Coagulation and platelet biology at the intersection of health and disease: illustrated capsules of the 11th Symposium on Hemostasis at the University of North Carolina.

The University of North Carolina Symposia on Hemostasis began in 2002, with The First Symposium on Hemostasis with a Special Focus on FVIIa and Tissue Factor. They have occurred biannually since and have maintained the primary goal of establishing a forum for the sharing of outstanding advances made in the basic sciences of hemostasis. The 2024 11th Symposium on Hemostasis will bring together leading scientists from around the globe to present and discuss the latest research related to coagulation factors and platelet biology. In keeping with the tradition of the conference, we expect novel cross-disciplinary collaborations to result from bringing together fundamental scientists and physician-scientists from different backgrounds and perspectives. The aim of these collaborations is to springboard the next generation of important advances in the field. This year's program was designed to discuss Coagulation and Platelet Biology at the Intersection of Health and Disease. The goal is to develop a better understanding of the pathophysiologic mechanisms leading to hemostatic and thrombotic disorders as this understanding is critical for the continued development of safe and efficacious therapeutics. Included in this review article are illustrated capsules provided by our speakers that highlight the main conclusions of the invited talks.

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.

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

Heparin-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.

Clot Imaging Using Photostable Nanodiamond.

While thrombosis is the leading cause of morbidity and mortality in the United States, an understanding of its triggers, progression, and response to anticoagulant therapy is lacking. Intravital fluorescence microscopy has advanced the study of thrombus formation by providing targeted, multi-color contrast. However, photodegradation of fluorophores limits the application in longitudinal studies (e.g., clot progression and/or dissolution). Fluorescent nanodiamond (FND) is a fluorophore which utilizes intrinsic fluorescence of chromogenic centers within and protected by the diamond crystalline lattice. Recent developments in diamond processing have allowed for the controlled production of nanodiamonds emitting in green or red. Here, the use of FND to label blood clots and/or clot lysis is demonstrated and compared to commonly used organic fluorophores. Model ex vivo clots were formed with incorporated labeled fibrinogen to allow imaging. FND was shown to match the morphology of organic fluorophore labels absent of photobleaching over time. The addition of tissue plasminogen activator (tPa) allowed visualization of the clot lysis stage, which is vital to studies of both DVT and pulmonary embolism resolution.

Modulation of ultralarge immune complexes in heparin-induced thrombocytopenia.

BACKGROUND: Heparin-induced thrombocytopenia (HIT) is a serious thrombotic disorder caused by ultralarge immune complexes (ULICs) containing platelet factor 4 (PF4) and heparin that form the HIT antigen, together with a subset of anti-PF4 antibodies. ULICs initiate prothrombotic responses by engaging Fcγ receptors on platelets, neutrophils, and monocytes. Contemporary anti-thrombotic therapy for HIT is neither entirely safe nor entirely successful and acts downstream of ULIC formation and Fcγ receptor-initiated generation of thrombin. OBJECTIVES: To determine whether HIT antigen and ULIC formation and stability could be modified favorably by inhibiting PF4-heparin interactions with fondaparinux, together with blocking formation of PF4 tetramers using a humanized monoclonal anti-PF4 antibody (hRTO). METHODS: Results: The combination of fondaparinux and hRTO inhibited HIT antigen formation, promoted antigen dissociation, inhibited ULIC formation, and promoted ULIC disassembly at concentrations below the effective concentration of either alone and blocked Fcγ receptor-dependent induction of factor Xa activity by monocytic THP1 cells and activation of human platelets in whole blood. Combined with hRTO, fondaparinux inhibited HIT antigen and immune complex formation and activation through Fcγ receptors at concentrations at or below those used clinically to inhibit FXa coagulant activity. CONCLUSIONS: HIT antigen and immune complexes are dynamic and amenable to modulation. Fondaparinux can be converted from an anticoagulant that acts at a downstream amplification step into a rationale, disease-specific intervention that blocks ULIC formation. Interventions that prevent ULIC formation and stability might increase the efficacy, permit use of lower doses, shorten the duration of antithrombotic therapy, and help prevent this serious thrombotic disorder.

Minimal role for the alternative pathway in complement activation by HIT immune complexes.

BACKGROUND: Anti-platelet factor 4 (PF4)/heparin immune complexes that cause heparin-induced thrombocytopenia (HIT) activate complement via the classical pathway. Previous studies have shown that the alternative pathway of complement substantially amplifies the classical pathway of complement activation through the C3b feedback cycle. OBJECTIVES: These studies sought to examine the contributions of the alternative pathway to complement activation by HIT antibodies. METHODS: Using IgG monoclonal (KKO) and/or patient-derived HIT antibodies, we compared the effects of classical pathway (BBK32 and C1-esterase inhibitor [C1-INH]), alternative pathway (anti-factor B [fB] or factor D [fD] inhibitor) or combined classical and alternative pathway inhibition (soluble complement receptor 1 [sCR1]) in whole blood or plasma. RESULTS: Classical pathway inhibitors BBK32 and C1-INH and the combined classical/alternative pathway inhibitor sCR1 prevented KKO/HIT immune complex-induced complement activation, including release of C3 and C5 activation products, binding of immune complexes to B cells, and neutrophil activation. The alternative pathway inhibitors fB and fD, however, did not affect complement activation by KKO/HIT immune complexes. Similarly, alternative pathway inhibition had no effect on complement activation by unrelated immune complexes consisting of anti-dinitrophenyl (DNP) antibody and the multivalent DNP--keyhole limpet hemocyanin antigen. CONCLUSIONS: Collectively, these findings suggest the alternative pathway contributes little in support of complement activation by HIT immune complexes. Additional in vitro and in vivo studies are required to examine if this property is shared by most IgG-containing immune complexes or if predominance of the classic pathway is limited to immune complexes composed of multivalent antigens.

Neutrophil functional heterogeneity is a fixed phenotype and is associated with distinct gene expression profiles.

Differences in the ability of neutrophils to perform relevant effector functions has been identified in a variety of disease states. Although neutrophil functional heterogeneity is increasingly recognized during disease, few studies have examined neutrophil functional heterogeneity during periods of health. In this study, we systematically characterize neutrophil functional heterogeneity in a cohort of healthy human subjects using a range of biologically relevant agonists including immune complexes, bacterial ligands, and pathogens. With repeated testing over several years, we show that neutrophil functional capability represents a fixed phenotype for each individual. This neutrophil phenotype is preserved across a range of agonists and extends to a variety of effector functions including degranulation, neutrophil extracellular trap release, reactive oxygen species generation, phagocytosis, and bacterial killing. Using well-phenotyped healthy human subjects, we demonstrate that neutrophil functional heterogeneity is characterized by differences in neutrophil gene expression patterns. Altogether, our findings demonstrate that while neutrophil function is highly heterogeneous among healthy subjects, each individual's functional capability represents a fixed phenotype defined by a distinct neutrophil gene expression profile. These findings may be relevant during disease states where the ability to perform relevant neutrophil effector functions may impact disease course and/or clinical outcome.

Aptamer-based factor IXa inhibition preserves hemostasis and prevents thrombosis in a piglet model of ECMO.

Extracorporeal membrane oxygenation (ECMO) requires anticoagulation to prevent clotting when the patient's blood contacts the circuit. Unfractionated heparin (UFH) usually prevents clotting but can cause life-threatening bleeding. An anticoagulant that selectively inhibits the contact activation (intrinsic) pathway while sparing the tissue factor (extrinsic) pathway of coagulation might prevent clotting triggered by the circuit while permitting physiologic coagulation at surgical sites. DTRI-178 is an RNA anticoagulant aptamer conjugated to polyethylene glycol that increases its half-life in circulation. This aptamer is based on a previously described molecule (9.3t) that inhibits intrinsic tenase activity by binding to factor IXa on an exosite. Using a piglet model of pediatric venoarterial (VA) ECMO, we compared thromboprevention and blood loss using a single dose of DTRI-178 versus UFH. In each of five experiments, we subjected two litter-matched piglets, one anticoagulated with DTRI-178 and the other with UFH, to simultaneous 12-h periods of VA ECMO. Both anticoagulants achieved satisfactory and comparable thromboprotection. However, UFH piglets had increased surgical site bleeding and required significantly greater blood transfusion volumes than piglets anticoagulated with DTRI-178. Our results indicate that DTRI-178, an aptamer against factor IXa, may be feasible, safer, and result in fewer transfusions and clinical bleeding events in ECMO.

C3 complement inhibition prevents antibody-mediated rejection and prolongs renal allograft survival in sensitized non-human primates.

Sensitized kidney transplant recipients experience high rates of antibody-mediated rejection due to the presence of donor-specific antibodies and immunologic memory. Here we show that transient peri-transplant treatment with the central complement component C3 inhibitor Cp40 significantly prolongs median allograft survival in a sensitized nonhuman primate model. Despite donor-specific antibody levels remaining high, fifty percent of Cp40-treated primates maintain normal kidney function beyond the last day of treatment. Interestingly, presence of antibodies of the IgM class associates with reduced median graft survival (8 vs. 40 days; p = 0.02). Cp40 does not alter lymphocyte depletion by rhesus-specific anti-thymocyte globulin, but inhibits lymphocyte activation and proliferation, resulting in reduced antibody-mediated injury and complement deposition. In summary, Cp40 prevents acute antibody-mediated rejection and prolongs graft survival in primates, and inhibits T and B cell activation and proliferation, suggesting an immunomodulatory effect beyond its direct impact on antibody-mediated injury.

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.

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.

The Developing Balance of Thrombosis and Hemorrhage in Pediatric Surgery: Clinical Implications of Age-Related Changes in Hemostasis.

Bleeding and thrombosis in critically ill infants and children is a vexing clinical problem. Despite the relatively low incidence of bleeding and thrombosis in the overall pediatric population relative to adults, these critically ill children face unique challenges to hemostasis due to extreme physiologic derangements, exposure of blood to foreign surfaces and membranes, and major vascular endothelial injury or disruption. Caring for pediatric patients on extracorporeal support, recovering from solid organ transplant or invasive surgery, and after major trauma is often complicated by major bleeding or clotting events. As our ability to care for the youngest and sickest of these children increases, the gaps in our understanding of the clinical implications of developmental hemostasis have become increasingly important. We review the current understanding of the development and function of the hemostatic system, including the complex and overlapping interactions of coagulation proteins, platelets, fibrinolysis, and immune mediators from the neonatal period through early childhood and to young adulthood. We then examine scenarios in which our ability to effectively measure and treat coagulation derangements in pediatric patients is limited. In these clinical situations, adult therapies are often extrapolated for use in children without taking age-related differences in pediatric hemostasis into account, leaving clinicians confused and impacting patient outcomes. We discuss the limitations of current coagulation testing in pediatric patients before turning to emerging ideas in the measurement and management of pediatric bleeding and thrombosis. Finally, we highlight opportunities for future research which take into account this developing balance of bleeding and thrombosis in our youngest patients.

Pathogenesis of heparin-induced thrombocytopenia.

There are currently no effective substitutes for high intensity therapy with unfractionated heparin (UFH) for cardiovascular procedures based on its rapid onset of action, ease of monitoring and reversibility. The continued use of UFH in these and other settings requires vigilance for its most serious nonhemorrhagic complication, heparin induced thrombocytopenia (HIT). HIT is an immune prothrombotic disorder caused by antibodies that recognize complexes between platelet factor 4 (PF4) and polyanions such as heparin (H).The pathogenicity of anti-PF4/H antibodies is likely due to the formation of immune complexes that initiate intense procoagulant responses by vascular and hematopoietic cells that lead to the generation of platelet microparticles, monocyte and endothelial cell procoagulant activity, and neutrophil extracellular traps, among other outcomes. The development of anti-PF4/H antibodies after exposure to UFH greatly exceeds the incidence of clinical disease, but the biochemical features that distinguish pathogenic from nonpathogenic antibodies have not been identified. Diagnosis relies on pretest clinical probability, screening for anti-PF4/H antibodies and documentation of their platelet activating capacity. However, both clinical algorithms and test modalities have limited predictive values making diagnosis and management challenging. Given the unacceptable rates of recurrent thromboembolism and bleeding associated with current therapies, there is an unmet need for novel rational nonanticoagulant therapeutics based on the pathogenesis of HIT. We will review recent developments in our understanding of the pathogenesis of HIT and its implications for future approaches to diagnosis and management.

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.

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.

Heterogeneity in neutrophil responses to immune complexes.

Immune complexes (ICs) can trigger inflammation and thrombosis, in part, by activating neutrophils. Much attention has focused on the serologic characteristics of ICs and Fc receptors associated with cellular activation, but few studies have examined host susceptibility to neutrophil activation by ICs. Here, we use a novel whole blood system to investigate the ability of ICs to cause neutrophil activation and degranulation. Using monoclonal anti-platelet factor 4/heparin (PF4/heparin), anti-protamine/heparin antibodies, patient-derived anti-PF4/heparin antibodies, and heat-aggregated immunoglobulin G as model ICs, we demonstrate that heparin-containing ICs cause robust, heparin-dependent neutrophil activation and degranulation which is mediated by both FcγRIIa and complement. Longitudinal testing over a 1-year period shows that an individual's neutrophil response to ICs represents a fixed phenotype resulting in high, intermediate, or low reactivity. Examination of individuals at the extremes of reactivity (high vs low) shows that phenotypic variation resides in the cellular compartment and is correlated with host white blood cell count and absolute neutrophil count, but not age, sex, race, polymorphisms in neutrophil Fcγ receptors, or CR1, CR3, and Fcγ receptor expression on neutrophils. Together, these studies demonstrate that susceptibility to neutrophil activation by ICs is intrinsic to the host and is likely genetic in origin. These findings may be relevant to the heterogeneous clinical outcomes seen in patients with heparin-induced thrombocytopenia and other IC-mediated disorders and could potentially identify patients at high risk for thrombotic and inflammatory complications.