Sirtuin 3 (SIRT3) is a nicotinamide adenine dinucleotide (NAD)-dependent mitochondrial deacetylase that regulates protein acetylation and maintains mitochondrial homeostasis in nucleated cells. By deacetylating cyclophilin D (CypD), SIRT3 limits mitochondrial permeability transition pore (mPTP) opening and protects against mitochondrial dysfunction. Although SIRT3 is present in murine and human platelets, its contribution to platelet mitochondrial regulation and procoagulant platelet formation remains unknown. This study investigated whether platelet SIRT3 modulates CypD-dependent procoagulant platelet formation. Platelets obtained from platelet-specific Sirt3 knockout mice (Sirt3plt-/-) and littermate controls (Sirt3plt+/+) were analyzed under resting conditions and after activation with CRP-XL and thrombin. Flow cytometry was used to analyze platelet (activation) markers and procoagulant platelet formation, and mitochondrial respiration was assessed using a Seahorse extracellular flux analyzer. Platelets lacking SIRT3 showed no alterations in basal or agonist-stimulated mitochondrial respiration. Likewise, platelet Sirt3 deletion did not affect the generation of procoagulant platelets in response to strong dual agonist stimulation. These findings indicate that, despite its role in regulating mPTP opening in nucleated cells, platelet SIRT3 is not required for procoagulant platelet formation, suggesting that platelets rely on distinct mechanisms for mPTP regulation.

Open ADAMTS13 conformation is gaining clinical interest as a biomarker for diagnosing immune-mediated thrombotic thrombocytopenic purpura (iTTP) and monitoring patients in remission for increased risk of relapse. Nevertheless, little is known about how open the structure of ADAMTS13 is exactly in iTTP patients. In this study, we aimed to assess the uniformity of open ADAMTS13 across iTTP patients. To do this, we identified four monoclonal antibodies that recognize epitopes that are cryptic in closed ADAMTS13 from healthy donors but accessible upon antibody-mediated ADAMTS13 opening. Distributed across the D, T7, T8 and CUB1 domains, these cryptic epitopes indicate ADAMTS13 closure through multiple interdomain contacts extending beyond the well-described S-CUB interaction. Interestingly, all acute iTTP patients consistently present one distinct open ADAMTS13 in which all novel cryptic epitopes are accessible. During remission, closed ADAMTS13 with all epitopes being cryptic is predominantly found in patients with restored activity, whereas distinct open ADAMTS13 is present in patients with subclinical disease. Furthermore, IgG from iTTP patients opened the conformation of ADAMTS13, corroborating the role of pathogenic autoantibodies in opening ADAMTS13 in iTTP. These new cryptic epitope-recognizing monoclonal antibodies hold promise to further enhance our understanding of compactly closed conformation of ADAMTS13 and may support the prediction of early relapses in the future.

BACKGROUND:During shock, neutrophil-mediated glycocalyx degradation can lead to exposure of the procoagulant endothelial surface and consequent organ injury. Resuscitation using crystalloid fluids may augment this endothelial damage, while resuscitation using plasma may preserve glycocalyx health. We hypothesised that resuscitation with plasma would preserve glycocalyx integrity by reducing inflammation, glycocalyx shedding, and dyscoagulation in a controlled model of systemic inflammation in human volunteers. METHODS:Twelve healthy male volunteers were injected with 2 ng kg-1 lipopolysaccharide (LPS) to induce systemic inflammation. Thirty minutes after LPS administration, participants were randomised to receive 10 ml kg-1 of either balanced salt solution or solvent detergent plasma (SDP). Plasma syndecan-1, a marker of glycocalyx degradation, was the primary outcome. Additionally, plasma biomarkers of inflammation, neutrophil extracellular trap formation, glycocalyx degradation, endothelial injury, and coagulation were measured serially, by bead-based or enzyme-linked immunoassays. RESULTS:LPS induced a systemic inflammatory response, accompanied by endothelial injury as indicated by increased levels of syndecan-1 (median increase: 2920-4430 pg ml-1, P<0.05). Participants receiving SDP had lower levels of neutrophils and plasma matrix metalloproteinase-9, compared with those receiving balanced salt solution. Neutrophil extracellular trap formation, inflammation, and glycocalyx degradation were not affected by fluid therapy. SDP reduced LPS-induced prolonged prothrombin time, but had no impact on other biomarkers of coagulation. CONCLUSIONS:In human endotoxaemia, plasma resuscitation reduced leucocyte and neutrophil levels but did not reduce glycocalyx degradation, when compared with equal volume resuscitation with a balanced crystalloid solution.

Allosteric regulation of ADAMTS13 (A Disintegrin And Metalloproteinase with ThromboSpondin type-1 motif, member 13) activity involves an interaction between its Spacer (S) and CUB1-2 domains to keep the enzyme in a closed, latent conformation. Monoclonal antibodies (mAb) uncouple the S-CUB interaction to open the ADAMTS13 conformation and thereby disrupt the global enzyme latency. The molecular mechanism behind this mAb-induced allostery remains poorly understood. To gain insights in the mAb-induced S-CUB uncoupling and global latency disruption, we combined hydrogen/deuterium exchange mass spectrometry (HDX MS) with structural analysis of ADAMTS13 CUB1-2 mutants. Thereby, the CUB1 L3 and L9 loops were fine-mapped as the 17G2 mAb binding epitope. Indirect S-CUB uncoupling was observed as mAb binding induced extensive structural dynamics within both CUB1-2 domains without directly targeting the contiguous CUB1 surface that engages with the ADAMTS13 S domain. HDX MS analysis revealed the short interdomain linker to structurally cover the central CUB1-2 domain interface, which also showed some protein regions that became more exposed upon mAb binding. Therefore, repositioning of the central CUB1-2 interface appears crucial to transfer structural dynamics between both domains. Nevertheless, mutagenesis of the short linker did not disrupt the ADAMTS13 global latency as its closed conformation was preserved. Presumably, allosteric disruption of the global latency requires a structural impact extending beyond the central interface repositioning. As anti-ADAMTS13 autoantibodies from immune-mediated thrombotic thrombocytopenic purpura (iTTP) patients also induce an open ADAMTS13 conformation, our novel insights in the antibody-mediated global latency disruption boost our understanding of the iTTP disease pathology.

The rapid transportation of blood samples and blood products using drones has high potential in the medical sector. However, before this can be implemented, sufficient evidence that drone transportation is not affecting clinical outcomes is needed. Currently, limited data on the stability of blood, and specifically on platelet activation, after transport using drones are available. Therefore, the impact of drone transportation on platelet activation parameters was analyzed. Blood was drawn from 20 healthy volunteers and lactate dehydrogenase (LDH), potassium, free hemoglobin, PFA-100 closure time, platelet factor 4 (PF4) plasma levels and platelet activation membrane markers were determined in blood that was transported by drone and compared to non-transported samples. In addition, a control group was included where blood samples were transported by car. Transport by both drone and car increased LDH and potassium levels, but the values mostly remained within the total allowable error. Both drone and car transportation impacted platelet activation, as indicated by a small increase in the baseline P-selectin expression and increased PF4 plasma levels. To our knowledge, this is the first study assessing the impact of drone transport on platelet activation. Transportation of blood tubes from healthy individuals using drones has only minimal impacts on blood stability and platelet activation parameters, and is comparable to blood transportation by car. Therefore, the effects observed as a result of drone transportation will likely not impact clinical decision making.

Results: Eighteen studies were included in the final review for a total of 45 patients.The median age was 78 years (range: 21-93).The vast majority (75.6%) were males.Presenting bleeding symptoms were widely variable; the most common were subcutaneous bleeding and muscle hematomas.In forty patients the FVIII levels at diagnosis were ≤1% or undetectable.The median inhibitor level was 40 BU/mL (1.8 to 2000 BU/mL).Kaplan-Meier analysis showed that around 56% of patients have been reported to achieve bleed control in one day after the start of treatment and almost 90% of patients had no bleeding by day 5 of starting Emicizumab (Figure 2).Side effects were infrequently reported; nevertheless, the treatment appeared to be well-tolerated.Figure: Time-to-bleed control after starting Emicizumab. Conclusion(s):Emicizumab appears to be an effective and safe hemostatic treatment for AHA.Bleeding control is rapidly achieved after initiating emicizumab.Clinical trials comparing emicizumab versus standard hemostatic agents are needed to confirm these findings.

BACKGROUND:Acquired von Willebrand syndrome (aVWS) is common in patients with mechanical circulatory support (MCS) devices. In these patients, the high shear stress in the device leads to increased shear-induced proteolysis of von Willebrand factor (VWF) by A Disintegrin And Metalloprotease with Thrombospondin type 1 repeats, number 13 (ADAMTS13). As a result, the high molecular weight (HMW) VWF multimers are lost, leading to a decreased VWF function and impaired hemostasis that could explain the bleeding complications that are frequently observed in these patients. To counteract this abnormal VWF degradation by ADAMTS13, we developed a novel targeted therapy, using an anti-ADAMTS13 monoclonal antibody (mAb) that inhibits the shear-induced proteolysis of VWF by ADAMTS13.METHODS:Human or bovine blood was circulated through in vitro MCS device systems with either inhibitory anti-ADAMTS13 mAb 3H9 or 17C7 (20 μg/ml) or control anti-ADAMTS13 mAb 5C11 or phosphate buffered saline (PBS). VWF multimers and function (collagen binding activity) were determined at different time points. Next, Impella pumps were implanted in calves and the calves were injected with PBS and subsequently treated with mAb 17C7. VWF, ADAMTS13, and blood parameters were determined.RESULTS:We demonstrated that blocking ADAMTS13 could prevent the loss of HMW VWF multimers in in vitro MCS device systems. Importantly, our antibody could reverse aVWS in a preclinical Impella-induced aVWS calf model.CONCLUSION:Hence, inhibition of ADAMTS13 could become a novel therapeutic strategy to manage aVWS in MCS device patients.

BACKGROUND:Hereditary antithrombin deficiency is a rare autosomal-dominant disorder predisposing to recurrent venous thromboembolism (VTE). To date, only two founder mutations have been described.OBJECTIVES:We investigated the antithrombin p.Thr147Ala variant, found in 12 patients of African origin. This variant is known as rs2227606 with minor allele frequency of 0.5% in Africans and absent in Europeans. A possible founder effect was investigated.METHODS:Phenotypical characterization was established through immunological and functional methods, both under basal and stress conditions. Recombinant antithrombin molecules were constructed by site-directed mutagenesis and expressed in HEK-293T cells. Secreted antithrombin was purified and functionally characterized. Structural modeling was performed to predict the impact of the mutation on protein structure. A novel nanopore sequencing approach was used for haplotype investigation.RESULTS:Ten patients experienced VTE, stroke, or obstetric complications. Antithrombin antigen levels and anti-IIa activity were normal or slightly reduced while anti-Xa activity was reduced with only one commercial assay. On crossed immunoelectrophoresis, an increase of antithrombin fractions with reduced heparin affinity was observed under high ionic strength conditions but not under physiological conditions. The recombinant p.Thr147Ala protein displayed a reduced anti-Xa activity. Structural modeling revealed that residue Thr147 forms three hydrogen bonds that are abolished when mutated to alanine. The investigated patients shared a common haplotype involving 13 SERPINC1 intragenic single nucleotide polymorphisms.CONCLUSION:Antithrombin p.Thr147Ala, responsible for antithrombin type II heparin binding site deficiency, is the first founder mutation reported in people of African ancestry. This study further emphasizes the limitations of commercial methods to diagnose this specific subtype.

Plasma ADAMTS13 circulates in a folded conformation that is stabilized by an interaction between the central Spacer domain and the C-terminal CUB (complement components C1r and C1s, sea urchin protein Uegf, and bone morphogenetic protein-1) domains. Binding of ADAMTS13 to the VWF D4(-CK) domains or to certain activating murine monoclonal antibodies (mAbs) induces a structural change that extends ADAMTS13 into an open conformation that enhances its function. The objective was to characterize the mechanism by which conformational activation enhances ADAMTS13-mediated proteolysis of VWF. The activating effects of a novel anti-Spacer (3E4) and the anti-CUB1 (17G2) mAbs on the kinetics of proteolysis of VWF A2 domain fragments by ADAMTS13 were analyzed. mAb-induced conformational changes in ADAMTS13 were investigated by enzyme-linked immunosorbent assay. Both mAbs enhanced ADAMTS13 catalytic efficiency (kcat/Km) by ∼twofold (3E4: 2.0-fold; 17G2: 1.8-fold). Contrary to previous hypotheses, ADAMTS13 activation was not mediated through exposure of the Spacer or cysteine-rich domain exosites. Kinetic analyses revealed that mAb-induced conformational extension of ADAMTS13 enhances the proteolytic function of the metalloprotease domain (kcat), rather than augmenting substrate binding (Km). A conformational effect on the metalloprotease domain was further corroborated by the finding that incubation of ADAMTS13 with either mAb exposed a cryptic epitope in the metalloprotease domain that is normally concealed when ADAMTS13 is in a closed conformation. We show for the first time that the primary mechanism of mAb-induced conformational activation of ADAMTS13 is not a consequence of functional exosite exposure. Rather, our data are consistent with an allosteric activation mechanism on the metalloprotease domain that augments active site function.

Background The biological diagnosis of immune-mediated thrombotic thrombocytopenic purpura (iTTP) is based on determination of ADAMTS13 activity (<10%) and anti-ADAMTS13 autoantibodies. ADAMTS13 antigen levels are not routinely measured in iTTP patients, but studies have shown that antigen levels are a valuable prognostic factor. Objectives To (a) report the validation of our in-house developed ADAMTS13 antigen enzyme-linked immunosorbent assay (ELISA) and determine ADAMTS13 antigen in a large cohort of healthy donor and iTTP patient plasma samples; and (b) to investigate whether ADAMTS13 antigen determination is not disturbed by the presence of anti-ADAMTS13 autoantibodies. Methods Our in-house ADAMTS13 antigen ELISA was validated in terms of sensitivity, repeatability, and reproducibility. ADAMTS13 antigen levels were determined in plasma samples from 423 healthy donors and 112 acute iTTP patients. Purified IgGs from iTTP patients were added to normal human plasma to determine whether anti-ADAMTS13 autoantibodies hampered ADAMTS13 antigen determination. Results Our in-house ADAMTS13 antigen ELISA has a detection limit of 3% and low intra-assay (coefficient of variation, %CV < 10%) and inter-assay (%CV < 18%) variability. ADAMTS13 antigen levels were significantly reduced (P < .0001) in acute iTTP patients (15 +/- 18%) compared to healthy donors (101 +/- 18%). The anti-ADAMTS13 autoantibodies in plasma of iTTP patients did not impede ADAMTS13 antigen determinations using our in-house ELISA. Conclusions Our in-house ADAMT13 antigen ELISA is a powerful tool to correctly determine ADAMTS13 antigen levels in iTTP patients, which supports routine ADAMTS13 antigen measurements in these patients to have better insight into disease prognosis.

Background Cerebral malaria (CM) is the most severe complication of malaria. Endothelial activation, cytokine release, and vascular obstruction are essential hallmarks of CM. Clinical studies have suggested a link between von Willebrand factor (VWF) and malaria pathology. Objectives To investigate the contribution of VWF in the pathogenesis of experimental cerebral malaria (ECM). Methods BothVwf(+/+)andVwf(-/-)mice were infected withPlasmodium bergheiANKA (PbANKA) to induce ECM. Alterations of plasma VWF and ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13), platelet count, neurological features, and accumulation of platelets and leukocytes in the brain were examined following infection. Results Plasma VWF levels significantly increased uponPbANKA infection inVwf(+/+)animals. While ADAMTS13 activity was not affected, high molecular weight VWF multimers disappeared at the end-stage ECM, possibly due to an ongoing hypercoagulability. Although the number of reticulocytes, a preferential target for the parasites, was increased inVwf(-/-)mice compared toVwf(+/+)mice early after infection, parasitemia levels did not markedly differ between the two groups. Interestingly,Vwf(-/-)mice manifested overall clinical ECM features similar to those observed inVwf(+/+)animals. At day 8.5 post-infection, however, clinical ECM features inVwf(-/-)mice were slightly more beneficial than inVwf(+/+)animals. Despite these minor differences, overall survival was not different betweenVwf(-/-)andVwf(+/+)mice. Similarly,PbANKA-induced thrombocytopenia, leukocyte, and platelet accumulations in the brain were not altered by the absence of VWF. Conclusions Our study suggests that increased VWF concentration is a hallmark of ECM. However, VWF does not have a major influence in modulating late-stage ECM pathogenesis.

Recently, we showed that ADAMTS13 circulates in an open conformation during the acute phase of immune-mediated thrombotic thrombocytopenic purpura (iTTP). Although the cause of this conformational change remains elusive, ADAMTS13 is primarily closed in iTTP patients in remission with ADAMTS13 activity >50% and undetectable anti-ADAMTS13 autoantibodies, as well as after rituximab treatment, suggesting a role for anti-ADAMTS13 autoantibodies. Therefore, immunoglobulin G from 18 acute iTTP patients was purified and added to closed ADAMTS13 in healthy donor plasma. This resulted in open ADAMTS13 in 14 of 18 (78%) samples, proving that anti-ADAMTS13 autoantibodies can induce an open ADAMTS13 conformation. To further elucidate the conformation of ADAMTS13 in iTTP patients, we studied a novel iTTP patient cohort (n = 197) that also included plasma samples from iTTP patients in remission in whom ADAMTS13 activity was <50%. The open ADAMTS13 conformation was found during acute iTTP, as well as in patients in remission with ADAMTS13 activity <50% and in half of the patients with ADAMTS13 activity >50%, although free anti-ADAMTS13 autoantibodies were not always detected. Thus, open ADAMTS13 is a hallmark of acute iTTP, as well as a novel biomarker that can be used to detect subclinical iTTP in patients in remission. Finally, a long-term follow-up study in 1 iTTP patient showed that the open conformation precedes a substantial drop in ADAMTS13 activity. In conclusion, we have shown that anti-ADAMTS13 autoantibodies from iTTP patients induce an open ADAMTS13 conformation. Most importantly, an open ADAMTS13 conformation is a biomarker for subclinical iTTP and could become an important tool in TTP management.

Bleeding is the most frequent adverse event in patients with continuous flow mechanical circulatory support (CF-MCS) and has been linked to the occurrence of acquired von Willebrand syndrome (aVWS). MCS devices cause an increased shear-induced proteolysis of von Willebrand factor (VWF) by ADAMTS13, leading to aVWS. Hence, specifically blocking ADAMTS13 might be an efficient way to rescue the loss of HMW VWF multimers in CF-MCS patients. To investigate if blocking ADAMTS13, using an in-house developed inhibitory anti-ADAMTS13 monoclonal antibody (mAb), prevents the loss of high molecular weight (HMW) VWF multimers in in vitro CF-MCS systems and to determine the efficacy of this therapy in a CF-MCS calf model. Human blood was perfused through in vitro CF-MCS systems (Heartmate II and Impella CP, axial flow heart pumps) in the presence of the inhibitory or control mAb (20 μg/mL). Bovine blood was perfused through an in vitro Impella 5.5 system with the inhibitory mAb (20 μg/mL) or PBS. Next, Impella 5.5 pumps were implanted in calves. One dose of the inhibitory mAb (600 μg/kg) or PBS was injected eight days after Impella implantation. Plasma samples were analysed for VWF multimers, VWF antigen (VWF:Ag) and VWF collagen binding activity (VWF:CB). A time-dependent decrease in HMW VWF multimers was observed in both in vitro CF-MCS systems in the presence of the control mAb, leading to a 70% reduction of HMW VWF multimers, 180 minutes (min) after blood perfusion (p=0.01 for HM II and p=0.0003 for Impella). This was also reflected by a severely decreased VWF:CB/VWF:Ag ratio (0.59±0.11 and 0.52±0.10 at 180 min versus 1.00±0.06 and 1.07±0.09 before perfusion, for the HM II (p=0.03) and Impella (p=0.001) respectively). Interestingly, blocking ADAMTS13 using the inhibitory mAb prevented the loss of HMW VWF multimers in both systems (p=0.50 for the HM II and p=0.06 for the Impella, 180 min after the start of perfusion). The preservation of HMW VWF multimers was also reflected by normal VWF:CB/VWF:Ag ratios (0.92±0.16 and 0.97±0.11 at 180 min versus 0.93±0.09 and 1.19±0.12 before perfusion for the HM II (p=0.75) and Impella (p=0.06) respectively). Blocking bovine ADAMTS13 using the inhibitory mAb could prevent the loss of HMW VWF multimers in the in vitro Impella 5.5 system, showing that the calf is a good preclinical animal model to study the in vivo effect of this novel therapy. Impella implantation in the calves led to a decrease in HMW VWF multimers (Figure 1A and B). Hence, this animal model represents the VWF laboratory features of MCS-induced aVWS. Moreover, the loss of HMW VWF multimers after pump implantation could be rescued after injection of the inhibitory mAb (Figure 1A and B). Blocking ADAMTS13 rescues MCS-induced VWF proteolysis in calves. Hence, inhibiting ADAMTS13 function could become a promising therapeutic strategy to rescue aVWS-induced bleeding in MCS patients. Figure 1. Impella calf model Type of funding source: Foundation. Main funding source(s): Fund for Scientific Research Flanders