Interaction Of Von Willebrand Factor Research Articles

Shear Stress Accumulation Enhances von Willebrand Factor-Induced Platelet P-Selectin Translocation in a PI3K/Akt Pathway-Dependent Manner.

Platelet adhesion and activation through the interaction of von Willebrand factor (VWF) with platelet glycoprotein (GP) Ibα are the early key events in hemostasis and thrombosis especially under high blood shear stress. P-selectin translocation from α granule to the cell surface is a typical platelet function phenotype, which makes the platelet-induced inflammatory response of flowing leukocytes possible and can be induced by either chemical agonists (thrombin, ADP, etc.) or high blood shear stress, but regulations of VWF mutation and blood shear stress on VWF-induced P-selectin translocation remain unclear. With flow cytometry, parallel plate flow chamber, and immunofluorescence staining techniques, we examined the P-selectin translocation of platelets on immobilized wild-type (WT) VWF-A1 domain and its two mutants, the gain-of-function (GOF) mutant R1308L and the loss-of-function (LOF) mutant G1324S, respectively. The results showed that the VWF-A1-induced platelet P-selectin translocation was triggered, accelerated, and enhanced by fluid shear stress and could be correlated with shear stress accumulation (SSA, the product of fluid shear stress and mechanical stimulus time), and the PI3K/Akt axis was involved in the platelet P-selectin translocation. The force-triggered P-selectin translocation occurred quickly on partial platelet surface first and then extended gradually to the whole platelet surface as SSA increased. The P-selectin translocation process would be promoted by the GOF mutation (R1308L) but slowed down by the LOF mutation (G1324S). These findings demonstrated a force-enhanced regulation mechanism for the VWF-induced platelet P-selectin translocation through the PI3K/Akt pathway and provided a novel insight into the mechano-chemical regulation mechanism for the key events, such as platelet activation and functional phenotype change in hemostasis and thrombosis.

Molecular determinants of the factor VIII/von Willebrand factor complex revealed by BIVV001 cryo-electron microscopy

AbstractInteraction of factor VIII (FVIII) with von Willebrand factor (VWF) is mediated by the VWF D′D3 domains and thrombin-mediated release is essential for hemostasis after vascular injury. VWF-D′D3 mutations resulting in loss of FVIII binding are the underlying cause of von Willebrand disease (VWD) type 2N. Furthermore, the FVIII–VWF interaction has significant implications for the development of therapeutics for bleeding disorders, particularly hemophilia A, in which endogenous VWF clearance imposes a half-life ceiling on replacement FVIII therapy. To understand the structural basis of FVIII engagement by VWF, we solved the structure of BIVV001 by cryo-electron microscopy to 2.9 Å resolution. BIVV001 is a bioengineered clinical-stage FVIII molecule for the treatment of hemophilia A. In BIVV001, VWF-D′D3 is covalently linked to an Fc domain of a B domain–deleted recombinant FVIII (rFVIII) Fc fusion protein, resulting in a stabilized rFVIII/VWF-D′D3 complex. Our rFVIII/VWF structure resolves BIVV001 architecture and provides a detailed spatial understanding of previous biochemical and clinical observations related to FVIII–VWF engagement. Notably, the FVIII acidic a3 peptide region (FVIII-a3), established as a critical determinant of FVIII/VWF complex formation, inserts into a basic groove formed at the VWF–D′/rFVIII interface. Our structure shows direct interaction of sulfated Y1680 in FVIII-a3 and VWF-R816 that, when mutated, leads to severe hemophilia A or VWD type 2N, respectively. These results provide insight on this key coagulation complex, explain the structural basis of many hemophilia A and VWD type 2N mutations, and inform studies to further elucidate how VWF dissociates rapidly from FVIII upon activation.

In silico features of ADAMTS13 contributing to plasmatic ADAMTS13 levels in neonates with congenital heart disease

IntroductionRisk factors contributing to heightened thrombosis in pediatric congenital heart disease (CHD) patients are not fully understood. Among the neonatal CHD population, those presenting with single ventricular physiology are at the highest risk for perioperative thrombosis. The von Willebrand factor and ADAMTS13 interactions have emerged as causative risk factors for pediatric stroke and could contribute to heightened thrombosis in CHD neonates. MethodsThis study investigates a cohort of children with single ventricle physiology and undergoing cardiac surgery, during which some patients developed thrombosis. In this cohort, we analyzed the relationship of several molecular features of ADAMTS13 with the plasma and activity levels in patients at risk of thrombosis. Additionally, in light of the natural antithrombotic activity of ADAMTS13, we have sequenced the ADAMTS13 gene for each patient and evaluated the role of genetic variants in determining the plasma ADAMTS13 levels using a series of in silico tools including Hidden Markov Models, EVmutation, and Rosetta. ResultsLower ADAMTS13 levels were found in patients that developed thrombosis. A novel in silico analysis to assess haplotype effect of co-occurring variants identified alterations in relative surface area and solvation energy as important contributors. Our analysis suggested that beneficial or deleterious effect of a variant can be reasonably predicted by comprehensive analysis of in silico assessment and in vitro and/or in vivo data. ConclusionFindings from this study add to our understanding the role of genetic features of ADAMTS13 in patients at high risk of thrombosis related to an imbalanced relation between VWF and ADAMTS13.

Unique surface‐exposed hydrophobic residues in the C1 domain of factor VIII contribute to cofactor function and von Willebrand factor binding

BackgroundThe identity of the amino acid regions of factor VIII (FVIII) that contribute to factor IXa (FIXa) and von Willebrand factor (VWF) binding has not been fully resolved. Previously, we observed that replacing the FVIII C1 domain for the one of factor V (FV) markedly reduces VWF binding and cofactor function. Compared to the FV C1 domain, this implies that the FVIII C1 domain comprises unique surface‐exposed elements involved in VWF and FIXa interaction. ObjectiveThe aim of this study is to identify residues in the FVIII C1 domain that contribute to VWF and FIXa binding. MethodsStructures and primary sequences of FVIII and FV were compared to identify surface‐exposed residues unique to the FVIII C1 domain. The identified residues were replaced with alanine residues to identify their role in FIXa and VWF interaction. This role was assessed employing surface plasmon resonance analysis studies and enzyme kinetic assays. ResultsFive surface‐exposed hydrophobic residues unique to the FVIII C1 domain, ie, F2035, F2068, F2127, V2130, I2139 were identified. Functional analysis indicated that residues F2068, V2130, and especially F2127 contribute to VWF and/or FIXa interaction. Substitution into alanine of the also surface‐exposed V2125, which is spatially next to F2127, affected only VWF binding. ConclusionThe surface‐exposed hydrophobic residues in C1 domain contribute to cofactor function and VWF binding. These findings provide novel information on the fundamental role of the C1 domain in FVIII life cycle.

Antithrombotic effect of SP-8008, a benzoic acid derivative, through the selective inhibition of shear stress-induced platelet aggregation.

Background and PurposeBleeding is one of the most critical adverse effects of antithrombotic drugs, and many efforts have been made to discover novel antiplatelet agents without bleeding complications. Shear stress‐induced platelet aggregation (SIPA), where the interaction of von Willebrand factor (vWF) and platelet glycoprotein (GP) Ib constitutes the initial step, is a promising target to overcome bleeding problems, as SIPA occurs only in pathological conditions. Here, we describe SP‐8008, a novel modulator of vWF–GP Ib interactions and evaluated its antiplatelet/antithrombotic effects.Experimental ApproachNewly synthesized compounds were screened for antiplatelet effects in vitro, using human platelets exposed to high shear stress. Aggregation, intracellular calcium level, granule secretion, and integrin activation were assessed. Molecular modelling using virtual docking and flow cytometry were used to evaluate effects on vWF–GP Ib interactions. Antithrombotic effects in vivo were determined in rats, using arterial thrombosis and shear stress‐specific thrombosis. Transection tail bleeding time was used to evaluate adverse effects.Key ResultsSP‐8008 was a potent inhibitor of SIPA, with IC50 of 1.44 ± 0.09 μM. SP‐8008 effectively and broadly blocked shear stress‐induced platelet activation events, without any significant toxicity. Importantly, SP‐8008 was highly selective against SIPA, effectively interfering with vWF–GP Ib engagement. Most importantly, SP‐8008 exerted significant antithrombotic effects in vivo in both shear stress‐specific and arterial thrombosis, without prolonging bleeding time.Conclusions and ImplicationsOur results demonstrated that SP‐8008 can be a novel selective antiplatelet agent with improved safety profile.

Platelets as Modulators of Cerebral Ischemia/Reperfusion Injury.

Ischemic stroke is among the leading causes of disability and death worldwide. In acute ischemic stroke, the rapid recanalization of occluded cranial vessels is the primary therapeutic aim. However, experimental data (obtained using mostly the transient middle cerebral artery occlusion model) indicates that progressive stroke can still develop despite successful recanalization, a process termed “reperfusion injury.” Mounting experimental evidence suggests that platelets and T cells contribute to cerebral ischemia/reperfusion injury, and ischemic stroke is increasingly considered a thrombo-inflammatory disease. The interaction of von Willebrand factor and its receptor on the platelet surface, glycoprotein Ib, as well as many activatory platelet receptors and platelet degranulation contribute to secondary infarct growth in this setting. In contrast, interference with GPIIb/IIIa-dependent platelet aggregation and thrombus formation does not improve the outcome of acute brain ischemia but dramatically increases the susceptibility to intracranial hemorrhage. Here, we summarize the current understanding of the mechanisms and the potential translational impact of platelet contributions to cerebral ischemia/reperfusion injury.

Von Willebrand Factor Mediates Pneumococcal Aggregation and Adhesion in Blood Flow.

Streptococcus pneumoniae is a major cause of community acquired pneumonia and septicaemia in humans. These diseases are frequently associated with thromboembolic cardiovascular complications. Pneumococci induce the exocytosis of endothelial Weibel-Palade Bodies and thereby actively stimulate the release of von Willebrand factor (VWF), which is an essential glycoprotein of the vascular hemostasis. Both, the pneumococcus induced pulmonary inflammation and the thromboembolytic complications are characterized by a dysbalanced hemostasis including a marked increase in VWF plasma concentrations. Here, we describe for the first time VWF as a novel interaction partner of capsulated and non-encapsulated pneumococci. Moreover, cell culture infection analyses with primary endothelial cells characterized VWF as bridging molecule that mediates bacterial adherence to endothelial cells in a heparin-sensitive manner. Due to the mechanoresponsive changes of the VWF protein conformation and multimerization status, which occur in the blood stream, we used a microfluidic pump system to generate shear flow-induced multimeric VWF strings on endothelial cell surfaces and analyzed attachment of RFP-expressing pneumococci in flow. By applying immunofluorescence visualization and additional electron microscopy, we detected a frequent and enduring bacterial attachment to the VWF strings. Bacterial attachment to the endothelium was confirmed in vivo using a zebrafish infection model, which is described in many reports and acknowledged as suitable model to study hemostasis mechanisms and protein interactions of coagulation factors. Notably, we visualized the recruitment of zebrafish-derived VWF to the surface of pneumococci circulating in the blood stream and detected a VWF-dependent formation of bacterial aggregates within the vasculature of infected zebrafish larvae. Furthermore, we identified the surface-exposed bacterial enolase as pneumococcal VWF binding protein, which interacts with the VWF domain A1 and determined the binding kinetics by surface plasmon resonance. Subsequent epitope mapping using an enolase peptide array indicates that the peptide 181YGAEIFHALKKILKS195 might serve as a possible core sequence of the VWF interaction site. In conclusion, we describe a VWF-mediated mechanism for pneumococcal anchoring within the bloodstream via surface-displayed enolase, which promotes intravascular bacterial aggregation.

Analytical characterization and reference interval of an enzyme-linked immunosorbent assay for active von Willebrand factor.

BackgroundInteraction of von Willebrand factor (VWF) with platelets requires a conformational change that exposes an epitope within the VWF A1 domain, enabling platelet glycoprotein Ibα binding. Quantification of this ‘‘active” conformation of VWF has been shown to provide pathophysiological insight into conditions characterized by excessive VWF-platelet interaction.MethodsWe developed an immunosorbent assay based on a variable heavy chain antibody fragment against the VWF A1 domain as a capture antibody. Assay performance in terms of specificity (binding to active R1306W- and sheared VWF), precision, accuracy, linearity, limits of detection and stability were determined. Active VWF, VWF antigen, VWF ristocetin cofactor activity, VWF:GP1bM and VWF propeptide were measured in citrated plasma and platelet-VWF binding in whole blood from 120 healthy individuals to establish a reference interval for active VWF and to assess associations with other VWF parameters.ResultsIntra- and inter-assay CVs were between 2.4–7.2% and 4.1–9.4%, depending on the level. Mean recovery of spiked recombinant R1306W VWF was 103±3%. The assay was linear in the range of 90.1–424.5% and had a limit of quantification of 101%. The reference interval for active VWF was 91.6–154.8% of NPP. Significant, positive correlations between active VWF and all other VWF parameters were found, with the strongest correlation with VWF:GP1bM binding.ConclusionsWe developed and validated an immunosorbent assay for the accurate detection of active VWF levels in plasma. The assay fulfilled all analytical criteria in this study and a reference interval was established, allowing its use to quantify active VWF in pathological conditions for future research.

Short-Acting Anti-VWF Aptamer Improves the Recovery and Survival, and Hemostatic Functions of Refrigerated Platelets

Background:Refrigerated platelets are rapidly cleared upon transfusion. The mechanism of this fast clearance is not fully understood. It was recently reported that refrigeration-induced binding of von Willebrand factor (VWF) to platelets contributed to the rapid clearance of platelet after transfusion. Adding a peptide that blocks the binding of VWF to platelet GPIbα during refrigeration can improve the post-transfusion recovery and survival of refrigerated murine platelets in vivo. However, since the interaction of VWF with GPIbα is required for primary hemostasis, adding a long-acting inhibitory peptide to stored platelets may impede the hemostatic function of these platelets after transfusion.DNA aptamer ARC1779 binds VWF and inhibits its binding to GPIbα. It has previously been developed as an anti-thrombotic agent. Its half-life is relatively fast, at a reported 2 hours in human.Objective:We aimed to test whether ARC1779 can improve the post-transfusion recovery and survival of refrigerated platelets, and, due to its fast clearance rate, without significantly impeding the hemostatic functions of these platelets in vivo.Methods:Human or murine platelets were stored at 4°C with or without ARC1779 for 48 hours. VWF binding was measured by flow cytometry. After refrigeration, human platelets were retro-orbitally transfused into NOD-SCID mice, while murine platelets into wild-type mice. Post-transfusion, periodic blood draw was performed, and the percentage of infused platelets in the total platelet population was measured by flow cytometry to calculate the recovery and survival over time. To monitor the hemostatic function, refrigerated murine platelets were transfused into transgenic IL4R-IbαTg mice, and a tail bleeding time assay was performed at four hours after transfusion. IL4R-IbαTg platelets lack the extracellular domain of GPIbα and cannot bind VWF and other GPIbα ligands. As a result, hemostasis in IL4R-IbαTg mice is severely impaired.Results and conclusion:Treatment of ARC1779 during 48-hour refrigeration efficiently inhibited VWF binding to platelets. ARC1779-treated refrigerated murine platelets exhibit increased post-transfusion recovery and survival rates compared to untreated refrigerated ones. (Recovery of ARC1779-treated platelets: 76.7±5.5%; Untreated: 63.7±0.8%, N=6, p<0.01. Half-life time of ARC1779-treated platelets: 31.4±2.36 hr; Untreated: 28.1±0.86 hr, N=6, p<0.05). A similar increase was also observed for refrigerated human platelets (Recovery: 49.4±4.4% vs. 36.8±2.1%, N=6, p<0.01; Half-life time: 9.2±1.5 hr vs. 8.7±0.9 hr, N=6, n.s.). Additionally, the tail bleeding time in ARC1779-treated group was significantly shorter than no ARC1779-treated group (160±65 vs. 373±96 seconds, N=7, p<0.01). For comparison, un-transfused IL4R-IbαTg mice have a tail bleeding time longer than 1200 s, and those transfused with freshly prepared platelets 155±80 s. Overall, these results demonstrate that inhibiting the GPIbα-VWF interaction by ARC1779 during cold storage of platelets improves platelet post-transfusion recovery. The treatment also helps to preserve the hemostatic function of refrigerated platelets. These results suggest that an inhibitor of the GPIbα-VWF interaction but with a limited half-life may be a potential therapeutic option to enable or improve the refrigeration of platelets for transfusion treatment. DisclosuresLi:Neoletix: Consultancy, Equity Ownership.

Who Are the Type 2 Von Willebrand Disease Patients? Results from Interim Analysis of the French HRQoL Study: Wish-Qol.

Type 2 von Willebrand disease (VWD) includes a wide range of qualitative abnormalities of von Willebrand factor (VWF) function and structure resulting in various bleeding phenotypes. Variants 2A, 2B and 2M are mainly related to abnormal interaction of VWF with platelets and/or the subendothelium. Mucocutaneous bleeds such as epistaxis, menorrhagia and gastro-intestinal bleedings are the most frequently reported in these subtypes, while in patients with type 2N VWD characterized by a defective binding of VWF to FVIII, a phenotype with symptoms suggestive of mild/moderate hemophilia A is common.Are type 2 VWD female patients' bleeding score, clinical phenotype and impairment of Quality of Life (QoL) more severe than the males' ones? To answer this question, we report here results of VWD type 2 patients enrolled in the French Heath related Quality of Life study: WiSH-QoL.This study is conducted with the French Reference Centre for von Willebrand Disease (CRMW). Clinical phenotype such as bleeding score (Tosetto score), biological profile and genotype are documented. Clinical characteristics and therapeutic approach are also recorded. Patients are treated with WILFACTIN®, triple-secured plasma-derived VWF with a low factor VIII content. Information on socio-demographic and resources consumption are gathered by patients in their diary. HRQoL is assessed with the generic SF-36 (for adults), the chronic-generic DISABKIDS Short Form (for children and adolescents), the VWD-specific HRQoL questionnaire VWD-QoL (for adults, children, adolescents and parents with a proxy version), the VWD-specific treatment satisfaction assessment VWD-Sat (for adults and parents with a proxy version). The Impact on Family (IOF) Scale is dedicated to caregivers of children with VWD. At least 350 patients will be followed during 2 years.During the recruitment period (Oct. 2014 - Nov. 2017), 357 patients were enrolled. At the time of this analysis, data at inclusion on 355 patients were evaluable. Focus was made on the 226 type 2 VWD patients: 141 (62.4%) females and 85 (37.6%) males, median age and range at study entry 32.7 (1-78) and 26.4 (1-83) years, respectively. The majority of them were adults with 101 (71.6%) females and 49 (57.6%) males. 76 patients were below 18 years (40 (28.4%) females and 36 (42.4%) males). In the female subgroup, 81 were women of child-bearing age (15-50 y.o), 27 under 15 years with 21 prepubertal children and 33 above 50 years.The median age at diagnosis was 11.5 years (range 0-78) associated with a family history of VWD in the majority of patients (178, 78.8%), earlier in males' life (6 y) than in females (15 y). The first substitutive treatment by VWF was administered at a median age of 19.0 years (range 0-78), with 13.0 years for males compared to 23.5 y for females.At baseline, 23 (10.2%) patients (12 females and 11 males) had reported GI manifestations.The patients were 64 patients with type 2A, 52 with type 2B, 68 with type 2M, 12 with type 2N and 30 with type 2 unspecified.The median Tosetto bleeding score (BS) reported for 202 patients was +7.0 (range -1 to 25), higher +8.0 (range -1 to 25) in female compared to +5.0 (range 0 to 21) in male patients. It was also able to distinguish disease severity by variants as shown in table1. Whatever the variants (2A, 2B, 2M and 2N VWD,) the women of child-bearing age group had the highest BS.At enrollment, 62 female patients (44.0%) have already received a concomitant treatment with Iron, oral contraceptive, intra-uterine device. In the group of 64 women who had experienced a total of 136 deliveries (mainly vaginal deliveries (107, 78.7%) and C. Section (29, 21.3%)), the mean number of childbirth was 2.1 (range 1-5). Post-Partum Hemorrhages occured in 42 of these cases (31.3%), mainly within 48 hours after giving birth in 27 cases (67.5%) out of the 40 cases with available data.Were also found differences in both Physical and Mental Component Score of the generic SF-36 with higher impairments in HRQoL in adults females than in males (PCS 54.04 vs 55.22) MCS (44.71 vs. 47.85). The same tendency was seen with the Normalized Global score VWD-Specific in adults with higher score in females than in males (17.26 vs 13.91).This study with HRQoL results will bring a deeper insight into type 2 VWD patients' real daily life. These findings may teach us what would be the best care for VWD patients and especially for females who need definitely specific health care throughout their fertile life. [Display omitted] DisclosuresBorel-Derlon:Octapharma: Other: Clinical Research Investigator; LFB: Membership on an entity's Board of Directors or advisory committees; Shire: Other: Principal investigator; Novonordisk: Membership on an entity's Board of Directors or advisory committees; Sobi: Membership on an entity's Board of Directors or advisory committees. Desprez:LFB: Other: Investigator. Volot:LFB: Other: Investigator. Pan-Petesch:LFB: Other: Investigator. Chatelanaz:LFB: Employment. Doriat-Robin:LFB: Employment. Veyradier:LFB: Other: Investigator. Von Mackensen:LFB: Membership on an entity's Board of Directors or advisory committees.

Platelet and von Willebrand factor interactions at the vessel wall.

The process of platelet thrombus formation contributes to the haemostatic response that prevents excessive blood loss after tissue injury, but may become a life-threatening disease mechanism by causing the acute thrombotic occlusion of atherosclerotic arteries. The participation of platelets in the formation of thrombi is centered on their adhesive properties and the ability to respond to stimuli with rapid activation. Platelet adhesion and activation are multifaceted and modulated by different environmental conditions, suggesting that it should be possible to obtain a selective pharmacological inhibition of the pathways more relevant to athero-thrombosis than to haemostasis. In particular, progress in understanding the structure and function of von Willebrand factor and the mechanisms that underlie its interactions with vascular surfaces and platelets can elucidate important differentiating aspects of normal haemostasis and pathological arterial thrombosis.

Platelet activation and erythrocyte lysis during brief exposure of blood to pathophysiological shear stress in vitro.

Interaction of von Willebrand factor (VWF) with circulating platelets is the trigger for thrombosis in a region of arterial stenosis. These events are typically studied in vitro under conditions where platelets adhere to a VWF-coated surface. Our approach assesses platelet responses in the absence of adhesion. To characterize extent of platelet activation and erythrocyte lysis in an artificial stenosis model. Whole blood is perfused through a length of polyetheretherketone tubing that includes an artificial stenosis, comprising narrow-bore (89-381 μm) tubing. Secretion of [14C] serotonin and hemoglobin release was measured to evaluate platelet activation and hemolysis respectively at various perfusion rates and different stenosis dimensions. Platelet activation and erythrocyte lysis increased progressively with increasing perfusion rate and decreasing stenosis diameter; the length of the stenosis had negligible influence. Modest platelet activation (5-10% secretion of [14C] serotonin) occurred without significant erythrocyte lysis under a limited range of perfusion conditions (4-6 mL/min flow through a 127 μm stenosis). Our experimental approach mimics conditions in severe arterial stenosis or a mechanical heart valve. It could be a valuable aid in the development of novel drugs to treat arterial thrombosis and in the design of heart valves.

Stasis Promotes Erythrocyte Adhesion to von Willebrand Factor.

Venous thromboembolism is a major contributor to global disease burden. Leukocytes and platelets initiate thrombogenesis on blood stasis and initiate the formation of a fibrin, VWF (von Willebrand factor), and neutrophil extracellular trap scaffold for erythrocytes. However, there is little knowledge on how erythrocytes become stably incorporated into this scaffold. Recently, we described the adhesion of calcium-loaded erythrocytes to endothelial-derived VWF strings. Because VWF is part of the scaffold of venous thrombi, we questioned whether reduced flow or stasis promotes the adhesion of normal erythrocytes to VWF and whether venous thrombi show evidence of erythrocyte-VWF interactions. In the present work, we perfused, under controlled shear conditions, washed, normal erythrocytes over surface-immobilized plasma and extracellular matrix proteins and showed that normal erythrocytes specifically bind to VWF. The interaction between erythrocytes and VWF significantly increased when the wall shear stress was reduced. Next, we investigated whether erythrocyte-VWF interactions support the structure of venous thrombi. High-resolution immunofluorescence imaging of human venous thrombi showed a striking pattern between erythrocytes, VWF, and fibrin, which suggests that VWF plays a supporting role, linking erythrocytes to fibrin in the thrombus. Our data suggest that erythrocyte retention in venous thrombi is mediated by erythrocyte-VWF or erythrocyte-VWF-fibrin interactions. Targeting erythrocyte retention could be a new strategy in the treatment or prevention of venous thrombosis.

Plasmin Cleaves Von Willebrand Factor at K1491-R1492 in the A1-A2 Linker Region in a Shear- and Glycan-Dependent Manner In Vitro.

Previous studies have demonstrated a role for plasmin in regulating plasma von Willebrand factor (VWF) multimer composition. Moreover, emerging data have shown that plasmin-induced cleavage of VWF is of particular importance in specific pathological states. Interestingly, plasmin has been successfully used as an alternative to ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin type 1 motif) in a mouse model of thrombotic thrombocytopenic purpura. Consequently, elucidating the molecular mechanisms through which plasmin binds and cleaves VWF is not only of basic scientific interest but also of direct clinical importance. Our aim was to investigate factors that modulate the susceptibility of human VWF to proteolysis by plasmin. We have adapted the VWF vortex proteolysis assay to allow for time-dependent shear exposure studies. We show that globular VWF is resistant to plasmin cleavage under static conditions, but is readily cleaved by plasmin under shear. Although both plasmin and ADAMTS13 cleave VWF in a shear-dependent manner, plasmin does not cleave at the Tyr1605-Met1606 ADAMTS13 proteolytic site in the A2 domain. Rather under shear stress conditions, or in the presence of denaturants, such as urea or ristocetin, plasmin cleaves the K1491-R1492 peptide bond within the VWF A1-A2 linker region. Finally, we demonstrate that VWF susceptibility to plasmin proteolysis at K1491-R1492 is modulated by local N-linked glycan expression within A1A2A3, and specifically inhibited by heparin binding to the A1 domain. Improved understanding of the plasmin-VWF interaction offers exciting opportunities to develop novel adjunctive therapies for the treatment of refractory thrombotic thrombocytopenic purpura.

PLATELETS AND VON WILLEBRAND FACTOR INTERACTIONS AFTER TRANSCATHETER AORTIC VALVE REPLACEMENT (TAVR): ASSOCIATION WITH THROMBOCYTOPENIA AND THROMBOSIS

Introduction: Thrombocytopenia and thrombotic events are common after TAVR. Changes in shear forces during TAVR and the subsequent platelet activation may explain these phenomena Methods: Flow cytometry with dual staining (PE-anti-CD41 and FITC-anti-vWF) was used to assess vWF binding to platelets

Platelet receptors as therapeutic targets: Past, present and future.

Anti-platelet drugs reduce arterial thrombosis after plaque rupture and erosion, prevent stent thrombosis and are used to prevent and treat myocardial infarction and ischaemic stroke. Some of them may also be helpful in treating less frequent diseases such as thrombotic thrombocytopenic purpura. The present concise review aims to cover current and future developments of anti-platelet drugs interfering with the interaction of von Willebrand factor (VWF) with glycoprotein (GP) Ibα, and directed against GPVI, GPIIb/IIIa (integrin αIIbβ3), the thrombin receptor PAR-1, and the ADP receptor P2Y12. The high expectations of having novel antiplatelet drugs which selectively inhibit arterial thrombosis without interfering with normal haemostasis could possibly be met in the near future.

Ristocetin-Induced Platelet Aggregation (RIPA) and RIPA Mixing Studies.

Ristocetin-induced platelet aggregation (RIPA) is used as an in vitro test to determine the presence and integrity of the platelet glycoprotein (GP) Ibα-V-IX complex and von Willebrand factor (VWF) interaction and is usually performed using platelet-rich plasma (PRP). Impairment in the response of VWF/GPIbα-V-IX is measured with reference to several established concentrations of ristocetin and may indicate defects in VWF or in GPIbα-V-IX function. RIPA-based mixing studies comprise an additional approach to testing this interaction to help define whether defects identified by RIPA lie in VWF or in GPIbα-V-IX. For example, the correction of an abnormal RIPA trace after mixing PRP with normal plasma and rechallenging with ristocetin at 1.0mg/mL suggests VWF function/quantity defect. RIPA mixing studies at lower doses of ristocetin (0.5mg/mL) are recommended for discrimination of von Willebrand disease type 2B (VWD2B) from the rarer platelet-type (PT) VWD and for the phenotypic laboratory diagnosis of VWD2B. The demonstration of a plasma factor capable of inducing platelet aggregation at such low doses of ristocetin represents the hallmark for the phenotypic laboratory diagnosis of VWD2B. Moreover, since both VWD2B and PT-VWD may present with thrombocytopenia, RIPA-based mixing studies are also useful in thrombocytopenic patients in whom RIPA testing is difficult to assess.

Activated FVIII Released from FVIII/VWF Complex Facilitates Thrombus Development Under High Shear Flow Condition

Activated FVIII Released from FVIII/VWF Complex Facilitates Thrombus Development Under High Shear Flow Condition

Von Willebrand Factor Interaction with FVIII: Development of Long Acting FVIII Therapies

Coagulation factor VIII (FVIII) and von Willebrand factor (VWF) both play a centrol role in hemostasis, illustrated by the severe bleeding disorders associated with their functional absence. Despite their different functionalities in hemostasis and being products from two different genes, both proteins circulate in a tight, non-covalently linked complex. The physiological concequences of complex formation are many, including stabilization of FVIII heterodimeric structure, protection of FVIII from protelytic degradation, and modulation of FVIII immunogenicity.Another relevant issue relates to the chaperone function of VWF, allowing FVIII to survive in the circulation. FVIII levels are markedly reduced in patients with no detectable VWF protein or with a defect in VWF-FVIII complex formation, indicating that VWF prevents FVIII from premature clearance. Moreover, evidence points to FVIII actually being predominantly cleared as part of the VWF-FVIII complex rather than as a separate protein. First, it is possible to predict FVIII half-life fairly accurately by knowing antigen levels of VWF and its propeptide in combination with blood group. Second, when FVIII and VWF are co-injected in Vwf-deficient mice, FVIII is targeted to the same macrophages as is VWF.Since the end of the 1990s, our knowledge on the clearance mechanism of FVIII and VWF has started to emerge, and multiple clearance receptors for both proteins have now been identified. Interestingly, there exists a large overlap in receptor-repertoire between FVIII and VWF. These findings have taught us that it will be difficult to design single-mutant FVIII or VWF variants that have prolonged half-lives.How then to prolong the half-life of FVIII to improve treatment of hemophilia A? Several novel bioengineered FVIII variants have been developed, including PEGylation, Fc fusion and single-chain design, aiming to increase FVIII half-life. These approaches have so far achieved only moderate increases in half-life (1.5- to 2-fold compared to marketed FVIII products), significantly less than when similar modifications are being applied to factor IX. Indeed, it seems as if in designing these FVIII variants, the role of the significant other in the complex has been overlooked, since FVIII clearance is principally determined by VWF.Could we instead use VWF as a tool to prolong half-life of FVIII? This option is actually limited by the nature of the interaction between VWF and FVIII. Although of high affinity, the interaction is characterized by high association- and dissociation-rates. Infusing FVIII in combination with long-acting VWF variants will therefore result in a rapid redistribution of FVIII to endogenous VWF, as has elegantly been shown by the group of Ginsburg. To overcome this limitation, we have designed a FVIII variant (FVIII-KB013bv) in which we have replaced the B-domain by a single-domain, llama-derived antibody fragment (nanobody) that recognizes the D'D3-region of VWF. Consequently, the dissociation-rate of the VWF/FVIII complex is reduced 100-fold. Preliminary studies revealed that FVIII-KB013bv has a two-fold prolonged half-life compared to FVIII, likely due to improved VWF binding properties. Combination of the FVIII-nanobody fusion protein with long-acting VWF variants is anticipated to prolong its half-life well beyond the limit of the current long-acting FVIII variants. DisclosuresLenting:NovoNordisk: Consultancy, Research Funding.

Humanized Von Willebrand Factor-Glycoprotein Ibα Interaction in Mouse Models of Hemostasis and Thrombosis

Introduction: The interaction between von Willebrand Factor (VWF) and platelet glycoprotein (GP) Ibα is key for initiating the response to vascular injury that leads to hemostasis or, in pathological conditions, may be a cause of thrombosis. VWF binding to GPIbα occurs through the A1 domain (VWFA1) and its role in platelet adhesion and aggregation becomes progressively more important with increasing shear rates, i.e., in arterioles or pathologically stenosed arteries. Owing to the key role in platelet adhesion/aggregation under arterial flow conditions, VWFA1 has been considered an obvious target for antithrombotic intervention. However, efforts to develop this concept have been complicated by the lack of suitable animal models due to species-specificity in VWFA1-GPIb binding. To obviate the problem, we have generated new mouse strains with humanized VWF-GPIb interaction and characterized the resulting phenotypes in experimental ex vivo and in vivo models of hemostasis and thrombosis.Methods: In the human VWF gene, and in the mouse Vwf ortholog, exon 28 encodes domains A1 and A2, including the VWFA1 GPIb-binding site. We generated a knock-in mouse by targeted insertion of human VWF exon 28 (VWFh28) into the mouse Vwf exon28 locus such that mouse platelet GPIbα (M1) interacted with mouse VWF containing human A1 domain (HA); the strain was designated M1HA (Table). These mice were cross-bred with the previously described transgenic strain (mGPIbαnull;hIbαTg) in which human GPIbα is expressed on the platelet surface in the context of the mouse GPIb-IX-V complex (strain designation: H1MA). The resulting strain was thus designated H1HA, with humanized GPIb-VWF interaction. The unmodified wild type mouse strain used for reference was designated M1MA (Table). All mouse strains were in C57BL/6 genetic background. VWF plasma concentration was measured by ELISA and function ex vivo was evaluated by ristocetin-induced platelet aggregation. In vivo, we measured the tail bleeding time to gauge hemostatic efficiency as well as the propensity to support pathological thrombosis in the carotid artery injured by exposure to ferric chloride.Results: Plasma of VWFh28 mice expressing mouse or human platelet GPIbα had VWF levels (M1HA: 876.4 ± 209.5 mU/ml, n = 16; H1HA: 848.9 ± 121.0 mU/ml, n = 15) not significantly different from wild type mice (M1MA: 1022 ± 267.4 mU/ml, n = 23). Addition of 1.5 mg/ml ristocetin into platelet-rich plasma (PRP) from M1MA, M1HA and H1MA mice elicited no response; only in PRP of H1HA mice did ristocetin cause platelet aggregation that, as in human PRP, was inhibited by the anti-human VWFA1 monoclonal antibody, NMC-4. The tail bleeding time was abnormally prolonged in the M1HA strain expressing human VWFA1/mouse GPIbα, but normal in the H1HA strain expressing human VWFA1/human GPIbα (6.0 ± 3.8 min vs. 1.5 ± 0.9 min; n = 12); the latter was similar to the bleeding time in the M1MA strain (1.0 ± 0.1 min, n = 10). Thrombus formation (time to occlusion) following a carotid artery injury with 9% FeCl3·6H2O was greatly delayed in the M1HA strain (1760.0 ± 538.5 s, n = 6) but similar to WT M1MA in the H1HA strain (485.2 ± 63.7 s vs. 598.3 ± 84.0 s, n = 6, respectively).Conclusions: Knock-in of human VWF exon 28 into the mouse Vwf locus led to successful biosynthesis of chimeric mouse-human VWF protein in vivo under endogenous promoter control, maintaining normal physiologic expression in endothelial cells and megakaryocytes/platelets. Tail bleeding time and in vivo thrombosis assays confirmed the normal functional interaction of mouse VWF containing human A1 domain with hGPIbα. Moreover, the normal response to ristocetin with platelet aggregation, and inhibition of the latter by the specific NMC4 antibody, indicates that the "humanized" H1HA mouse strain could be a powerful tool to select and develop new interventions for the diagnosis and treatment of hemostatic and thrombotic disorders. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.