A novel leukocyte adhesion deficiency type III mutation manifests functional importance of the compact FERM domain in kindlin-3.

BACKGROUND: Leukocyte adhesion deficiency III (LAD-III) is a rare autosomal recessive syndrome characterized by functional deficiencies of platelets and leukocytes that occurs due to mutations in the FERMT3 gene encoding kindlin-3. Kindlin-3 is a FERM domain-containing adaptor protein that is essential in integrin activation. We have previously demonstrated that the FERM domain of kindlin-3 is structurally compact and plays an important role in supporting integrin activation in a mouse model. The impact of destabilizing the compact FERM domain in kindlin-3 on the development of LAD-III in humans remains uncertain. OBJECTIVES: To use primary cells from a patient with LAD-III to validate the role of the compact FERM domain in kindlin-3 function in platelets and leukocytes. METHODS: The patient is a 4-year-old girl who since infancy has displayed clinical features of LAD-III. Patient platelets and leukocytes were functionally analyzed, and structural analysis of the kindlin-3 variant was conducted. RESULTS: We identified a novel homozygous missense mutation in the FERMT3 (c.412G>A, p.E138K) FERM domain. Substantially reduced levels of kindlin-3 were detected in the proband's platelets and leukocytes. Functional evaluation verified that integrin αIIbß3-mediated platelet activation, spreading, and aggregation and ß2-integrin-mediated neutrophil adhesion and spreading were significantly compromised. Structural analysis revealed that this newly identified E138K substitution in kindlin-3 destabilizes the compacted FERM domain, resulting in poor expression of kindlin-3 in blood cells and subsequent LAD-III. CONCLUSION: We have identified a novel missense mutation and verified the functional significance of the compact kindlin-3 FERM domain in supporting integrin functions in platelets and leukocytes.

Plasma tissue-type plasminogen activator is associated with lipoprotein(a) and clinical outcomes in hospitalized patients with COVID-19.

Background: Patients with COVID-19 have a higher risk of thrombosis and thromboembolism, but the underlying mechanism(s) remain to be fully elucidated. In patients with COVID-19, high lipoprotein(a) (Lp(a)) is positively associated with the risk of ischemic heart disease. Lp(a), composed of an apoB-containing particle and apolipoprotein(a) (apo(a)), inhibits the key fibrinolytic enzyme, tissue-type plasminogen activator (tPA). However, whether the higher Lp(a) associates with lower tPA activity, the longitudinal changes of these parameters in hospitalized patients with COVID-19, and their correlation with clinical outcomes are unknown. Objectives: To assess if Lp(a) associates with lower tPA activity in COVID-19 patients, and how in COVID-19 populations Lp(a) and tPA change post infection. Methods: Endogenous tPA enzymatic activity, tPA or Lp(a) concentration were measured in plasma from hospitalized patients with and without COVID-19. The association between plasma tPA and adverse clinical outcomes was assessed. Results: In hospitalized patients with COVID-19, we found lower tPA enzymatic activity and higher plasma Lp(a) than that in non-COVID-19 controls. During hospitalization, Lp(a) increased and tPA activity decreased, which associates with mortality. Among those who survived, Lp(a) decreased and tPA enzymatic activity increased during recovery. In patients with COVID-19, tPA activity is inversely correlated with tPA concentrations, thus, in another larger COVID-19 cohort, we utilized plasma tPA concentration as a surrogate to inversely reflect tPA activity. The tPA concentration was positively associated with death, disease severity, plasma inflammatory, and prothrombotic markers, and with length of hospitalization among those who were discharged. Conclusion: High Lp(a) concentration provides a possible explanation for low endogenous tPA enzymatic activity, and poor clinical outcomes in patients with COVID-19.

Implementation of a Bleeding Risk Screening Tool and Hematology Referral Process Prior to Pediatric Endoscopy.

OBJECTIVES: Gastrointestinal (GI) endoscopic procedures are considered low risk with an overall bleeding risk for upper and lower endoscopies of 0.11%. However, a certain population of patients may have a higher risk for bleeding, and there is not a standardized process for screening patients to determine who these patients are. METHODS: At Children's Wisconsin, our gastroenterology and hematology divisions adapted an abbreviated version of a validated, history-based bleeding risk screening tool and implemented a hematology referral process to identify those at risk for bleeding prior to their first endoscopy. Provider compliance with the bleeding screen, referral to hematology, time to be seen in hematology clinic, new diagnoses of bleeding disorders, and bleeding complications were assessed from 2019 to 2021 across 3 phases. RESULTS: Provider compliance with the bleeding screen improved throughout our study from 48% (120/251) to 75% (189/253). For those who screened positive, compliance with referral to hematology ranged from 38% to 74% across our phases. The overall time to be seen by hematology decreased from 30 days to 7.5 days. Eighteen patients ultimately screened positive and were seen in hematology clinic, of whom 22% (4/18) were diagnosed with a new bleeding disorder. No bleeding complications were seen in our study population. CONCLUSIONS: Our quality improvement project provided a standardized screening tool to assess preoperative bleeding risk and reinforced the value of a history-based screening tool. This modified screening tool identified those with an undiagnosed bleeding disorder and preventative measures were undertaken to prevent procedural bleeding complications.

Building the foundation for a community-generated national research blueprint for inherited bleeding disorders: facilitating research through infrastructure, workforce, resources and funding.

BACKGROUND: The National Hemophilia Foundation (NHF) conducted extensive, inclusive community consultations to guide prioritization of research in coming decades in alignment with its mission to find cures and address and prevent complications enabling people and families with blood disorders to thrive. RESEARCH DESIGN AND METHODS: With the American Thrombosis and Hemostasis Network, NHF recruited multidisciplinary expert working groups (WG) to distill the community-identified priorities into concrete research questions and score their feasibility, impact, and risk. WG6 was charged with identifying the infrastructure, workforce development, and funding and resources to facilitate the prioritized research. Community input on conclusions was gathered at the NHF State of the Science Research Summit. RESULTS: WG6 detailed a minimal research capacity infrastructure threshold, and opportunities to enable its attainment, for bleeding disorders centers to participate in prospective, multicenter national registries. They identified challenges and opportunities to recruit, retain, and train the diverse multidisciplinary care and research workforce required into the future. Innovative collaborative approaches to trial design, resource networking, and funding to surmount obstacles facing research in rare disorders were elucidated. CONCLUSIONS: The innovations in infrastructure, workforce development, and resources and funding proposed herein may contribute to facilitating a National Research Blueprint for Inherited Bleeding Disorders.

Research is critical to advancing the diagnosis and care of people with inherited bleeding disorders (PWIBD). This research requires significant infrastructure, including people and resources. Hemophilia treatment centers (HTC) need many different skilled care professionals including doctors, nurses, and other providers; also statisticians, data managers, and other experts to process patients' clinical information into research. Attracting diverse qualified professionals to the clinical and research work requires long-term planning, recruiting individuals in training programs and retaining them as they become experts. Research infrastructure includes physical servers running database software, networks that link them, and the environment in which these components function. US Centers for Disease Control and Prevention (CDC) and American Thrombosis and Hemostasis Network (ATHN) coordinate and fund data collection at HTCs on the health and well-being of thousands of PWIBD into a registry used in research studies.National Hemophilia Foundation (NHF) and ATHN asked our group of health care professionals, technology experts, and lived experience experts (LEE) to identify the infrastructure, workforce, and resources needed to do the research most important to PWIBD. We identified the types of CDC/ATHN studies all HTCs should be able to perform, and the physical and human infrastructure this requires. We prioritized finding the best clinical trial designs to study inherited bleeding disorders, identifying ways to share personnel and tools between HTCs, and innovating how research is governed and funded. Involving LEEs in designing, managing, and carrying out research will be key in conducting research to improve the lives of PWIBD.

SARS-CoV-2 receptor binding domain-specific antibodies activate platelets with features resembling the pathogenic antibodies in heparin-induced thrombocytopenia.

Severe COVID-19 is associated with unprecedented thromboembolic complications. We found that hospitalized COVID-19 patients develop immunoglobulin Gs (IgGs) that recognize a complex consisting of platelet factor 4 and heparin similar to those developed in heparin-induced thrombocytopenia and thrombosis (HIT), however, independent of heparin exposure. These antibodies activate platelets in the presence of TLR9 stimuli, stimuli that are prominent in COVID-19. Strikingly, 4 out of 42 antibodies cloned from IgG1+ RBD-binding B cells could activate platelets. These antibodies possessed, in the heavy-chain complementarity-determining region 3, an RKH or Y5 motif that we recently described among platelet-activating antibodies cloned from HIT patients. RKH and Y5 motifs were prevalent among published RBD-specific antibodies, and 3 out of 6 such antibodies tested could activate platelets. Features of platelet activation by these antibodies resemble those by pathogenic HIT antibodies. B cells with an RKH or Y5 motif were robustly expanded in COVID-19 patients. Our study demonstrates that SARS-CoV-2 infection drives the development of a subset of RBD-specific antibodies that can activate platelets and have activation properties and structural features similar to those of the pathogenic HIT antibodies.

Nitric oxide added to the sweep gas of the oxygenator during cardiopulmonary bypass in infants: A pilot randomized controlled trial.

Our objective was to assess the effect of nitric oxide added to the sweep gas of the oxygenator during cardiopulmonary bypass (CPB) in infants on platelet count, platelet function, clinical outcomes, and safety. A randomized, double-blinded, placebo-controlled clinical trial in infants less than a year of age undergoing cardiac surgery requiring CPB was undertaken. Nitric oxide at a dose of 20 ppm was added to the sweep gas in the treatment group. Blood was collected at baseline and prior to separation from CPB to measure platelet count and function as determined by responsiveness to specific agonists. Clinical outcomes were observed through hospital discharge. Methemoglobin levels were measured preoperatively, at the conclusion of CPB, and upon admission to the ICU. Forty patients consented and were randomized in the trial. Eighteen patients were randomized to the treatment group and 22 were included in the placebo group. The groups were similar in terms of age, weight, gender, and surgical complexity. No significant differences were found in measures of platelet count, platelet response to agonist, or clinical outcomes. Patients in the treatment group had higher methemoglobin levels after receiving nitric oxide, but no levels approached toxicity (maximum 2.4%). Nitric oxide added to the sweep gas of the oxygenator during CPB in infants did not have an appreciable effect on the preservation of platelet count, platelet responsiveness to agonist, or clinical outcomes. Methemoglobin levels were increased after receiving nitric oxide but were far below a toxic level of 15%.

Platelet CD36 signaling through ERK5 promotes caspase-dependent procoagulant activity and fibrin deposition in vivo.

Dyslipidemia is a risk factor for clinically significant thrombotic events. In this condition, scavenger receptor CD36 potentiates platelet reactivity through recognition of circulating oxidized lipids. CD36 promotes thrombosis by activating redox-sensitive signaling molecules, such as the MAPK extracellular signal-regulated kinase 5 (ERK5). However, the events downstream of platelet ERK5 are not clear. In this study, we report that oxidized low-density lipoprotein (oxLDL) promotes exposure of procoagulant phosphatidylserine (PSer) on platelet surfaces. Studies using pharmacologic inhibitors indicate that oxLDL-CD36 interaction-induced PSer exposure requires apoptotic caspases in addition to the downstream CD36-signaling molecules Src kinases, hydrogen peroxide, and ERK5. Caspases promote PSer exposure and, subsequently, recruitment of the prothrombinase complex, resulting in the generation of fibrin from the activation of thrombin. Caspase activity was observed when platelets were stimulated with oxLDL. This was prevented by inhibiting CD36 and ERK5. Furthermore, oxLDL potentiates convulxin/glycoprotein VI-mediated fibrin formation by platelets, which was prevented when CD36, ERK5, and caspases were inhibited. Using 2 in vivo arterial thrombosis models in apoE-null hyperlipidemic mice demonstrated enhanced arterial fibrin accumulation upon vessel injury. Importantly, absence of ERK5 in platelets or mice lacking CD36 displayed decreased fibrin accumulation in high-fat diet-fed conditions comparable to that seen in chow diet-fed animals. These findings suggest that platelet signaling through CD36 and ERK5 induces a procoagulant phenotype in the hyperlipidemic environment by enhancing caspase-mediated PSer exposure.

Congenital Disorders of Platelet Function and Number.

Mucocutaneous bleeding symptoms and/or persistent thrombocytopenia occur in individuals with congenital disorders of platelet function and number. Apart from bleeding, these disorders are often associated with additional hematologic and clinical manifestations, including auditory, immunologic, and oncologic disease. Autosomal recessive, dominant, and X-linked inheritance patterns have been demonstrated. Precise delineation of the molecular cause of the platelet disorder can aid the pediatrician in the detection and prevention of specific disorder-associated manifestations and guide appropriate treatment and anticipatory care for the patient and family.

A microengineered vascularized bleeding model that integrates the principal components of hemostasis.

Hemostasis encompasses an ensemble of interactions among platelets, coagulation factors, blood cells, endothelium, and hemodynamic forces, but current assays assess only isolated aspects of this complex process. Accordingly, here we develop a comprehensive in vitro mechanical injury bleeding model comprising an "endothelialized" microfluidic system coupled with a microengineered pneumatic valve that induces a vascular "injury". With perfusion of whole blood, hemostatic plug formation is visualized and "in vitro bleeding time" is measured. We investigate the interaction of different components of hemostasis, gaining insight into several unresolved hematologic issues. Specifically, we visualize and quantitatively demonstrate: the effect of anti-platelet agent on clot contraction and hemostatic plug formation, that von Willebrand factor is essential for hemostasis at high shear, that hemophilia A blood confers unstable hemostatic plug formation and altered fibrin architecture, and the importance of endothelial phosphatidylserine in hemostasis. These results establish the versatility and clinical utility of our microfluidic bleeding model.

Inner Mitochondrial Membrane Disruption Links Apoptotic and Agonist-Initiated Phosphatidylserine Externalization in Platelets.

OBJECTIVE: Phosphatidylserine exposure mediates platelet procoagulant function and regulates platelet life span. Apoptotic, necrotic, and integrin-mediated mechanisms have been implicated as intracellular determinants of platelet phosphatidylserine exposure. Here, we investigate (1) the role of mitochondrial events in platelet phosphatidylserine exposure initiated by these distinct stimuli and (2) the cellular interactions of the procoagulant platelet in vitro and in vivo. APPROACH AND RESULTS: Key mitochondrial events were examined, including cytochrome c release and inner mitochondrial membrane (IMM) disruption. In both ABT-737 (apoptotic) and agonist (necrotic)-treated platelets, phosphatidylserine externalization was temporally correlated with IMM disruption. Agonist stimulation resulted in rapid cyclophilin D-dependent IMM disruption that coincided with phosphatidylserine exposure. ABT-737 treatment caused rapid cytochrome c release, eventually followed by caspase-dependent IMM disruption that again closely coincided with phosphatidylserine exposure. A nonmitochondrial and integrin-mediated mechanism has been implicated in the formation of a novel phosphatidylserine-externalizing platelet subpopulation. Using image cytometry, this subpopulation is demonstrated to be the result of the interaction of an aggregatory platelet and a procoagulant platelet rather than indicative of a novel intracellular mechanism regulating platelet phosphatidylserine externalization. Using electron microscopy, similar interactions between aggregatory and procoagulant platelets are demonstrated in vitro and in vivo within a mesenteric vein hemostatic thrombus. CONCLUSIONS: Platelet phosphatidylserine externalization is closely associated with the mitochondrial event of IMM disruption identifying a common pathway in phosphatidylserine-externalizing platelets. The limited interaction of procoagulant platelets and integrin-active aggregatory platelets identifies a potential mechanism for procoagulant platelet retention within the hemostatic thrombus.

Mitochondrially mediated integrin αIIbß3 protein inactivation limits thrombus growth.

When platelets are strongly stimulated, a procoagulant platelet subpopulation is formed that is characterized by phosphatidylserine (PS) exposure and epitope modulation of integrin αIIbß3 or a loss of binding of activation-dependent antibodies. Mitochondrial permeability transition pore (mPTP) formation, which is essential for the formation of procoagulant platelets, is impaired in the absence of cyclophilin D (CypD). Here we investigate the mechanisms responsible for these procoagulant platelet-associated changes in integrin αIIbß3 and the physiologic role of procoagulant platelet formation in the regulation of platelet aggregation. Among strongly stimulated adherent platelets, integrin αIIbß3 epitope changes, mPTP formation, PS exposure, and platelet rounding were closely associated. Furthermore, platelet mPTP formation resulted in a decreased ability to recruit additional platelets. In the absence of CypD, integrin αIIbß3 function was accentuated in both static and flow conditions, and, in vivo, a prothrombotic phenotype occurred in mice with a platelet-specific deficiency of CypD. CypD-dependent proteolytic events, including cleavage of the integrin ß3 cytoplasmic domain, coincided closely with integrin αIIbß3 inactivation. Calpain inhibition blocked integrin ß3 cleavage and inactivation but not mPTP formation or PS exposure, indicating that integrin inactivation and PS exposure are mediated by distinct pathways subsequent to mPTP formation. mPTP-dependent alkalinization occurred in procoagulant platelets, suggesting a possible alternative mechanism for enhancement of calpain activity in procoagulant platelets. Together, these results indicate that, in strongly stimulated platelets, mPTP formation initiates the calpain-dependent cleavage of integrin ß3 and associated regulatory proteins, resulting in integrin αIIbß3 inactivation, and demonstrate a novel CypD-dependent negative feedback mechanism that limits platelet aggregation and thrombotic occlusion.