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.

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.

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.

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.

Dual mechanism of integrin αIIbß3 closure in procoagulant platelets.

BACKGROUND: Inactivation of integrin αIIbß3 reverses platelet aggregate formation upon coagulation. RESULTS AND CONCLUSION: Platelets from patient (Scott) and mouse (Capn1(-/-) and Ppif(-/-)) blood reveal a dual mechanism of αIIbß3 inactivation: by calpain-2 cleavage of integrin-associated proteins and by cyclophilin D/TMEM16F-dependent phospholipid scrambling. SIGNIFICANCE: These data provide novel insight into the switch mechanisms from aggregating to procoagulant platelets. Aggregation of platelets via activated integrin αIIbß3 is a prerequisite for thrombus formation. Phosphatidylserine-exposing platelets with a key role in the coagulation process disconnect from a thrombus by integrin inactivation via an unknown mechanism. Here we show that αIIbß3 inactivation in procoagulant platelets relies on a sustained high intracellular Ca(2+), stimulating intracellular cleavage of the ß3 chain, talin, and Src kinase. Inhibition of calpain activity abolished protein cleavage, but only partly suppressed αIIbß3 inactivation. Integrin αIIbß3 inactivation was unchanged in platelets from Capn1(-/-) mice, suggesting a role of the calpain-2 isoform. Scott syndrome platelets, lacking the transmembrane protein TMEM16F and having low phosphatidylserine exposure, displayed reduced αIIbß3 inactivation with the remaining activity fully dependent on calpain. In platelets from Ppif(-/-) mice, lacking mitochondrial permeability transition pore (mPTP) formation, agonist-induced phosphatidylserine exposure and αIIbß3 inactivation were reduced. Treatment of human platelets with cyclosporin A gave a similar phenotype. Together, these data point to a dual mechanism of αIIbß3 inactivation via calpain(-2) cleavage of integrin-associated proteins and via TMEM16F-dependent phospholipid scrambling with an assistant role of mPTP formation.

Mitochondrial calcium and reactive oxygen species regulate agonist-initiated platelet phosphatidylserine exposure.

OBJECTIVE: To study the interactions of cytoplasmic calcium elevation, mitochondrial permeability transition pore (mPTP) formation, and reactive oxygen species formation in the regulation of phosphatidylserine (PS) exposure in platelets. METHODS AND RESULTS: mPTP formation, but not the degree of cytoplasmic calcium elevation, was associated with PS exposure in wild-type, cyclophilin D-null, ionomycin-treated, and reactive oxygen species-treated platelets. In the absence of the mPTP regulator cyclophilin D, agonist-initiated mPTP formation and high-level PS exposure were markedly blunted, but cytoplasmic calcium transients were unchanged. Mitochondrial calcium (Ca(2+)(mit)) transients and reactive oxygen species, key regulators of mPTP formation, were examined in strongly stimulated platelets. Increased reactive oxygen species production occurred in strongly stimulated platelets and was dependent on extracellular calcium entry, but not the presence of cyclophilin D. Ca(2+)(mit) increased significantly in strongly stimulated platelets. Abrogation of Ca(2+)(mit) entry, either by inhibition of the Ca(2+)(mit) uniporter or mitochondrial depolarization, prevented mPTP formation and exposure but not platelet aggregation or granule release. CONCLUSIONS: Sustained cytoplasmic calcium levels are necessary, but not sufficient, for high-level PS exposure in response to agonists. Increased Ca(2+)(mit) levels are a key signal initiating mPTP formation and PS exposure. Blockade of Ca(2+)(mit) entry allows the specific inhibition of platelet procoagulant activity.

Platelet biogenesis and functions require correct protein O-glycosylation.

Platelets express a variety of membrane and secreted glycoproteins, but the importance of glycosylation to platelet functions is poorly understood. To explore the importance of O-glycosylation, we generated mice with a targeted deletion of Cosmc in murine endothelial/hematopoietic cells (EHC) (EHC Cosmc(-/y)). X-linked Cosmc encodes an essential chaperone that regulates protein O-glycosylation. This targeted mutation resulted in lethal perinatal hemorrhage in the majority of mice, and the surviving mice displayed severely prolonged tail-bleeding times and macrothrombocytopenia. EHC Cosmc(-/y) platelets exhibited a marked decrease in GPIb-IX-V function and agonist-mediated integrin αIIbß3 activation, associated with loss of interactions with von Willebrand factor and fibrinogen, respectively. Significantly, three O-glycosylated glycoproteins, GPIbα, αIIb, and GPVI normally on platelet surfaces that play essential roles in platelet functions, were partially proteolyzed in EHC Cosmc(-/y) platelets. These results demonstrate that extended O-glycans are required for normal biogenesis of the platelets as well as the expression and functions of their essential glycoproteins, and that variations in O-glycosylation may contribute to altered hemostasis.

Mutations in NBEAL2, encoding a BEACH protein, cause gray platelet syndrome.

Next-generation RNA sequence analysis of platelets from an individual with autosomal recessive gray platelet syndrome (GPS, MIM139090) detected abnormal transcript reads, including intron retention, mapping to NBEAL2 (encoding neurobeachin-like 2). Genomic DNA sequencing confirmed mutations in NBEAL2 as the genetic cause of GPS. NBEAL2 encodes a protein containing a BEACH domain that is predicted to be involved in vesicular trafficking and may be critical for the development of platelet α-granules.

Critical role for the mitochondrial permeability transition pore and cyclophilin D in platelet activation and thrombosis.

Many of the cellular responses that occur in activated platelets resemble events that take place following activation of cell-death pathways in nucleated cells. We tested the hypothesis that formation of the mitochondrial permeability transition pore (MPTP), a key signaling event during cell death, also plays a critical role in platelet activation. Stimulation of murine platelets with thrombin plus the glycoprotein VI agonist convulxin resulted in a rapid loss of mitochondrial transmembrane potential (Deltapsi(m)) in a subpopulation of activated platelets. In the absence of cyclophilin D (CypD), an essential regulator of MPTP formation, murine platelet activation responses were altered. CypD-deficient platelets exhibited defects in phosphatidylserine externalization, high-level surface fibrinogen retention, membrane vesiculation, and procoagulant activity. Also, in CypD-deficient platelet-rich plasma, clot retraction was altered. Stimulation with thrombin plus H(2)O(2), a known activator of MPTP formation, also increased high-level surface fibrinogen retention, phosphatidylserine externalization, and platelet procoagulant activity in a CypD-dependent manner. In a model of carotid artery photochemical injury, thrombosis was markedly accelerated in CypD-deficient mice. These results implicate CypD and the MPTP as critical regulators of platelet activation and suggest a novel CypD-dependent negative-feedback mechanism regulating arterial thrombosis.

Role of FcRgamma and factor XIIIA in coated platelet formation.

Platelet activation in response to dual stimulation with collagen and thrombin results in the formation of a subpopulation of activated platelets known as coated platelets. Coated platelets are characterized by high surface levels of alpha-granule proteins and phosphatidylserine, which support the assembly of procoagulant protein complexes. Using murine models, we tested the hypothesis that the collagen receptor-associated molecule FcRgamma and the transglutaminase factor XIIIA are required for the formation of coated platelets. Following dual stimulation with the collagen receptor agonist convulxin and thrombin, 68% of platelets from C57BL/6 mice acquired the coated platelet phenotype, defined by high surface levels of fibrinogen and von Willebrand factor and decreased binding of the alphaIIbbeta3 activation-dependent antibody PE-JON/A. In FcRgamma-/- mice, only 10% of platelets became "coated" after dual stimulation with convulxin plus thrombin (P < .05 vs C57BL/6 platelets). Decreased coated platelet formation in FcRgamma-/- platelets was accompanied by decreased annexin V binding (P < .01) and decreased platelet procoagulant activity (P < .05). Platelets from FXIIIA-/- mice did not differ from control platelets in coated platelet formation or annexin V binding. We conclude that FcRgamma, but not factor XIIIA, is essential for formation of highly procoagulant coated platelets following dual stimulation with collagen and thrombin.