Computer based visualization of clot structures in extracorporeal membrane oxygenation and histological clot investigations for understanding thrombosis in membrane lungs.

Extracorporeal membrane oxygenation (ECMO) was established as a treatment for severe cardiac or respiratory disease. Intra-device clot formation is a common risk. This is based on complex coagulation phenomena which are not yet sufficiently understood. The objective was the development and validation of a methodology to capture the key properties of clots deposed in membrane lungs (MLs), such as clot size, distribution, burden, and composition. One end-of-therapy PLS ML was examined. Clot detection was performed using multidetector computed tomography (MDCT), microcomputed tomography (µCT), and photography of fiber mats (fiber mat imaging, FMI). Histological staining was conducted for von Willebrand factor (vWF), platelets (CD42b, CD62P), fibrin, and nucleated cells (4', 6-diamidino-2-phenylindole, DAPI). The three imaging methods showed similar clot distribution inside the ML. Independent of the imaging method, clot loading was detected predominantly in the inlet chamber of the ML. The µCT had the highest accuracy. However, it was more expensive and time consuming than MDCT or FMI. The MDCT detected the clots with low scanning time. Due to its lower resolution, it only showed clotted areas but not the exact shape of clot structures. FMI represented the simplest variant, requiring little effort and resources. FMI allowed clot localization and calculation of clot volume. Histological evaluation indicated omnipresent immunological deposits throughout the ML. Visually clot-free areas were covered with leukocytes and platelets forming platelet-leukocyte aggregates (PLAs). Cells were embedded in vWF cobwebs, while vWF fibers were negligible. In conclusion, the presented methodology allowed adequate clot identification and histological classification of possible thrombosis markers such as PLAs.

[Advances in the treatment of thrombotic thrombocytopenic purpura].

Thrombotic thrombocytopenic purpura (TTP) is a fatal thrombotic disease caused by a marked decrease in the activity of ADAMTS13, a von Willebrand factor cleaving protease. In congenital TTP, ADAMTS13 activity is decreased by an abnormality in ADAMTS13, and in acquired TTP, by anti-ADAMTS13 autoantibody. Death from thrombosis in the acute phase has been an issue with conventional treatment of acquired TTP by plasma exchange or immunosuppressive therapy. However, the advent of caplacizumab, an anti-VWF nanobody, has made it possible to suppress thrombus formation and is expected to improve survival rates. In addition, some case series have shown the efficacy of caplacizumab without plasma exchange for acquired TTP, and this approach is being investigated in clinical trials. Fresh-frozen plasma is transfused to supply ADAMTS13 for congenital TTP, but frequent transfusions over a long period of time can lead to problems such as infection and allergic reactions. Novel therapies such as recombinant ADAMTS13 products and gene therapy are now under development, and show promise for future clinical use.

Imbalance in the vWF - ADAMTS13 axis exists early in acute pancreatitis and predicts persistent organ failure and pancreatic necrosis-a prospective study.

BACKGROUND: The pathophysiology of Acute Pancreatitis (AP) may be complicated by endothelial activation. von Willebrand Factor (vWF)- ADAMTS13 axis is a marker of endothelial activation. The study aimed to investigate the axis in AP, comparing it in patients with and without persistent organ failure (OF), with and without pancreatic necrosis, and correlating it with the standard severity scores (CRP, APACHE II, BISAP, SOFA, and qSOFA) METHODS: vWF-Antigen (vWF:Ag), vWF-Collagen-Binding-Assay (vWF:CBA), and ADAMTS13 activity (ADAMTS13:act) levels were measured within 5 days of symptom onset in consecutive patients (n = 98), who were admitted with a first episode of AP (Dec 2021-May 2023). RESULTS: Of the 98 patients admitted with AP, 78(79.6 %) had no or transient OF; 20(20.4 %) had persistent OF. Age was comparable (43.73 ± 15.36 vs 38.65 ± 13.69) [mean ± SD](years), and males were predominant in both groups (70.5 % vs 80 %). Patientswith persistent OF had higher vWF:CBA(%)[323(279-486.5) vs 199.5(159.1-295.75)] and lower ADAMTS13:act(%)[35.4(23.8-56.85) vs 56.35(44.1-71.9)][median (25th - 75th percentile)](P = 0.001) than those with no or transient OF. Patients with pancreatic necrosis (n = 19) had lower ADAMTS13:act(%)[42.79 ± 18.69] than those without pancreatic necrosis (n = 18) [62.49 ± 22.64] (P < 0.01). ADAMTS13:act had a negative correlation(r = -0.2), whereas vWF:Ag and vWF:CBA had a positive correlation (r = 0.2) with the standard severity scores (P < 0.05). ADAMTS13:act could predict pancreatic necrosis [AUROC-0.737, P < 0.05] and persistent OF [AUROC-0.746, P < 0.001], while vWF:CBA could predict persistent OF [AUROC- 0.73, P < 0.001]. CONCLUSION: vWF-ADAMTS13 axis helps to predict severe disease and is associated with poor outcomes in acute pancreatitis.

Plasma von Willebrand factor levels in patients with cancer: A meta­analysis.

von Willebrand Factor (VWF) is well recognized for being dysregulated in various malignancies and has emerged as a potential biomarker for cancer detection. The present meta-analysis aimed to elucidate the association between plasma VWF and the incidence and metastasis of cancer. For this purpose, a comprehensive search was conducted across multiple databases from their inception until March 3, 2023. This culminated in the selection of 15 original studies on various types of cancer, including a collective sample of 1,403 individuals. The standardized mean difference (SMD) and 95% confidence intervals (CIs) were employed as statistical parameters to determine the association between plasma VWF and the incidence and metastasis of cancer. These were estimated using a random-effects model. The pooled data revealed that the plasma VWF levels of patients with cancer were significantly elevated compared with those of healthy controls (SMD, 0.98; 95% CI, 0.59-1.36), and a significant association was observed between plasma VWF levels and cancer metastasis (SMD, 0.69; 95% CI, 0.33-1.06). The symmetry of the Begg's funnel plots indicated that no significant bias was present in the analyses of VWF in cancer and its metastasis. In summary, the results of the present meta-analysis support the hypothesis that increased plasma VWF levels may serve as a biomarker for cancer and metastatic progression.

IL-6 Released from Hepatic Stellate Cells Promotes Glycolysis and Migration of HCC Through the JAK1/vWF/TGFB1 Axis.

Purpose: The crosstalk between hepatocellular carcinoma (HCC) cells and hepatic stellate cells (HSCs) is one of the important mechanisms of liver cancer metastasis. The relationship between liver cancer metastasis and glycolysis has been extensively studied recently. However, the role of von Willebrand factor (vWF) mediated glycolysis mechanism in liver cancer metastasis is currently unknown. Methods: Western blot was used to verify the expression of vWF in HCC cells. PAS staining, glycogen and L-lactate content assays were used to reflect cellular glycolysis levels. The ability of cell migration was explored by Wound-healing and Transwell assays. Besides, the effect of vWF on the progression of HCC in vivo was also studied using subcutaneous xenograft model. Results: vWF derived from HCC cells promoted tumor migration by mediating glycolysis. Besides, vWF participated in the crosstalk between HCC cells and HSCs. HCC cells activated HSCs through vWF-mediated TGFB1 expression and secretion, and activated HSCs upregulated vWF expression in HCC cells through IL-6 secretion feedback. Further, in vitro and in vivo experiments also confirmed the importance of the JAK1/vWF/TGFB1 axis in regulating HSCs-derived IL-6 mediated HCC migration and growth. Conclusion: In summary, this article demonstrated that IL-6 released from hepatic stellate cells enhanced glycolysis and migration ability of liver cancer cells by activating JAK1/vWF/TGFB1 axis which may also be a potential target for inhibiting liver cancer metastasis.

R1205H (Vicenza) causes conformational changes in the VWF-D'D3 domains and enhances VWF binding to clearance receptors LRP1 and SR-A1.

INTRODUCTION: Von Willebrand factor (VWF)-R1205H variant (Vicenza) results in markedly enhanced VWF clearance in humans that has been shown to be largely macrophage-mediated. However, the biological mechanisms underlying this enhanced clearance remain poorly understood. This study aimed to investigate the roles of (i) specific VWF domains and (ii) different macrophage receptors in regulating enhanced VWF-R1205H clearance. METHODS: In vivo clearance of full-length and truncated wild-type (WT)-VWF and VWF with R1205 substitutions was investigated in VWF-/- mice. Plate-binding assays were employed to characterize VWF binding to purified scavenger receptor class A member 1 (SR-A1), low-density lipoprotein receptor-related protein-1 (LRP1) cluster II or cluster IV receptors, and macrophage galactose-type lectin (MGL). RESULTS: In full-length VWF missing the A1 domain (VWF-ΔA1), introduction of R1205H led to significantly enhanced clearance in VWF-/- mice compared to WT-VWF-ΔA1. Importantly, R1205H in a truncated VWF-D'D3 fragment also triggered increased clearance compared to WT-VWF-D'D3. Additional in vivo studies demonstrated that VWF-R1205K (which preserves the positive charge at 1205) exhibited normal clearance, whereas VWF-R1205E (which results in loss of the positive charge) caused significantly enhanced clearance, pinpointing the importance of the positive charge at VWF-R1205. In vitro plate-binding studies confirmed increased VWF-R1205H interaction with SR-A1 compared to WT-VWF. Furthermore, significantly enhanced VWF-R1205H binding to LRP1 cluster IV (p<0.001) and less marked enhanced binding to LRP1 cluster II (p=0.034) was observed. In contrast, VWF-R1205H and WT-VWF demonstrated no difference in binding affinity to MGL. CONCLUSION: Disruption of the positive charge at amino acid 1205 causes conformational changes in the VWF-D'D3 domains, and triggers enhanced LRP1 and SR-A1 mediated clearance.

Prediction model for leaflet thrombosis in patients undergoing transcatheter aortic valve implantation: the EFFORT study.

BACKGROUND: Leaflet thrombosis (LT) is a multifaceted and underexplored condition that can manifest following transcatheter aortic valve implantation (TAVI). The objective of this study was to formulate a prediction model based on laboratory assessments and clinical parameters, providing additional guidance and insight into this relatively unexplored aspect of post-TAVI complications. METHODS: The present study was an observational prospective hypothesis-generating study, including 101 patients who underwent TAVI and a screening for LT (the primary endpoint) by multidetector computed tomography (MDCT). All images were acquired on a third-generation dual-source CT system. Levels of von Willebrand factor (vWF) activity, hemoglobin (Hb), and lactate dehydrogenase (LDH) were measured among other parameters. A predictive score utilizing binary logistic regression, Kaplan-Meier time-to-event analysis, and receiver operating characteristics (ROC) analysis was established. RESULTS: LT (11 subclinical and 2 clinical) was detected in 13 of 101 patients (13%) after a median time to screening by MDCT of 105 days (IQR, 98-129 days). Elevated levels of vWF activity (> 188%) pre-TAVI, decreased Hb values (< 11.9 g/dL), as well as increased levels of LDH (> 312 U/L) post-TAVI and absence of oral anticoagulation (OAC) were found in patients with subsequent LT formation as compared to patients without LT. The established EFFORT score ranged from - 1 to 3 points, with an increased probability for LT development in patients with ≥ 2 points (85.7% of LT cases) vs < 2 points (14.3% of LT cases; p < 0.001). Achieving an EFFORT score of ≥ 2 points was found to be significantly associated with a 10.8 times higher likelihood of developing an LT (p = 0.001). The EFFORT score has an excellent c-statistic (area under the curve (AUC) = 0.89; 95% CI 0.74-1.00; p = 0.001) and a high negative predictive value (98%). CONCLUSION: An EFFORT score might be a helpful tool to predict LT development and could be used in risk assessment, if validated in confirmatory studies. Therefore, the score has the potential to guide the stratification of individuals for the planning of subsequent MDCT screenings.

Exposure-Response Relationship between VWF/FVIII Activity and Spontaneous Bleeding Events Following Recombinant VWF Prophylaxis in Severe VWD.

Background Recombinant von Willebrand factor (rVWF, vonicog alfa, Takeda Pharmaceuticals USA) is indicated in adults diagnosed with von Willebrand disease (VWD). In this study, the exposure-response (ER) relationship between VWF activity (VWF:RCo) or factor VIII activity (FVIII:C) and spontaneous bleeding events (BEs) was evaluated in adults with severe VWD receiving rVWF prophylaxis for up to 1 year. Methods This secondary analysis included 23 patients receiving rVWF prophylaxis in the open-label, phase 3 prophylaxis trial (NCT02973087). Population pharmacokinetic (PK) and PK/pharmacodynamic (PD) models were used to characterize VWF activity and endogenous FVIII:C, and PK/PD simulations were linked to spontaneous BEs to develop an ER model. Results None of the five patients with VWD types 1 or 2A/B experienced spontaneous BEs. Five of 18 patients with VWD type 3 experienced ≥1 spontaneous BEs. An ER relationship was observed whereby higher VWF:RCo levels were associated with a numerically lower spontaneous BE risk ( p < 0.10). This relationship was independent of patients' pretrial VWF treatment. A statistically significant ER relationship was observed after accounting for relevant data (average ± standard error exposure estimate for VWF:RCo over 24 hours prior to the spontaneous BE: -0.043 ± 0.021, p = 0.041). The model-generated hazard ratio for a 10 IU/dL increment in the average exposure of VWF:RCo 24 hours before a spontaneous BE was 0.651 (95% confidence interval: 0.431-0.982). Conclusions This ER analysis suggests a causal association between VWF:RCo and spontaneous BEs, with an increase of VWF:RCo exposure leading to a decrease in spontaneous BE risk.

Shiga toxin down-regulates ERG protein in endothelial cells and impairs angiogenesis.

BACKGROUND: Shiga toxin (Stx) can activate inflammatory signaling, leading to vascular dysfunction and promotion of a pro-thrombotic tissue microenvironment. Stx can trigger the development of the enterohemorrhagic (childhood) hemolytic uremic syndrome (eHUS), a triad of thrombocytopenia, hemolytic anemia, and acute kidney injury, often requiring dialysis. Additional features may include damage to other organs, including the gastrointestinal tract, pancreas, brain and cardiovascular system; death occurs in 2-5 %. eHUS is a thrombotic microangiopathy; thus, endothelial cell (EC) injury and platelet fibrin thrombus formation in glomerular arterioles and in the arterioles of other affected organs are likely. To elucidate mechanisms of this microangiopathy, we examined in human ECs the regulation of the platelet adhesion proteins P-selectin and von Willebrand factor (VWF), along with the downregulation of erythroblast-transformation-specific transcription factor (ERG) a key regulator of angiogenesis and megakaryocyte development. METHODS: VWF, P-selectin, and ERG levels were determined using immunofluorescence and Western blot in human umbilical endothelial cells (HUVECs). HUVECs were treated with tumor necrosis factor-alpha (TNF-α), Stx-1 or both, versus normal controls. Capillary morphogenesis on Matrigel was performed using HUVECs treated, for 22 h with TNF-α, Stx-1, or both, or treated 4 h with Stx-1 alone or in combination with TNF-α for 22 h. RESULTS: Stx-1 significantly reduced ERG and VWF expression on HUVECs, but upregulated P-selectin expression. ERG levels decreased with Stx-1 alone or in combination with TNF-α, in the nuclear, perinuclear and cytoplasmatic regions. Stx-1 reduced capillary morphogenesis, while Stx-1-TNF-α combined treatment reduced capillary morphogenesis still further. CONCLUSIONS: In the presence of Stx-1 or TNF-α or both treatments, ECs were activated, expressing higher levels of P-selectin and lower levels of VWF. Our findings, further, provide evidence that Stx-1 downregulates ERG, repressing angiogenesis in vitro.

In vitro study on device-induced damage to blood cellular components and degradation of von Willebrand factor in a CentriMag pump-assisted circulation.

BACKGROUND: High mechanical shear stress (HMSS) generated by blood pumps during mechanical circulatory support induces blood damage (or function alteration) not only of blood cell components but also of plasma proteins. METHODS: In the present study, fresh, healthy human blood was used to prime a blood circuit assisted by a CentriMag centrifugal pump at a flow rate of 4.5 L/min under three pump pressure heads (75, 150, and 350 mm Hg) for 4 h. Blood samples were collected for analyses of plasma-free hemoglobin (PFH), von Willebrand factor (VWF) degradation and platelet glycoprotein (GP) IIb/IIIa receptor shedding. RESULTS: The extent of all investigated aspects of blood damage increased with increasing cross-pump pressure and duration. Loss of high-molecular-weight multimers (HMWM)-VWF in Loop 2 and Loop 3 significantly increased after 2 h. PFH, loss of HMWM-VWF, and platelet GPIIb/IIIa receptor shedding showed a good linear correlation with mean shear stress corresponding to the three pump pressure heads. CONCLUSIONS: HMSS could damage red blood cells, cause pathological VWF degradation, and induce platelet activation and platelet receptor shedding. Different blood components can be damaged to different degrees by HMSS; VWF and VWF-enhanced platelet activation may be more susceptible to HMSS.

Application of genetic testing for the diagnosis of von Willebrand disease.

von Willebrand disease (VWD) is the most frequent inherited bleeding disorder, with an estimated symptomatic prevalence of 1 per 1000 in the general population. VWD is characterized by defects in the quantity, quality, or multimeric structure of von Willebrand factor (VWF), a glycoprotein being hemostatically essential in circulation. VWD is classified into 3 principal types: low VWF/type 1 with partial quantitative deficiency of VWF, type 3 with virtual absence of VWF, and type 2 with functional abnormalities of VWF, being classified as 2A, 2B, 2M, and 2N. A new VWD type has been officially recognized by the ISTH SSC on von Willebrand factor which has also been discussed by the joint ASH/ISTH/NHF/WFH 2021 guidelines (ie, type 1C), indicating patients with quantitative deficiency due to an enhanced VWF clearance. With the advent of next-generation sequencing technologies, the process of genetic diagnosis has substantially changed and improved accuracy. Therefore, nowadays, patients with type 3 and severe type 1 VWD can benefit from genetic testing as much as type 2 VWD. Specifically, genetic testing can be used to confirm or differentiate a VWD diagnosis, as well as to provide genetic counseling. The focus of this manuscript is to discuss the current knowledge on VWD molecular pathophysiology and the application of genetic testing for VWD diagnosis.

ADAMTS13, von Willebrand Factor, Platelet Microparticles, Factor VIII, and Impact of Somatic Mutations in the Pathogenesis of Splanchnic Vein Thrombosis Associated with BCR-ABL-Negative Myeloproliferative Neoplasms.

BACKGROUND: Myeloproliferative neoplasms (MPNs) are often associated with splanchnic vein thrombosis (SVT). Not all the factors involved in the thrombotic tendency are currently known. OBJECTIVES: This study aims to evaluate a possible association between ADAMTS13, von Willebrand factor (VWF), platelet microvesicles (MV), and factor VIII activity (FVIII:C) with thrombotic events in MPN patients. MATERIALS AND METHODS: In total, 36 consecutive MPN patients with SVT were enrolled. The MPNs were diagnosed based on clinical characteristics and one or more gene mutations among JAK-2, CALR, and MPL. As controls, 50 randomly selected patients with MPN without thrombosis, 50 patients with deep vein thrombosis without MPNs, and 50 healthy blood donors were evaluated. Complete blood count, ADAMTS13, VWF, MV, and FVIII:C in plasma were measured in all the subjects. RESULTS: The JAK-2 mutation was found in 94% of the patients with SVT, but none were triple-negative for genetic mutations (JAK2 V617F, CALR, MPL, and exon 12). Compared to the normal subjects, in all the MPN patients (with or without SVT), the levels of ADAMTS13 were found to be significantly lower (p < 0.001) and the MV concentrations were significantly higher (p < 0.001). Among the MPN patients, the VWF and FVIII:C levels were significantly higher in the patients with SVT than those without thrombosis (p = 0.007 and p = 0.04, respectively). Splenomegaly was present in 78% of MPN patients with SVT and in 30% of those without SVT (p < 0.001). The ADAMTS13/VWF ratio was reduced in all the patients, but not in the healthy blood donors (p < 0.001). CONCLUSIONS: The significant increase in circulating MV, VWF, and FVIII:C in the MPN patients and in the patients with thrombosis supports the role of endothelium damage in promoting thrombotic events. In particular, a significant increase in VWF and FVIII:C levels was found in the MPN patients with SVT.

Modern diagnostic methods for early assessment of the abdominal involvement in Schönlein-Henoch disease.

INTRODUCTION: Schönlein-Henoch disease is a small vessel vasculitis resulting from IgA-mediated inflammation. It is the most common acute systemic vasculitis in childhood, mainly affecting the skin, gastrointestinal tract, joints, and kidneys. Although the prognosis of Schönlein-Henoch is generally good, gastrointestinal tract involvement is a potential complication, presenting as massive gastrointestinal bleeding, bowel infarction, perforation, as well as intussusception and peritonitis.

A career in solving clinical-pathological conundrums: Heyde syndrome, anti-platelet factor 4 disorders, and microvascular limb ischemic necrosis.

Hematology is a clinical specialty with strong roots in the laboratory; accordingly, the lab can help solve perplexing clinical problems. This review highlights clinical-pathological conundrums addressed during my 35-year hematology career at McMaster University. Heyde syndrome is the association between aortic stenosis and bleeding gastrointestinal (GI) angiodysplasia where the bleeding is usually cured by aortic valve replacement; the chance reading of a neonatal study showing reversible deficiency of high-molecular-weight (HMW) multimers of von Willebrand factor (vWF) following surgical correction of congenital heart disease provided the key insight that a subtle deficiency of HMW multimers of vWF explains Heyde syndrome. The unusual immunobiology of heparin-induced thrombocytopenia (HIT)-a highly prothrombotic, antibody-mediated, anti-platelet factor 4 (PF4) disorder featuring rapid appearance and then disappearance (seroreversion) of the pathological heparin-dependent platelet-activating antibodies-permitted identification of key clinical features that informed development of a scoring system (4Ts) to aid in HIT diagnosis. Atypical clinical presentations of HIT prompted identification of heparin-independent anti-PF4 antibodies, now recognized as the explanation for vaccine-induced immune thrombotic thrombocytopenia (VITT), as well as VITT-like disorders triggered by adenovirus infection. Another unusual feature of HIT is its strong association with limb ischemia, including limb necrosis secondary to deep-vein/microvascular thrombosis (venous limb gangrene). The remarkable observation that supratherapeutic warfarin anticoagulation predisposes to HIT- and cancer-associated venous limb gangrene provided insight into disturbed procoagulant/anticoagulant balance; these concepts are relevant to microvascular thrombosis in critical illness (symmetrical peripheral gangrene), including a pathophysiological role for proximate "shock liver" (impaired hepatic synthesis of natural anticoagulants).

VWF/ADAMTS13 Ratio as a Potential Predictive Biomarker for Acute Kidney Injury Onset in Cirrhosis.

AIM: We investigated the von Willebrand factor to ADAMTS13 ratio (von Willebrand factor [VWF]:Ag/ADAMTS13:AC) as a potential biomarker for the outcomes of acute kidney injury (AKI) in liver cirrhosis (LC). METHODS: This retrospective cross-sectional study included patients with LC who developed AKI (AKI group: n = 91) and patients with LC who did not develop AKI [non-AKI (NAKI) group, n = 91] as a control group. Plasma levels of the von Willebrand factor antigen (Ag) and ADAMTS13 activity (AC) were measured in patients with AKI or NAKI. Moreover, risk factors for onset of AKI, AKI-associated 90-day mortality, and poor AKI treatment response were identified. RESULTS: The AKI group had a significantly higher VWF:Ag/ADAMTS13:AC than the NAKI group. Values of VWF:Ag/ADAMTS13:AC ≥ 5.7 were identified as risk factors for AKI onset in patients with LC (odds ratio [OR] 2.56; 95% CI 1.26-4.99; p < 0.001). Among patients with AKI, values of VWF:Ag/ADAMTS13:AC ≥ 9.0 were identified as risk factors for 90-day mortality (OR 6.83; 95% CI 2.32-20.10; p < 0.001). Cumulative survival was significantly lower in those with high (≥ 9.0) than in those with low (< 9.0) VWF:Ag/ADAMTS13:AC. Furthermore, values of VWF:Ag/ADAMTS13:AC ≥ 7.4 were identified as risk factors for poor treatment response (OR 4.2; 95% CI 1.39-12.70; p < 0.001). The treatment response rates were significantly higher in those with low (< 7.4) VWF:Ag/ADAMTS13:AC than in those with high (≥ 7.4) VWF:Ag/ADAMTS13:AC. CONCLUSION: VWF:Ag/ADAMTS13:AC potentially predicts the onset, prognosis, and treatment response of AKI in patients with LC.

Numerical study of ultra-large von Willebrand factor multimers in coagulopathy.

An excessive von Willebrand factor (VWF) secretion, coupled with a moderate to severe deficiency of ADAMTS13 activity, serves as a linking mechanism between inflammation to thrombosis. The former facilitates platelet adhesion to the vessel wall and the latter is required to cleave VWF multimers. As a result, the ultra-large VWF (UL-VWF) multimers released by Weibel-Palade bodies remain uncleaved. In this study, using a computational model based on first principles, we quantitatively show how the uncleaved UL-VWF multimers interact with the blood cells to initiate microthrombosis. We observed that platelets first adhere to unfolded and stretched uncleaved UL-VWF multimers anchored to the microvessel wall. By the end of this initial adhesion phase, the UL-VWF multimers and platelets make a mesh-like trap in which the red blood cells increasingly accumulate to initiate a gradually growing microthrombosis. Although high-shear rate and blood flow velocity are required to activate platelets and unfold the UL-VWFs, during the initial adhesion phase, the blood velocity drastically drops after thrombosis, and as a result, the wall shear stress is elevated near UL-VWF roots, and the pressure drops up to 6 times of the healthy condition. As the time passes, these trends progressively continue until the microthrombosis fully develops and the effective size of the microthrombosis and these flow quantities remain almost constant. Our findings quantitatively demonstrate the potential role of UL-VWF in coagulopathy.

Late transplant-associated thrombotic microangiopathy verified in bone marrow biopsy specimens is associated with chronic GVHD and viral infections.

OBJECTIVES: To describe late transplant-associated thrombotic microangiopathy (TA-TMA) as chronic endothelial complication in bone marrow (BM) after allogeneic hematopoietic stem cell transplantation (HSCT). METHODS: BM specimens along with conventional diagnostic parameters were assessed in 14 single-institutional patients with late TA-TMA (more than 100 days after HCST), including 11 late with history of early TA-TMA, 10 with early TA-TMA (within 100 days), and 12 non TA-TMA patients. Three non-HSCT patients served as control. The time points of BM biopsy were +1086, +798, +396, and +363 days after HSCT, respectively. RESULTS: Late TA-TMA patients showed an increase of CD34+ and von Willebrand Factor (VWF)+ microvascular endothelial cells with atypical VWF+ conglomerates forming thickened VWF+ plaque sinus in the BM compared to patients without late TA-TMA and non-HSCT. Severe chronic (p = .002), steroid-refractory GVHD (p = .007) and reactivation of HHV6 (p = .002), EBV (p = .003), and adenovirus (p = .005) were pronounced in late TA-TMA. Overall and relapse-free survival were shorter in late TA-TMA than in patients without late TA-TMA (5-year OS and RFS: 78.6% vs. 90.2%, 71.4% vs. 86.4%, respectively). CONCLUSION: Chronic allo-immune microangiopathy in BM associated with chronic, steroid-refractory GVHD and/or viral infections are key findings of late, high-risk TA-TMA, which deserves clinical attention.

von Willebrand factor Ristocetin co-factor activity to von Willebrand factor antigen level ratio for diagnosis of acquired von Willebrand syndrome caused by aortic stenosis.

Background: Severe aortic stenosis (AS) causes acquired von Willebrand syndrome by the excessive shear stress-dependent cleavage of high molecular weight multimers of von Willebrand factor (VWF). While the current standard diagnostic method is so-called VWF multimer analysis that is western blotting under nonreducing conditions, it remains unclear whether a ratio of VWF Ristocetin co-factor activity (VWF:RCo) to VWF antigen levels (VWF:Ag) of <0.7, which can be measured with an automated coagulation analyzer in clinical laboratories and is used for the diagnosis of hereditary von Willebrand disease. Objectives: To evaluated whether the VWF:RCo/VWF:Ag is useful for the diagnosis of AS-induced acquired von Willebrand syndrome. Methods: VWF:RCo and VWF:Ag were evaluated with the VWF large multimer index as a reference, which represents the percentage of a patient's VWF high molecular weight multimer ratio to that of standard plasma in the VWF multimer analysis. Results: We analyzed 382 patients with AS having transaortic valve maximal pressure gradients of >30 mmHg, 27 patients with peripheral artery disease, and 46 control patients free of cardiovascular disease with osteoarthritis, diabetes, and so on. We assumed a large multimer index of <80% as loss of VWF large multimers since 59.0% of patients with severe AS had the indices of <80%, while no control patients or patients with peripheral artery disease, except for 2 patients, exhibited the indices of <80%. The VWF:RCo/VWF:Ag ratios, measured using an automated blood coagulation analyzer, were correlated with the indices (rs = 0.470, P < .001). When the ratio of <0.7 was used as a cut-off point, the sensitivity and specificity to VWF large multimer indices of <80% were 0.437 and 0.826, respectively. Conclusion: VWF:RCo/VWF:Ag ratios of <0.7 may indicate loss of VWF large multimers with high specificity, but low sensitivity. VWF:RCo/VWF:Ag ratios in patients with AS having a ratio of <0.7 may be useful for monitoring the loss of VWF large multimers during their clinical courses.

Comparison of thrombomodulin, vWF, and ADAMTS13 levels between preeclampsia and normal pregnancy

Objectives: To determine and compare plasma thrombomodulin, von Willebrand factor and von Willebrand factorcleaving protease levels between pre-eclamptic and healthy pregnant females. METHODS: The cross-sectional, comparative study was conducted at the Department of Haematology, University of Health Sciences, Lahore, Pakistan, from November 2019 to December 2020, and comprised pregnant females who were divided into healthy pregnant group A and pre-eclamptic group B. Plasma thrombomodulin and von Willebrand factor-cleaving protease levels were determined by using commercially available enzyme-linked immunosorbent assay kit, and von Willebrand factor level was determined by using immuno-turbidimetric assay kit. Data was analysed using SPSS 25. RESULTS: Of the 88 participants, there were 44(50%) females with mean age 25.5±6 years in group A and 44(50%) in group B with mean age 26±5 years. Median thrombomodulin level in group B was significantly higher than group A (p=0.003). Median von Willebrand factor-cleaving protease levels were lower in group B compared to group A (p=0.838). A significant difference in von Willebrand factor level was observed between the groups (p=0.038). Conclusion: Females with pre-eclampsia had significantly higher plasma levels of von Willebrand factor and thrombomodulin than healthy pregnant subjects.