Microvascular Thrombus Formation Research Articles

Interferons prime the endothelium for toll-like receptor–mediated thrombin generation

BackgroundRespiratory infection is associated with microvascular thrombus formation and marked elevation in cytokine levels. The role of cytokines elaborated by the pulmonary epithelium in thrombotic responses is poorly understood. ObjectivesOur goal was to identify cytokines of pulmonary epithelial cell origin that enhance thrombin generation in the endothelium at concentrations equal to or less than those found in the circulation during infection. MethodsWe screened multiple cytokines produced by the pulmonary epithelium for the ability to enhance toll-like receptor (TLR)–mediated endothelial thrombin generation. Effects of cytokines on tissue factor and thrombomodulin expression, cytokine selectivity for different TLRs, and prothrombotic activity of endogenous cytokines in conditioned medium from pulmonary human epithelial cells were evaluated. ResultsMIP-1β, MCP-1, IL-10, IL-6, IL-1β, TNFα, IFNα, IFNβ, and IFNγ were tested for their ability to enhance TLR3-mediated thrombin generation on endothelial cells. Only interferons (IFNs) and TNFα promoted TLR3-mediated thrombin generation at levels that circulate during infection. IFNs robustly enhanced tissue factor expression when used in conjunction with TLR agonists and reduced thrombomodulin expression in the endothelium independently of TLRs. IFNα, which is typically elevated with viral infection, only synergized with TLR3 agonists mimicking viral pathogen-associated molecular patterns. In contrast, IFNγ, which is typically observed in bacterial infection, synergized more effectively with TLR4 agonists released by bacteria. Conditioned media from inflamed pulmonary epithelial cells primed the endothelium for TLR-mediated thrombin generation. Anti-IFN type I antibodies blocked this effect, indicating that endogenous IFNs prime the endothelium for TLR-mediated thrombin generation. ConclusionIFNs elaborated by the pulmonary epithelium are necessary and sufficient to enhance TLR-mediated thrombin generation.

Endothelial ADAM10 utilization defines a molecular pathway of vascular injury in mice with bacterial sepsis.

The endothelium plays a critical role in the host response to infection and has been a focus of investigation in sepsis. While it is appreciated that intravascular thrombus formation, severe inflammation, and loss of endothelial integrity impair tissue oxygenation during sepsis, the precise molecular mechanisms that lead to endothelial injury remain poorly understood. We demonstrate here that endothelial ADAM10 was essential for the pathogenesis of Staphylococcus aureus sepsis, contributing to α-toxin-mediated (Hla-mediated) microvascular thrombus formation and lethality. As ADAM10 is essential for endothelial development and homeostasis, we examined whether other major human sepsis pathogens also rely on ADAM10-dependent pathways in pathogenesis. Mice harboring an endothelium-specific knockout of ADAM10 were protected against lethal Pseudomonas aeruginosa and Streptococcus pneumoniae sepsis, yet remained fully susceptible to group B streptococci and Candida albicans sepsis. These studies illustrate a previously unknown role for ADAM10 in sepsis-associated endothelial injury and suggest that understanding pathogen-specific divergent host pathways in sepsis may enable more precise targeting of disease.

ATYPICAL HEMOLYTIC UREMIC SYNDROME – A RARE CASE REPORT

Atypical hemolytic uremic syndrome (aHUS), a rare variant of thrombotic microangiopathy, is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. The condition is associated with poor clinical outcomes with high morbidity and mortality. Atypical HUS predominantly affects the kidneys but has the potential to cause multi-organ system dysfunction. This uncommon disorder is caused by a genetic abnormality in the complement alternative pathway resulting in over-activation of the complement system and formation of microvascular thrombi. Abnormalities of the complement pathway may be in the form of mutations in key complement genes or autoantibodies against specic complement factors. We discuss , clinical manifestations, diagnosis, complications, and management of aHUS.

Intrapulmonary shunt and alveolar dead space in a cohort of patients with acute COVID-19 pneumonitis and early recovery.

Pathological evidence suggests that coronavirus disease 2019 (COVID-19) pulmonary infection involves both alveolar damage (causing shunt) and diffuse microvascular thrombus formation (causing alveolar dead space). We propose that measuring respiratory gas exchange enables detection and quantification of these abnormalities. We aimed to measure shunt and alveolar dead space in moderate COVID-19 during acute illness and recovery. We studied 30 patients (22 males; mean±sd age 49.9±13.5 years) 3-15 days from symptom onset and again during recovery, 55±10 days later (n=17). Arterial blood (breathing ambient air) was collected while exhaled oxygen and carbon dioxide concentrations were measured, yielding alveolar-arterial differences for each gas (P A-aO2 and P a-ACO2 , respectively) from which shunt and alveolar dead space were computed. For acute COVID-19 patients, group mean (range) for P A-aO2 was 41.4 (-3.5-69.3) mmHg and for P a-ACO2 was 6.0 (-2.3-13.4) mmHg. Both shunt (% cardiac output) at 10.4% (0-22.0%) and alveolar dead space (% tidal volume) at 14.9% (0-32.3%) were elevated (normal: <5% and <10%, respectively), but not correlated (p=0.27). At recovery, shunt was 2.4% (0-6.1%) and alveolar dead space was 8.5% (0-22.4%) (both p<0.05 versus acute). Shunt was marginally elevated for two patients; however, five patients (30%) had elevated alveolar dead space. We speculate impaired pulmonary gas exchange in early COVID-19 pneumonitis arises from two concurrent, independent and variable processes (alveolar filling and pulmonary vascular obstruction). For most patients these resolve within weeks; however, high alveolar dead space in ∼30% of recovered patients suggests persistent pulmonary vascular pathology.

Promoting the Resolution of Thromboinflammation

BackgroundThrombosis is viewed as a multi‐step, multi‐cellular process driven by inflammatory stimuli and leukocyte (e.g. neutrophil) activation, and is a major pathophysiological contributor of stroke. Accumulating data supports the hypothesis that anti‐inflammatory therapies may limit thrombosis and anti‐thrombotic therapies may reduce vascular inflammation. One such target is the anti‐inflammatory and pro‐resolving endogenous mediator Annexin A1 (AnxA1). The development of therapies that temper inflammation and enhance resolution offer potential therapeutic strategies for the treatment and management of stroke and other thromboinflammatory diseases. Here we sought to comprehensively elucidate the functional significance of targeting the AnxA1/Fpr2/ALX pathway in thrombosis.Material and MethodsWe used a well‐characterized light/dye thrombosis model, coupled with pharmacological and genetic approaches. Male and female adult wild type (WT, C57/BL6) or AnxA1 knock‐out (AnxA1‐/‐) mice were used. Fluorescence intravital microscopy (IVM) was used to monitor cerebral thrombosis, along with flow cytometry. Mice were treated with AnxA1 (1 µg/mouse) or saline vehicle 20 minutes before the onset of cerebral thrombus formation. Thrombogenesis and blood flow cessation times were quantified. (All animal experiments were approved by relevant institutional animal welfare panels and were conducted in accordance with the American Association for Accreditation of Laboratory Animal Care guidelines and complied with ARRIVE [Animal Research: Reporting In Vivo Experiments]).ResultsIVM revealed that AnxA1 treatment was able to prolong blood flow cessation times in both cerebral arterioles and venules, via regulation of the key inflammation resolution receptor: FPR2/ALX, as evidenced by the co‐administration of the FPR2/ALX antagonist WRW4. Using AnxA1−/− mice, we found that a deletion of AnxA1 impacts thrombosis as shown by the accelerated cerebral microvascular thrombus formation (decrease in blood flow cessation time) observed in both arterioles and venules of these mice compared to their WT counterparts (arterioles: 17.9 ± 2.3 vs 33.2 ± 1.9 min and venules: 13.2 ± 2.4 vs 20.9 ± 2.2 min. p&lt;0.05). Furthermore, we found that in an inflammatory backdrop (induced by the administration of lipopolysaccharide [LPS]), AnxA1 treatment was able to suppress both platelet αIIbβ3 and P‐selectin expression.ConclusionHerein, we found a previously unknown phenomenon that the anti‐inflammatory protein AnxA1 can modify a thromboinflammatory environment to promote resolution. These findings have far‐reaching applicability for drug discovery programs focused on resolution biologics targeting thromboinflammatory diseases such as cardiovascular disease and stroke.

P-Selectin Deficiency Reduces Acute Vascular Complications but Not Chronic Organ Damage in Sickle Cell Mice

P-selectin (Psel) is an adhesion molecule expressed on platelets and endothelial cells, which interacts with its ligand PSGL-1 on leukocytes and a PSGL-1-like molecule on sickle red blood cells (RBCs). Psel mediates the formation of the multicellular aggregates that promote vaso-occlusive crisis (VOC) and acute painful episodes in sickle cell disease (SCD). Crizanlizumab, a monoclonal antibody blocking Psel, reduces the frequency of VOC in SCD patients. In SCD mice, Psel contributes to heme-induced microvascular stasis, and thrombus and platelet-neutrophil aggregate formation in the lung and liver. Unexpectedly, Psel deficiency promotes liver senescence in a mouse model of SCD, suggesting a possible detrimental effect of Psel inhibition. In this study, we further investigated the long-term effects of Psel deficiency on thromboinflammation and end-organ damage in murine SCD.We used Townes wild type (AA) and sickle (SS) mice (n=12-27) that either express Psel (Psel +/+) or lack Psel (Psel -/-) at 11-12 months of age. Kidney damage was evaluated by histology and urine analysis. Heart function was determined using echocardiography. Complete blood counts and plasma biomarkers of thrombin generation (thrombin anti-thrombin [TAT] complexes) and inflammation (interleukin [IL]-6) were also analyzed. In addition, in a pilot experiment, we analyzed the effect of Psel deficiency on experimental venous thrombosis in SS mice.SS Psel +/+ mice exhibited renal damage, urinary concentrating defect, and reduced creatinine clearance, which was not improved in SS Psel -/- mice. Psel deficiency attenuated glomerular (GLM) congestion (0.17±0.05 vs 0.39±0.06, p<0.05) but failed to prevent GLM injury, as evidenced by similar extent of GLM sclerosis, hypertrophy and podocyte loss in SS mice. Interestingly, only SS P-sel -/- mice presented withdistinct differences in GLM structure, demonstrated by extensive hypercellularity, mesangial expansion and mesangiolysis. We also observed tubular injury concomitant with brush border loss and interstitial fibrosis in SS mice, regardless of Psel expression. Additionally, there was a significant reduction in CD3 + T cells (5.0±1.2 vs 9.5±1.6 cells/field; p<0.05) and macrophages (1.0±0.1 vs 1.6±0.2 % of area; p<0.05) infiltration in the kidney cortex of SS Psel -/- compared to SS Psel +/+ mice. This corresponded to a greater renal iron accumulation in SS Psel -/- mice (59.9±4.2 vs 48.2±3.2 Mpix/μm; p<0.05).We observed cardiac hypertrophy (elevated heart weight to tibia length ratio) in SS mice, regardless of Psel expression. Echocardiography of left ventricle (LV) revealed that SS mice had increased LV mass and LV internal diameter with no change in ejection fraction or fractional shortening compared to AA mice. None of these parameters were affected by Psel expression. Furthermore, hypertrophy observed in other organs (kidney, liver, lung and spleen) of SS Psel +/+ mice was also not affected by Psel deficiency.Consistent with previous studies, SS Psel +/+ mice had elevated plasma levels of TAT and IL-6 compared to AA Psel +/+ mice. Psel deficiency significantly reduced IL-6 (47±15 vs 20±10 ng/mL, P<0.05), but had no effect on elevated plasma TAT levels in SS mice. Despite the lack of effect of Psel deficiency on systemic thrombin generation in SS mice, femoral vein thrombi formed after electrolytic injury exhibited a strong trend in reduced fibrin and platelet content compared to SS Psel+/+ mice (n=5 per group).As expected, SS Psel +/+ mice exhibited anemia as shown by reduced RBC number, hemoglobin (Hb) and hematocrit (Hct) compared to AA Psel +/+ animals. Interestingly, these parameters were further decreased in SS Psel -/- mice (p<0.01 for all); RBC (5.6±0.2 vs 4.4±0.4 x 10 6/mL), Hg (6.7±0.2 vs 5.7±0.2 g/dL) and Hct (25.1±1.1 vs 19.7±1.1%). This was accompanied by a significant increase in mean corpuscular Hb concentration (26.9±0.5 vs 29.2±0.5 g/dL p< 0.01) but no changes in RBC mean corpuscular volume in SS Psel -/-mice compared to SS Psel +/+ mice.Our data suggest that despite improving acute vascular pathologies, including VOC and thromboinflammation, long term deficiency of Psel does not prevent end-organ damage. If fact, it may contribute to organ dysfunction and enhanced anemia. More mechanistic studies are needed to better understand the long-term effects of anti-Psel treatment in SCD patients. DisclosuresReeves: Incyte Corporation: Honoraria; Takeda: Honoraria; Bristol-Myers Squibb: Speakers Bureau; Pharma Essentia: Consultancy, Honoraria. Sundd: CSL Behring Inc: Research Funding; Bayer: Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding.

The Resolution Mediator Annexin A1 Affords Protection Against Thromboinflammation

Stroke is a leading cause of death and disability worldwide, with the majority (~85 %) being ischemic in origin. Age is the most important non-modifiable risk factor for acute ischemic stroke (AIS). While inflammation with ageing is a well-known complication of AIS, a new model is emerging in which ageing-associated thrombosis is being viewed as a multi-step, multi-cellular process driven by inflammatory stimuli and recruitment/activation of leukocytes. The ideal outcome of inflammation is resolution, an active process involving specific endogenous mediators (e.g. annexin A1 [AnxA1]) and related pathways (e.g. formyl peptide receptor-2 [Fpr2/ALX] pathway).[1,2] The development of therapies that temper inflammation and enhance resolution offer potential therapeutic strategies for the treatment and management of thromboinflammation associated with AIS. We have shown that the AnxA1 mimetic peptide AnxA1 Ac2-26 ameliorates thrombotic responses in thromboinflammatory conditions such as Sickle Cell Disease,[3] however, the role that AnxA1 plays in age-related thrombosis is currently unknown. Here we sought to comprehensively elucidate the functional significance of targeting the AnxA1/Fpr2/ALX pathway in age-related thrombosis.Initially, to evaluate the role of AnxA1, thrombosis in cerebral vessels was induced using the light/dye thrombosis model.[2] Male and female adult (10-14 weeks) and ageing (18-24 months) wild type (WT, C57/BL6) or AnxA1 knock-out (AnxA1 -/-) mice were used. WT mice received AnxA1 (1 µg/mouse), or saline vehicle injected 20 min before the onset of thrombus formation in cerebral pial vessels. Thrombogenesis and blood flow cessation times were quantified. AnxA1 treatment was able to prolong blood flow cessation times in both cerebral arterioles and venules, an effect which was more pronounced in ageing mice (p<0.05) via regulation of the FPR2/ALX-pathway.Next, to investigate the mechanism of action of AnxA1 in an inflammatory backdrop (i.e. lipopolysaccharide [LPS]), the effect of AnxA1 on platelet P‐selectin and αIIbβ3 receptor expression, following stimulation with the GPVI collagen receptor agonist convulxin (CVX), was performed. CVX treatment increased platelet activation, which was suppressed by AnxA1 co‐administration (100 ng. p<0.05). CVX+LPS increased platelet αIIbβ3 or P‐selectin levels, which were inhibited by the administration of AnxA1.Finally, to determine whether a deletion of AnxA1 impacts thrombosis, we performed the light/dye thrombosis model in AnxA1 −/− mice. These mice displayed accelerated cerebral microvascular thrombus formation (decrease in blood flow cessation time) compared to WT mice in both arterioles and venules (arterioles: 17.9 ± 2.3 vs 33.2 ± 1.9 min and venules: 13.2 ± 2.4 vs 20.9 ± 2.2 min. p<0.05).In conclusion, these results demonstrate the ability of AnxA1 to modify the thromboinflammatory environment, including reducing platelet activation under inflammatory conditions via GPVI. Collectively, these data show the importance of the AnxA1/Fpr2/ALX system in effecting the resolution of cerebral thromboinflammation in ageing and may provide a novel therapeutic strategy for AIS and other thromboinflammatory conditions. DisclosuresNo relevant conflicts of interest to declare.

Association of von Willebrand factor Ag-to-ADAMTS13 ratio with early sepsis-related mortality

IntroductionSepsis is a highly complex syndrome with highly heterogeneous clinical manifestations, which makes it difficult to detect and treat. Von Willebrand factor (vWF) functions differently depending on its multimeric size and adhesive properties, which are regulated by ADAMTS 13. Thus, a decrease in ADAMTS 13 activity results in the persistence of ultralarge vWF and the formation of microvascular thrombi, ischaemia, and organ failure. The aim of the study was to identify the role of von Willebrand factor antigen-to-ADAMTS 13 ratio in predicting early sepsis-related mortality.Material and methodsThis is a cohort of 70 sequentially selected adults with sepsis. The patients were classified into two groups: A (survivors) and B (non-survivors) based upon their survival within 7 days of hospital admission.ResultsVWF Ag, ADAMTS13, and vWF/ADAMTS13 ratio were significant predictors of early hospital mortality. For vWF Ag at a cut-off level of ≥ 3560 ng/l, sensitivity was 76% and specificity was 88.9%. For ADAMTS at a cut-off level of ≤ 210 ng/l, sensitivity was 84% and specificity was reported as 68.9%. For vWF/ADAMTS ratio at a cut off level of ≥ 17, sensitivity was 80% and specificity was 84.4%. There was a statistically highly significant positive correlation between non-survival and levels of vWF and vWF/ADAMTS 13 ratio, and there was a statistically highly significant negative correlation between non-survival and ADAMTS 13 level.ConclusionsHigh vWF/Ag and vWF/Ag/ADAMTS13 ratios on day 1 of admission are associated with increased early (7 days) sepsis-related mortality.

Edoxaban, a direct oral factor Xa inhibitor, ameliorates coagulation, microvascular thrombus formation, and acute liver injury in a lipopolysaccharide-induced coagulopathy model in rats

Infection increases the risk of thrombosis through the activation of inflammation and coagulation. Edoxaban, a direct oral factor Xa inhibitor, is used for the prevention and treatment of thrombotic diseases. The aim of this study was to determine the effects of edoxaban on microvascular thrombus formation in a rat model of lipopolysaccharide (LPS)-induced coagulopathy. Rats were intravenously injected with 7.5 mg/kg of LPS (Escherichia coli 055:B5). Immediately after LPS injection, the rats were treated with subcutaneous injection of edoxaban. At 2 and 6 h after the injection of LPS, biomarkers of coagulation and organ damages and inflammatory cytokines were measured. Microvascular thrombus formation in organs was evaluated using 125I-fibrinogen (human) or by the pathological analysis. Mortality was examined 24 h after LPS injection. After the injection of LPS, D-dimer and thrombin-antithrombin complex increased and platelet numbers decreased, indicating the activation of coagulation. Microvascular thrombi were found in the liver. Markers of liver injury (aspartate aminotransferase and alanine aminotransferase) also increased. Treatment with edoxaban attenuated the changes in the coagulation markers and microvascular thrombus formation in the liver. Edoxaban suppressed the increase in the liver injury markers and reduced the mortality. Edoxaban did not affect the levels of inflammatory cytokines. In conclusions, edoxaban significantly inhibited the activation of coagulation, the formation of microvascular thrombus in the liver and the liver damage, and reduced mortality in rats injected with LPS. These results suggest that the FXa inhibition by edoxaban might be a beneficial therapy for the management of infection-associated thrombosis.

ZESPÓŁ HEMOLITYCZNO-MOCZNICOWY ZALEŻNY OD ZABURZEŃ UKŁADU DOPEŁNIACZA – PODEJŚCIE DIAGNOSTYCZNE

Complement-mediated hemolytic uremic syndrome (a-HUS), an uncommon variant of thrombotic microangiopathy, is characterized by hemolytic anemia, thrombocytopenia and renal impairment. This disorder might be inherited or/and acquired and leads to dysregulation of the alternative complement pathway at the endothelial cell surface and formation of microvascular thrombi. The differential diagnosis includes other forms of hemolytic syndrome (eg. Shiga-toxin-producing E.coli or S. dysenteriae -associated HUS - STEC-HUS), thrombotic thrombocytopenic purpura (TTP) and congenital errors of vitamin B12 metabolism. The diagnostic approach is presented below.

Developing Quasi-Steady Model for Studying Hemostatic Response Using Supercomputer Technologies

Formation of the platelet plug represents a primary response to the vessel wall injury, but may also result in vessel occlusion. The decrease of the local blood flow due to platelet thrombus formation may lead to serious complications, such as ischemic stroke and myocardial infarction. However, mechanisms responsible for regulation of thrombus dynamics are not clear. In order to get a deeper insight into the role of blood flow and platelet interactions in the formation of the primary platelet plug we developed a particle-based model of microvascular thrombosis using quasisteady flow approximation. In order to simulate thrombus dynamics at physiologically relevant timescales of several minutes, we took advantage of the supercomputer technologies. Our in silico analysis revealed the importance of platelet size heterogeneity for describing experimental data on microvascular thrombus formation. Thus, our model represents a useful tool for the supercomputeraided computational analysis of thrombus dynamics in the microvessels on physiologically relevant timescales.

Atypical Hemolytic-Uremic Syndrome: An Update on Pathophysiology, Diagnosis, and Treatment.

Atypical hemolytic uremic syndrome (aHUS), a rare variant of thrombotic microangiopathy, is characterized by microangiopathic hemolytic anemia, thrombocytopenia, and renal impairment. The condition is associated with poor clinical outcomes with high morbidity and mortality. Atypical HUS predominantly affects the kidneys but has the potential to cause multi-organ system dysfunction. This uncommon disorder is caused by a genetic abnormality in the complement alternative pathway resulting in over-activation of the complement system and formation of microvascular thrombi. Abnormalities of the complement pathway may be in the form of mutations in key complement genes or autoantibodies against specific complement factors. We discuss the pathophysiology, clinical manifestations, diagnosis, complications, and management of aHUS. We also review the efficacy and safety of the novel therapeutic agent, eculizumab, in aHUS, pregnancy-associated aHUS, and aHUS in renal transplant patients.

Propagation of thrombosis by neutrophils and extracellular nucleosome networks.

Neutrophils, early mediators of the innate immune defense, are recruited to developing thrombi in different types of thrombosis. They amplify intravascular coagulation by stimulating the tissue factor-dependent extrinsic pathway via inactivation of endogenous anticoagulants, enhancing factor XII activation or decreasing plasmin generation. Neutrophil-dependent prothrombotic mechanisms are supported by the externalization of decondensed nucleosomes and granule proteins that together form neutrophil extracellular traps. These traps, either in intact or fragmented form, are causally involved in various forms of experimental thrombosis as first indicated by their role in the enhancement of both microvascular thrombosis during bacterial infection and carotid artery thrombosis. Neutrophil extracellular traps can be induced by interactions of neutrophils with activated platelets; vice versa, these traps enhance adhesion of platelets via von Willebrand factor. Neutrophil-induced microvascular thrombus formation can restrict the dissemination and survival of blood-borne bacteria and thereby sustain intravascular immunity. Dysregulation of this innate immune pathway may support sepsis-associated coagulopathies. Notably, neutrophils and extracellular nucleosomes, together with platelets, critically promote fibrin formation during flow restriction-induced deep vein thrombosis. Neutrophil extracellular traps/extracellular nucleosomes are increased in thrombi and in the blood of patients with different vaso-occlusive pathologies and could be therapeutically targeted for the prevention of thrombosis. Thus, during infections and in response to blood vessel damage, neutrophils and externalized nucleosomes are major promoters of intravascular blood coagulation and thrombosis.

Analysis of Clinical Features, Laboratory Characteristics and Therapeutic Outcome in Patients with Thrombotic Thrombocytopenic Purpura Treated at King Fahad Medical City, Riyadh, Saudi Arabia

BackgroundThrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterized by microvascular platelet deposition and thrombus formation with resulting microangiopathic hemolytic anemia and thrombocytopenia. Deficiency of the von Willebrand factor cleavage metalloprotease, also known as ADAMTS 13, has been implicated as an important etiological factor in TTP. Few small studies have been reported on Saudi patients with TTP until now. Our aim was to analyze the clinical features, laboratory characteristics and treatment outcomes with TTP patients treated at our large tertiary care center.MethodsThis is retrospective data of 24 patients with diagnosed of TTP who were treated at King Fahad Medical City, Riyadh, Saudi Arabia between October 2006 and April 2015. Patient suspected as a case of TTP on the basis of clinical features with the evidence of microangiopathic hemolysis and thrombocytopenia were included in this study although data related to pentad of TTP was collected and wherever logistically possible ADAMTS13 levels and inhibitor titer were determined. The primary aim was outcome assessment by overall response rate (ORR) in the treated patients through Kaplan-Meier method. Paired sample t-test was applied to determine the mean significant difference among platelets (plt), hemoglobin (Hgb) & LDH on day 1 and day 7 of treatment.ResultsTwenty-four TTP patients (18 females; 6 males) admitted to our hospital from 2006 to 2015 were analyzed. The mean age was 33.5±13.9 years. Twenty-two (91%) of the patients presented with neurologic features, seven (29%) had fever, ten (42%) had renal impairment that normalized with treatment and four (20.83%) had increased troponin-T or cardiac symptoms. There were 22 patients (91.7%) with the triad of TTP, including hemolytic anemia, thrombocytopenia and neurologic abnormalities; only 2 (8.2%) had the classical pentad of TTP. Among the plausible etiology, idiopathic (51.8%) was the most common followed by acquired autoimmune abnormalities (29.2%). Plasma ADAMTS 13 activity was determined in 19 patients. Eight patients (42.1%) had severe ADAMTS 13 deficiency (activity< 5%); 5 (26.3%) had moderate decrease of ADAMTS 13 activity (activity: 5-10%); another 3 (15.8%) had low ADAMTS 13 activity and 3 (15.8%) patients had normal ADAMTS 13 (>50%) most likely due to sampling post plasma infusion in emergency situations. Median platelet count on Day 1 was 14x10^9/L, and Day 7 was 119x10^9/L (P value< 0.001), Median Hgb on Day1 was 8.25 gm/dl and Day 7 was 9.35 gm/dl (P value< 0.007), Median LDH on Day 1 was 1211 IU/L and Day7 was 278.92 IU/L (P value< 0.001) respectively. All patients received plasma exchange whereas 23 (95.8%) patients received adjunctive corticosteroids. Five patients (20.8%) were early refractory to standard treatment with therapeutic plasma exchange (TPE). Thirteen (54.2%) patients received rituximab either due to refractoriness to TPE on ~ day 7, or earlier due to cardiac or neurological manifestations at treating physician’s discretion. Average hospital stay was 27 days (range 1-131). Twenty-one out of 24 (87.5%) achieved complete remission (CR) without any subsequent relapse. On long term follow up of 22 months (median, Range 1-113), overall survival was 80%. Three patients died during acute episode because of very sever disease or delayed arrival to our center. One patient died later on because of other comorbidities while in CR.ConclusionThrombotic thrombocytopenic purpura is a life threatening condition and immediate treatment with plasma exchange along with steroids and or rituximab was very effective in preventing high risk of mortality and achieving durable CR in 87.5% of our patients. Combination of very severe CNS manifestations and delayed arrival contributed to mortality significantly. More awareness is needed for early diagnosis and early referral to higher centers. DisclosuresNo relevant conflicts of interest to declare.

Dynamics of blood flow and thrombus formation in a multi-bypass microfluidic ladder network.

The reaction dynamics of a complex mixture of cells and proteins, such as blood, in branched circulatory networks within the human microvasculature or extravascular therapeutic devices such as extracorporeal oxygenation machine (ECMO) remains ill-defined. In this report we utilize a multi-bypass microfluidics ladder network design with dimensions mimicking venules to study patterns of blood platelet aggregation and fibrin formation under complex shear. Complex blood fluid dynamics within multi-bypass networks under flow were modeled using COMSOL. Red blood cells and platelets were assumed to be non-interacting spherical particles transported by the bulk fluid flow, and convection of the activated coagulation factor II, thrombin, was assumed to be governed by mass transfer. This model served as the basis for predicting formation of local shear rate gradients, stagnation points and recirculation zones as dictated by the bypass geometry. Based on the insights from these models, we were able to predict the patterns of blood clot formation at specific locations in the device. Our experimental data was then used to adjust the model to account for the dynamical presence of thrombus formation in the biorheology of blood flow. The model predictions were then compared to results from experiments using recalcified whole human blood. Microfluidic devices were coated with the extracellular matrix protein, fibrillar collagen, and the initiator of the extrinsic pathway of coagulation, tissue factor. Blood was perfused through the devices at a flow rate of 2 µL/min, translating to physiologically relevant initial shear rates of 300 and 700 s-1 for main channels and bypasses, respectively. Using fluorescent and light microscopy, we observed distinct flow and thrombus formation patterns near channel intersections at bypass points, within recirculation zones and at stagnation points. Findings from this proof-of-principle ladder network model suggest a specific correlation between microvascular geometry and thrombus formation dynamics under shear. This model holds potential for use as an integrative approach to identify regions susceptible to intravascular thrombus formation within the microvasculature as well as extravascular devices such as ECMO.

Targeting coagulation factor XII as a novel therapeutic option in brain trauma.

ObjectiveTraumatic brain injury is a major global public health problem for which specific therapeutic interventions are lacking. There is, therefore, a pressing need to identify innovative pathomechanism‐based effective therapies for this condition. Thrombus formation in the cerebral microcirculation has been proposed to contribute to secondary brain damage by causing pericontusional ischemia, but previous studies have failed to harness this finding for therapeutic use. The aim of this study was to obtain preclinical evidence supporting the hypothesis that targeting factor XII prevents thrombus formation and has a beneficial effect on outcome after traumatic brain injury.MethodsWe investigated the impact of genetic deficiency of factor XII and acute inhibition of activated factor XII with a single bolus injection of recombinant human albumin‐fused infestin‐4 (rHA‐Infestin‐4) on trauma‐induced microvascular thrombus formation and the subsequent outcome in 2 mouse models of traumatic brain injury.ResultsOur study showed that both genetic deficiency of factor XII and an inhibition of activated factor XII in mice minimize trauma‐induced microvascular thrombus formation and improve outcome, as reflected by better motor function, reduced brain lesion volume, and diminished neurodegeneration. Administration of human factor XII in factor XII–deficient mice fully restored injury‐induced microvascular thrombus formation and brain damage.InterpretationThe robust protective effect of rHA‐Infestin‐4 points to a novel treatment option that can decrease ischemic injury after traumatic brain injury without increasing bleeding tendencies. Ann Neurol 2016;79:970–982

Analysis of the influence of antithrombin on microvascular thrombosis: anti-inflammation is crucial for anticoagulation.

PurposeMicrovascular thrombosis during septic conditions is of essential clinical relevance, but the pathomechanisms are not yet completely understood. The purpose of this study was to study the distinguished differentiation of the interactions of inflammation and coagulation using antithrombin (AT), a mediator of anticoagulation and anti-inflammation.MethodsUsing a thrombosis model in a cremaster muscle preparation of male C57Bl/6J mice (n = 83), we quantitatively assessed microvascular thrombus formation by using intravital fluorescence microscopy. Experimental groups consisted of animals treated with AT or with tryptophan49-blocked AT (TrypAT), which exerts only anticoagulant but no anti-inflammatory effects. To further see whether endothelial glycosaminoglycan (GAG) binding with consecutive prostacyclin (PGI2) release is mandatory for the anticoagulant process of AT, animals were administered heparin or indomethacin either alone or in combination with AT.ResultsThe antithrombotic capacity of AT significantly differs in the experimental groups in which anti-inflammation was antagonized. This is given by the significantly prolonged occlusion times (p < 0.05) and higher patency rates in case of application of AT alone; while all other groups in which the anti-inflammatory action of AT was blocked by TrypAT, heparin or indomethacin revealed thrombus kinetics comparable to controls.ConclusionsThe anti-inflammatory influence of AT is essentially linked to its anticoagulant effect in the microvascular system. Those specifications of the active profile of AT characterize the intimate interactions of the anticoagulant and anti-inflammatory pathways. This might be of relevance for AT as a therapeutic agent in critically diseased patients and the clinical understanding of microvascular thrombosis.

Analysis of clinical and laboratory characteristics in 42 patients with thrombotic thrombocytopenic purpura from a single center in China

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterized by microvascular platelet deposition and thrombus formation with resulting microangiopathic hemolytic anemia. Deficiency of the von Willebrand factor cleavage protease, also known as ADAMTS 13, has been implicated as an important etiological factor in TTP. Little studies were obtained on Chinese patients with TTP until now. Our aim was to analyze the clinical features, outcome and laboratory characteristics of Chinese TTP patients, and determine whether plasma ADAMTS 13 activity is decreased in TTP and its diagnostic value for TTP. Forty-two TTP patients (29 females; 13 males) admitted to our hospital from 1998 to 2010 were analyzed. There were 34 patients (81%) with the triad of TTP, including hemolytic anemia, thrombocytopenia and neurologic abnormalities; 7 (16.7%) had the classical pentad of TTP. Major etiologic factors were acquired autoimmunological abnormalities (31%); no familial TTP was identified in this series. The schistocytes of peripheral blood smears were present in all cases with a mean frequency of 4.6% (range from 0.3% to 13.4%). Plasma ADAMTS 13 activity was determined in 22 patients with the FRET-vWF86 assay. Only 4 idiopathic TTP patients (18.2%) had severe ADAMTS 13 deficiency (activity<10%); 9 (40.9%) had moderate decrease of ADAMTS 13 activity (activity: 10-40%); another 9 (40.91%) had normal ADAMTS 13 activity (>40%). T lymphocyte subpopulation was measured in 23 TTP patients with FACS Calibur; 14 of the 23 (60.9%) had significantly decreased CD4 cells count and CD4/CD8 ratio, suggesting cellular immune dysfunction may be involved in the pathogenesis of TTP. In the studies, plasmapheresis is the main therapeutic method. 26 of 31 patients (83.9%) accepting plasmapheresis achieved complete remission; those patients who only underwent plasma infusion had low remission rate (18.2%) and high mortality (9/11; 81.8%). Four patients with packed RBC infusion manifested transient exacerbation of neurologic or psychiatric symptoms. In conclusion, the diagnosis of TTP in China is still based on clinical features including evidence of microangiopathic hemolysis. Severe ADAMTS 13 activity deficiency might be a valuable indicator for idiopathic TTP diagnosis. Further studies are needed to determine the real value of ADAMTS 13 activity for TTP diagnosis and whether T lymphocytes subset dysregulation plays important role in TTP pathogenesis.

CON: Regulatory T Cells Are Protective in Ischemic Stroke

For many years ischemic stroke was regarded as a purely thrombotic disease resulting from the occlusion of cerebral arteries and subsequent breakdown of energy supply to neuronal tissues. Back in the late 1970, however, some pioneers in experimental stroke research, above all John Hallenbeck at the National Institute of Neurological Disorders and Stroke recognized the existence of polymorphonuclear leukocytes and macrophages in brain specimen from animals and patients with cerebral ischemia, thereby establishing the conceptual basis for the immunobiology of stroke.1 Since then a plethora of experimental studies stressed the importance of these typically innate immune cells in brain ischemia but clinical trials testing, for example, neutrophil blocking agents, ultimately bombed. Several years later Stoll and Jander2 from Dusseldorf, Germany, were among the first to describe the infiltration of T lymphocytes, which per definition belong to the adaptive immune system, into ischemic brains of rodents. Of note, this happened accidentally while searching for a central nervous system lesion paradigm devoid of inflammation which could be used as control for experimental …

Effect of the hydrogen sulfide donor GYY4137 on platelet activation and microvascular thrombus formation in mice

This study evaluates the effect of the H2S donor GYY4137 (GYY) on adhesion molecule expression, protein S-sulfhydration and morphology of platelets in vitro and on kinetics of microvascular thrombus formation in vivo. Using flowcytometry, untreated resting, TRAP-activated, or TRAP-activated and GYY-exposed human platelets were studied for expression of P-selectin, GPIb and GPIIb/IIIa as well as for fibrinogen binding. By means of electron microscopy, platelet morphology and intracellular granule numbers were assessed. Platelet shape change was studied using immunohistochemistry for P-selectin, NSF and F-actin by SR-SIM. Biotin switch assay served for the analysis of platelet protein S-sulfhydration by GYY. Using the FeCl3 and the light/dye model in dorsal skinfold chamber-equipped mice, the effect of GYY and its vehicle DMSO was studied on venular thrombus formation and tail-vein bleeding time. Soluble (s)P-selectin plasma concentrations were measured in GYY- or DMSO-treated animals. Exposure to GYY increased the S-sulfhydration of platelet proteins. GYY reduced dose-dependently the TRAP-induced adhesion molecule expression and attenuated the morphological signs of TRAP-associated platelet activation. In mice, GYY caused a significant prolongation of venular thrombus formation and tail-vein bleeding time. Application of an anti-P-selectin antibody in DMSO-exposed animals prolonged thrombosis formation comparably as GYY did. GYY reversed the TRAP-induced distribution of P-selectin at the plasma membrane of platelets. This indicates reduced exocytosis and shedding of P-selectin, which is supported by significantly lower sP-selectin concentrations in GYY- vs. DMSO-treated mice. H2S acts anti-thrombotic and seems to regulate thrombogenesis by interference with platelet activation and adhesion molecule-mediated aggregation.