VWF-mediated Platelet Adhesion Research Articles

Removal of endothelial surface-associated von villebrand factor suppresses accelerate datherosclerosis after myocardial infarction

BackgroundThromboinflammation involving platelet adhesion to endothelial surface-associated von Willebrand factor (VWF) has been implicated in the accelerated progression of non-culprit plaques after MI. The aim of this study was to use arterial endothelial molecular imaging to mechanistically evaluate endothelial-associated VWF as a therapeutic target for reducing remote plaque activation after myocardial infarction (MI).MethodsHyperlipidemic mice deficient for the low-density lipoprotein receptor and Apobec-1 underwent closed-chest MI and were treated chronically with either: (i) recombinant ADAMTS13 which is responsible for proteolytic removal of VWF from the endothelial surface, (ii) N-acetylcysteine (NAC) which removes VWF by disulfide bond reduction, (iii) function-blocking anti-factor XI (FXI) antibody, or (iv) no therapy. Non-ischemic controls were also studied. At day 3 and 21, ultrasound molecular imaging was performed with probes targeted to endothelial-associated VWF A1-domain, platelet GPIbα, P-selectin and vascular cell adhesion molecule-1 (VCAM-1) at lesion-prone sites of the aorta. Histology was performed at day 21.ResultsAortic signal for P-selectin, VCAM-1, VWF, and platelet-GPIbα were all increased several-fold (p < 0.01) in post-MI mice versus sham-treated animals at day 3 and 21. Treatment with NAC and ADAMTS13 significantly attenuated the post-MI increase for all four molecular targets by > 50% (p < 0.05 vs. non-treated at day 3 and 21). On aortic root histology, mice undergoing MI versus controls had 2–4 fold greater plaque size and macrophage content (p < 0.05), approximately 20-fold greater platelet adhesion (p < 0.05), and increased staining for markers of platelet transforming growth factor-β1 signaling. Accelerated plaque growth and inflammatory activation was almost entirely prevented by ADAMTS13 and NAC. Inhibition of FXI had no significant effect on molecular imaging signal or plaque morphology.ConclusionsPlaque inflammatory activation in remote arteries after MI is strongly influenced by VWF-mediated platelet adhesion to the endothelium. These findings support investigation into new secondary preventive therapies for reducing non-culprit artery events after MI.

Influence of Hematocrit Level and Integrin αIIbβIII Function on vWF-Mediated Platelet Adhesion and Shear-Induced Platelet Aggregation in a Sudden Expansion.

Shear-mediated thrombosis is a clinically relevant phenomenon that underlies excessive arterial thrombosis and device-induced thrombosis. Red blood cells are known to mechanically contribute to physiological hemostasis through margination of platelets and vWF, facilitating the unfurling of vWF multimers, and increasing the fraction of thrombus-contacting platelets. Shear also plays a role in this phenomenon, increasing both the degree of margination and the near-wall forces experienced by vWF and platelets leading to unfurling and activation. Despite this, the contribution of red blood cells in shear-induced platelet aggregation has not been fully investigated-specifically the effect of elevated hematocrit has not yet been demonstrated. Here, a microfluidic model of a sudden expansion is presented as a platform for investigating platelet adhesion at hematocrits ranging from 0 to 60% and shear rates ranging from 1000 to 10,000 s-1. The sudden expansion geometry models nonphysiological flow separation characteristic to mechanical circulatory support devices, and the validatory framework of the FDA benchmark nozzle. PDMS microchannels were fabricated and coated with human collagen. Platelets were fluorescently tagged, and blood was reconstituted at variable hematocrit prior to perfusion experiments. Integrin function of selected blood samples was inhibited by a blocking antibody, and platelet adhesion and aggregation over the course of perfusion was monitored. Increasing shear rates at physiological and elevated hematocrit levels facilitate robust platelet adhesion and formation of large aggregates. Shear-induced platelet aggregation is demonstrated to be dependent on both αIIbβIII function and the presence of red blood cells. Inhibition of αIIbβIII results in an 86.4% reduction in overall platelet adhesion and an 85.7% reduction in thrombus size at 20-60% hematocrit. Hematocrit levels of 20% are inadequate for effective platelet margination and subsequent vWF tethering, resulting in notable decreases in platelet adhesion at 5000 and 10,000 s-1 compared to 40% and 60%. Inhibition of αIIbβIII triggered dramatic reductions in overall thrombus coverage and large aggregate formation. Stability of platelets tethered by vWF are demonstrated to be αIIbβIII-dependent, as adhesion of single platelets treated with A2A9, an anti-αIIbβIII blocking antibody, is transient and did not lead to sustained thrombus formation. This study highlights driving factors in vWF-mediated platelet adhesion that are relevant to clinical suppression of shear-induced thrombosis and in vitro assays of platelet adhesion. Primarily, increasing hematocrit promotes platelet margination, permitting shear-induced platelet aggregation through αIIbβIII-mediated adhesion at supraphysiological shear rates. The online version contains supplementary material available at 10.1007/s12195-024-00796-0.

Conformational activation and inhibition of von Willebrand factor by targeting its autoinhibitory module

AbstractActivation of von Willebrand factor (VWF) is a tightly controlled process governed primarily by local elements around its A1 domain. Recent studies suggest that the O-glycosylated sequences flanking the A1 domain constitute a discontinuous and force-sensitive autoinhibitory module (AIM), although its extent and conformation remains controversial. Here, we used a targeted screening strategy to identify 2 groups of nanobodies. One group, represented by clone 6D12, is conformation insensitive and binds the N-terminal AIM (NAIM) sequence that is distal from A1; 6D12 activates human VWF and induces aggregation of platelet-rich plasma at submicromolar concentrations. The other group, represented by clones Nd4 and Nd6, is conformation sensitive and targets the C-terminal AIM (CAIM). Nd4 and Nd6 inhibit ristocetin-induced platelet aggregation and reduce VWF-mediated platelet adhesion under flow. A crystal structure of Nd6 in complex with AIM-A1 shows a novel conformation of both CAIM and NAIM that are primed to interact, providing a model of steric hindrance stabilized by the AIM as the mechanism for regulating GPIbα binding to VWF. Hydrogen-deuterium exchange mass spectrometry analysis shows that binding of 6D12 induces the exposure of the GPIbα-binding site in the A1 domain, but binding of inhibitory nanobodies reduces it. Overall, these results suggest that the distal portion of NAIM is involved in specific interactions with CAIM, and binding of nanobodies to the AIM could either disrupt its conformation to activate VWF or stabilize its conformation to upkeep VWF autoinhibition. These reported nanobodies could facilitate future studies of VWF functions and related pathologies.

Coarse-grained simulations of von Willebrand factor adsorption to collagen with consequent platelet recruitment.

A multimeric glycoprotein of blood plasma-Von Willebrand factor (VWF)-mediates platelet adhesion to the fibrillar collagen of the subendothelial matrix if the blood vessel walls are damaged. The adsorption of VWF to collagen is thus essential for the initial stages of platelet hemostasis and thrombosis, as it plays a role of a molecular bridge between the injury and platelet adhesion receptors. Biomechanical complexity and sensitivity to the hydrodynamics are inherent in this system, therefore, modern computational methods supplement experimental studies of biophysical and molecular mechanisms that underlie platelet adhesion and aggregation in the blood flow. In the present paper, we propose a simulation framework for the VWF-mediated platelet adhesion to a plane wall with immobilized binding sites for VWF under the action of shear flow. VWF multimers and platelets are represented in the model by particles connected by elastic bonds and immersed in a viscous continuum fluid. This work complements the scientific field by taking into account the shape of a flattened platelet, but keeping a compromise between the detail of the description and the computational complexity of the model.

Inhibition of Shear-Induced Platelet Aggregation by Xueshuantong via Targeting Piezo1 Channel-Mediated Ca2+ Signaling Pathway.

XueShuanTong (XST) comprising therapeutically active ginsenosides, a lyophilized extract of Panax notoginseng roots, is extensively used in traditional Chinese medicine to treat ischemic heart and cerebrovascular diseases. Our recent study shows that treatment with XST inhibits shear-induced thrombosis formation but the underlying mechanism remained unclear. This study aimed to investigate the hypothesis that XST inhibited shear-induced platelet aggregation via targeting the mechanosensitive Ca2+-permeable Piezo1 channel by performing platelet aggregation assay, Ca2+ imaging and Western blotting analysis. Exposure to shear at physiologically (1,000–2000 s−1) and pathologically related rates (4,000–6,000 s−1) induced platelet aggregation that was inhibited by treatment with GsMTx-4. Exposure to shear evoked robust Ca2+ responses in platelets that were inhibited by treatment with GsMTx-4 and conversely enhanced by treatment with Yoda1. Treatment with XST at a clinically relevant concentration (0.15 g L−1) potently inhibited shear-induced Ca2+ responses and platelet aggregation, without altering vWF-mediated platelet adhesion and rolling. Exposure to shear, while resulting in no effect on the calpain-2 expression in platelets, induced calpain-2-mediated cleavage of talin1 protein, which is known to be critical for platelet activation. Shear-induced activation of calpain-2 and cleavage of talin1 were attenuated by treatment with XST. Taken together, our results suggest that XST inhibits shear-induced platelet aggregation via targeting the Piezo1 channel to prevent Piezo1-mediated Ca2+ signaling and downstream calpain-2 and talin1 signal pathway, thus providing novel insights into the mechanism of the therapeutic action of XST on platelet aggregation and thrombosis formation.

Shear stress induced activation of von Willebrand factor may predispose to gastrointestinal bleeding in syndrome of Heyde

Abstract Introduction Von Willebrand factor (vWF) changes conformation from globular to fibrillar in the range of shear rates above 5000 s-1. High shear rates, observed in severe aortic stenosis, create conditions for activation of vWF, which opens up access for platelets and coagulation factors to the previously hidden domains of the molecule. At the same time, vWF undergoes increased degradation by metalloproteinase ADAMTS13. Proteolytic cleavage of vWF leads to deficiency of hemostatically active high molecular weight multimers (HMWM) of vWF, while its mass concentration remains unaltered. This results in the development of acquired von Willebrand disease type 2A (vWD) and concomitant gastrointestinal bleeding. The combination of acquired vWD 2A and gastrointestinal bleeding, developed due to severe aortic stenosis, is called Heyde's syndrome. The correlation of shear stress activation of vWF and gastrointestinal bleeding in patients with Heyde's syndrome remains poorly studied. The aim of the study was to measure vWF-mediated platelet adhesion to fibrinogen-coated surfaces under shear rates higher than 5000 s-1 in whole blood samples of healthy volunteers and patients with Heyde's syndrome. Methods A microfluidic system simulating blood flow in vessels was used to assess platelet adhesion. Platelet adhesion was measured by an increase in the intensity of laser radiation scattered from a fibrinogen-coated surface during a 15 minutes circulation of whole blood samples through a flow chamber under shear rates higher than 5000 s-1. Platelets in whole blood samples were activated with 5 μM ADP prior to measurement. The study included 5 patients with Heyde's syndrome 55–80 years old. The control group included 6 healthy volunteers 25–55 years old. vWF-mediated platelet adhesion was detected by blocking platelet-vWF binding with anti-GPIb monoclonal antibody (mAb). Fibrinogen-mediated platelet adhesion was detected by blocking platelet GPIIb/IIIa receptors with mAb. Result The inhibition of GPIb vWF-receptor reduced platelet adhesion by 7.6±3.5% (p&amp;lt;0.05) in patients with Heyde's syndrome, and by 16.5±3.3% (p&amp;lt;0.05) in healthy volunteers. The inhibition of GPIIb/IIIa fibrinogen receptor reduced platelet adhesion by 96±7% (p&amp;lt;0.05) in patients with Heyde's syndrome, and by 80.2±6.6% (p&amp;lt;0.05) in healthy volunteers. Conclusion Significantly reduced contribution of vWF to platelet adhesion under shear rates higher than 5000 s-1 may indicate a decrease in hemostatically active HMWM of vWF. Shear stress activation of vWF in the range of high shear rates and its subsequent inactivation by ADAMTS13 may lead to functional vWF deficiency and the development of gastrointestinal bleeding in Heyde's syndrome. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): The Russian Science Foundation

Abstract WP97: The Aptamer BT200 Blocks Von Willebrand Factor and Platelet Function in Blood of Stroke Patients

Background: The effect of conventional anti-platelet agents is limited in secondary stroke prevention, and their effects are further blunted under conditions of high shear stress in the presence of increased levels of circulating VWF. VWF mediates platelet adhesion to collagen under high shear stress and is thereby critically involved in thrombus formation at sites of stenotic extracranial intracranial arteries (reviewed by Buchtele et al. 2018). We have created a novel anti-VWF aptamer (BT200) which could be useful for secondary stroke prevention, because the anti-VWF aptamer ARC1779 effectively reduced cerebral embolization after carotid endarterectomy (Markus et al. 2011). Aims: To characterize the effects of BT200 in blood of patients with large artery atherosclerosis stroke. Methods: Blood samples were obtained from 30 patients with acute stroke Inhibition of VWF activity by BT200 was quantified by REAADS ELISA and VWF ristocetin cofactor activity (VWF:RCo), platelet function under high shear rates with the PFA-100, and ristocetin-induced platelet aggregation in whole blood. Results: The majority of stroke patients had elevated VWF:RCo levels (mean: 198%; range 55-330%). Of 15 patients receiving clopidogrel with or without aspirin, only two had a prolonged collagen adenosine diphosphate closure time (CADP-CT) &gt;123s, and only one patient had a ristocetin induced aggregation of &lt;20U. BT200 concentration dependently inhibited VWF activity to &lt;3% and VWF dependent platelet function (p&lt;0.001): BT200 invariably prolonged CADP-CT to target levels of &gt;300s, and decreased aggregation to &lt;20U in blood samples from all patients. Conclusions: BT200 effectively inhibits VWF activity and VWF-dependent platelet function in blood from patients with acute stroke. Results from this study proved useful for planning of the ongoing phase I and planned phase II trial.

Thrombotic microangiopathy as a cause of cardiovascular toxicity from the BCR-ABL1 tyrosine kinase inhibitor ponatinib

AbstractThe third-generation tyrosine kinase inhibitor (TKI) ponatinib has been associated with high rates of acute ischemic events. The pathophysiology responsible for these events is unknown. We hypothesized that ponatinib produces an endothelial angiopathy involving excessive endothelial-associated von Willebrand factor (VWF) and secondary platelet adhesion. In wild-type mice and ApoE−/− mice on a Western diet, ultrasound molecular imaging of the thoracic aorta for VWF A1-domain and glycoprotein-Ibα was performed to quantify endothelial-associated VWF and platelet adhesion. After treatment of wild-type mice for 7 days, aortic molecular signal for endothelial-associated VWF and platelet adhesion were five- to sixfold higher in ponatinib vs sham therapy (P < .001), whereas dasatinib had no effect. In ApoE−/− mice, aortic VWF and platelet signals were two- to fourfold higher for ponatinib-treated compared with sham-treated mice (P < .05) and were significantly higher than in treated wild-type mice (P < .05). Platelet and VWF signals in ponatinib-treated mice were significantly reduced by N-acetylcysteine and completely eliminated by recombinant ADAMTS13. Ponatinib produced segmental left ventricular wall motion abnormalities in 33% of wild-type and 45% of ApoE−/− mice and corresponding patchy perfusion defects, yet coronary arteries were normal on angiography. Instead, a global microvascular angiopathy was detected by immunohistochemistry and by intravital microscopy observation of platelet aggregates and nets associated with endothelial cells and leukocytes. Our findings reveal a new form of vascular toxicity for the TKI ponatinib that involves VWF-mediated platelet adhesion and a secondary microvascular angiopathy that produces ischemic wall motion abnormalities. These processes can be mitigated by interventions known to reduce VWF multimer size.

Preclinical Development of a vWF Aptamer to Limit Thrombosis and Engender Arterial Recanalization of Occluded Vessels.

Endothelial surface and circulating glycoprotein von Willebrand factor (vWF) regulates platelet adhesion and is associated with thrombotic diseases, including ischemic stroke, myocardial infarction, and peripheral vascular disease. Thrombosis, as manifested in these diseases, is the leading cause of disability and death in the western world. Current parenteral antithrombotic and thrombolytic agents used to treat these conditions are limited by a short therapeutic window, irreversibility, and major risk of hemorrhage. To overcome these limitations, we developed a novel anti-vWF aptamer, called DTRI-031, that selectively binds and inhibits vWF-mediated platelet adhesion and arterial thrombosis while enabling rapid reversal of this antiplatelet activity by an antidote oligonucleotide (AO). Aptamer DTRI-031 exerts dose-dependent inhibition of platelet aggregation and thrombosis in whole blood and mice, respectively. Moreover, DTRI-031 can achieve potent vascular recanalization of platelet-rich thrombotic occlusions in murine and canine carotid arteries. Finally, DTRI-031 activity is rapidly (<5min) and completely reversed by AO administration in a murine saphenous vein hemorrhage model, and murine toxicology studies indicate the aptamer is well tolerated. These findings suggest that targeting vWF with an antidote-controllable aptamer potentially represents an effective and safer treatment for thrombosis patients having platelet-rich arterial occlusions in the brain, heart, or periphery.

Spray-dried plasma deficient in high-molecular-weight multimers of von Willebrand factor retains hemostatic properties.

Dried plasmas can overcome logistical barriers that prevent fresh frozen plasma (FFP) usage in acute resuscitation, but processing of these products can detrimentally alter the composition. Spray-dried plasma (SpDP) from single units is deficient in high-molecular-weight multimers of von Willebrand factor (vWF), a critical facilitator of platelet adhesion and thrombus formation. We hypothesized that converting high-molecular-weight multimers to smaller-molecular-weight multimers would retain vWF's capacity to mediate platelet adhesion. SpDP obtained from untreated FFP was reconstituted with glycine-hydrochloric acid (HCl) and glycine (20 mM:50 mM) or pretreated with glycine-HCl (20 mM) or glycine-glycine-HCl (20 mM:50 mM) and reconstituted with water. In vitro hemostatic potential of SpDPs versus FFP or FFP spiked with 70 mM of glycine was evaluated, leading to a more detailed in vitro study of glycine-HCl-glycine (20 mM:50 mM) pretreated SpDP. Plasmas were combined with RBCs and platelets to observe global coagulation response. While vWF-ristocetin cofactor activity is significantly decreased (-41.13%; p < .0001) in SpDP, a model of vWF-mediated platelet adhesion to collagen under flow showed enhanced function (+13%; p < .01). Fewer microparticles, particularly of platelet origin, were observed in SpDP versus FFP (p < .0001). Small but significant differences in thromboelastography results were observed, although SpDP and FFP were within normal ranges. Comparable coagulability was observed in FFP and SpDP. The apparent paradox between vWF-ristocetin cofactor assay and vWF-mediated platelet adhesion may be explained by the increase in smaller multimers of vWF in SpDP, producing different outcomes in these assays.

Insights into 3D Structure of ADAMTS13: A Stepping Stone towards Novel Therapeutic Treatment of Thrombotic Thrombocytopenic Purpura.

ADAMTS13 (A D: isintegrin A: nd M: etalloprotease with a T: hromboS: pondin type-1 motif, member 13: ) and von Willebrand factor (VWF) can be considered as scale weights which control platelet adhesion during primary haemostasis. In a very uncommon condition designated thrombotic thrombocytopenic purpura (TTP), functional absence of ADAMTS13 tips the balance toward VWF-mediated platelet adhesion in the microcirculation. TTP is associated with a high mortality and arises from either a congenital or acquired autoimmune deficiency of the plasma enzyme ADAMTS13. In case of acquired ADAMTS13 deficiency, autoantibodies bind to and inhibit the function of ADAMTS13. Currently available treatments of TTP aim to supply ADAMTS13 through plasma exchange or are aimed at B-cell depletion with rituximab. None of the available therapeutics, however, aims at protection of ADAMTS13 from circulating autoantibodies. In this review, our aim is to describe the structure-function relationship of ADAMTS13 employing homology models and previously published crystal structures. Structural bioinformatics investigation of ADAMTS13 reveals many insights and explains how mutations and autoantibodies may lead to the pathophysiology of TTP. The results of these studies provide a roadmap for the further development of rationally designed therapeutics for the treatment of patients with acquired TTP. In addition, we share our opinion on the state of the art of the open-closed conformations of ADAMTS13 which regulate the activity of this highly specific VWF cleaving protease.

The role of von Willebrand factor in primary haemostasis under conditions of haemodilution

IntroductionSevere blood loss and related haemodilution during cardiac surgery result in a reduced platelet count which may lead to impaired primary haemostasis. Additionally, the reduced haematocrit lowers rheological forces in circulation and may account for lowered platelet adhesiveness and potentially reduced von Willebrand factor (VWF) activity. These mechanisms may lead to postoperative bleeding. Aim of this study was the examination of VWF activity and VWF-mediated platelet adhesion to collagen under conditions of haemodilution. Materials and methodsAn in vitro flow chamber was utilized to examine the primary haemostasis under a high arterial shear rate of 1500s−1 at variable VWF concentrations, platelet counts and haematocrit levels. ResultsUnder a high arterial shear rate, VWF activity is highly dependent on blood viscosity. Both VWF-collagen binding and VWF-mediated platelet adhesion to collagen were significantly increased with increasing haematocrit. Interestingly, we found slight differences in the VWF multimer sizes able to bind collagen under different shear stress conditions. Under conditions of haemodilution, platelet adhesion was strongly dependent on VWF concentration. Increasing VWF concentration improved platelet adhesiveness under low haematocrit conditions (30%) and variable platelet counts (80, 150 and 250×109/L). This effect was nearly abolished at very low platelet count levels of 50×109/L. ConclusionsVWF improves platelet function under conditions of haemodilution. Therefore, increasing VWF concentration may represent a complementary strategy to administration of platelet concentrates for the management of bleeding in thrombocytopenia.

Abstract WMP83: Extracellular Vimentin Contributes to Von Willebrand Factor (VWF) String Formation in the Cerebrovasculature Following Endothelial Activation

Introduction: VWF strings can form within the vascular lumen following endothelial activation and have been implicated in platelet adhesion and worsened outcome following stroke. However, the molecular interactions facilitating VWF string anchorage to the endothelial surface are currently unknown. Here we examined the novel role of endothelial vimentin in mediating the anchorage of VWF strings within the cerebrovasculature. We hypothesize that VWF released from activated endothelium remains anchored at the luminal surface (i.e. VWF strings) through direct interaction with extracellular vimentin. Methods: Cultured endothelial cell (EC) experiments were performed with human umbilical vein EC (HUVECs) and human brain microvascular EC (HBMVECs). EC were stimulated with histamine (10 uM) under flow conditions. Specific protein interactions were probed with recombinant vimentin and A2 domain of VWF. Mouse middle cerebral and superior cerebellar arteries from WT and vimentin KO mice were set up in a pressurized artery chamber, stimulated with histamine, and then processed for VWF immunofluorescence to quantify VWF strings. VWF string formation was further evaluated by cranial window preparation using labeled platelets to detect real-time in vivo string formation. Results: In cultured EC, histamine stimulation promoted the production of long VWF strings that were significantly attenuated in the presence of recombinant vimentin or A2 domain of VWF. In pressurized cerebral arteries, histamine stimulation promoted VWF strings that aligned along the luminal endothelial surface. VWF string formation was reduced 2.9 fold in arteries from vimentin knockout mice (P=0.02). Histamine stimulation in the cranial window produced platelet-adherent strings in the pial microvasculature. Conclusions: These studies provide in vitro and in vivo evidence for a novel interaction between vimentin and the A2 domain of VWF at the endothelial surface which contributes to the anchorage of VWF strings in the microvasculature. This vimentin/VWF interaction critically regulates VWF-mediated platelet adhesion at the surface of activated endothelium and could thus provide a novel therapeutic target in the treatment of acute ischemic stroke.

Vimentin Is a Novel Molecule Required for the Formation of Von Willebrand Factor Strings from the Vascular Endothelium

Background: Von Willebrand factor (VWF) is a multimeric plasma and subendothelial glycoprotein which is produced and secreted by endothelial cells. With intense stimulation (e.g. after vascular injury), endothelial cells secrete unusually large multimers of VWF in a hyper-adhesive string arrangement. Upon secretion, these long multimers or strings remain anchored to the cell surface and are capable of quickly attracting circulating platelets through interaction with the receptor GPIbα. In the absence of the VWF-protease, the VWF strings attached to the endothelium mediate spontaneous platelet adhesion that leads to the formation of microthrombi on the endothelial surface, resulting in vessel occlusion. To date, it is not clear which molecules allow VWF strings to remain docked on the surface of the endothelium once secreted. Vimentin is a cytoskeletal molecule and its extracellular form has been shown to be expressed on the surface of various cell types, including endothelial cells. Recent work from our lab has highlighted the role of extracellular vimentin in mediating platelet adhesion to VWF and that anti-vimentin antibodies inhibit this interaction. We have also found that vimentin binds the A2 domain of VWF, which is exposed on the newly secreted VWF strings. Therefore, we hypothesize that vimentin mediates the anchorage of VWF strings to the vascular endothelium. Understanding these interactions is important as VWF strings have been implicated in the pathophysiology of several disease states, such as sickle cell disease and malaria.Methods: Commercial human umbilical vein endothelial cells (HUVECs) were used. Cells were stimulated with histamine and analyzed under flow conditions to assess the quantity of VWF strings in the presence of soluble recombinant A2 domain, soluble recombinant vimentin, or anti-vimentin antibodies versus control buffer. VWF strings were visualized by tagging with commercial fluorescent-conjugated antibody. We also evaluated VWF string adherence to the endothelium of intact pressurized cerebral arteries from vimentin knockout mice versus wild-type (WT) mice ex vivo. Cerebral middle cerebral artery and parenchymal arterioles from mice were isolated, pressurized, and luminally perfused in a perfusion chamber. Histamine was applied to activate the endothelium and elicit VWF string formation. The negative control was an irrelevant isotype antibody. After histamine treatment, the arteries/arterioles were processed for VWF immunofluorescence to assess VWF string formation. VWF strings were quantified as length normalized to endothelial surface area.Results: As expected, HUVECs expressed surface vimentin as determined using flow cytometry and confocal microscopy. The presence of either soluble A2 or soluble vimentin significantly reduced the amount of VWF string formation from histamine-stimulated HUVECs in comparison to control. In some experiments, anti-vimentin antibodies decreased VWF string formation but findings were not significant. Vascular endothelial cells from vimentin knockout mice failed to form VWF strings after histamine stimulation in comparison to vimentin WT mice.Conclusions: These novel findings show that extracellular vimentin appears to play a role in VWF string formation likely via A2 domain binding. Further studies are necessary to shed light on the intricate pathways regulating VWF-mediated platelet adhesion. Our long term goals are to understand the novel interactions between vimentin and VWF strings in governing hemostasis and thrombosis. DisclosuresNo relevant conflicts of interest to declare.

The Role Of High Von Willebrand Factor and Low ADAMTS13 Levels In The Risk Of Venous Thromboembolism

BackgroundVenous thromboembolism (VTE) is a common multicausal disease in the general population. von Willebrand factor (VWF) has been associated with VTE in epidemiological studies and recently VWF-mediated platelet adhesion has been shown to be critical for deep vein thrombosis (DVT) in mouse models. The large VWF multimers are cleaved by ADAMTS13 into smaller, less active multimers. Because of the key role of ADAMTS13, VWF and factor VIII (FVIII) in hemostasis and their close biological relationship, we aimed at investigating in the same population the effect of these proteins on VTE risk. Materials and MethodsWe included consecutive patients admitted to our anticoagulation clinic with a first objectively confirmed VTE event between January 2007 and July 2011. The study included only patients with DVT of the lower limbs and/or pulmonary embolism aged between 18-70 years, and without medical history of overt malignancy, arterial thrombosis, liver cirrhosis, renal failure on hemodialysis, connective tissue disease and other conditions characterized by chronic inflammation. Blood was collected at least 1 month after stopping anticoagulation and ≥ 6 months after VTE. Controls were recruited from friends or partners of patients, had no personal history of VTE and were matched to patients by gender and age. The other exclusion criteria for controls were the same as for patients, and in both groups pregnant or postpartum women at blood collection were excluded. ADAMTS13 and VWF antigens were determined by commercial ELISA and FVIII activity was measured using FVIII deficient plasma. High VWF (>150%) and FVIII (>150%) were defined by plasma levels of these proteins exceeding the 88th and 94th percentiles of the control group, respectively. To define low ADAMTS13 levels we used the 10th percentile of the controls (≤0.64 μg/ml). Odds ratios (OR) and 95% confidence intervals (CI) were presented adjusted for gender and age and medians with interquartile variation (25th-75th percentiles). Results358 patients with a first objectively confirmed VTE event were admitted to our clinic, of whom 282 did not participate in the study due to the exclusion criteria (n= 249) and loss of follow-up or refusal (n= 33). Therefore, we included 76 patients (53 women, 70%) with a median age of 43 years (33-55 years) and 96 controls (66 women, 69%), with a median age of 42 years (31-52 years). In controls, there was a negative albeit weak correlation between ADAMTS13 levels and VWF (rs= -0.213 by Spearman coefficient, p= 0.037) and FVIII (rs= -0.251, p= 0.014). As expected, VTE was associated with high levels of VWF (OR 2.80, 95% CI 1.20-6.54) and FVIII (OR 3.02, 95% CI 1.08-8.43). Low ADAMTS13 levels were detected in 12 patients and 9 controls (OR 1.76, 95% CI 0.70-4.46). The population was dichotomized according to the 88th percentile of VWF of the controls. Median ADAMTS13 was lower in the group with VWF >p88 compared to the group with VWF ≤p88 in controls (0.80μg/ml, 0.63-0.94μg/ml vs. 0.98μg/ml, 0.81-1.23μg/ml, p= 0.051) and in patients (0.75μg/ml, 0.53-0.95μg/ml vs. 0.95μg/ml, 0.81-1.14μg/ml, p= 0.001). Next the population was categorized into subjects with VWF ≤p88 and ADAMTS13 >p10 (reference category: 52 patients, 79 controls), VWF ≤p88 and ADAMTS13 ≤p10 (4 patients, 6 controls), VWF >p88 and ADAMTS13 >p10 (12 patients, 8 controls) and VWF >p88 and ADAMTS13 ≤p10 (8 patients, 3 controls). The combination of high VWF and low ADAMTS13 increased VTE risk when compared to the reference category (OR 4.14; 95% CI 1.03-16.71). The risk did not substantially change with adjustment for acute phase proteins and ABO group, and it was attenuated when further adjusted for high FVIII (OR 3.41, 95% CI 0.80-14.70). No correlation was found between the time since the VTE event and levels of VWF, FVIII and ADAMTS13 (p>0.05). Conclusionsour data suggest a potential effect of high VWF and low ADAMTS13 on VTE risk. An unbalance between these two interconnected proteins might have a role in the pathophysiology of VTE. Results should be interpreted with caution due to the wide 95% CI and to the attenuation of VTE risk with further adjustment for FVIII. In order to better explain our results studies with larger sample size are warranted. Disclosures:No relevant conflicts of interest to declare.

Free Thiol Groups In Von Willebrand Factor (VWF) Are Required For Its Proper Function Under Physiological Flow Conditions

BackgroundThe multimeric plasma glycoprotein von Willebrand factor (VWF) is exceptionally rich in cysteine, and its structure is largely determined by inter- and intramolecular disulfide bonding. Additionally, VWF was shown to contain unpaired cysteine residues potentially affecting protein function. The significance of free thiols on the surface of plasmatic VWF has been confirmed previously with respect to platelet binding under pathologically high shear stress. Furthermore their potential involvement in functional VWF self-association occurring at elevated shear stress has been suggested. AimsObjective of the present study was to investigate whether free thiol groups of plasma VWF contribute to the physiological VWF function under high physiological arterial shear stress conditions. Furthermore, we aimed to elucidate possible underlying mechanisms involved in this regulation. MethodsFree and accessible thiol groups of plasma-derived VWF were blocked with N-ethylmaleimide (NEM). Derivatization was followed by detailed structural and functional examination including multimer analysis (MMA) and Fourier transform infrared spectroscopy (FTIR). Functional differences between the NEM-derivatized sample and the control sample were detected using an in vitro flow chamber system with respect to VWF-mediated platelet adhesion to collagen. Interactions with collagen type III and platelet glycoprotein (GP)Ib receptor were investigated using surface plasmon resonance (SPR). Identification of accessible cysteine residues was accomplished using biotin-linked maleimide (MPB) followed by analysis of multimer and domain incorporation as well as mass spectrometry. ResultsBlocking free thiol groups provoked substantial loss of VWF activity with respect to platelet recruitment to collagen type III under flow. The lowered platelet adhesion to collagen type III was shown to be a combined effect of inhibition of (i) the initial VWF binding to collagen type III as well as (ii) VWF-platelet GPIb interaction. Free thiol groups were accessible for derivatization solely on the surface of coiled multimers. Domain incorporation studies revealed a high level of derivatization in VWF N- and C-terminus. This was in accordance with the mass spectrometric analysis, where 19 MPB-derivatized peptides, predominantly located at the N- and C-terminus, could be identified. ConclusionBlocking free thiol groups in VWF significantly impaired mediation of platelet adhesion under physiological shear stress conditions. This result suggests an essential functional role of free thiol groups in VWF regarding binding to subendothelial matrix as well as platelet recruitment. Disclosures:Solecka:Octapharma AG: Employment. Fuchs:Octapharma AG: Employment. Kannicht:Octapharma AG: Employment.

The GPVI-Fc Fusion Protein Revacept Improves Cerebral Infarct Volume and Functional Outcome in Stroke

ObjectivesWe examined the effect of Revacept, an Fc fusion protein which is specifically linked to the extracellular domain of glycoprotein VI (GPVI), on thrombus formation after vessel wall injury and on experimental stroke in mice.BackgroundSeveral antiplatelet drugs for the treatment of myocardial infarction or ischemic stroke with potent anti-ischemic effects have been developed, but all incur a significant risk of bleeding.MethodsPlatelet adhesion and thrombus formation after endothelial injury was monitored in the carotid artery by intra-vital fluorescence microscopy. The morphological and clinical consequences of stroke were investigated in a mouse model with a one hour-occlusion of the middle cerebral artery.ResultsThrombus formation was significantly decreased after endothelial injury by 1 mg/kg Revacept IV, compared to Fc only. 1 mg/kg Revacept IV applied in mice with ischemic stroke immediately before reperfusion significantly improved functional outcome, cerebral infarct size and edema compared to Fc only. Also treatment with 10 mg/kg rtPA was effective, and functional outcome was similar in both treatment groups. The combination of Revacept with rtPA leads to increased reperfusion compared to treatment with either agent alone. In contrast to rtPA, however, there were no signs of increased intracranial bleeding with Revacept. Both rtPA and Revacept improved survival after stroke compared to placebo treatment. Revacept and vWF bind to collagen and Revacept competitively prevented the binding of vWF to collagen.ConclusionsRevacept reduces arterial thrombus formation, reduces cerebral infarct size and edema after ischemic stroke, improves functional and prognostic outcome without intracranial bleeding. Revacept not only prevents GPVI-mediated, but probably also vWF-mediated platelet adhesion and aggregate formation. Therefore Revacept might be a potent and safe tool to treat ischemic complications of stroke.

Von Willebrand Factor

Thrombus formation is of paramount importance in the pathophysiology of acute ischemic stroke. Current antithrombotics used to treat or prevent cerebral ischemia are only moderately effective or bear an increased risk of severe bleeding. von Willebrand factor (VWF) has long been known to be a key player in thrombus formation at sites of vascular damage. While the association between VWF and coronary heart disease has been well studied, knowledge about the role of VWF in stroke is much more limited. However, in recent years, an increasing amount of clinical and preclinical evidence has revealed the critical involvement of VWF in stroke development. This review summarizes the latest insights into the pathophysiologic role of VWF-related processes in ischemic brain injury under experimental conditions and in humans. Potential clinical merits of novel inhibitors of VWF-mediated platelet adhesion and activation as powerful and safe tools to combat thromboembolic disorders including ischemic stroke are discussed. Preclinical and clinical evidence illustrates an important role of VWF in ischemic stroke, suggesting that VWF could become a promising target in stroke therapy.

ADAMTS13 Cleavage of Von Willebrand Factor (VWF) in Commercial Concentrates and Effect on VWF Function

Abstract 4354 Objective:The characteristic multimeric pattern of plasmatic VWF results from asymmetric cleavage by ADAMTS13. Regulation of VWF multimer distribution is critical for its physiological function. In human plasma, VWF multimer gels reveal species of various multimeric sizes with flanking satellite bands (triplets). The faster and slower migrating bands encompassing a VWF multimer lack one N-terminal fragment or possess an additional N-terminal fragment, respectively. Defects in VWF secretion, impaired assembly of multimers, or increased proteolysis can cause von Willebrand Disease (VWD). Distribution of VWF triplet bands is significantly altered in some plasma-derived VWF concentrates. The impact of triplet structure on VWF function has not been investigated so far. Methods:Four commercially available VWF concentrates were analyzed for ADAMTS13 content as well as VWF multimer- and triplet structure using agarose gel electrophoresis. ADAMTS13 activity was quantified by fluorescence resonance energy transfer (FRET) assay. Samples composed of different VWF triplet distribution but comparable multimers were obtained by heparin affinity chromatography. Platelet adhesion under flow was determined using a flow-chamber model. Results:VWF concentrates markedly differed with respect to their content of ADAMTS13 antigen and activity. A higher content of ADAMTS13 correlated with an altered triplet structure reflected by an increased presence of the faster migrating triplet band, indicating VWF proteolysis. VWF-mediated platelet adhesion under flow over time was increased using a VWF fraction predominantly containing the slower migrating triplet band. Conclusion:These findings suggest that an intact triplet structure has an impact on platelet adhesion at physiological high arterial shear rate conditions. The relevance of VWF N-terminal domains for platelet binding and potential clinical consequences of enhanced proteolysis in commercial concentrates has to be further evaluated. Disclosures:Fuchs:octapharma: Employment. Solecka:Octapharma: Employment. Kröning:Octapharma: Employment. Kannicht:Octapharma: Employment. Schwartz:Octapharma: Employment.

VWF-Mediated Platelet Adhesion is Required for Deep Vein Thrombosis in a Flow Restriction Model.

Abstract 473Deep vein thrombosis (DVT) and its life-threatening complication, pulmonary embolism, are wide-spread in the Western world. Disturbance of blood flow without substantial endothelial denudation is a leading pathogenic factor for non-cancer related DVT. Von Willebrand Factor (VWF), a large multimeric protein, facilitates hemostasis via two separate pathways by stabilizing coagulation Factor VIII (FVIII) and by recruiting platelets to injured vessel wall or thrombi through the interaction with GPIb-alpha. Whereas the role of FVIII in DVT has been suggested by clinical studies (Koster T et al., Lancet, 345(8943):152-5,1995), whether VWF-platelet interaction is implicated in venous thrombosis remains unclear. We utilized murine models of partial and complete flow restriction in the inferior vena cava (IVC) in mice to mimic clinical conditions in which thrombus develops in deep veins. In 8-10 week old C57BL/6 male mice anesthetized by isoflurane-oxygen mixture, IVC and two side branches were ligated by a polypropylene suture immediately below the renal veins to obtain complete blood stasis. For partial flow restriction (stenosis), IVC ligation was performed over a 30G needle and then the needle was removed. Mice were euthanized after 48 h and thrombi from the IVC were taken for analysis. Results were evaluated using the chi-square test. The VWF-/- mice were completely protected from thrombosis in the stenosis model: none of the 14 VWF-/- mice developed a thrombus compared to 6/6 wild-type (WT) mice (p<0.001). In the stasis model, a similar albeit less pronounced phenotype was observed (33% of VWF-/- mice with thrombus, n=9, versus 82% in WT mice, n=11; p<0.03). Stenosis-induced thrombi in WT mice contained abundant amounts of VWF, as was shown by immunostaining. To delineate the involvement of VWF-platelet interactions, we infused WT mice with GPG-290, a recombinant GPIb-alpha N-terminal domain conjugated with human IgG1 Fc fragment. This compound has been shown to inhibit VWF-GPIb-alpha interaction (Hennan JK et al., Thromb Haemost, 95(3):469-75, 2006), but does not interfere with FVIII binding and turnover. Infusion of GPG-290 markedly reduced thrombus development in the stenosis model (3/9 GPG-290-treated WT mice developed DVT versus 9/9 vehicle-treated control mice; p=0.003). Notably, in the absence of blood flow (stasis model), GPG-290 was less effective (83% thrombosis development in vehicle-treated WT, n=6, versus 55.6% thrombosis in GPG-290-treated WT group, n=9; p=0.26). We next addressed the events preceding thrombus formation in the DVT stenosis model using intravital microscopy on living mice. We observed accumulation of fluorescently labeled platelets and leukocytes in the IVC in the area below the suture 6 h after stenosis induction. The amount of both adhering platelets and leukocytes was substantially reduced in VWF-/- mice compared to WT (approx. 20-fold, p<0.005 and 11-fold, p<0.001, respectively). In conclusion, VWF mediates platelet and leukocyte recruitment to the vessel wall. This initiates thrombus development in the absence of major endothelial injury. Interference with the VWF-GPIb-alpha axis may be a potential target for prophylaxis of deep vein thrombosis. Disclosures:No relevant conflicts of interest to declare.