Soluble Von Willebrand Factor Research Articles

COVID-19 Biomarkers for Critically Ill Patients: A Compendium for the Physician.

SARS-CoV-2 clinical manifestation and progression are variable and unpredictable, hence the importance of considering biomarkers in clinical practice that can be useful for both diagnosis and prognostic evaluation. This review aims to summarize, for intensive care physicians, the most recent state of knowledge regarding known COVID-19 in critical patients. We searched PubMed® using the Boolean operators and identified all results on the PubMed® database of all studies regarding COVID-19 biomarkers. We selected studies regarding endothelium, cytokines, bacterial infection, coagulation, and cardiovascular biomarkers. We divided the results into four essential paragraphs: "Cytokine storm", "Endothelium dysfunction and coagulation biomarkers in COVID-19", "Biomarker of sepsis", and Cardiovascular lung and new perspectives. The assessments of the severe COVID-19 prognosis should monitor, over time, IL-6, soluble Von Willebrand factor (VWF), P-selectin, sCD40L, thrombomodulin, VCAM-1, endothelin- Troponin, D-dimer, LDH, CRP, and procalcitonin. Metabolomic alterations and ACE2 receptors represent new perspectives. Early identification of critically ill patients has been crucial in the first COVID-19 pandemic wave for the sustainability of the healthcare emergency system and clinical management. Only through the early identification of the most severe patients can they be provided with the most appropriate treatments.

SIPA in 10 milliseconds: VWF tentacles agglomerate and capture platelets under high shear

AbstractShear-induced platelet aggregation (SIPA) occurs under elevated shear rates (10 000 s−1) found in stenotic coronary and carotid arteries. The pathologically high shear environment can lead to occlusive thrombosis by SIPA from the interaction of nonactivated platelets and von Willebrand factor (VWF) via glycoprotein Ib–A1 binding. This process under high shear rates is difficult to visualize experimentally with concurrent molecular- and cellular-resolutions. To understand this fast bonding, we employ a validated multiscale in silico model incorporating measured molecular kinetics and a thrombosis-on-a-chip device to delineate the flow-mediated biophysics of VWF and platelets assembly into mural microthrombi. We show that SIPA begins with VWF elongation, followed by agglomeration of platelets in the flow by soluble VWF entanglement before mural capture of the agglomerate by immobilized VWF. The entire SIPA process occurs on the order of 10 milliseconds with the agglomerate traveling a lag distance of a few hundred microns before capture, matching in vitro results. Increasing soluble VWF concentration by ∼20 times in silico leads to a ∼2 to 3 times increase in SIPA rates, matching the increase in occlusion rates found in vitro. The morphology of mural aggregates is primarily controlled by VWF molecular weight (length), where normal-length VWF leads to cluster or elongated aggregates and ultra-long VWF leads to loose aggregates seen by others' experiments. Finally, we present phase diagrams of SIPA, which provides biomechanistic rationales for a variety of thrombotic and hemostatic events in terms of platelet agglomeration and capture.

Platelet adhesion and aggregate formation controlled by immobilised and soluble VWF

BackgroundIt has been demonstrated that von Willebrand factor (VWF) mediated platelet-endothelium and platelet-platelet interactions are shear dependent. The VWF’s mobility under dynamic conditions (e.g. flow) is pivotal to platelet adhesion and VWF-mediated aggregate formation in the cascade of VWF-platelet interactions in haemostasis.ResultsCombining microfluidic tools with fluorescence and reflection interference contrast microscopy (RICM), here we show, that specific deletions in the A-domains of the biopolymer VWF affect both, adhesion and aggregation properties independently. Intuitively, the deletion of the A1-domain led to a significant decrease in both adhesion and aggregate formation of platelets. Nevertheless, the deletion of the A2-domain revealed a completely different picture, with a significant increase in formation of rolling aggregates (gain of function). We predict that the A2-domain effectively ‘masks’ the potential between the platelet glycoprotein (GP) Ib and the VWF A1-domain. Furthermore, the deletion of the A3-domain led to no significant variation in either of the two functional characteristics.ConclusionsThese data demonstrate that the macroscopic functional properties i.e. adhesion and aggregate formation cannot simply be assigned to the properties of one particular domain, but have to be explained by cooperative phenomena. The absence or presence of molecular entities likewise affects the properties (thermodynamic phenomenology) of its neighbours, therefore altering the macromolecular function.

Formation and Resolution of Pial Microvascular Thrombosis in a Mouse Model of Thrombotic Thrombocytopenic Purpura.

Microvascular thrombosis is the hallmark pathology of thrombotic thrombocytopenic purpura (TTP), a rare life-threatening disease. Neurological dysfunction is present in over 90% of patients with TTP, and TTP can cause long-lasting neurological damage or death. However, the pathophysiology of microvascular thrombosis in the brain is not well studied to date. Here, we investigate the formation and resolution of thrombosis in pial microvessels. Approach and Results: Using a cranial intravital microscopy in well-established mouse models of congenital TTP induced by infusion of recombinant VWF (von Willebrand factor), we found that soluble VWF, at high concentration, adheres to the endothelium of the vessel wall, self-associates, and initiates platelet adhesion resulting in the formation of pial microvascular thrombosis in ADAMTS13-/- (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) mice. Importantly, VWF-mediated pial microvascular thrombosis occurred without vascular injury to the brain, and thrombi consisted of resting platelets adhered onto ultra-large VWF without fibrin in the brain in rVWF (recombinant VWF) challenged ADAMTS13-/- mice. Prophylactic treatment with recombinant ADAMTS13 (BAX930) effectively prevented the onset of the VWF-mediated microvascular thrombosis and therapeutic treatment with BAX930 acutely resolved the preexisting or growing thrombi in the brain of ADAMTS13-/- mice after rVWF challenge. The absence of platelet activation and fibrin formation within VWF-mediated thrombi and efficacy of BAX930 was confirmed with an endothelial-driven VWF-mediated microvascular thrombosis model in mice. Our results provide important insight into the initiation and development of microvascular thrombi in mouse models that mimics TTP and indicate that rADAMTS13 could be an effective interventional therapy for microvascular thrombosis, the hallmark pathology in TTP.

Platelet Signaling in Primary Haemostasis and Arterial Thrombus Formation: Part 1.

Platelets react immediately in response to traumatic vascular injury by adhesion, activation, aggregation and subsequent haemostatic plug formation. While this reaction pattern is essential for haemostasis, platelet responses can also cause occlusive thrombi in diseased arteries, leading to myocardial infarction or stroke. Initially, flowing platelets are captured from the circulation to vascular lesions. This step is mediated by glycoprotein (GP) Ib-IX-V interacting with immobilized von Willebrand factor (VWF) on exposed subendothelial components. Tethered platelets can now bind to collagen through GPVI and integrin α2β1. Outside-in signals from the adhesion receptors act synergistically with inside-out signals from soluble stimuli and induce platelet activation. These mediators operate through G protein-coupled receptors and reinforce adhesion and activation. Typical manifestations of activated platelets include calcium mobilization, procoagulant activity, cytoskeletal reorganization, granule secretion and aggregation. This requires activation of integrin αIIbβ3 with shifting into a high-affinity state and is indispensable to bind soluble fibrinogen, VWF and fibronectin. The multiple interactions and the impact of thrombin result in firm adhesion and recruitment of circulating platelets into growing aggregates. A fibrin meshwork supports stabilization of haemostatic thrombi and prevents detachment by the flowing blood. This two-part review provides an overview of platelet activation and signal transduction mechanisms with a focus on αIIbβ3-mediated outside-in signaling in integrin variants. In the first part, a three-stage model of platelet recruitment and activation in vivo is presented. Along with that, platelet responses upon exposure to thrombogenic surfaces followed by platelet-to-platelet interactions and formation of haemostatic thrombi are discussed. Moreover, several determinants involved in pathological thrombosis will be reviewed.

Endothelial damage in major depression patients is modulated by SSRI treatment, as demonstrated by circulating biomarkers and an in vitro cell model.

There is a link between depression, cardiovascular events and inflammation. We have explored this connection through endothelial dysfunction, using in vivo and in vitro approaches. We evaluated circulating biomarkers of endothelial dysfunction in patients with major depression at their diagnosis (MD-0) and during antidepressant treatment with the selective serotonin reuptake inhibitor escitalopram, for 8 and 24 weeks (MD-8 and MD-24). Results were always compared with matched healthy controls (CON). We measured in vivo circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) in blood samples, and assessed plasma levels of soluble von Willebrand factor (VWF) and vascular cell adhesion molecule-1 (VCAM-1). CEC counts, soluble VWF and VCAM-1 were statistically elevated in MD-0 (P<0.01 versus CON) and gradually decreased during treatment. Conversely, EPC levels were lower in MD-0, tending to increase throughout treatment. In vitro studies were performed in human endothelial cells cultured in the presence of sera from each study group. Elevated expression of the inflammation marker intercellular adhesion molecule-1 and oxidative stress, with lower presence of endothelial nitric oxide synthase and higher reactive oxygen species production, were found in cells exposed to MD-0 sera (P<0.05 versus CON). These results were normalized in cells exposed to MD-24 sera. Thrombogenicity of extracellular matrices generated by these cells, measured as expression of VWF, tissue factor and platelet reactivity, showed non-significant differences. We provide a model of cultured endothelial cells reproducing endothelial dysfunction in naive patients with major depression, demonstrating endothelial damage and inflammation at diagnosis, and recovering with selective serotonin reuptake inhibitor treatment for 24 weeks.

Human neutrophil peptides inhibit cleavage of von Willebrand factor by ADAMTS13: a potential link of inflammation to TTP

AbstractInfection or inflammation may precede and trigger formation of microvascular thrombosis in patients with acquired thrombotic thrombocytopenic purpura (TTP). However, the mechanism underlying this clinical observation is not fully understood. Here, we show that human neutrophil peptides (HNPs) released from activated and degranulated neutrophils inhibit proteolytic cleavage of von Willebrand factor (VWF) by ADAMTS13 in a concentration-dependent manner. Half-maximal inhibitory concentrations of native HNPs toward ADAMTS13-mediated proteolysis of peptidyl VWF73 and multimeric VWF are 3.5 μM and 45 μM, respectively. Inhibitory activity of HNPs depends on the RRY motif that is shared by the spacer domain of ADAMTS13. Native HNPs bind to VWF73 (KD = 0.72 μM), soluble VWF (KD = 0.58 μM), and ultra-large VWF on endothelial cells. Enzyme-linked immunosorbent assay (ELISA) demonstrates markedly increased plasma HNPs1-3 in most patients with acquired autoimmune TTP at presentation (median, ∼170 ng/mL; range, 58-3570; n = 19) compared with healthy controls (median, ∼23 ng/mL; range, 6-44; n = 18) (P < .0001). Liquid chromatography plus tandem mass spectrometry (LC-MS/MS) reveals statistically significant increases of HNP1, HNP2, and HNP3 in patient samples (all P values <.001). There is a good correlation between measurement of HNPs1-3 by ELISA and by LC-MS/MS (Spearman ρ = 0.7932, P < .0001). Together, these results demonstrate that HNPs1-3 may be potent inhibitors of ADAMTS13 activity, likely by binding to the central A2 domain of VWF and physically blocking ADAMTS13 binding. Our findings may provide a novel link between inflammation/infection and the onset of microvascular thrombosis in acquired TTP and potentially other immune thrombotic disorders.

Recombinant HSA-CMG2 Is a Promising Anthrax Toxin Inhibitor.

Anthrax toxin is the major virulence factor produced by Bacillus anthracis. Protective antigen (PA) is the key component of the toxin and has been confirmed as the main target for the development of toxin inhibitors. The inhibition of the binding of PA to its receptor, capillary morphogenesis protein-2 (CMG2), can effectively block anthrax intoxication. The recombinant, soluble von Willebrand factor type A (vWA) domain of CMG2 (sCMG2) has demonstrated potency against anthrax toxin. However, the short half-life of sCMG2 in vivo is a disadvantage for its development as a new anthrax drug. In the present study, we report that HSA-CMG2, a protein combining human serum albumin (HSA) and sCMG2, produced in the Pichia pastoris expression system prolonged the half-life of sCMG2 while maintaining PA binding ability. The IC50 of HSA-CMG2 is similar to those of sCMG2 and CMG2-Fc in in vitro toxin neutralization assays, and HSA-CMG2 completely protects rats from lethal doses of anthrax toxin challenge; these same challenge doses exceed sCMG2 at a sub-equivalent dose ratio and overwhelm CMG2-Fc. Our results suggest that HSA-CMG2 is a promising inhibitor of anthrax toxin and may contribute to the development of novel anthrax drugs.

The function of ultra-large von Willebrand factor multimers in high shear flow controlled by ADAMTS13.

The paradigm that platelet aggregation, which contributes to bleeding arrest and also to thrombovascular disorders, initiates after signaling-induced platelet activation has been refuted in past recent years. Platelets can form aggregates independently of activation when soluble von Willebrand factor (VWF) is present and the shear rate exceeds a certain threshold where active A1 domains become exposed in soluble VWF multimers and can bind to platelet glycoprotein Ib. Subsequently - fostering each other - VWF can self-assemble into large nets combining with platelets into large conglomerates, which are entirely reversible when they enter a flow region with shear rates below the threshold. In addition the threshold changes from approximately 20 000 s⁻¹ in wall parallel flow to approximately 10 000 s⁻¹ in stagnation point flow. VWF containing ultra-large multimers - as when just released from endothelial storage sites - has been shown to have the highest binding potential to platelets and to each other, thus facilitating rapid platelet accrual to sites of vessel injury and exposed subendothelial structures, i.e. collagen. The VWF nets as well as the platelet-VWF conglomerates are controlled by the cleaving protease ADAMTS13 within minutes under high shear flow. Therewith the hemostatic potential is delivered where needed and the thrombogenic potential is highly controlled twofold: by flow and enzymatic proteolytic cleavage.

Anti-β2-GPI Antibodies Induce Von Willebrand Factor Release, Modulate Platelet Binding, and Affect VWF Proteolysis

Background:The antiphospholipid syndrome (APS) is characterized by predisposition to thrombosis. The cause for this pathology is poorly understood but is likely multifactorial, involving activation of blood cells and vasculature. The role that anti-β2-GPI antibodies play in von Willebrand factor (VWF) release from endothelial cells, VWF-platelet binding, and VWF cleavage by ADAMTS13 has not been well characterized in APS. We decided to study the effect of these antibodies on expressed ultra large VWF strings (ULVWF strings) that bind platelets (VWF-PLT strings) under flow to better understand platelet–VWF binding and ADAMTS13 regulation in APS.Hypothesis:We hypothesized that Anti-β2-GPI antibodies could induce VWF release from endothelial cells and modulate VWF’s prothombotic effect through alterations in VWF-Platelet binding and VWF cleavage by ADAMTS13.Methods:Human umbilical vein endothelial cells were seeded in 96-well plates/flow chambers prepared with a collagen Type I substrate for static/flow experiments, respectively.Static assays: Cells were incubated for 1 hr with Anti-β2-GPI or control antibodies and the conditioned media was assayed for VWF by ELISA, normalized to normal plasma.Flow Assay Analysis: After stimulation with agonist and perfusion with a platelet suspension, platelets bound to ULVWF in a string pattern were quantified via brightfield microscopy. Images of chambers were captured and VWF-PLT string-units (defined as a string length of 25μM) per slide were quantified. To minimize bias, image acquisition was standardized and the investigator was blinded at time of image acquisition/analysis.β2-GPI Flow assays: Endothelial cells in flow chambers were stimulated with 50ng/mL of phorbol myristate acetate (PMA), and a solution of fixed platelets with β2-GPI or β2-GPI+Anti-β2-GPI were perfused prior to image acquisition.ADAMTS13 assays: After stimulation with 25ng/mL PMA and perfusion with fixed platelets, images were acquired. Then control/patient plasma was perfused over formed strings. Images taken after plasma perfusion were quantified and compared to images prior to plasma perfusion.Data are shown as mean +/- SEM, and significance was determined as p<0.05 by student’s t-test or Mann-Whitney U Test, when appropriate.Results:Static Assays: Compared to control human IgG (8.28 +/- 3.34 mU/mL), VWF release was increased in the presence of two patient-derived Anti-β2-GPI antibodies, APS25-6 Anti-β2-GPI, 35.73 +/- 7.83 mU/mL (P = 0.008) and APS203-2 Anti-β2-GPI, 34.08 +/- 7.119 mU/mL (P = 0.039). As compared to control rabbit IgG (15.80 +/- 7.12 mU/mL), a rabbit polyclonal Anti-β2-GPI antibody, R24-6, also demonstrated increased soluble VWF (43.16 +/- 9.60 mU/mL, P = 0.013) release.β2GPI Flow Assays:The presence of β2GPI (2µM) reduced String-unit formation from 50.10 +/-5.57 Sting-units/image to 20.98 +/- 2.05 String Units/image (P < 0.0001) as compared to buffer. Addition of goat Anti-β2-GPI antibody (1µM) increased the VWF-PLT string observed as compared to β2GPI (2µM), 30.09 +/- 1.83 String Units to 20.98 +/- 2.05 String Units (P = 0.012) indicating that an Anti-β2-GPI antibody partially reverses the effect of β2GPI on reducing VWF-PLT string formation.ADAMTS13 Assay:Compared to pooled normal plasma (ADAMTS13 Activity 100%) (4.57 +/- 0.60 String Units/image cleaved), there was a significant decrease in the amount of string units/image cleaved in two APS plasmas with Anti-β2-GPI antibodies, APS232-9 (-0.23 +/- 0.98, P = 0.0003) and APS227-9 (2.23 +/- 0.73, P = 0.0009). ADAMTS13 Activity of patient plasma was 98.37% and 83.97%, respectively. These results suggest an inhibitory role of APS plasma on the cleavage of ULVWF strings.Conclusions:Anti-β2-GPI antibodies and antiphospholipid syndrome plasma may contribute to the prothrombotic phenotype observed in APS by three mechanisms: 1) the increased release of VWF from endothelial cells after incubation with Anti-β2-GPI, 2) increased platelet binding to ULVWF strings likely mediated by interfering with β2GPI’s known inhibition of Gp1bα VWF-platelet binding, and 3) a reduced ability to cleave VWF-PLT strings by APS plasma, suggestive of ADAMTS13 inhibition that does not correlate with ADAMTS13 activity. Taken together, our results suggest that VWF and its modulation may contribute to the prothrombotic phenotype observed in the antiphospholipid syndrome. DisclosuresNo relevant conflicts of interest to declare.

Thrombotic microangiopathies and the linkage between von Willebrand factor and the alternative complement pathway.

Molecular linkages between von Willebrand factor (VWF) and the alternative complement pathway (AP) have recently been discovered. Endothelial cell (EC)-anchored ultra-large (UL) VWF multimeric strings function as an activating surface for the AP. C3 (in active C3b form) binds to the EC-anchored ULVWF strings, and promotes the assembly of C3bBb (C3 convertase) and C3bBbC3b (C5 convertase). These linkages may help to explain enigmatic clinical problems related to thrombotic microangiopathies, including some cases of refractory thrombotic thrombocytopenic purpura (TTP), TTP associated with only mild-modest deficiencies of ADAMTS-13, the provocation (or exacerbation) of acute episodes in patients with the atypical hemolytic uremic syndrome, and thrombosis in paroxysmal nocturnal hemoglobinuria. Recent experiments have also demonstrated that complement factor H performs a dual role: participating in regulation of the AP by binding to EC-anchored ULVWF strings; and functioning as a reductase to decrease the size of soluble VWF multimers.

High Shear Dependent von Willebrand Factor Self-assembly Fostered by Platelet Interaction and Controlled by ADAMTS13

IntroductionThe paradigm of activation induced platelet aggregation has recently been refuted under blood flow conditions with shear rates exceeding 20,000s-1. These lead to reversible rolling platelet aggregates, which were dependent on the presence of immobilized and soluble von Willebrand factor. Material and MethodsIn vitro experiments using direct fluorescence video-microscopy were performed in wall parallel and stagnation point flow chambers with shear rates raised from 20,000 to 50,000s-1. Washed blood cell suspension containing recombinant von Willebrand factor (rVWF) was perfused over rVWF or collagen coated surfaces. ResultsHere we show for the first time with the visualization of rVWF that not only colloid and polymer, i.e. platelets and VWF, form a composite, but that VWF itself is capable of entirely reversible self-assembly. On a collagen surface the platelet-VWF-conglomerates did not roll but VWF nets bound permanently to the collagen fibers and captured and immobilized platelets from the flow. Lowering the shear rate below the threshold of 20,000s-1 no longer dissolved these deposits. Ultralarge multimer containing rVWF was most effective compared to normal sized rVWF. The presence of ADAMTS13 limited rolling aggregate and platelet-VWF-conglomerate formation to a time window of 7-8minutes. Changing wall parallel flow to stagnation point flow halved the required shear rate threshold. ConclusionWe conclude that flow dynamics can trigger reversible von Willebrand factor self-assembly and platelet-VWF-conglomerate accrual, which are regulated by ADAMTS13 to a time span needed by coagulation to stabilize it, e.g. in case of vessel injury.

The various states of von Willebrand factor and their function in physiology and pathophysiology

The specific interactions of von Willebrand factor (VWF) with the vessel wall, platelets or other interfaces strongly depend on (a shear-induced) VWF activation. Shear flow has been shown to induce a conformational transition of VWF, but is modulated by its thermodynamic state (state-function relationship). The state in turn is determined by physical (e.g. vessel geometry), physico-chemical (e.g. pH) and molecular-biological (e.g. mutants, binding) factors. Combining established results with recent insights, we reconstruct VWF biology and its state-function relationship from endothelial cell release to final degradation in the human vasculature. After VWF secretion, endothelial-anchored and shear activated VWF multimers can rapidly interact with surrounding colloids, typically with platelets. Simultaneously, this VWF activation enables ADAMTS13 to cleave VWF multimers thereby limiting VWF binding capacity. The subsequent cell-surface dissociation leads to a VWF recoiling to a globular conformation, shielding from further degradation by ADAMTS13. High local concentrations of these soluble VWF multimers, transported to the downstream vasculature, are capable for an immediate reactivation and re-polymerisation initiating colloid-binding or VWF-colloid aggregation at the site of inflamed endothelium, vessel injuries or pathological high-shear areas. Focusing on these functional steps in the lifecycle of VWF, its qualitative and quantitative deficiencies in the different VWD types will facilitate more precise diagnostics and reliable risk stratification for prophylactic therapies. The underlying biophysical principles are of general character, which broadens prospective studies on the physiological and pathophysiological impact of VWF and VWF-associated diseases and beares hope for a more universal understanding of an entire class of phenomena.

Carboxyl Terminal Domains Of ADAMTS13 Inhibit The Formation Of Ultra Large VWF Strings and Platelet/Leukocyte Aggregates On Endothelial Cells In Murine Mesenteric Venules Via a Disulfide Bond Reduction Mechanism

von Willebrand factor (VWF) undergoes self-association mediated by free cysteine residues between two apposed VWF multimers. This process occurs in solution or between soluble and ultra large (UL) VWF on endothelial cells or matrix under flow. ADAMTS13 is a key regulator for the ULVWF string formation and adhesive function, primarily through its proteolytic cleavage at the Tyr1605-Met1606 bond in the central A2 domain of VWF. The proteolytic cleavage of VWF by ADAMTS13 rapidly removes newly secreted ULVWF on activated endothelial cell surface, thereby preventing excessive platelet aggregation and thrombus formation under physiological conditions. Previous studies have shown that the proteolytic cleavage of both cell-bound and soluble ULVWF by ADAMTS13 depends on its amino terminal MDTCS domains. However, the role of more distal carboxyl-terminal domains is not fully established. Here, we show by N-ethylmaleimide (NEM) labeling and mass spectrometry that TSP1 2-8 repeats and CUB domains of human recombinant ADAMTS13 contain multiple free cysteine residues (-SH). We hypothesize that these free thiols may directly interact with those on the surface of VWF multimers, thereby preventing their lateral association, thereby inhibiting thrombus formation under flow. Using an intravital microscopic imaging technique we demonstrate that even in the absence of circulating ADAMTS13 (Adamts13-/-) the formation and elongation of ULVWF strings on endothelial cells of mesenteric venules after FeCl3 injury are highly dynamic over time (180 seconds), suggesting other proteases or reductases in blood are able cleave or reduce ULVWF strings under flow. However, the average length of ULVWF strings remains relatively constant. Recombinant ADAMTS13 (rA13) at physiological concentration (10 nM), when infused, rapidly cleaves or eliminates preformed ULVWF strings and platelet/leukocyte aggregates on endothelial cells. The efficacy of rA13 in reducing the average length of ULVWF strings and platelet/leukocyte aggregation decreases by ∼50% if rA13 is pretreated with NEM. Moreover, a C-terminal fragment of ADAMTS13 comprising 2-8 TSP1 repeats and two CUB domains (T2C) or containing 5-8 TSP1 repeats and two CUB domains (T5C), when infused, also significantly attenuates the formation and/or elongation of ULVWF strings/bundles and platelet/leukocyte aggregation on endothelial cells under the same conditions. The inhibitory activity of T2C and T5C is dose-dependent. At the concentration of 0.5 µM, the average length of the ULVWF strings and the surface coverage of platelets/leukocytes decrease by ∼50% and ∼40% (p&lt;0.001), respectively. Also, treatment with NEM abolishes the inhibitory effect of T2C and T5C on the ULVWF string formation and platelet/leukocyte aggregation on activated endothelial cells surface (Fig. 1). In conclusion, our results demonstrate for the first time that the distal C-terminal fragments of ADAMTS13 lacking the protease domain are of direct anti-thrombotic activity, which is mediated through its disulfide-bond reduction mechanism. Furthermore, the carboxyl-terminal disulfide bond reducing activity may contribute to the overall anti-thrombotic activity of ADAMTS13 in vivo. Disclosures: No relevant conflicts of interest to declare.

Antiphospholipid Antibodies Increase Von Willebrand Factor-Platelet String Formation From Human Endothelial Cells Under Physiologic Flow Conditions

BackgroundThe antiphospholipid syndrome (APS) is characterized by a predisposition to arterial and venous thromboembolism. The underlying mechanism of thrombosis in APS is uncertain, but is thought to involve a multifactorial activation of various components of the blood and vasculature. The role of von Willebrand factor (VWF), a plasma protein expressed by endothelial cells in response to endothelial cell damage or activation, is not clearly defined in APS. Previous studies have demonstrated that antiphospholipid antibodies increase the release of soluble VWF from human umbilical vein endothelial cells. However, these experiments were performed in static conditions and not in a flow based model. Recent investigations of expressed ultra large VWF (ULVWF) under flow have led to a new model of VWF-platelet strings that remain anchored to endothelial cells and provide a nidus for the formation of thrombi. Consistent with this, increases in VWF and variations in ADAMTS13, the enzyme that modulates ULVWF, have been associated with venous and arterial thrombosis. ObjectivesWe hypothesized that antiphospholipid antibodies would induce VWF-platelet strings on endothelial cells and that this mechanism contributes to APS-associated thrombosis. MethodsHuman umbilical vein endothelial cells were seeded in flow chambers prepared with a collagen substrate. All experiments were performed with cells of passage 5 or under. Confluent cells were incubated in serum-free medium with APS-derived IgG or anti-β2-GPI antibodies vs. control IgG. After incubation, the slides were perfused with lyophilized platelets and washed with Tyrode's buffer prior to image acquisition. Shear stresses were varied from 2-10 dyn/cm2 to assess for shear stress dependent variations in string formation. Pre-determined sites of imaging were set via a standardized method with relief contrast brightfield microscopy. Between 50-100 images were collected per condition. A VWF-platelet string-unit was defined as a 25 µm length of an individual VWF-platelet string. Images were analyzed for the number of VWF-platelet string units per image; quantification of string units was performed blinded to sample vs. control IgG treatment status. Data are shown as means +/- SEM, and significance was determined as p<0.05 by paired t-test. ResultsEndothelial cells treated with human antiphospholipid antibodies demonstrated increased VWF-platelet strings at two different shear stresses, one mimicking venous flow and one mimicking arterial flow. At 2 dyn/cm2, cells incubated for 1 hour with 100 µM of APS patient–derived IgG demonstrated 23.04 +/- 2.139 (SEM) VWF-platelet string units/image vs. 17.64 +/- 1.203 in cells treated with 100 µM control human IgG (p=0.0247). At 10 dyn/cm2, cells incubated for 6 hours with 600 nM of human anti-β2-GPI antibodies demonstrated 1.848 +/- 0.2866 (SEM) VWF-platelet string units/image vs 1.048 +/- 0.2350 in cells treated with 600 nM control human IgG (p=0.0395). ConclusionsAntiphospholipid antibodies, in aggregate (IgG from an APS patient) or specific (anti-β2-GPI), induce the formation of VWF-platelet strings from human endothelial cells. This finding suggests the potential role of VWF-platelet strings in thrombosis associated with APS. The clinical predisposition to arterial and venous thrombi in APS and the observation of increased VWF-platelet strings at arterial and venous shear stresses suggest that modulation of VWF-platelet strings may provide a future therapeutic target. Disclosures:Manco-Johnson:Bayer HealthCare: Membership on an entity's Board of Directors or advisory committees, Research Funding; CSL Behring: Membership on an entity's Board of Directors or advisory committees, Research Funding; Baxter BioScience: Membership on an entity's Board of Directors or advisory committees; Biogen Idec: Membership on an entity's Board of Directors or advisory committees; Novo Nordisk: Membership on an entity's Board of Directors or advisory committees; Eisai: Research Funding. Di Paola:Pfizer: DSMB, DSMB Other; CSL Behring: Consultancy.

Markers of Vascular Perturbation Correlate with Airway Structural Change in Asthma

Air trapping and ventilation defects on imaging are characteristics of asthma. Airway wall thickening occurs in asthma and is associated with increased bronchial vascularity and vascular permeability. Vascular endothelial cell products have not been explored as a surrogate to mark structural airway changes in asthma. Determine whether reporters of vascular endothelial cell perturbation correlate with airway imaging metrics in patients with asthma of varying severity. Plasma from Severe Asthma Research Program subjects was analyzed by ELISAs for soluble von Willebrand factor mature protein (VWF:Ag) and propeptide (VWFpp), P-selectin, and platelet factor 4. Additional subjects were analyzed over 48 hours after whole-lung antigen challenge. We calculated ventilation defect volume by hyperpolarized helium-3 magnetic resonance imaging and areas of low signal density by multidetector computed tomography (less than -856 Hounsfield units [HU] at functional residual capacity and -950 HU at total lung capacity [TLC]). VWFpp and VWFpp/Ag ratio correlated with and predicted greater percentage defect volume on hyperpolarized helium-3 magnetic resonance imaging. P-selectin correlated with and predicted greater area of low density on chest multidetector computed tomography less than -950 HU at TLC. Platelet factor 4 did not correlate. Following whole-lung antigen challenge, variation in VWFpp, VWFpp/Ag, and P-selectin among time-points was less than that among subjects, indicating stability and repeatability of the measurements. Plasma VWFpp and P-selectin may be useful as surrogates of functional and structural defects that are evident on imaging. The results raise important questions about why VWFpp and P-selectin are associated specifically with different imaging abnormalities.

Light Chain of Factor VIII Is Sufficient for Accelerating Cleavage of von Willebrand Factor by ADAMTS13 Metalloprotease

We previously demonstrated that coagulation factor VIII (FVIII) accelerates proteolytic cleavage of von Willebrand factor (VWF) by A disintegrin and metalloprotease with thrombospondin type 1 repeats (ADAMTS13) under fluid shear stress. In this study, the structural elements of FVIII required for the rate-enhancing effect and the biological relevance of this cofactor activity are determined using a murine model. An isolated light chain of human FVIII (hFVIII-LC) increases proteolytic cleavage of VWF by ADAMTS13 under shear in a concentration-dependent manner. The maximal rate-enhancing effect of hFVIII-LC is ∼8-fold, which is comparable with human full-length FVIII and B-domain deleted FVIII (hFVIII-BDD). The heavy chain (hFVIII-HC) and the light chain lacking the acidic (a3) region (hFVIII-LCΔa3) have no effect in accelerating VWF proteolysis by ADAMTS13 under the same conditions. Although recombinant hFVIII-HC and hFVIII-LCΔa3 do not detectably bind immobilized VWF, recombinant hFVIII-LC binds VWF with high affinity (K(D), ∼15 nM). Moreover, ultra-large VWF multimers accumulate in the plasma of fVIII(-/-) mice after hydrodynamic challenge but not in those reconstituted with either hFVIII-BDD or hFVIII-LC. These results suggest that the light chain of FVIII, which is not biologically active for clot formation, is sufficient for accelerating proteolytic cleavage of VWF by ADAMTS13 under fluid shear stress and (patho) physiological conditions. Our findings provide novel insight into the molecular mechanism of how FVIII regulates VWF homeostasis.

Thrombogenic changes in young and old mice upon subchronic exposure to air pollution in an urban roadside tunnel

Epidemiological studies indicate that elderly persons are particularly susceptible to the cardiovascular health complications of air pollution, but pathophysiological mechanisms behind the increased susceptibility remain unclear. Therefore, we investigated how continuous traffic-related air pollution exposure affects haemostasis parameters in young and old mice. Young (10 weeks) and old (20 months) mice were placed in an urban roadside tunnel or in a clean environment for 25 or 26 days and markers of inflammation and endothelial cells or blood platelet activation were measured, respectively. Plasma microvesicles and pro/anticoagulant factors were analysed, and thrombin generation analysis was performed. Despite elevated macrophage carbon load, tunnel mice showed no overt pulmonary or systemic inflammation, yet manifested reduced pulmonary thrombomudulin expression and elevated endothelial von Willebrand factor (VWF) expression in lung capillaries. In young mice, soluble P-selectin (sP-sel) increased with exposure and correlated with soluble E-selectin and VWF. Baseline plasma factor VIII (FVIII), sP-sel and VWF were higher in old mice, but did not pronouncedly increase further with exposure. Traffic-related air pollution markedly raised red blood cell and blood platelet numbers in young and old mice and procoagulant blood platelet-derived microvesicle numbers in old animals. Changes in coagulation factors and thrombin generation were mild or absent. Hence, continuous traffic-related air pollution did not trigger overt lung inflammation, yet modified pulmonary endothelial cell function and enhanced platelet activity. In old mice, subchronic exposure to polluted air raised platelet numbers, VWF, sP-sel and microvesicles to the highest values presently recorded, collectively substantiating a further elevation of thrombogenicity, already high at old age.

Cleavage of von Willebrand Factor by Granzyme M Destroys Its Factor VIII Binding Capacity

Von Willebrand factor (VWF) is a pro-hemostatic multimeric plasma protein that promotes platelet aggregation and stabilizes coagulation factor VIII (FVIII) in plasma. The metalloproteinase ADAMTS13 regulates the platelet aggregation function of VWF via proteolysis. Severe deficiency of ADAMTS13 is associated with thrombotic thrombocytopenic purpura, but does not always correlate with its clinical course. Therefore, other proteases could also be important in regulating VWF activity. In the present study, we demonstrate that VWF is cleaved by the cytotoxic lymphocyte granule component granzyme M (GrM). GrM cleaved both denaturated and soluble plasma-derived VWF after Leu at position 276 in the D3 domain. GrM is unique in that it did not affect the multimeric size and pro-hemostatic platelet aggregation ability of VWF, but instead destroyed the binding of VWF to FVIII in vitro. In meningococcal sepsis patients, we found increased plasma GrM levels that positively correlated with an increased plasma VWF/FVIII ratio in vivo. We conclude that, next to its intracellular role in triggering apoptosis, GrM also exists extracellularly in plasma where it could play a physiological role in controlling blood coagulation by determining plasma FVIII levels via proteolytic processing of its carrier VWF.

Soluble Monomeric Von Willebrand Factor A1A2A3 (R1450E) Mutant Fragment Enhances Shear-Induced Platelet Aggregation and Platelet Adhesion to Fibrin(ogen) Via αIIbβ3 Under High Shear Stress.

Abstract 4025Poster Board III-961Recently, we described that the gain of function mutation R1450E in the A1 domain of von Willebrand factor (VWF) eliminates the formation of catch bond with glycoprotein (GP)Ibα, prolonging the bond lifetimes at low forces. Because those studies were performed with the mutant immobilized on a plastic surface, we further characterize the effect of this mutant on platelet function in solution and under shear stress. Both wild type (WT) and mutant A1A2A3 proteins were expressed in HEK293 cells and purified to homogeneity. The monomeric state of A1A2A3 proteins were assessed by gel filtration chromatography and neither of the proteins had formed dimers or any higher order aggregates. The recombinant A1A2A3 mutant bound spontaneously to GPIbα without the modulator ristocetin with a half-maximal binding observed at 65 ± 8 nM. This apparent dissociation constant was comparable to that of WT (50 ± 10 nM) in the presence of ristocetin. The mutant failed to induce spontaneous platelet aggregation under stirring conditions, and blocked 100% ristocetin-induced platelet agglutination (RIPA) at concentration of 250 nM. At the same concentration, the mutant increased shear-induced platelet aggregation (SIPA) at 500s-1 and 5000s-1 shear rates, reaching 42% and 66%, respectively, while SIPA did not exceed 18% in the presence of WT. The anti-αIIbβ3 antibody 7E3 blocked the effect of the mutant on SIPA. Blood was then incubated with the mutant (250 nM) and perfused over a surface coated with fibrin(ogen) at different shear rates. Blood containing WT resulted in <10% surface coverage by platelets after 1.5 minutes while platelets from blood containing the mutant rapidly bound covering 100% of the fibrin(ogen) surface area at 1500s-1. At shear rate of 2500s-1, surface coverage was 20% for the mutant and 0% for WT fragment. EDTA and antibodies 6D1 (GPIbα) and 10E5 (αIIbβ3) effectively blocked mutant-mediated platelet adhesion and thrombus formation under high shear rates. The addition of ristocetin (0.5 mg/ml) to whole blood prior perfusion reproduced the effect of the mutant. Here, we describe an A1A2A3 mutant that bound spontaneously to GPIbα but affected differently RIPA and SIPA. These results suggest that hydrodynamic forces directly act on the GPIbα-mutant A1A2A3 complex, regulating signaling. In addition, platelet activation induced by the binding of soluble mutant A1A2A3 or plasma VWF results in αIIbβ3-mediated platelet adhesion to fibrin(ogen) under high shear rates. Disclosures:No relevant conflicts of interest to declare.