medical patients.

To determine incidence, risk factors, hematologic complications, and prognostic significance of thrombocytopenia in the general medicine population, we performed a single-institutional, retrospective study of all adult patients admitted to a general medical ward from January 1st, 2014 to December 31st, 2014 with hospital-acquired thrombocytopenia. Those with moderate thrombocytopenia, defined as a platelet count nadir of <100 × 10^9/L and/or a >50% relative decline, were compared to those with less severe thrombocytopenia. Of the 7420 patients admitted, 465 (6.3%) developed hospital-acquired thrombocytopenia. Infection and moderate thrombocytopenia were present in 56 and 23%, respectively. Severe sepsis and antibiotic use were both associated with moderate thrombocytopenia, and proton pump inhibitor use was statistically significant in both univariate and multivariate analysis. Hematologic complications were more frequent with moderate thrombocytopenia, including frequency of HIT testing and red blood cell transfusions. Outcome metrics including transfer to an intensive care unit (OR 6.78), death during admission (OR 6.85), and length of stay (10.6 vs. 5.1 days) were all associated with moderate thrombocytopenia. Thrombocytopenia is associated with poor prognosis, and the association between moderate thrombocytopenia and proton pump inhibitor use is relatively novel and should be validated in prospective studies.

Thrombocytopenia in hospitalized patients with severe clostridium difficile infection.

Clostridium difficile infection (CDI) is a common cause of nosocomial diarrhea and colitis. The incidence and prognostic significance of thrombocytopenia as related to mode of acquisition (hospital vs. community), NAP1/027 strain, and disease severity has not been examined. We performed a single-institution retrospective analysis of all adult inpatients from 2013 to 2014 diagnosed with CDI during their hospitalization to document the incidence/prevalence of thrombocytopenia and associated outcomes. Severe disease was defined by a composite endpoint of inpatient death, death within 30 days of discharge, presence of septic shock, or need for colectomy during hospitalization. Of the 533 patients diagnosed with CDI, moderate thrombocytopenia (platelet count <100 × 109/L at time of CDI diagnosis) was present in 15 % of the total cohort and incident thrombocytopenia developed in 3 % of patients after admission. Thrombocytopenia was more common in hospital-acquired disease and associated with increased length of stay, but was not associated with treatment failure. Those with moderate thrombocytopenia were more likely to have severe disease, after controlling for white blood cell count, albumin, and creatinine. Moderate thrombocytopenia is associated with poor prognosis and is a potential risk stratification tool for severe CDI.

The thrombotic microangiopathy Registry of North America: A United States multi-institutional TMA network.

The thrombotic microangiopathy (TMA) Registry Network of North America (TRNA) is a collaborative network organized for the purpose of developing a multi-institutional registry and network to conduct clinical studies in a rare patient population. The TRNA was founded in 2013 by four academic medical centers (Columbia University Medical Center, Duke University Medical Center, University of Alabama at Birmingham, and University of Pennsylvania) to develop a national and demographically diverse dataset of patients with TMA. A clinical database was developed by network members using REDCap (Research Electronic Data Capture), a web-based database developed for clinical research. To facilitate rapid Institutional Review Board (IRB) approval at multiple sites, the TRNA utilized IRBshare, a streamlined IRB process to allow patient recruitment and enrollment into the TMA registry. This article reviews the process used to establish the TRNA network and discusses the significance of the first multi-institutional clinical apheresis network developed in the United States. J. Clin. Apheresis 31:448-453, 2016. © 2015 Wiley Periodicals, Inc.