Large Multimers Research Articles

Hierarchical assembly and environmental enhancement of bacterial ice nucleators

Bacterial ice nucleating proteins (INPs) are exceptionally effective in promoting the kinetically hindered transition of water to ice. Their efficiency relies on the assembly of INPs into large functional aggregates, with the size of ice nucleation sites determining activity. Experimental freezing spectra have revealed two distinct, defined aggregate sizes, typically classified as class A and C ice nucleators (INs). Despite the importance of INPs and years of extensive research, the precise number of INPs forming the two aggregate classes, and their assembly mechanism have remained enigmatic. Here, we report that bacterial ice nucleation activity emerges from more than two prevailing aggregate species and identify the specific number of INPs responsible for distinct crystallization temperatures. We find that INP dimers constitute class C INs, tetramers class B INs, and hexamers and larger multimers are responsible for the most efficient class A activity. We propose a hierarchical assembly mechanism based on tyrosine interactions for dimers, and electrostatic interactions between INP dimers to produce larger aggregates. This assembly is membrane-assisted: Increasing the bacterial outer membrane fluidity decreases the population of the larger aggregates, while preserving the dimers. Inversely, Dulbecco's Phosphate-Buffered Saline buffer increases the population of multimeric class A and B aggregates 200-fold and endows the bacteria with enhanced stability toward repeated freeze-thaw cycles. Our analysis suggests that the enhancement results from the better alignment of dimers in the negatively charged outer membrane, due to screening of their electrostatic repulsion. This demonstrates significant enhancement of the most potent bacterial INs.

Self-limiting multimerization of α-synuclein on membrane and its implication in Parkinson's diseases.

α-Synuclein (α-syn), a crucial molecule in Parkinson's disease (PD), is known for its interaction with lipid membranes, which facilitates vesicle trafficking and modulates its pathological aggregation. Deciphering the complexity of the membrane-binding behavior of α-syn is crucial to understand its functions and the pathology of PD. Here, we used single-molecule imaging to show that α-syn forms multimers on lipid membranes with huge intermultimer distances. The multimers are characterized by self-limiting growth, manifesting in concentration-dependent exchanges of monomers, which are fast at micromolar concentrations and almost stop at nanomolar concentrations. We further uncovered movement patterns of α-syn's occasional trapping on membranes, which may be attributed to sparse lipid packing defects. Mutations such as E46K and E35K may disrupt the limit on the growth, resulting in larger multimers and accelerated amyloid fibril formation. This work emphasizes sophisticated regulation of α-syn multimerization on membranes as a critical underlying factor in the PD pathology.

DockQ v2: improved automatic quality measure for protein multimers, nucleic acids, and small molecules.

It is important to assess the quality of modeled biomolecules to benchmark and assess the performance of different prediction methods. DockQ has emerged as the standard tool for assessing the quality of protein interfaces in model structures against given references. However, as predictions of large multimers with multiple chains become more common, DockQ needs to be updated with more functionality for robustness and speed. Moreover, as the field progresses and more methods are released to predict interactions between proteins and other types of molecules, such as nucleic acids and small molecules, it becomes necessary to have a tool that can assess all types of interactions. Here, we present a complete reimplementation of DockQ in pure Python. The updated version of DockQ is more portable, faster and introduces novel functionalities, such as automatic DockQ calculations for multiple interfaces and automatic chain mapping with multi-threading. These enhancements are designed to facilitate comparative analyses of protein complexes, particularly large multi-chain complexes. Furthermore, DockQ is now also able to score interfaces between proteins, nucleic acids, and small molecules. DockQ v2 is available online at: https://wallnerlab.org/DockQ.

Intracellular levels of biologically active large multimers of von Willebrand factor are increased in response to aging

Intracellular levels of biologically active large multimers of von Willebrand factor are increased in response to aging

Excessive cleavage of von Willebrand factor multimers by ADAMTS13 may predict the progression of transplant-associated thrombotic microangiopathy

BackgroundTransplant-associated thrombotic microangiopathy (TA-TMA) is a fatal complication of hematopoietic stem cell transplantation and is characterized by severe thrombocytopenia, hemolytic anemia, and organ dysfunction. In response to several possible triggers, dynamic multimetric change in von Willebrand factor (VWF) may contribute to inducing microthrombi in circulation in TA-TMA. ObjectivesBy performing VWF multimer analysis and measuring VWF-degradation product (DP), we unraveled the relationship between multimeric changes in circulating VWF and the pathogenesis of TA-TMA. MethodsThis study analyzed 135 plasma samples from 14 patients who underwent allogeneic hematopoietic stem cell transplantation at a single institute. VWF-associated markers, namely VWF:antigen (VWF:Ag), VWF-DP/VWF:Ag ratio, VWF:ristocetin cofactor activity, VWF:ristocetin cofactor activity/VWF:Ag ratio, and ADAMTS13 activity, were analyzed in these samples collected every 7 days. ResultsThere were 2 patients with definite thrombotic microangiopathy (TMA) and 6 patients who presented with probable TMA that did not progress to definite TMA. Each plasma sample was classified into 3 groups: definite TMA, probable TMA, and non-TMA. VWF multimer analysis showed the absence of high-molecular-weight VWF multimers in probable TMA, whereas the appearance of unusually large VWF multimers was observed in definite TMA. The median value of the VWF-DP/VWF:Ag ratio in probable TMA was elevated to 4.17, suggesting that excessive cleavage of VWF multimers by VWF cleaving enzyme, ADAMTS13, resulted in the loss of high-molecular-weight VWF multimers. ConclusionDuring the transition from probable to definite TMA, drastic VWF multimer changes imply a switch from bleeding to thrombotic tendencies. Extensive VWF-DP and VWF multimer analyses provided novel insights.

Impact of extracorporeal membrane oxygenation treatments on acquired von Willebrand syndrome in patients with out-of-hospital cardiac arrest: a retrospective observational study

BackgroundVon Willebrand factor (vWF) plays a crucial role in hemostasis, acting as a key factor for platelet adhesion/aggregation and as a transport protein for coagulation factor VIII. vWF is secreted as a giant multimer, and it undergoes shear stress-dependent cleavage by a specific metalloproteinase in plasma. Among vWF multimers, high-molecular-weight (large) multimers are essential for hemostasis. Acquired von Willebrand syndrome, linked to various conditions, is a hemostatic disorder due to reduced vWF activity. Extracorporeal membrane oxygenation (ECMO), utilized recently for out-of-hospital cardiac arrest patients, generates high shear stress inside the pump. This stress may induce a conformational change in vWF, enhancing cleavage by a specific metalloproteinase and thereby reducing vWF activity. However, no study has investigated the effects of ECMO on vWF-related factors in patients receiving or not receiving ECMO. This study aimed to elucidate the relationship between ECMO treatment and acquired von Willebrand syndrome-related factors in patients with out-of-hospital cardiac arrest.MethodsThis study included patients with cardiogenic out-of-hospital cardiac arrest admitted to our hospital. The patients were categorized into two groups (ECMO and non-ECMO) based on the presence or absence of ECMO treatment. Plasma samples were collected from patients admitted to the emergency department (days 0–4). The vWF antigen (vWF: Ag), vWF ristocetin cofactor activity (vWF: RCo), and factor VIII activity were measured. Additionally, a large multimer of vWF was evaluated through vWF multimer analysis, utilizing western blotting to probe vWF under non-reducing conditions.ResultsThe ECMO and non-ECMO groups included 10 and 22 patients, respectively. The median ECMO treatment in the ECMO group was 64.6 h. No differences in vWF: Ag or factor VIII activity were observed between the two groups during the observation period. However, the ECMO group exhibited a decrease in large vWF multimers and vWF: RCo during ECMO. Strong correlations were observed between vWF: RCo and vWF: Ag in both groups, although the relationships were significantly different between the two groups.ConclusionsECMO treatment in patients with out-of-hospital cardiac arrest resulted in the loss of large vWF multimers and decreased vWF activity. Hence, decreased vWF activity should be considered as a cause of bleeding during ECMO management.

A Targetable Self-association Surface of the Huntingtin exon1 Helical Tetramer Required for Assembly of Amyloid Pre-nucleation Oligomers

Polyglutamine (polyQ) sequences undergo repeat-length dependent formation of disease-associated, amyloid-like cross-β core structures with kinetics and aggregate morphologies often influenced by the flanking sequences. In Huntington’s disease (HD), the httNT segment on the polyQ’s N-terminal flank enhances aggregation rates by changing amyloid nucleation from a classical homogeneous mechanism to a two-step process requiring an ɑ-helix-rich oligomeric intermediate. A folded, helix-rich httNT tetrameric structure suggested to be this critical intermediate was recently reported. Here we employ single alanine replacements along the httNT sequence to assess this proposed structure and refine the mechanistic model. We find that Ala replacement of hydrophobic residues within simple httNT peptides greatly suppresses helicity, supporting the tetramer model. These same helix-disruptive replacements in the httNT segment of an exon-1 analog greatly reduce aggregation kinetics, suggesting that an ɑ-helix rich multimer – either the tetramer or a larger multimer – plays an on-pathway role in nucleation. Surprisingly, several other Ala replacements actually enhance helicity and/or amyloid aggregation. The spatial localization of these residues on the tetramer surface suggests a self-association interface responsible for formation of the octomers and higher-order multimers most likely required for polyQ amyloid nucleation. Multimer docking of the tetramer, using the protein–protein docking algorithm ClusPro, predicts this symmetric surface to be a viable tetramer dimerization interface. Intriguingly, octomer formation brings the emerging polyQ chains into closer proximity at this tetramer-tetramer interface. Further supporting the potential importance of tetramer super-assembly, computational docking with a known exon-1 aggregation inhibitor predicts ligand contacts with residues at this interface.

Mitral regurgitation is associated with similar loss of von Willebrand factor large multimers but lower frequency of anemia compared with aortic stenosis

BackgroundVarious cardiovascular diseases cause acquired von Willebrand syndrome (AVWS), which is characterized by a decrease in high-molecular-weight (large) von Willebrand factor (VWF) multimers. Mitral regurgitation (MR) has been reported as a cause of AVWS. However, much remains unclear about AVWS associated with MR. ObjectivesTo evaluate VWF multimers in MR patients and examine their impact on clinical characteristics. MethodsModerate or severe MR patients (n = 84) were enrolled. VWF parameters such as the VWF large multimer index (VWF-LMI), a quantitative value that represents the amount of VWF large multimers, and clinical data were prospectively analyzed. ResultsAt baseline, the mean hemoglobin level was 12.9 ± 1.9 g/dL and 58 patients (69.0%) showed loss of VWF large multimers defined as VWF-LMI < 80%. VWF-LMI in patients with degenerative MR was lower than in those with functional MR. VWF-LMI appeared to be restored the day after mitral valve intervention, and the improvement was maintained 1 month after the intervention. Seven patients (8.3%) had a history of bleeding, 6 (7.1%) of whom had gastrointestinal bleeding. Gastrointestinal endoscopy was performed in 23 patients (27.4%) to investigate overt gastrointestinal bleeding, anemia, etc. Angiodysplasia was detected in 2 of the 23 patients (8.7%). ConclusionModerate or severe MR is frequently associated with loss of VWF large multimers, and degenerative MR may cause more severe loss compared with functional MR. Mitral valve intervention corrects the loss of VWF large multimers. Gastrointestinal bleeding may be relatively less frequent and hemoglobin level remains stable in MR patients.

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

BackgroundSevere 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. ObjectivesTo evaluated whether the VWF:RCo/VWF:Ag is useful for the diagnosis of AS-induced acquired von Willebrand syndrome. MethodsVWF: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. ResultsWe 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. ConclusionVWF: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.

PB0853 Comparison of VWF:RCo/VWF:Ag Ratios with the VWF Large Multimer Indices in Patients with Aortic Stenosis

PB0853 Comparison of VWF:RCo/VWF:Ag Ratios with the VWF Large Multimer Indices in Patients with Aortic Stenosis

Impact of intraoperative use of venovenous extracorporeal membrane oxygenation on the status of von Willebrand factor large multimers during single lung transplantation.

von Willebrand factors (vWFs), hemostatic factors, are produced as large multimers and are shear stress-dependently cleaved to become the appropriate size. A reduction in vWF large multimers develops in various conditions including the use of extracorporeal life support, which can cause excessive-high shear stress in the blood flow and result in hemostatic disorders. The objective of this prospective study was to investigate the impact of venovenous extracorporeal membrane oxygenation (VV ECMO) use on the status of vWF large multimers and hemostatic disorders during single lung transplantation (SLT). We prospectively enrolled 12 patients who underwent SLT at our center. Among them, seven patients were supported by VV ECMO intraoperatively (ECMO group) and the remaining five patients underwent SLT without ECMO support (control group). The vWF large multimer index (%) was defined as the ratio of the large multimer proportion in total vWF (vWF large multimer ratio) derived from a patient's plasma to that from standard human plasma. The vWF large multimer index at the end of the surgery was significantly lower in the ECMO group than in the control group (112.6% vs. 75.8%, respectively; P<0.05). The intraoperative blood loss and the amounts of intraoperative transfusion products in the ECMO group tended to be greater than those in the control group; however, the differences were not significant. During SLT, the use of VV ECMO caused a decrease in the vWF large multimer index. The short duration of time of VV ECMO use in our study did not significantly affect the intra- and postoperative outcomes including blood loss, blood transfusion, and re-exploration thoracotomy for bleeding. Nevertheless, to comprehensively evaluate the actual influence of this decrease in the vWF large multimer index on intra- and postoperative outcomes, a multicenter larger-scale study is warranted.

Molecular basis of Q-length selectivity for the MW1 antibody–huntingtin interaction

Huntington's disease is caused by a polyglutamine (polyQ) expansion in the huntingtin protein. Huntingtin exon 1 (Httex1), as well as other naturally occurring N-terminal huntingtin fragments with expanded polyQ are prone to aggregation, forming potentially cytotoxic oligomers and fibrils. Antibodies and other N-terminal huntingtin binders are widely explored as biomarkers and possible aggregation-inhibiting therapeutics. A monoclonal antibody, MW1, is known to preferentially bind to huntingtin fragments with expanded polyQ lengths, but the molecular basis of the polyQ length specificity remains poorly understood. Using solution NMR, electron paramagnetic resonance, and other biophysical methods, we investigated the structural features of the Httex1-MW1 interaction. Rather than recognizing residual α-helical structure, which is promoted by expanded Q-lengths, MW1 caused the formation of a new, non-native, conformation in which the entire polyQ is largely extended. This non-native polyQ structure allowed the formation of large mixed Httex1-MW1 multimers (600-2900 kD), when Httex1 with pathogenic Q-length (Q46) was used. We propose that these multivalent, entropically favored interactions, are available only to proteins with longer Q-lengths and represent a major factor governing the Q-length preference of MW1. The present study reveals that it is possible to target proteins with longer Q-lengths without having to stabilize a natively favored conformation. Such mechanisms could be exploited in the design of other Q-length specific binders.

Hidden behind thromboinflammation: revealing the roles of von Willebrand factor in sickle cell disease pathophysiology.

This review provides an update on the pathophysiology of sickle cell disease (SCD) with a particular focus on the dysregulation of the von Willebrand factor (VWF) - ADAMTS13 axis that contributes to its pathogenesis. In discussing recent developments, we hope to encourage new and ongoing discussions surrounding therapeutic targets for SCD. Within the last 5 years, the role of VWF in the pathophysiology of SCD has been further elucidated and is now a target of study in ongoing clinical trials. The pathophysiology of SCD is multifaceted, as it involves systemwide vascular activation, altered blood rheology, and the activation of immune responses and coagulative pathways. The presence of VWF in excess in SCD, particularly in its largest multimeric form, greatly contributes to its pathogenesis. Understanding the molecular mechanisms that underly the presence of large VWF multimers in SCD will provide further insight into the pathogenesis of SCD and provide specific targets for therapy.

Transcatheter aortic valve implantation in patient with Heyde’s syndrome

Clinical case of an 85-year-old patient with Heid’s syndrome is being reported: recurrent gastrointestinal bleeding due to angiodysplasia of the stomach and intestines in combination with acquired destruction of large von Willebrand factor multimers in severe aortic stenosis. Actually it remains difficult to make a diagnosis in these patients and perform timely surgical intervention. Considering the age of the patient, severity of manifestations of heart failure and high risk of surgical complications, transcatheter aortic valve implantation (TAVI) was the method of choice. Surgical correction of heart disease in this case is aimed not only at treating its clinical manifestations, but also at eliminating the cause of recurrent gastrointestinal bleeding in Hyde’s syndrome.

Stability of (N+1) -body fermion clusters in a multiband Hubbard model

We start with a variational approach and derive a set of coupled integral equations for the bound states of $N$ identical spin-$\ensuremath{\uparrow}$ fermions and a single spin-$\ensuremath{\downarrow}$ fermion in a generic multiband Hubbard Hamiltonian with an attractive on-site interaction. As an illustration, we apply our integral equations to the one-dimensional sawtooth lattice up to $N\ensuremath{\le}3$, i.e., to the $(3+1)$-body problem, and we reveal not only the presence of tetramer states in this two-band model but also their quasiflat dispersion when formed in a flat band. Furthermore, for $N={4,5,\ensuremath{\cdots},10}$, our density-matrix renormalization-group simulations and exact diagonalization suggest the presence of larger and larger multimers with lower and lower binding energies, conceivably without an upper bound on $N$. These peculiar $(N+1)$-body clusters are in sharp contrast with the exact results on the single-band linear-chain model where none of the $N\ensuremath{\ge}2$ multimers appear. Hence their presence must be taken into account for a proper description of the many-body phenomena in flat-band systems, e.g., they may suppress superconductivity especially when there exists a large spin imbalance.

Von Willebrand Factor and Myeloperoxidase as Laboratory Predictive Markers of In-Hospital Survival in Severe COVID-19 Patients

Background. The severe acute respiratory syndrome of the SARS-CoV-2 virus-mediated coronavirus disease 2019 (COVID-19) highlighted the central role of immunothrombosis. Severe endothelial damage with the release of unusually large multimers of von Willebrand factor (vWF) and subsequent consumption of ADAMTS-13 is described during severe COVID-19. The activation of innate immune cells among which neutrophils contribute to the formation of extracellular neutrophil traps (NETs) and to the release of myeloperoxidase (MPO) potentially contributing to the spread of inflammation and microvascular thrombosis. Objective to evaluate the ability of vWF, ADAMTS-13 and MPO to predict in-hospital mortality in severe COVID-19 patients needing mechanical ventilation. Methods. We performed a one-center observational study of 79 severe COVID-19 patients entering intensive care unit for mechanical ventilation, examining vWF, ADAMTS-13 and MPO among other potential predictors for in-hospital death. Results. After multivariate analysis, vWF antigen (vWF:Ag) and MPO antigen (MPO:Ag) were finally the single two parameters which increasing values were independently associated with non-survival; vWF:Ag (U/dL): adjusted OR 3.360, 95% CI 1.5627.228, p = 0.0019; MPO:Ag (ng/ml): adjusted OR 1.062, 95% CI 1.0241.101, p = 0.0011. From these results a simplified mortality score was derived and patients categorized as having a score value higher or lower that the median value of the score: a high score value was associated with a lower cumulative survival rate (p 0.0001), 50% of the cases being dead at day 13 post-hospital admission. Conclusions. In severe COVID-19 necessitating mechanical ventilation, increasing values of MPO activity and of vWF antigen tested at admission are associated with poor survival.

COVID-19 microthrombosis: unusually large VWF multimers are a platform for activation of the alternative complement pathway under cytokine storm.

ADAMTS13, a metalloproteinase, specifically cleaves unusually large multimers of von Willebrand factor (VWF), newly released from vascular endothelial cells. The ratio of ADAMTS13 activity to VWF antigen (ADAMTS13/VWF) and indicators of the alternative complement pathway (C3a and sC5b-9) are both related to the severity of COVID-19. The ADAMTS13/VWF ratio is generally moderately decreased (0.18–0.35) in patients with severe COVID-19. When these patients experience cytokine storms, both interleukin-8 and TNFα stimulate VWF release from vascular endothelial cells, while interleukin-6 inhibits both production of ADAMTS13 and its interaction with VWF, resulting in localized severe deficiency of ADAMTS13 activity. Platelet factor 4 and thrombospondin-1, both released upon platelet activation, bind to the VWF-A2 domain and enhance the blockade of ADAMTS13 function. Thus, the released unusually-large VWF multimers remain associated with the vascular endothelial cell surface, via anchoring with syndecan-1 in the glycocalyx. Unfolding of the VWF-A2 domain, which has high sequence homology with complement factor B, allows the domain to bind to activated complement C3b, providing a platform for complement activation of the alternative pathway. The resultant C3a and C5a generate tissue factor-rich neutrophil extracellular traps (NETs), which induce the mixed immunothrombosis, fibrin clots and platelet aggregates typically seen in patients with severe COVID-19.

Individual Variability in von Willebrand Factor Fragility in Response to Shear Stress: A Possible Clue for Predicting Bleeding Risk.

Acquired von Willebrand syndrome (AVWS), characterized by reduced von Willebrand factor (VWF) large multimers, has recently been implicated as the principal mechanism underlying bleeding in patients implanted with left ventricular assist devices (LVADs). Hematological severity of AVWS varies among patients, even if an identical device is implanted. We investigated whether this diversity in hematological severity is due to individual variability in VWF fragility, according to responses to incremental shear stress. Whole-blood samples were sheared at 20,000-40,000 s -1 shear rate, an index of shear stress, using a custom-made shear stressor that could generate shear stress compatible with that produced by an LVAD. The degree of VWF large multimers degradation was evaluated using the VWF large multimer index. A significant inverse correlation was observed between the VWF large multimer index and LVAD-compatible magnitudes of shear stress: the VWF large multimer indices were 68.5 ± 18.3, 48.0 ± 13.9, 33.9 ± 12.1, 23.7 ± 7.9, and 18.7% ± 8.7% at 20,000, 25,000, 30,000, 35,000, and 40,000 s -1 of shear rates, respectively ( P < 0.0001). Furthermore, experimental VWF large multimer index values were compatible with those derived from patients with implanted LVADs (median; 28.9%). Finally, reduction in the VWF large multimer index corresponding to shear stress showed individual variation. We demonstrated that the combined use of a novel high shear stress loading device and quantitative evaluation of VWF large multimers may predict risk of bleeding before LVAD implantation.

A Rare Case of Mild COVID-19 Disease Associated with Type 1 Cryoglobulinemia and Thrombotic Thrombocytopenic Purpura

Introduction:Type I cryoglobulinemia (Type I CG) usually develops in the setting of protein-secreting monoclonal gammopathies [1,2]. The estimated frequency of renal involvement is 30% in Type 1 CG. Thrombotic Thrombocytopenic Purpura (TTP) is a life-threatening disease in which there is a deficiency in ADAMTS13, a protease which normally functions to cleave von Willebrand factor (vWF). A deficiency leads to persistence of large vWF multimers and the formation of platelet-rich thrombi in the microvasculature. We present a case of 52 years old female with Type 1 CG and acquired autoimmune TTP secondary to mild Covid-19 disease.Case Report:A 52-year-old woman with medical history significant for CKD stage 2, with baseline creatinine (Cr) of 2.5, Diffuse Large B-Cell Gastric Lymphoma (DLBCL), in complete remission, and recent diagnosis with COVID-19 presented to the ED for evaluation of three days of a petechial rash on her bilateral extremities. Significant labs on arrival included hemoglobin (Hgb) 8.9, platelet count 65, Cr of 3.65, and K+ 6.6, meeting criteria for acute kidney injury (AKI). Peripheral blood smear showedthrombocytopenia and normochromic normocytic anemia with no schistocytes or microangiopathic changes. ADAMTS13 was 43% and there was no clinical suspicion for TTP. The patient's Cr continued to worsen up to 8.1 during admission, prompting renal biopsy. Renal biopsy showed Type 1 cryoglobulinemic glomerulonephritis with vasculitis and 30 to 40% fibrosis of glomeruli. The patient was started on systemic steroids, plasma exchange every 2-3 day (completed 6 sessions) and hemodialysis. Her platelet counts steadily increased to 212 but had a significant drop to 32 when rechecked seven days later, prompting further investigation. ADAMTS13 was rechecked and found to be 5% with presence of inhibitor, Hgb was 7.7, haptoglobin was <20, and a diagnosis of TTP was made. The patient was started on daily plasmapheresis, rituximab, Caplacizumab and high dose systemic steroid therapy. Our patient had prior history of DLBCL and was treated with radiation therapy. Restaging PET/CT and EGD with biopsy showed no evidence of recurrence. Bone marrow biopsy was negative for evidence of lymphoma. Several serologic tests were used to rule out other etiologies of Type I CG and came back negative including: SLE, HIV, hepatitis C, multiple myeloma, and Waldenstrom's macroglobulinemia. The patient had elevations in multiple immune markers including IgG, IgM, free kappa light chains, and free lambda light chains. She also had hypocomplementemia of both C3 and C4.Discussion:The relationship of Type I CG and COVID-19 is unclear, though viral infections can trigger autoimmune diseases [3]. Previously, all patients with Type I CG had either ahematologic malignancy, solid-organ malignancy, infection, or autoimmune syndromes. Interestingly, in our patient, several serologic tests were performed and negative, which ruled out these etiologies of Type I CG. Our patient's manifestation of Type I CG was petechial rash on extremities and acute on chronic renal disease. Specific treatment of CG can include plasma exchange, corticosteroids, rituximab, and hemodialysis, depending on underlying cause. Our patient was started on systemic steroids, plasma exchange every 2-3 days, and hemodialysis due to worsening renal functions. Despite cases of covid-19 and TTP being reported, the relationship between the disease processes remains unclear. COVID-19 infection can cause disproportionate activation of the complement system and lead to excessive coagulation and thrombotic events. Systemic infection can decrease ADAMTS-13 activity, and this has also been noted in COVID studies. ADAMTS-13 deficiency has no pathophysiologic relevance specific to Type 1 CG. Our patient developed anemia and thrombocytopenia, and ADAMTS13 activity testing was found to be 5% with presence of an inhibitor. Considered as high-risk disease, our patient was started on steroids, rituximab, Caplacizumab and daily plasmapheresis. Repeated weekly ADAMTS13 level was 26% and platelets were stabilized at 69k/microL.Conclusion:Suggests that Covid-19 virus has the capability to induce cryoglobulinemia through an unknown mechanism• There are two other case reports of an association between Covid-19 and TTP.• May be the first case indicating a possible association of Covid-19 with cryoglobulinemia. DisclosuresNo relevant conflicts of interest to declare.

Analysis focusing on plasma von Willebrand factor in pachychoroid neovasculopathy and age-related macular degeneration

Pachychoroid neovasculopathy (PNV) is a new concept of macular disorder. Some cases diagnosed as age-related macular degeneration (AMD) have been re-diagnosed as PNV. However, the biological features of PNV are still uncertain. The purpose of this study was to compare PNV and AMD by analyses focusing on von Willebrand factor (VWF) and complement factor H (CFH). Ninety-seven patients who were previously diagnosed with treatment naïve AMD were enrolled in this study. They were re-classified as either PNV or AMD based on the clinical criteria and 33 patients were classified as PNV and 64 patients as AMD. We examined the clinical data, analyzed VWF multimer and two genetic polymorphisms (I62V and Y402H) in the CFH. PNV group was significantly younger than AMD group (P = 0.001). In both I62V and Y402H, there were no significant differences between PNV and AMD while the recessive homozygous (AA) was found only in PNV group in I62V. The presence of unusually large VWF multimers (UL-VWFMs) and subretinal hemorrhages were significantly higher in PNV than in AMD (P = 0.045, P = 0.020, respectively). Thus, the residual UL-VWFMs may result in platelet thrombosis and hemorrhages in the choriocapillaris of PNV. In conclusion, our results suggest the biological differences between PNV and AMD.