Presence of procoagulant peripheral blood mononuclear cells in severe COVID-19 patients relate to ventilation perfusion mismatch and precede pulmonary embolism.

PURPOSE: Pulmonary emboli (PE) contribute substantially to coronavirus disease 2019 (COVID-19) related mortality and morbidity. Immune cell-mediated hyperinflammation drives the procoagulant state in COVID-19 patients, resulting in immunothrombosis. To study the role of peripheral blood mononuclear cells (PBMC) in the procoagulant state of COVID-19 patients, we performed a functional bioassay and related outcomes to the occurrence of PE. Secondary aims were to relate this functional assay to plasma D-dimer levels, ventilation perfusion mismatch and TF expression on monocyte subsets. METHODS: PBMC from an ICU biobank were obtained from 20 patients with a computed tomography angiograph (CTA) proven PE and compared to 15 COVID-19 controls without a proven PE. Functional procoagulant properties of PBMC were measured using a modified fibrin generation time (MC-FGT) assay. Tissue factor (TF) expression on monocyte subsets were measured by flow cytometry. Additional clinical data were obtained from patient records including end-tidal to arterial carbon dioxide gradient. RESULTS: MC-FGT levels were highest in the samples taken closest to the PE detection, similar to the end-tidal to arterial carbon dioxide gradient (ETCO2 - PaCO2), a measurement to quantify ventilation-perfusion mismatch. In patients without proven PE, peak MC-FGT relates to an increase in end-tidal to arterial carbon dioxide gradient. We identified non-classical, CD16 positive monocytes as the subset with increased TF expression. CONCLUSION: We show that the procoagulant state of PBMC could aid in early detection of PE in COVID-19 ICU patients. Combined with end-tidal to ETCO2 - PaCO2 gradient, these tests could improve early detection of PE on the ICU.

Abnormal coagulation and enhanced fibrinolysis due to lysinuric protein intolerance associates with bleeds and renal impairment.

INTRODUCTION: Lysinuric protein intolerance (LPI), a rare autosomal recessive transport disorder of cationic amino acids lysine, arginine and ornithine, affects intestines, lungs, liver and kidneys. LPI patients may display potentially life-threatening bleeding events, which are poorly understood. AIMS: To characterize alterations in haemostatic and fibrinolytic variables associated with LPI. METHODS: We enrolled 15 adult patients (8 female) and assessed the clinical ISTH/SSC-BAT bleeding score (BS). A variety of metabolic and coagulation assays, including fibrin generation test derivatives, clotting time (CT) and clot lysis time (CLT), thromboelastometry (ROTEM), and PFA-100 and Calibrated Automated Thrombogram (CAT), were used. RESULTS: All patients had mild-to-moderate renal insufficiency, and moderate bleeding tendency (BS 4) without spontaneous bleeds. Mild anaemia and thrombocytopenia occurred. Traditional clotting times were normal, but in contrast, CT in fibrin generation test, and especially ROTEM FIBTEM was abnormal. The patients showed impaired primary haemostasis in PFA, irrespective of normal von Willebrand factor activity, but together with lowered fibrinogen and FXIII. Thrombin generation (TG) was reduced in vitro, according to CAT-derived endogenous thrombin potential, but in vivo TG was enhanced in the form of circulating prothrombin fragment 1 and 2 values. Very high D-dimer and plasmin-α2-antiplasmin (PAP) complex levels coincided with shortened CLT in vitro. CONCLUSIONS: Defective primary haemostasis, coagulopathy, fibrin abnormality (FIBTEM, CT and CLT), low TG in vitro and clearly augmented fibrinolysis (PAP and D-dimer) in vivo were all detected in LPI. Altered fibrin generation and hyperfibrinolysis were associated with the metabolic and renal defect, suggesting a pathogenetic link in LPI.

Factor XI promotes hemostasis in factor IX-deficient mice.

Essentials Mice lacking factor IX (FIX) or factor XI (FXI) were tested in a saphenous vein bleeding model. FIX-deficient mice displayed a hemostatic defect and FXI-deficient mice were similar to wild type mice. Infusion of FXI or over-expression of FXI in FIX-deficient mice improved hemostasis. FXI may affect the phenotype of FIX-deficiency (hemophilia B). SUMMARY: Background In humans, deficiency of coagulation factor XI may be associated with a bleeding disorder, but, until recently, FXI-deficient mice did not appear to have a hemostatic abnormality. A recent study, however, indicated that FXI-deficient mice show a moderate hemostatic defect in a saphenous vein bleeding (SVB) model. Objectives To study the effect of FXI on bleeding in mice with normal levels of the FXI substrate FIX and in mice lacking FIX (a murine model of hemophilia B). Methods Wild-type mice and mice lacking either FIX (F9- ) or FXI (F11-/- ) were tested in the SVB model. The plasma levels of FXI in F11-/- mice were manipulated by infusion of FXI or its active form FXIa, or by overexpressing FXI by the use of hydrodynamic tail vein injection. Results F9- mice showed a significant defect in the SVB model, whereas F11-/- mice and wild-type mice were indistinguishable. Intravenous infusion of FXI or FXIa into, or overexpression of FXI in, F9- mice improved hemostasis in the SVB model. Overexpression of a FXI variant lacking a FIX-binding site also improved hemostasis in F9- mice. Conclusions Although we were unable to demonstrate a hemostatic defect in F11-/- mice in the SVB model, our results support the premise that supraphysiological levels of FXI improve hemostasis in F9- mice through FIX-independent pathways.

Role of von Willebrand factor and ADAMTS-13 in early brain injury after experimental subarachnoid hemorrhage.

Essentials von Willebrand Factor (VWF) and ADAMTS13 may affect early injury after subarachnoid hemorrhage (SAH). Early brain injury was assessed in VWF-/- , ADAMTS13-/- and recombinant (r) ADAMTS13 treated mice. VWF-/- and rADAMTS13 treated mice had less brain injury than ADAMTS13-/- and wild-type mice. Early administration of rADAMTS13 may improve outcome after SAH by reducing early brain injury. SUMMARY: Background Early brain injury is an important determinant of poor functional outcome and case fatality after aneurysmal subarachnoid hemorrhage (SAH), and is associated with early platelet aggregation. No treatment exists for early brain injury after SAH. We investigated whether von Willebrand factor (VWF) is involved in the pathogenesis of early brain injury, and whether ultra-early treatment with recombinant ADAMTS-13 (rADAMTS-13) reduces early brain injury after experimental SAH. Methods Experimental SAH in mice was induced by prechiasmatic injection of non-anticoagulated blood from a littermate. The following experimental SAH groups were investigated: C57BL/6J control (n = 21), VWF-/- (n = 25), ADAMTS-13-/- (n = 23), and C57BL/6J treated with rADAMTS-13 (n = 26). Mice were killed at 2 h after SAH. Primary outcome measures were microglial activation (IBA-1 surface area) and neuronal injury (number of cleaved caspase-3-positive neurons). Results As compared with controls, microglial activation was decreased in VWF-/- mice (mean difference of - 20.0%, 95% confidence interval [CI] - 4.0% to - 38.6%), increased in ADAMTS-13-/- mice (mean difference of + 34.0%, 95% CI 16.2-51.7%), and decreased in rADAMTS-13-treated mice (mean difference of - 22.1%, 95% CI - 3.4% to - 39.1%). As compared with controls (185 neurons, interquartile range [IQR] 133-353), neuronal injury in the cerebral cortex was decreased in VWF-/- mice (63 neurons, IQR 25-78), not changed in ADAMTS-13-/- mice (53 neurons, IQR 26-221), and reduced in rADAMTS-13-treated mice (45 neurons, IQR 9-115). Conclusions Our findings suggest that VWF is involved in the pathogenesis of early brain injury, and support the further study of rADAMTS-13 as a treatment option for early brain injury after SAH.

Atherothrombosis and Thromboembolism: Position Paper from the Second Maastricht Consensus Conference on Thrombosis.

Atherothrombosis is a leading cause of cardiovascular mortality and long-term morbidity. Platelets and coagulation proteases, interacting with circulating cells and in different vascular beds, modify several complex pathologies including atherosclerosis. In the second Maastricht Consensus Conference on Thrombosis, this theme was addressed by diverse scientists from bench to bedside. All presentations were discussed with audience members and the results of these discussions were incorporated in the final document that presents a state-of-the-art reflection of expert opinions and consensus recommendations regarding the following five topics: 1. Risk factors, biomarkers and plaque instability: In atherothrombosis research, more focus on the contribution of specific risk factors like ectopic fat needs to be considered; definitions of atherothrombosis are important distinguishing different phases of disease, including plaque (in)stability; proteomic and metabolomics data are to be added to genetic information. 2. Circulating cells including platelets and atherothrombosis: Mechanisms of leukocyte and macrophage plasticity, migration, and transformation in murine atherosclerosis need to be considered; disease mechanism-based biomarkers need to be identified; experimental systems are needed that incorporate whole-blood flow to understand how red blood cells influence thrombus formation and stability; knowledge on platelet heterogeneity and priming conditions needs to be translated toward the in vivo situation. 3. Coagulation proteases, fibrin(ogen) and thrombus formation: The role of factor (F) XI in thrombosis including the lower margins of this factor related to safe and effective antithrombotic therapy needs to be established; FXI is a key regulator in linking platelets, thrombin generation, and inflammatory mechanisms in a renin-angiotensin dependent manner; however, the impact on thrombin-dependent PAR signaling needs further study; the fundamental mechanisms in FXIII biology and biochemistry and its impact on thrombus biophysical characteristics need to be explored; the interactions of red cells and fibrin formation and its consequences for thrombus formation and lysis need to be addressed. Platelet-fibrin interactions are pivotal determinants of clot formation and stability with potential therapeutic consequences. 4. Preventive and acute treatment of atherothrombosis and arterial embolism; novel ways and tailoring? The role of protease-activated receptor (PAR)-4 vis à vis PAR-1 as target for antithrombotic therapy merits study; ongoing trials on platelet function test-based antiplatelet therapy adjustment support development of practically feasible tests; risk scores for patients with atrial fibrillation need refinement, taking new biomarkers including coagulation into account; risk scores that consider organ system differences in bleeding may have added value; all forms of oral anticoagulant treatment require better organization, including education and emergency access; laboratory testing still needs rapidly available sensitive tests with short turnaround time. 5. Pleiotropy of coagulation proteases, thrombus resolution and ischaemia-reperfusion: Biobanks specifically for thrombus storage and analysis are needed; further studies on novel modified activated protein C-based agents are required including its cytoprotective properties; new avenues for optimizing treatment of patients with ischaemic stroke are needed, also including novel agents that modify fibrinolytic activity (aimed at plasminogen activator inhibitor-1 and thrombin activatable fibrinolysis inhibitor.

Venous thrombosis and coagulation parameters in patients with pure venous malformations.

INTRODUCTION: Venous malformations (VMs) are ubiquitous, low-flow vascular anomalies known to be occasionally painful due to thrombotic episodes within the lesion. The prevalence of superficial or deep vein thrombosis is unclear. METHODS: A cross-sectional study among outpatients aged ≥ 12 years with pure VMs was performed, quantifying the prevalence of thrombosis by screening all patients with compression ultrasonography (CUS). Additionally, we evaluated whether coagulation alterations were related to thrombosis observed with CUS. RESULTS: In total, 69 patients with pure VMs were eligible, median age was 30 years (range 12-63) and 52% were female. A total of 68 patients underwent CUS. Superficial vein thrombosis was observed in 10 (15%) cases; 1 patient had a current asymptomatic deep venous thrombosis. Residual superficial or deep thrombosis was observed in 25 patients (36%). In total, 49% had either a history or current signs of a thrombotic event and overall 10% had venous thromboembolism. In approximately 50% of the patients the D-dimer level was above 0.5 mg/l. Median P-selectin and Von Willebrand factor levels were 29 ng/ml (interquartile range (IQR) 21-34) and 108% (IQR 83-132), respectively. No differences were observed in the coagulation parameters between the patients with and without current clots in their VM. CONCLUSION: This study shows that superficial or deep vein thrombosis is common among patients with a pure VM. Physicians should be aware of this high incidence, especially if other risk factors for thrombosis are present.

Factor V Leiden is associated with increased sperm count.

STUDY QUESTION: Is the thrombophilia mutation factor V Leiden (FVL) associated with an increased total sperm count? SUMMARY ANSWER: Carriers of FVL have a higher total sperm count than non-FVL-carriers, which could not be explained by genetic linkage or by observations in a FVL-mouse model. WHAT IS KNOWN ALREADY: FVL has a high prevalence in Caucasians despite detrimental health effects. Carriers have been shown to have higher fecundity, which might partly explain this evolutionary paradox. STUDY DESIGN, SIZE, DURATION: We determined FVL status in two cohorts (Dutch, n = 627; Danish, n = 854) of consecutively included men without known causes for spermatogenic failure, and performed an individual patient data meta-analysis of these two cohorts together with one previously published (Dutch, n = 908) cohort. We explored possible biological underpinnings for the relation between sperm count and FVL, by use of a FVL-mouse model and investigations of genetic linkage. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were male partners of subfertile couples (two Dutch cohorts) and young men from the general population (Danish cohort): FVL carrier rate was 4.0%, 4.6% and 7.3%, respectively. There were differences in smoking, abstinence time and age between the cohorts. We corrected for these in the primary analysis, which consisted of a mixed linear effects model, also incorporating unobjectified population differences. In public haplotype data from subjects of European descent, we explored linkage disequilibrium of FVL with all known single nucleotide polymorphisms in a 1.5 MB region around the F5 gene with an R2 cutoff of 0.8. We sequenced exons of four candidate genes hypothesized to be linked to FVL in a subgroup of FVL carriers with extreme sperm count values. The animal studies consisted of never mated 15-18-week-old C57BL/J6 mice heterozygous and homozygous for FVL and wild-type mice. We compared spermatogenesis parameters (normalized internal genitalia weights, epididymis sperm content and sperm motility) between FVL and wild-type mice. MAIN RESULTS AND THE ROLE OF CHANCE: Human FVL carriers have a higher total sperm count than non-carriers, with an adjusted mean difference of 31 × 106 (95%CI 0.2-61.7; P = 0.048). Mice with the FVL mutation do not have increased spermatogenesis as compared to wildtype mice. None of the studied polymorphisms was in linkage disequilibrium, either in the public databases or in a subgroup of FVL carriers with extremely high sperm counts. LIMITATIONS, REASONS FOR CAUTION: The difference in total sperm count would benefit from confirmation in other cohorts. The finding of higher count in carriers was consistent however, with no heterogeneity between the cohorts. The lack of effect of murine FVL might suggest there is no direct causality. The exploratory efforts on genetic linkage do not rule out that the association is a reflection of FVL co-inheritance with a non-studied causative polymorphism. WIDER IMPLICATIONS OF THE FINDINGS: A high sperm count in FVL-carrying males contributes to understanding the high prevalence of this otherwise disadvantageous mutation. The findings might provide directions for future research on male fertility. STUDY FUNDING/COMPETING INTEREST(S): No conflicts of interest. Research was conducted with funding from the Netherlands Organisation for Scientific Research (NWO, VIDI innovative research grant 016.126.364 awarded to S. Middeldorp). The Danish cohort was supported by the Innovation Fund Denmark (InnovationsFonden, grant no. 14-2013-4), The Danish Ministry of Health and the Danish Environmental Protection Agency. TRIAL REGISTRATION NUMBER: Not applicable.

Dynamics of von Willebrand factor reactivity in sickle cell disease during vaso-occlusive crisis and steady state.

Essentials The role of von Willebrand Factor (VWF) in the pathophysiology of sickle cell disease is unclear. We assessed markers of VWF during admission for vaso-occlusive crisis (VOC) and steady state. VWF reactivity was higher during VOC and was associated with inflammation and neutrophil activation. Hyper-adhesive VWF may promote VOC in sickle cell disease. SUMMARY: Background Endothelial activation plays a central role in the pathophysiology of vaso-occlusion in sickle cell disease (SCD), facilitating adhesive interactions with circulating blood cells. Upon activation, various adhesive molecules are expressed, including von Willebrand factor (VWF). Increased VWF levels have been observed in patients with SCD during steady state. However, the role of VWF in the pathogenesis of SCD vaso-occlusion is unclear. Objectives To longitudinally assess the quantity and reactivity of VWF and its regulating protease ADAMTS-13 during vaso-occlusive crisis (VOC). Methods In this observational study, we obtained sequential blood samples in adult SCD patients during VOC. Results VWF reactivity was significantly higher during VOC (active VWF, VWF glycoprotein Ib-binding activity, and high molecular weight multimers), whereas platelet count and levels of ADAMTS-13 antigen and ADAMTS-13 activity were concomitantly lower than during steady state. Levels of VWF antigen, VWF propeptide (VWF:pp) and ADAMTS-13 specific activity did not change during VOC. VWF reactivity correlated strongly with markers of inflammation and neutrophil activation, and was inversely correlated with the platelet count. In patients who developed acute chest syndrome, levels of VWF, VWF:pp and active, hyperadhesive VWF were significantly higher, whereas ADAMTS-13 activity was lower, than in patients without this complication. Conclusions We provide the first evidence that VOC in SCD is associated with increased reactivity of VWF, without a pronounced ADAMTS-13 deficiency. This hyper-reactivity may be explained by resistance of VWF to proteolysis, secondary to processes such as inflammation and oxidative stress. Hyperadhesive VWF, scavenging blood cells in the microcirculation, may thereby amplify and sustain VOC in SCD.

Association between protein C levels and mortality in patients with advanced prostate, lung and pancreatic cancer.

INTRODUCTION: Procoagulant factors promote cancer progression and metastasis. Protein C is involved in hemostasis, inflammation and signal transduction, and has a protective effect on the endothelial barrier. In mice, administration of activated protein C reduced experimental metastasis. We assessed the association between protein C and mortality in patients with three types of cancer. METHODS: The study population consisted of patients with advanced prostate, non-small cell lung or pancreatic cancer, who participated in the INPACT trial (NCT00312013). The trial evaluated the addition of nadroparin to chemotherapy in patients with advanced malignancy. Patients were divided into tertiles based on protein C at baseline. The association between protein C levels and mortality was evaluated with Cox proportional hazard models. RESULTS: We analysed 477 patients (protein C tertiles: <97, 97-121 and ≥121%). Mean age was 65±9years; 390 (82%) were male; 191 patients (40%) had prostate cancer, 161 (34%) had lung cancer, and 125 (26%) pancreatic cancer. During a median follow-up of 10.4months, 291 patients (61%) died. Median protein C level was 107% (IQR 92-129). In the lowest tertile, 75 patients per 100 patient-years died, as compared to 60 and 54 in the middle and high tertile, respectively. Lower levels of protein C were associated with increased mortality (in tertiles: HR for trend 1.18, 95%CI 1.02-1.36, adjusted for age, sex and nadroparin use; as a continuous variable: HR 1.004, 95%CI 1.00-1.008, p=0.07). CONCLUSION: Protein C seems inversely associated with mortality in patients with advanced prostate, lung and pancreatic cancer. Further research should validate protein C as a biomarker for mortality, and explore the effects of protein C on progression of cancer.

Use of an absorbable embolization material for reversible portal vein embolization in an experimental model.

BACKGROUND: Portal vein embolization (PVE) is used to increase future remnant liver size in patients requiring major hepatic resection. PVE using permanent embolization, however, predisposes to complications and excludes the use of PVE in living donor liver transplantation. In the present study, an absorbable embolization material containing fibrin glue and different concentrations of the fibrinolysis inhibitor aprotinin was used in an experimental animal model. METHODS: PVE of the cranial liver lobes was performed in 30 New Zealand White rabbits, which were divided into five groups, fibrin glue + 1000, 700, 500, 300 or 150 kunits/ml aprotinin, and were compared with a previous series of permanent embolization using the same experimental set-up. Caudal liver lobe hypertrophy was determined by CT volumetry, and portal recanalization was identified on contrast-enhanced CT images. Animals were killed after 7 or 42 days, and the results were compared with those of permanent embolization. RESULTS: PVE using fibrin glue with aprotinin as embolic material was effective, with 500 kunits/ml providing the optimal hypertrophic response. Lower concentrations of aprotinin (150 and 300 kunits/ml) led to reduced hypertrophy owing to early recanalization of the embolized segments. The regeneration rate over the first 3 days was higher in the group with 500 kunits/ml aprotinin than in the groups with 300 or 150 kunits/ml or permanent embolization. In the 500-kunits/ml group, four of five animals showed recanalization 42 days after embolization, with minimal histological changes in the cranial lobes following recanalization. CONCLUSION: Fibrin glue combined with 500 kunits/ml aprotinin resulted in reversible PVE in 80 per cent of animals, with a hypertrophy response comparable to that achieved with permanent embolization material. Surgical relevance Portal vein embolization (PVE) is used to increase future remnant liver volume in patients scheduled for major liver resection who have insufficient future remnant liver size to perform a safe resection. The current standard is PVE with permanent embolization materials, which renders patients found to have unresectable disease prone to complications owing to the permanently deportalized liver segments. Absorbable embolization might prevent the PVE-associated morbidity and lower the threshold for its application. In this study, PVE using fibrin glue and aprotinin resulted in an adequate hypertrophy response with 80 per cent recanalization after 42 days. Considering the minor histological changes following recanalization of embolized segments and potentially preserved function, reversible PVE might also be applied in living donor liver transplantation.

Structure-function relationships in thrombin-activatable fibrinolysis inhibitor.

Thrombin-activatable fibrinolysis inhibitor (TAFI) is an important regulator in the balance of coagulation and fibrinolysis. TAFI is a metallocarboxypeptidase that circulates in plasma as zymogen. Activated TAFI (TAFIa) cleaves C-terminal lysine or arginine residues from peptide substrates. The removal of C-terminal lysine residues from partially degraded fibrin leads to reduced plasmin formation and thus attenuation of fibrinolysis. TAFI also plays a role in inflammatory processes via the removal of C-terminal arginine or lysine residues from bradykinin, thrombin-cleaved osteopontin, C3a, C5a and chemerin. TAFI has been studied extensively over the past three decades and recent publications provide a wealth of information, including crystal structures, mutants and structural data obtained with antibodies and peptides. In this review, we combined and compared available data on structure/function relationships of TAFI.

The influence of corticosteroids on hemostasis in healthy subjects.

BACKGROUND: Corticosteroids have been associated with an increased risk of venous thromboembolism in patients treated for inflammatory diseases. It is unclear whether the thrombotic risk is induced by the inflammation of the underlying inflammatory diseases or whether corticosteroids are prothrombotic as well. Considering the widespread use of corticosteroids in clinical practise, it is critical to know whether corticosteroids enhance coagulation. OBJECTIVE: To investigate whether a 10-day prednisolone burst therapy activates hemostasis in healthy individuals. METHODS: Healthy subjects received either 0.5 mg kg(-1) day(-1) of oral prednisolone or placebo. Venous blood was collected at baseline, day 1 and day 10 and tested for thrombin-antithrombin complexes (TATc), D-dimer, plasmin-alpha2-antiplasmin complexes (PAPc), plasminogen-activator inhibitor type-1 (PAI-1), von Willebrand factor (VWF) and thrombin generation (peak thrombin, velocity index and endogenous thrombin potential [ETP]). RESULTS: Fifteen subjects received prednisolone and 16 placebo (median age 29 vs. 22 years, female subjects 33% vs. 56%, respectively). Peak thrombin and velocity index were higher in the placebo group at baseline. After 10 days of treatment, peak thrombin, velocity index, PAI-1 and VWF increased in the oral prednisolone group as compared with the placebo group (15.8 [SD 16.3] vs. -0.1 [SD 16.1], 41.2 [SD 41.3] vs. -2.3 [SD 42.7], 18.0 [IQR 8.0-37.0] vs. 0.5 [IQR -18.5-13.0], 4.0 [IQR -1.0-12.0] vs. 0.0 [IQR -2.5-1.5], respectively). No changes were observed for TATc, ETP, PAPc and D-dimer. CONCLUSIONS: Oral prednisolone induces a procoagulant state in healthy subjects, suggesting that corticosteroid treatment may increase the thromboembolic risk in patients with inflammatory diseases.

Loss of asthma control and activation of coagulation and fibrinolysis.

BACKGROUND: Epidemiologic studies have shown that patients with severe asthma have increased risk of pulmonary embolism, in particular patients with frequent asthma exacerbations. Therefore, we hypothesized that asthma exacerbations are associated with increased haemostatic activity. OBJECTIVE: To investigate whether induced loss of asthma control is associated with changes in coagulation and fibrinolytic parameters in peripheral blood. METHODS: We performed a prospective, inhaled steroid withdrawal study in 23 patients with moderate to moderately severe asthma, consisting of a baseline visit and a visit after loss of asthma control. During the visits, we measured asthma control questionnaire (ACQ), atopy, lung function, inflammatory markers (eosinophils and neutrophils), and haemostatic parameters in plasma. RESULTS: Complete cessation of inhaled corticosteroids led to a loss of asthma control in 22 of 23 patients. We found increased asthma symptoms (ACQ 0.9 vs. 2.9, P < 0.01), significantly reduced lung function (forced expiratory volume in 1 s (FEV1) 3.51L vs. 3.13L, P < 0.01) and increased levels of eosinophils in plasma (0.26 × 10(E9)/L vs. 0.16 × 10(E9)/L, P = 0.03) in patients after loss of asthma control. However, we observed no significant changes in the coagulation and fibrinolysis parameters. CONCLUSION: Loss of asthma control after cessation of inhaled corticosteroids does not lead to increased haemostatic activation in patients with moderate to moderately severe asthma. This suggests that more severe inflammation or additional risk factors are required for activation of coagulation or reduction of fibrinolysis in asthma.

Thrombin-activatable fibrinolysis inhibitor influences disease severity in humans and mice with pneumococcal meningitis.

BACKGROUND: Mortality and morbidity in patients with bacterial meningitis result from the proinflammatory response and dysregulation of coagulation and fibrinolysis. Thrombin-activatable fibrinolysis inhibitor (TAFI) is activated by free thrombin or thrombin in complex with thrombomodulin, and plays an antifibrinolytic role during fibrin clot degradation, but also has an anti-inflammatory role by inactivating proinflammatory mediators, such as complement activation products. OBJECTIVE: To assess the role of TAFI in pneumococcal meningitis. METHODS: We performed a prospective nationwide genetic association study in patients with bacterial meningitis, determined TAFI and complement levels in cerebrospinal fluid (CSF), and assessed the function of TAFI in a pneumococcal meningitis mouse model by using Cpb2 (TAFI) knockout mice. RESULTS: Polymorphisms (reference sequences: rs1926447 and rs3742264) in the CPB2 gene, coding for TAFI, were related to the development of systemic complications in patients with pneumococcal meningitis. Higher protein levels of TAFI in CSF were significantly associated with CSF complement levels (C3a, iC3b, and C5b-9) and with more systemic complications in patients with bacterial meningitis. The risk allele of rs1926447 (TT) was associated with higher levels of TAFI in CSF. In the murine model, consistent with the human data, Cpb2-deficient mice had decreased disease severity, as reflected by lower mortality, and attenuated cytokine levels and bacterial outgrowth in the systemic compartment during disease, without differences in the brain compartment, as compared with wild-type mice. CONCLUSIONS: These findings suggest that TAFI plays an important role during pneumococcal meningitis, which is likely to be mediated through inhibition of the complement system, and influences the occurrence of systemic complications and inflammation.

Selective modulation of thrombin-activatable fibrinolysis inhibitor (TAFI) activation by thrombin or the thrombin-thrombomodulin complex using TAFI-derived peptides.

BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) is a risk factor for coronary heart disease. TAFI is proteolytically activated by thrombin, the thrombin-thrombomodulin complex and plasmin. Once active, it dampens fibrinolysis and inflammation. The aim of this study was to generate TAFI-derived peptides that specifically modulate TAFI activation and activity. METHODS: Thirty-four overlapping TAFI peptides, and modifications thereof, were synthesized. The effects of these peptides on TAFI activation and TAFIa activity were determined. In addition, the binding of the peptides to thrombin were determined. RESULTS: Four peptides (peptides 2, 18, 19 and 34) inhibited TAFI activation and two peptides (peptides 14 and 24) inhibited TAFIa activity directly. Peptide 2 (Arg12-Glu28) and peptide 34 (Cys383-Val401) inhibited TAFI activation by the thrombin-thrombomodulin complex with IC50 values of 7.3 ± 1.8 and 6.1 ± 0.9 µm, respectively. However, no inhibition was observed in the absence of thrombomodulin. This suggests that the regions Arg12-Glu28 and Cys383-Val401 in TAFI are involved in thrombomodulin-mediated TAFI activation. Peptide 18 (Gly205-Ser221) and peptide 19 (Arg214-Asp232) inhibited TAFI activation by thrombin and the thrombin-thrombomodulin complex. Furthermore, these peptides bound to thrombin (KD : 1.5 ± 0.4 and 0.52 ± 0.07 µm for peptides 18 and 19, respectively), suggesting that Gly205-Asp232 of TAFI is involved in binding to thrombin. Peptide 14 (His159-His175) inhibited TAFIa activity. The inhibition was TAFIa specific, because no effect on the homologous enzyme carboxypeptidase B was observed. CONCLUSIONS: Thrombin-activatable fibrinolysis inhibitor-derived peptides show promise as new tools to modulate TAFI activation and TAFIa activity. Furthermore, these peptides revealed potential binding sites on TAFI for thrombin and the thrombin-thrombomodulin complex.

In vivo increase in thrombin generation by four-factor prothrombin complex concentrate in apixaban-treated healthy volunteers.

BACKGROUND: Four-factor prothrombin complex concentrate (PCC) (Cofact; Sanquin Blood Supply) 50 IU kg(-1) increased thrombin generation beyond baseline values in healthy, rivaroxaban-treated subjects. OBJECTIVE: To assess whether infusion with doses of 37.5 IU kg(-1) and 25 IU kg(-1) PCC reverses the anticoagulant effect of high-dose apixaban, another oral direct factor Xa inhibitor. METHODS: In a randomized, double-blind, placebo-controlled, crossover study, six healthy subjects received twice-daily apixaban 10 mg for 3.5 days followed by a single bolus of 37.5 IU kg(-1) PCC, 25 IU kg(-1) PCC, or placebo. The primary outcome was the effect of PCC 15 min after infusion on thrombin generation (endogenous thrombin potential [ETP]); secondary outcomes were the immediate effect of PCC on prothrombin time (PT) and the effect of PCC as compared with placebo over a period of 24 h on ETP and PT. RESULTS: Fifteen minutes after infusion of 37.5 IU kg(-1) and 25 IU kg(-1) PCC, ETP increased from 41% ± 11% to 56% ± 23% (P = 0.06) and from 44% ± 12% to 51% ± 15% (P = 0.03), respectively. ETP significantly differed over time between 37.5 IU kg(-1) PCC and placebo during 24 h after infusion (P < 0.01). Both PCC doses restored apixaban-induced PT prolongation after 15 min (P < 0.01), and this was sustained over a period of 24 h. CONCLUSION: Both 37.5 IU kg(-1) PCC and 25 IU/kg PCC improved coagulation parameters in healthy subjects, suggesting partial reversal of the anticoagulant effect of apixaban. This implies that PCC might be considered in patients with apixaban-associated bleeding. However, ETP was not immediately restored to pre-apixaban levels, suggesting that these doses are too low to instantly and fully restore hemostasis at peak apixaban levels.

Lack of TAFI increases brain damage and microparticle generation after thrombolytic therapy in ischemic stroke.

BACKGROUND: Thrombin-activatable fibrinolysis inhibitor (TAFI) plays an important role in coagulation and fibrinolysis. Whereas TAFI deficiency may lead to a haemorrhagic tendency, data from TAFI knockout mice (TAFI-/-) are controversial and no differences have been reported in these animals after ischemic stroke. There are also no data regarding the role of circulating microparticles (MPs) in TAFI-/-. OBJECTIVES: to examine the effect of tPA on the rate of intracranial haemorrhage (ICH) and on MPs generated in a model of ischemic stroke in TAFI-/- mice. METHODS: Thrombin was injected into the middle cerebral artery (MCA) to analyse the effect of tPA (10mg/Kg) on the infarct size and haemorrhage in the absence of TAFI. Immunofluorescence for Fluoro-Jade C was performed on frozen brain slides to analyse neuronal degeneration after ischemia. MPs were isolated from mouse blood and their concentrations calculated by flow cytometry. RESULTS: Compared with saline, tPA significantly increased the infarct size in TAFI-/- mice (p<0.05). Although plasma fibrinolytic activity (fibrin plate assay) was higher in these animals, no macroscopic or microscopic ICH was detected. A positive signal for apoptosis and degenerating neurons was observed in the infarct area, being significantly higher in tPA treated TAFI-/- mice (p<0.05). Interestingly, higher numbers of MPs were found in TAFI-/- plasma as compared to wild type, after stroke (p<0.05). CONCLUSIONS: TAFI deficiency results in increased brain damage in a model of thrombolysis after ischemic stroke, which was not associated with bleeding but with neuronal degeneration and MP production.

Decreased plasma levels of activated factor VII in patients with deep vein thrombosis.

BACKGROUND: The initiating trigger in the development of deep vein thrombosis (DVT) remains unidentified. It has been suggested that tissue factor (TF)-bearing microparticles play a key role, which indicates a role for the TF pathway in the initiation of DVT. OBJECTIVE: To assess the role of the TF pathway in the initiation of venous thrombosis, we measured plasma levels of factor VII and VIIa in patients with acute DVT and in controls. METHODS: We included 148 patients diagnosed with acute DVT and 179 controls in this study. Antigen levels of FVII and FVIIa were measured by using assays recently developed in our laboratory. RESULTS: Median FVII levels in patients were 109.8% (interquartile range [IQR] 86.0-153.2) compared with 102.2% (IQR 76.1-141.7) in controls. Individuals with FVII levels in the upper quartile had a 1.6-fold increased risk for the presence of a DVT (odds ratio 1.6, 95% confidence interval 0.8-3.1). Median FVIIa levels in patients were 50.2 ng mL(-1) (IQR 25.2-86.1) compared with 96.6 ng mL(-1) (69.9-168.9) in controls. Individuals with FVIIa levels in the lowest quartile had a > 5-fold increased risk for the presence of a DVT (odds ratio 5.5, 95% confidence interval 2.8-10.6). Both risks did not change substantially after adjustment for potential confounders. CONCLUSION: Decreased plasma levels of FVIIa in patients with deep vein thrombosis may indicate ongoing consumption of FVIIa and suggest a contributory role for TF in venous thrombus formation.

Development of a Hyperactive Primary Hemostatic System During Off-Pump Lung Transplantation Resulting From an Unbalance Between von Willebrand Factor and Its Cleaving Protease ADAMTS13.

An unbalance between the platelet-adhesive protein von Willebrand factor (VWF) and its cleaving protease ADAMTS13 is a risk factor for thrombosis. Here, we assessed levels and functionality of VWF and ADAMTS13 in patients undergoing off-pump lung transplantation. We analyzed plasma of 10 patients and distinguished lung transplantation-specific effects from those generally accompanying open-chest surgeries by comparing results with 11 patients undergoing off-pump coronary bypass graft (CABG) surgery. Forty healthy volunteers were included for reference values. VWF antigen levels as well as the VWF ristocetin cofactor activity/VWF antigen ratio increased during lung transplantation and after CABG surgery. An increase in VWF propeptide levels was paralleled by a decrease in ADAMTS13 activity. This was more pronounced during lung transplantation. Similarly, the capacity of plasma to support platelet aggregation under shear flow conditions in vitro was more increased during lung transplantation. The proportion of high molecular weight VWF multimers was elevated in both groups without evidence for ultra-large VWF. VWF's collagen binding activity remained unchanged. In conclusion, a hyperactive primary hemostatic system develops during lung transplantation resulting both from a pronounced (functional) increase of the VWF molecule and decrease of ADAMTS13. This may increase the risk of platelet thrombosis within the allograft.