Interleukin-7 Reverses Lymphopenia and Improves T-Cell Function in Coronavirus Disease 2019 Patient With Inborn Error of Toll-Like Receptor 3: A Case Report.

BACKGROUND: Immunotherapy treatment for coronavirus disease 2019 combined with antiviral therapy and supportive care remains under intense investigation. However, the capacity to distinguish patients who would benefit from immunosuppressive or immune stimulatory therapies remains insufficient. Here, we present a patient with severe coronavirus disease 2019 with a defective immune response, treated successfully with interleukin-7 on compassionate basis with resultant improved adaptive immune function. CASE SUMMARY: A previously healthy 43-year-old male developed severe acute respiratory distress syndrome due to the severe acute respiratory syndrome coronavirus 2 virus with acute hypoxemic respiratory failure and persistent, profound lymphopenia. Functional analysis demonstrated depressed lymphocyte function and few antigen-specific T cells. Interleukin-7 administration resulted in reversal of lymphopenia and improved T-cell function. Respiratory function and clinical status rapidly improved, and he was discharged home. Whole exome sequencing identified a deleterious autosomal dominant mutation in TICAM1, associated with a dysfunctional type I interferon antiviral response with increased severity of coronavirus disease 2019 disease. CONCLUSIONS: Immunoadjuvant therapies to boost host immunity may be efficacious in life-threatening severe coronavirus disease 2019 infections, particularly by applying a precision medicine approach in selecting patients expressing an immunosuppressive phenotype.

A mathematical model of coagulation under flow identifies factor V as a modifier of thrombin generation in hemophilia A.

BACKGROUND: The variability in bleeding patterns among individuals with hemophilia A, who have similar factor VIII (FVIII) levels, is significant and the origins are unknown. OBJECTIVE: To use a previously validated mathematical model of flow-mediated coagulation as a screening tool to identify parameters that are most likely to enhance thrombin generation in the context of FVIII deficiency. METHODS: We performed a global sensitivity analysis (GSA) on our mathematical model to identify potential modifiers of thrombin generation. Candidates from the GSA were confirmed by calibrated automated thrombography (CAT) and flow assays on collagen-tissue factor (TF) surfaces at a shear rate of 100 per second. RESULTS: Simulations identified low-normal factor V (FV) (50%) as the strongest modifier, with additional thrombin enhancement when combined with high-normal prothrombin (150%). Low-normal FV levels or partial FV inhibition (60% activity) augmented thrombin generation in FVIII-inhibited or FVIII-deficient plasma in CAT. Partial FV inhibition (60%) boosted fibrin deposition in flow assays performed with whole blood from individuals with mild and moderate FVIII deficiencies. These effects were amplified by high-normal prothrombin levels in both experimental models. CONCLUSIONS: These results show that low-normal FV levels can enhance thrombin generation in hemophilia A. Further explorations with the mathematical model suggest a potential mechanism: lowering FV reduces competition between FV and FVIII for factor Xa (FXa) on activated platelet surfaces (APS), which enhances FVIII activation and rescues thrombin generation in FVIII-deficient blood.

Editor's Highlight: Pulmonary Vascular Thrombosis in Rats Exposed to Inhaled Sulfur Mustard.

Sulfur mustard (SM) is a chemical warfare agent. When inhaled, SM causes significant injury to the respiratory tract. Although the mechanism involved in acute airway injury after SM inhalation has been well described previously, the mechanism of SM's contribution to distal lung vascular injury is not well understood. We hypothesized that acute inhalation of vaporized SM causes activated systemic coagulation with subsequent pulmonary vascular thrombi formation after SM inhalation exposure. Sprague Dawley rats inhaled SM ethanolic vapor (3.8 mg/kg). Barium/gelatin CT pulmonary angiograms were performed to assess for pulmonary vascular thrombi burden. Lung immunohistochemistry was performed for common procoagulant markers including fibrin(ogen), von Willebrand factor, and CD42d in control and SM-exposed lungs. Additionally, systemic levels of d-dimer and platelet aggregometry after adenosine diphosphate- and thrombin-stimulation were measured in plasma after SM exposure. In SM-exposed lungs, chest CT angiography demonstrated a significant decrease in the distal pulmonary vessel density assessed at 6 h postexposure. Immunohistochemistry also demonstrated increased intravascular fibrin(ogen), vascular von Willebrand factor, and platelet CD42d in the distal pulmonary vessels (<200 µm diameter). Circulating d-dimer levels were significantly increased (p < .001) at 6, 9, and 12 h after SM inhalation versus controls. Platelet aggregation was also increased in both adenosine diphosphate - (p < .01) and thrombin- (p < .001) stimulated platelet-rich plasma after SM inhalation. Significant pulmonary vascular thrombi formation was evident in distal pulmonary arterioles following SM inhalation in rats assessed by CT angiography and immunohistochemistry. Enhanced systemic platelet aggregation and activated systemic coagulation with subsequent thrombi formation likely contributed to pulmonary vessel occlusion.

Mutations in the D'D3 region of VWF traditionally associated with type 1 VWD lead to quantitative and qualitative deficiencies of VWF.

Type 1 von Willebrand disease (VWD) is characterized by low plasma levels of von Willebrand factor (VWF) and clinical bleeding. Several mechanisms have been described that cause a decrease in plasma VWF levels in VWD, and the goal of this study was to elucidate the pathogenic origins of VWD for a group of mutations in the VWF D'D3 region traditionally associated with type 1 VWD. Varying ratios of mutant-to-wild-type VWF were expressed in two cell lines in order to study the intracellular location, multimer assembly, secretion and function of VWF. We identified four mutants (M771I, Y1146C, T1156M, R782Q) that caused defective intracellular packaging and markedly reduced VWF secretion. Consistent with previous reports, Y1146C and T1156M VWF led to a loss of high molecular weight multimers. In a functional analysis, Y1146C demonstrated a novel FVIII binding defect. Mutations R924W and I1094T were processed normally and did not show abnormal FVIII binding suggesting that other mechanisms such as plasma clearance or platelet binding defects may contribute to the pathogenicity of these mutants.

FVIII/VWF ratio is not a reliable predictor of VWD in children.

Adults with von Willebrand Disease (VWD) are known to have a ratio of factor VIII activity (FVIII:C) to von Willebrand factor antigen (VWF:Ag) greater than 1. We, however, noted healthy children with ratios that are unexpectedly high. Though the FVIII:C/VWF:Ag ratio differs significantly between healthy children and VWD patients in some age groups, the substantial overlap of observed ranges suggests that a ratio threshold-based screening approach alone cannot reliably discriminate between these groups. The diagnostic performance of this ratio is poor for VWD in children, which may decrease its value as a screening tool in the pediatric population.

Discovery of Mer specific tyrosine kinase inhibitors for the treatment and prevention of thrombosis.

The role of Mer kinase in regulating the second phase of platelet activation generates an opportunity to use Mer inhibitors for preventing thrombosis with diminished likelihood for bleeding as compared to current therapies. Toward this end, we have discovered a novel, Mer kinase specific substituted-pyrimidine scaffold using a structure-based drug design and a pseudo ring replacement strategy. The cocrystal structure of Mer with two compounds (7 and 22) possessing distinct activity have been determined. Subsequent SAR studies identified compound 23 (UNC2881) as a lead compound for in vivo evaluation. When applied to live cells, 23 inhibits steady-state Mer kinase phosphorylation with an IC50 value of 22 nM. Treatment with 23 is also sufficient to block EGF-mediated stimulation of a chimeric receptor containing the intracellular domain of Mer fused to the extracellular domain of EGFR. In addition, 23 potently inhibits collagen-induced platelet aggregation, suggesting that this class of inhibitors may have utility for prevention and/or treatment of pathologic thrombosis.

The effect of factor VIII deficiencies and replacement and bypass therapies on thrombus formation under venous flow conditions in microfluidic and computational models.

Clinical evidence suggests that individuals with factor VIII (FVIII) deficiency (hemophilia A) are protected against venous thrombosis, but treatment with recombinant proteins can increase their risk for thrombosis. In this study we examined the dynamics of thrombus formation in individuals with hemophilia A and their response to replacement and bypass therapies under venous flow conditions. Fibrin and platelet accumulation were measured in microfluidic flow assays on a TF-rich surface at a shear rate of 100 s⁻¹. Thrombin generation was calculated with a computational spatial-temporal model of thrombus formation. Mild FVIII deficiencies (5-30% normal levels) could support fibrin fiber formation, while severe (<1%) and moderate (1-5%) deficiencies could not. Based on these experimental observations, computational calculations estimate an average thrombin concentration of ∼10 nM is necessary to support fibrin formation under flow. There was no difference in fibrin formation between severe and moderate deficiencies, but platelet aggregate size was significantly larger for moderate deficiencies. Computational calculations estimate that the local thrombin concentration in moderate deficiencies is high enough to induce platelet activation (>1 nM), but too low to support fibrin formation (<10 nM). In the absence of platelets, fibrin formation was not supported even at normal FVIII levels, suggesting platelet adhesion is necessary for fibrin formation. Individuals treated by replacement therapy, recombinant FVIII, showed normalized fibrin formation. Individuals treated with bypass therapy, recombinant FVIIa, had a reduced lag time in fibrin formation, as well as elevated fibrin accumulation compared to healthy controls. Treatment of rFVIIa, but not rFVIII, resulted in significant changes in fibrin dynamics that could lead to a prothrombotic state.

No increase in bleeding identified in type 1 VWD subjects with D1472H sequence variation.

The diagnosis of von Willebrand disease (VWD) is complicated by issues with current laboratory testing, particularly the ristocetin cofactor activity assay (VWF:RCo). We have recently reported a sequence variation in the von Willebrand factor (VWF) A1 domain, p.D1472H (D1472H), associated with a decrease in the VWF:RCo/VWF antigen (VWF:Ag) ratio but not associated with bleeding in healthy control subjects. This report expands the previous study to include subjects with symptoms leading to the diagnosis of type 1 VWD. Type 1 VWD subjects with D1472H had a significant decrease in the VWF:RCo/VWF:Ag ratio compared with those without D1472H, similar to the findings in the healthy control population. No increase in bleeding score was observed, however, for VWD subjects with D1472H compared with those without D1472H. These results suggest that the presence of the D1472H sequence variation is not associated with a significant increase in bleeding symptoms, even in type 1 VWD subjects.

Sources of variability in platelet accumulation on type 1 fibrillar collagen in microfluidic flow assays.

Microfluidic flow assays (MFA) that measure shear dependent platelet function have potential clinical applications in the diagnosis and treatment of bleeding and thrombotic disorders. As a step towards clinical application, the objective of this study was to measure how phenotypic and genetic factors, as well as experimental conditions, affect the variability of platelet accumulation on type 1 collagen within a MFA. Whole blood was perfused over type 1 fibrillar collagen at wall shear rates of 150, 300, 750 and 1500 s⁻¹ through four independent channels with a height of 50 µm and a width of 500 µm. The accumulation of platelets was characterized by the lag time to 1% platelet surface coverage (Lag(T)), the rate of platelet accumulation (V(PLT)), and platelet surface coverage (SC). A cohort of normal donors was tested and the results were correlated to plasma von Willebrand factor (VWF) levels, platelet count, hematocrit, sex, and collagen receptors genotypes. VWF levels were the strongest determinant of platelet accumulation. VWF levels were positively correlated to V(PLT) and SC at all wall shear rates. A longer Lag(T) for platelet accumulation at arterial shear rates compared to venous shear rates was attributed to the time required for plasma proteins to adsorb to collagen. There was no association between platelet accumulation and hematocrit or platelet count. Individuals with the AG genotype of the GP6 gene had lower platelet accumulation than individuals with the AA genotype at 150 s⁻¹ and 300 s⁻¹. Recalcified blood collected into sodium citrate and corn trypsin inhibitor (CTI) resulted in diminished platelet accumulation compared to CTI alone, suggesting that citrate irreversibly diminishes platelet function. This study the largest association study of MFA in healthy donors (n = 104) and will likely set up the basis for the determination of the normal range of platelet responses in this type of assay.

Characterization of collagen thin films for von Willebrand factor binding and platelet adhesion.

Von Willebrand factor (VWF) binding and platelet adhesion to subendothelial collagens are initial events in thrombus formation at sites of vascular injury. These events are often studied in vitro using flow assays designed to mimic vascular hemodynamics. Flow assays commonly employ collagen-functionalized substrates, but a lack of standardized methods of surface ligation limits their widespread use as a clinical diagnostic. Here, we report the use of collagen thin films (CTF) in flow assays. Thin films were grown on hydrophobic substrates from type I collagen solutions of increasing concentration (10, 100, and 1000 µg/mL). We found that the corresponding increase in fiber surface area determined the amount of VWF binding and platelet adhesion. The association rate constant (k(a)) of plasma VWF binding at a wall shear stress of 45 dyn/cm(2) was 0.3 × 10(5), 1.8 × 10(5), and 1.6 × 10(5) M(-1) s(-1) for CTF grown from 10, 100, and 1000 µg/mL solutions, respectively. We observed a 5-fold increase in VWF binding capacity with each 10-fold increase in collagen solution concentration. The association rates of Ser1731Thr and His1786Asp VWF mutants with collagen binding deficiencies were 9% and 22%, respectively, of wild-type rates. Using microfluidic devices for blood flow assays, we observed that CTF supported platelet adhesion at a wall shear rate of 1000 s(-1). CTF grown from 10 and 100 µg/mL solutions had variable levels of platelet surface coverage between multiple normal donors. However, CTF substrates grown from 1000 µg/mL solutions had reproducible surface coverage levels (74 ± 17%) between normal donors, and there was significantly diminished surface coverage from two type 1 von Willebrand disease patients (8.0% and 24%). These results demonstrate that collagen thin films are homogeneous and reproducible substrates that can measure dysfunctions in VWF binding and platelet adhesion under flow in a clinical microfluidic assay format.

Common VWF exon 28 polymorphisms in African Americans affecting the VWF activity assay by ristocetin cofactor.

The diagnosis of von Willebrand disease relies on abnormalities in specific tests of von Willebrand factor (VWF), including VWF antigen (VWF:Ag) and VWF ristocetin cofactor activity (VWF:RCo). When examining healthy controls enrolled in the T. S. Zimmerman Program for the Molecular and Clinical Biology of von Willebrand disease, we, like others, found a lower mean VWF:RCo compared with VWF:Ag in African American controls and therefore sought a genetic cause for these differences. For the African American controls, the presence of 3 exon 28 single nucleotide polymorphisms (SNPs), I1380V, N1435S, and D1472H, was associated with a significantly lower VWF:RCo/VWF:Ag ratio, whereas the presence of D1472H alone was associated with a decreased ratio in both African American and Caucasian controls. Multivariate analysis comparing race, SNP status, and VWF:RCo/VWF:Ag ratio confirmed that only the presence of D1472H was significant. No difference was seen in VWF binding to collagen, regardless of SNP status. Similarly, no difference in activity was seen using a GPIb complex-binding assay that is independent of ristocetin. Because the VWF:RCo assay depends on ristocetin binding to VWF, mutations (and polymorphisms) in VWF may affect the measurement of "VWF activity" by this assay and may not reflect a functional defect or true hemorrhagic risk.

Immune thrombocytopenic purpura.

Although many advances have been achieved in the understanding of ITP, critical issues regarding the pathophysiology and biology of the disease remain to be elucidated. The recent characterization of the human genome along with new sophisticated molecular biology techniques will allow basic researchers to study genes that may affect the presentation and clinical course of the disease. Different patterns of gene expression in this population can be studied, leading to the identification of subsets of patients with ITP at higher risk of bleeding. The multigene patterns of expression might also provide clues about regulatory mechanisms and broader cellular functions. In order to answer essential clinical questions, like the incidence of ICH in relation to drug treatment or observation alone, clinical trials should be appropriately designed. More studies are necessary to better define the optimal treatment approach for each child with ITP. Even though the incidence of intracranial hemorrhage cannot be used as the primary outcome measure because of its rarity, numerous other outcomes, such as rate of rise in platelet count, cost and side effects of therapy, health related quality of life of the patient and family, and severity of hemorrhage can be measured and compared between treatment groups. Future investigators should find it attractive to conduct trials in children with this common hematological disease so that decision making can be based more on scientific evidence than on anecdote and opinion.