Diagnosis and treatment of von Willebrand disease in 2024 and beyond.

MANUSCRIPT BACKGROUND AND AIM: The diagnosis and clinical care of patients with von Willebrand disease (VWD) has continued to evolve since the characterization of the von Willebrand factor (VWF) gene in 1985. This condition is almost certainly the most common inherited bleeding disorder, and the major symptomatic burden of the disease is experienced by females during their reproductive years. Diagnosis relies on the identification of a personal and family history of excessive mucocutaneous bleeding, and laboratory features consistent with quantitative and/or qualitative abnormalities of VWF. This review focuses on three aspects of VWD management, with current updates and a look into the future. MANUSCRIPT THEMES: First, we will address the role of genetics in the diagnosis and possible therapies for VWD. With current technologies, VWD genetic diagnosis is usually confined to the confirmation of type 2 subtypes of the disease and type 3 VWD analysis for family planning. While type 3 VWD is a potential candidate for the application of gene therapy, no treatments are currently close to entering the clinic. Second, the peri-procedural management of patients with VWD remains an important element of care. The choice of product, its dose and schedule all require careful consideration depending upon the type and disruptive nature of the planned procedure. Lastly, in addition to gene therapy, several other novel therapeutic interventions are also being developed for bleeding and prophylaxis in VWD. These include a VWF aptamer interfering with VWF clearance and bioengineered forms of VWF.

Illustrated State-of-the-Art Capsules of the ISTH 2023 Congress.

This year's Congress of the International Society of Thrombosis and Haemostasis (ISTH) took place in person in Montréal, Canada, from June 24-28, 2023. The conference, held annually, highlighted cutting-edge advances in basic, translational, population and clinical sciences relevant to the Society. As for all ISTH congresses, we offered a special, congress-specific scientific theme; this year, the special theme was immunothrombosis. Certainly, over the last few years, COVID-19 infection and its related thrombotic and other complications have renewed interest in the concepts of thromboinflammation and immunothrombosis; namely, the relationship between inflammation, infection and clotting. Other main scientific themes of the Congress included Arterial Thromboembolism, Coagulation and Natural Anticoagulants, Diagnostics and Omics, Fibrinolysis and Proteolysis, Hemophilia and Rare Bleeding Disorders, Hemostatic System in Cancer, Inflammation and Immunity, Pediatrics, Platelet Disorders, von Willebrand Disease and Thrombotic Microangiopathies, Platelets and Megakaryocytes, Vascular Biology, Venous Thromboembolism and Women's Health. Among other sessions, the program included 28 State-of-the-Art (SOA) sessions with a total of 84 talks given by internationally recognized leaders in the field. SOA speakers were invited to prepare brief illustrated reviews of their talks that were peer reviewed and are included in this article. These illustrated capsules highlight the major scientific advances with potential to impact clinical practice. Readers are invited to take advantage of the excellent educational resource provided by these illustrated capsules. They are also encouraged to use the image in social media to draw attention to the high quality and impact of the science presented at the Congress.

Cerebral Microbleeds During Transcatheter Aortic Valve Replacement: A Prospective Magnetic Resonance Imaging Cohort.

BACKGROUND: Cerebral microbleeds (CMBs) have been observed in healthy elderly people undergoing systematic brain magnetic resonance imaging. The potential role of acute triggers on the appearance of CMBs remains unknown. We aimed to describe the incidence of new CMBs after transcatheter aortic valve replacement (TAVR) and to identify clinical and procedural factors associated with new CMBs including hemostatic measures and anticoagulation management. METHODS: We evaluated a prospective cohort of patients with symptomatic aortic stenosis referred for TAVR for CMBs (METHYSTROKE [Identification of Epigenetic Risk Factors for Ischemic Complication During the TAVR Procedure in the Elderly]). Standardized neurologic assessment, brain magnetic resonance imaging, and analysis of hemostatic measures including von Willebrand factor were performed before and after TAVR. Numbers and location of microbleeds on preprocedural magnetic resonance imaging and of new microbleeds on postprocedural magnetic resonance imaging were reported by 2 independent neuroradiologists blinded to clinical data. Measures associated with new microbleeds and postprocedural outcome including neurologic functional outcome at 6 months were also examined. RESULTS: A total of 84 patients (47% men, 80.9±5.7 years of age) were included. On preprocedural magnetic resonance imaging, 22 patients (26% [95% CI, 17%-37%]) had at least 1 microbleed. After TAVR, new microbleeds were observed in 19 (23% [95% CI, 14%-33%]) patients. The occurrence of new microbleeds was independent of the presence of microbleeds at baseline and of diffusion-weighted imaging hypersignals. In univariable analysis, a previous history of bleeding (P=0.01), a higher total dose of heparin (P=0.02), a prolonged procedure (P=0.03), absence of protamine reversion (P=0.04), higher final activated partial thromboplastin time (P=0.05), lower final von Willebrand factor high-molecular-weight:multimer ratio (P=0.007), and lower final closure time with adenosine-diphosphate (P=0.02) were associated with the occurrence of new postprocedural microbleeds. In multivariable analysis, a prolonged procedure (odds ratio, 1.22 [95% CI, 1.03-1.73] for every 5 minutes of fluoroscopy time; P=0.02) and postprocedural acquired von Willebrand factor defect (odds ratio, 1.42 [95% CI, 1.08-1.89] for every lower 0.1 unit of high-molecular-weight:multimer ratio; P=0.004) were independently associated with the occurrence of new postprocedural microbleeds. New CMBs were not associated with changes in neurologic functional outcome or quality of life at 6 months. CONCLUSIONS: One out of 4 patients undergoing TAVR has CMBs before the procedure and 1 out of 4 patients develops new CMBs. Procedural or antithrombotic management and persistence of acquired von Willebrand factor defect were associated with the occurrence of new CMBs. REGISTRATION: URL: https://www. CLINICALTRIALS: gov; Unique identifier: NCT02972008.

A thrombopoietin receptor agonist to rescue an unusual platelet transfusion-induced reaction in a p.V1316M-associated von Willebrand disease type 2B patient.

This report describes the first case of splenic injury in a patient with p.V1316M-associated von Willebrand disease type 2B (VWD2B) with chronic thrombocytopenia, successfully treated with nonoperative management including von Willebrand factor (VWF) replacement therapy, and platelet transfusions relayed by a thrombopoietin receptor agonist (TPO-RA, Eltrombopag). Eltrombopag was initially introduced to rescue an unusual post-platelet-transfusion reaction exacerbating the thrombocytopenia. In-depth analysis of the dramatic platelet count drop and VWF measurements timeline ruled out an allo-immune reaction and supported an alternative hypothesis of a sudden platelet clearance as a consequence of stress-induced release of abnormal VWF. One year later, a second life-threatening bleeding episode required urgent surgery successfully managed with VWF replacement therapy and platelet transfusions. Eltrombopag was further introduced in the post-surgery period to allow bleeding-free and platelet-transfusion-free successful recovery. Treatment decisions are particularly challenging in patients with VWD2B, and this case highlights how such decisions can benefit from understanding the molecular origin of platelet count fluctuations observed in these patients. Here, we successfully used a new therapeutic approach combining VWF-replacement therapy and initial platelet-transfusion relayed by TPO-RA to optimize patient management. PLAIN LANGUAGE SUMMARY: A combination of von Willebrand factor replacement and thrombopoietin receptor agonist in thrombocytopenic patients with von Willebrand disease type 2B: a new therapy approach to optimize patient management?Therapeutic management of patients with von Willebrand disease type 2B are particularly challenging in case of severe thrombocytopenia.Treatment includes von Willebrands factor replacement therapy and iterative platelet transfusions.We describe the first case of splenic injury in a patient with p.V1316M-associated von Willebrand disease type 2B successfully treated with nonoperative management including von Willebrand factor replacement therapy and platelet transfusions relayed by a thrombopoietin receptor agonist.We showed that the unusual post-platelet-transfusion reaction associated with a dramatic platelet count drop was a consequence of stress-induced release of abnormal von Willebrand factor.The combination of von Willebrand factor replacement therapy and thrombopoietin receptor agonist may offer a new therapeutic approach to optimize patient management.

A compact integrated microfluidic oxygenator with high gas exchange efficiency and compatibility for long-lasting endothelialization.

We have developed and tested a novel microfluidic device for blood oxygenation, which exhibits a large surface area of gas exchange and can support long-term sustainable endothelialization of blood microcapillaries, enhancing its hemocompatibility for clinical applications. The architecture of the parallel stacking of the trilayers is based on a central injection for blood and a lateral injection/output for gas which allows significant reduction in shear stress, promoting sustainable endothelialization since cells can be maintained viable for up to 2 weeks after initial seeding in the blood microchannel network. The circular design of curved blood capillaries allows covering a maximal surface area at 4 inch wafer scale, producing high oxygen uptake and carbon dioxide release in each single unit. Since the conventional bonding process based on oxygen plasma cannot be used for surface areas larger than several cm2, a new "wet bonding" process based on soft microprinting has been developed and patented. Using this new protocol, each 4 inch trilayer unit can be sealed without a collapsed membrane even at reduced 15 µm thickness and can support a high blood flow rate. The height of the blood channels has been optimized to reduce pressure drop and enhance gas exchange at a high volumetric blood flow rate up to 15 ml min-1. The simplicity of connecting different units in the stacked architecture is demonstrated for 3- or 5-unit stacked devices that exhibit remarkable performance with low primary volume, high oxygen uptake and carbon dioxide release and high flow rate of up to 80 ml min-1.

A Thrombin-Activatable Factor X Variant Corrects Hemostasis in a Mouse Model for Hemophilia A.

Engineered recombinant factor X (FX) variants represent a promising strategy to bypass the tenase complex and restore hemostasis in hemophilia patients. Previously, a thrombin-activatable FX variant with fibrinopeptide-A replacing the activation peptide (FX-delAP/FpA) has been described in this regard. Here we show that FX-delAP/FpA is characterized by a sixfold shorter circulatory half-life compared with wild-type FX, limiting its therapeutical applicability. We therefore designed a variant in which the FpA sequence is inserted C-terminal to the FX activation peptide (FX/FpA). FX/FpA displayed a similar survival to wt-FX in clearance experiments and could be converted into FX by thrombin and other activating agents. In in vitro assays, FX/FpA efficiently restored thrombin generation in hemophilia A and hemophilia B plasmas, even in the presence of inhibitory antibodies. Expression following hydrodynamic gene transfer of FX/FpA restored thrombus formation in FVIII-deficient mice in a laser-induced injury model as well as hemostasis in a tail-clip bleeding model. Hemostasis after tail transection in FVIII-deficient mice was also corrected at 5 and 90 minutes after injection of purified FX/FpA. Our data indicate that FX/FpA represents a potential tenase-bypassing agent for the treatment of hemophilia patients with or without inhibitors.

Weibel-Palade Bodies Orchestrate Pericytes During Angiogenesis.

Objective Weibel-Palade bodies (WPBs) are endothelial cell (EC)-specific organelles formed by vWF (von Willebrand factor) polymerization and that contain the proangiogenic factor Ang-2 (angiopoietin-2). WPB exocytosis has been shown to be implicated for vascular repair and inflammatory responses. Here, we investigate the role of WPBs during angiogenesis and vessel stabilization. Approach and Results WPB density in ECs decreased at the angiogenic front of retinal vascular network during development and neovascularization compared with stable vessels. In vitro, VEGF (vascular endothelial growth factor) induced a VEGFR-2 (vascular endothelial growth factor receptor-2)-dependent exocytosis of WPBs that contain Ang-2 and consequently the secretion of vWF and Ang-2. Blocking VEGF-dependant WPB exocytosis and Ang-2 secretion promoted pericyte migration toward ECs. Pericyte migration was inhibited by adding recombinant Ang-2 or by silencing Ang-1 (angiopoietin-1) or Tie2 (angiopoietin-1 receptor) in pericytes. Consistently, in vivo anti-VEGF treatment induced accumulation of WPBs in retinal vessels because of the inhibition of WPB exocytosis and promoted the increase of pericyte coverage of retinal vessels during angiogenesis. In tumor angiogenesis, depletion of WPBs in vWF knockout tumor-bearing mice promoted an increase of tumor angiogenesis and a decrease of pericyte coverage of tumor vessels. By another approach, normalized tumor vessels had higher WPB density. Conclusions We demonstrate that WPB exocytosis and Ang-2 secretion are regulated during angiogenesis to limit pericyte coverage of remodeling vessels by disrupting Ang-1/Tie2 autocrine signaling in pericytes.

Protein kinase C signaling dysfunction in von Willebrand disease (p.V1316M) type 2B platelets.

von Willebrand disease (VWD) type 2B is characterized by gain-of-function mutations in von Willebrand factor (VWF), enhancing its binding affinity for the platelet receptor glycoprotein (GP)Ibα. VWD type 2B patients display a bleeding tendency associated with loss of high-molecular-weight VWF multimers and variable thrombocytopenia. We recently demonstrated that a marked defect in agonist-induced activation of the small GTPase, Rap1, and integrin αIIbß3 in VWD (p.V1316M) type 2B platelets also contributes to the bleeding tendency. Here, we investigated the molecular mechanisms underlying impaired platelet Rap1 signaling in this disease. Two distinct pathways contribute to Rap1 activation in platelets: rapid activation mediated by the calcium-sensing guanine nucleotide exchange factor CalDAG-GEF-I (CDGI) and sustained activation that is dependent on signaling by protein kinase C (PKC) and the adenosine 5'-diphosphate receptor P2Y12. To investigate which Rap1 signaling pathway is affected, we expressed VWF/p.V1316M by hydrodynamic gene transfer in wild-type and Caldaggef1-/- mice. Using αIIbß3 integrin activation as a read-out, we demonstrate that platelet dysfunction in VWD (p.V1316M) type 2B affects PKC-mediated, but not CDGI-mediated, activation of Rap1. Consistently, we observed decreased PKC substrate phosphorylation and impaired granule release in stimulated VWD type 2B platelets. Interestingly, the defect in PKC signaling was caused by a significant increase in baseline PKC substrate phosphorylation in circulating VWD (p.V1316M) type 2B platelets, suggesting that the VWF-GPIbα interaction leads to preactivation and exhaustion of the PKC pathway. Consistent with PKC preactivation, VWD (p.V1316M) type 2B mice also exhibited marked shedding of platelet GPIbα. In summary, our studies identify altered PKC signaling as the underlying cause of platelet hypofunction in p.V1316M-associated VWD type 2B.

Macrophage scavenger receptor SR-AI contributes to the clearance of von Willebrand factor.

Previously, we found that LDL-receptor related protein-1 on macrophages mediated shear stress-dependent clearance of von Willebrand factor. In control experiments, however, we observed that von Willebrand factor also binds to macrophages independently of this receptor under static conditions, suggesting the existence of additional clearance-receptors. In search for such receptors, we focused on the macrophage-specific scavenger-receptor SR-AI. von Willebrand factor displays efficient binding to SR-AI (half-maximum binding 14±5 nM). Binding is calcium-dependent and is inhibited by 72±4% in the combined presence of antibodies against the A1- and D4-domains. Association with SR-AI was confirmed in cell-binding experiments. In addition, binding to bone marrow-derived murine SR-AI-deficient macrophages was strongly reduced compared to binding to wild-type murine macrophages. Following expression via hydrodynamic gene transfer, we determined ratios for von Willebrand factor-propeptide over von Willebrand factor-antigen, a marker of von Willebrand factor clearance. Propeptide/antigen ratios were significantly reduced in SR-AI-deficient mice compared to wild-type mice (0.6±0.2 versus 1.3±0.3; P<0.0001), compatible with a slower clearance of von Willebrand factor in SR-AI-deficient mice. Interestingly, mutants associated with increased clearance (von Willebrand factor/p.R1205H and von Willebrand factor/p.S2179F) had significantly increased binding to purified SR-AI and SR-AI expressed on macrophages. Accordingly, propeptide/antigen ratios for these mutants were reduced in SR-AI-deficient mice. In conclusion, we have identified SR-AI as a novel macrophage-specific receptor for von Willebrand factor. Enhanced binding of von Willebrand factor mutants to SR-AI may contribute to the increased clearance of these mutants.

Acquired platelet disorders.

In contrast to congenital platelet disorders, which are rare, acquired platelet dysfunctions are more common in clinical practice. Their main causes are medications and systemic/hematologic diseases. Typical clinical manifestations are mucosal bleeding, epistaxis, or superficial epidermal bleeding normally of modest entity. In most cases, the molecular mechanisms underlying impaired platelet function are not fully established, making it difficult to optimize patient care. We here provide a short overview of the various forms of acquired platelet disorders, with a particular focus on recent mechanistic studies on platelet dysfunction in von Willebrand disease.

CalDAG-GEFI Deficiency Reduces Atherosclerotic Lesion Development in Mice.

OBJECTIVE: Platelets are important for the development and progression of atherosclerotic lesions. However, relatively little is known about the contribution of platelet signaling to this pathological process. Our recent work identified 2 independent, yet synergistic, signaling pathways that lead to the activation of the small GTPase Rap1; one mediated by the guanine nucleotide exchange factor, CalDAG-GEFI (CDGI), the other by P2Y12, a platelet receptor for adenosine diphosphate and the target of antiplatelet drugs. In this study, we evaluated lesion formation in atherosclerosis-prone low-density lipoprotein receptor deficient (Ldlr(-/-)) mice lacking CDGI or P2Y12 in hematopoietic cells. APPROACH AND RESULTS: Lethally irradiated Ldlr(-/-) mice were reconstituted with bone marrow from wild-type (WT), Caldaggef1(-/-) (cdgI(-/-)), p2y12(-/-), or cdgI(-/-)p2y12(-/-) (double knockout [DKO]) mice and fed a high-fat diet for 12 weeks. Ldlr(-/-) chimeras deficient for CDGI or P2Y12 developed significantly smaller atherosclerotic lesions in the aortic sinus and in aortas when compared with the Ldlr(-/-)/WT controls. We also observed a significant reduction in platelet-leukocyte aggregates in blood from hypercholesterolemic Ldlr(-/-)/cdgI(-/-) and Ldlr(-/-)/p2y12(-/-) chimeras. Consistently, fewer macrophages and neutrophils were detected in the aortic sinus of Ldlr(-/-)/cdgI(-/-) and Ldlr(-/-)/ p2y12(-/-) chimeras. Compared with controls, the plaque collagen content was significantly higher in Ldlr(-/-) chimeras lacking CDGI. Interestingly, no statistically significant additive effects were seen in Ldlr(-/-)/DKO chimeras when compared with chimeras lacking only CDGI. CONCLUSIONS: Our findings suggest that CDGI is critical for atherosclerotic plaque development in hypercholesterolemic Ldlr(-/-) mice because of its contribution to platelet-leukocyte aggregate formation and leukocyte recruitment to the lesion area.

RASA3 is a critical inhibitor of RAP1-dependent platelet activation.

The small GTPase RAP1 is critical for platelet activation and thrombus formation. RAP1 activity in platelets is controlled by the GEF CalDAG-GEFI and an unknown regulator that operates downstream of the adenosine diphosphate (ADP) receptor, P2Y12, a target of antithrombotic therapy. Here, we provide evidence that the GAP, RASA3, inhibits platelet activation and provides a link between P2Y12 and activation of the RAP1 signaling pathway. In mice, reduced expression of RASA3 led to premature platelet activation and markedly reduced the life span of circulating platelets. The increased platelet turnover and the resulting thrombocytopenia were reversed by concomitant deletion of the gene encoding CalDAG-GEFI. Rasa3 mutant platelets were hyperresponsive to agonist stimulation, both in vitro and in vivo. Moreover, activation of Rasa3 mutant platelets occurred independently of ADP feedback signaling and was insensitive to inhibitors of P2Y12 or PI3 kinase. Together, our results indicate that RASA3 ensures that circulating platelets remain quiescent by restraining CalDAG-GEFI/RAP1 signaling and suggest that P2Y12 signaling is required to inhibit RASA3 and enable sustained RAP1-dependent platelet activation and thrombus formation at sites of vascular injury. These findings provide insight into the antithrombotic effect of P2Y12 inhibitors and may lead to improved diagnosis and treatment of platelet-related disorders.

von Willebrand factor mutation promotes thrombocytopathy by inhibiting integrin αIIbß3.

von Willebrand disease type 2B (vWD-type 2B) is characterized by gain-of-function mutations in von Willebrand factor (vWF) that enhance its binding to the glycoprotein Ib-IX-V complex on platelets. Patients with vWD-type 2B have a bleeding tendency that is linked to loss of vWF multimers and/or thrombocytopenia. In this study, we uncovered evidence that platelet dysfunction is a third possible mechanism for bleeding tendency. We found that platelet aggregation, secretion, and spreading were diminished due to inhibition of integrin αIIbß3 in platelets from mice expressing a vWD-type 2B-associated vWF (vWF/p.V1316M), platelets from a patient with the same mutation, and control platelets pretreated with recombinant vWF/p.V1316M. Impaired platelet function coincided with reduced thrombus growth. Further, αIIbß3 activation and activation of the small GTPase Rap1 were impaired by vWF/p.V1316M following exposure to platelet agonists (thrombin, ADP, or convulxin). Conversely, thrombin- or ADP-induced Ca2+ store release, which is required for αIIbß3 activation, was normal, indicating that vWF/p.V1316M acts downstream of Ca2+ release and upstream of Rap1. We found normal Syk phosphorylation and PLCγ2 activation following collagen receptor signaling, further implying that vWF/p.V1316M acts directly on or downstream of Ca2+ release. These data indicate that the vWD-type 2B mutation p.V1316M is associated with severe thrombocytopathy, which likely contributes to the bleeding tendency in vWD-type 2B.

Accelerated uptake of VWF/platelet complexes in macrophages contributes to VWD type 2B-associated thrombocytopenia.

Von Willebrand disease (VWD) type 2B is characterized by mutations causing enhanced binding of von Willebrand factor (VWF) to platelets. Bleeding tendency is associated with heterogeneous clinical manifestations, including moderate to severe thrombocytopenia. The underlying mechanism of the thrombocytopenia has remained unclear. Here, a mouse model of VWD type 2B was used to investigate pathways contributing to thrombocytopenia. Immunohistochemical analysis of blood smears revealed that mutant VWF was exclusively detected on platelets of thrombocytopenic VWD type 2B mice, suggesting that thrombocytopenic VWD type 2B mice were elevated two- to threefold upon chemical macrophage depletion. Colocalization of platelets with CD68-positive Kupffer cells and CD168-positive marginal macrophages in liver and spleen, respectively, confirmed the involvement of macrophages in the removal of VWF/platelet complexes. Significantly more platelets were found in liver and spleen of VWD type 2B mice compared with control mice. Finally, platelet survival was significantly shorter in VWD type 2B mice compared with control mice, providing a rationale for lower platelet counts in VWD type 2B mice. In conclusion, our data indicate that VWF type 2B binds to platelets and that this is a signal for clearance by macrophages, which could contribute to the thrombocytopenia in patients with VWD type 2B.

Factor VIII and von Willebrand factor are ligands for the carbohydrate-receptor Siglec-5.

BACKGROUND: Factor VIII (FVIII) and von Willebrand factor (VWF) circulate in plasma in a tight non-covalent complex, being critical to hemostasis. Although structurally unrelated, both share the presence of sialylated glycan-structures, making them potential ligands for sialic-acid-binding-immunoglobulin-like-lectins (Siglecs). DESIGN AND METHODS: We explored the potential interaction between FVIII/VWF and Siglec-5, a receptor expressed in macrophages using various experimental approaches, including binding experiments with purified proteins and cell-binding studies with Siglec-5 expressing cells. Finally, Siglec-5 was overexpressed in mice via hydrodynamic gene transfer. RESULTS: In different systems using purified proteins, saturable, dose-dependent and reversible interactions between a soluble Siglec-5 fragment and both hemostatic proteins were found. Sialidase treatment of VWF resulted in a complete lack of Siglec-5 binding. In contrast, sialidase treatment left interactions between FVIII and Siglec-5 unaffected. FVIII and VWF also bound to cellsurface exposed Siglec-5, as was visualized by classical immunostaining as well as by Duolinkproximity ligation assays. Co-localization of FVIII and VWF with early endosomal markers further suggested that binding to Siglec-5 is followed by endocytosis of the proteins. Finally, overexpression of human Siglec-5 in murine hepatocytes following hydrodynamic gene transfer resulted in a significant decrease in plasma levels of FVIII and VWF in these mice. CONCLUSIONS: Our data indicate that FVIII and VWF may act as a ligand for Siglec-5, and that Siglec-5 may contribute to the regulation of plasma levels of the FVIII/VWF complex.

Macrophage LRP1 contributes to the clearance of von Willebrand factor.

The relationship between low-density lipoprotein receptor-related protein-1 (LRP1) and von Willebrand factor (VWF) has remained elusive for years. Indeed, despite a reported absence of interaction between both proteins, liver-specific deletion of LRP1 results in increased VWF levels. To investigate this discrepancy, we used mice with a macrophage-specific deficiency of LRP1 (macLRP1(-)) because we previously found that macrophages dominate VWF clearance. Basal VWF levels were increased in macLRP1(-) mice compared with control mice (1.6 ± 0.4 vs 1.0 ± 0.4 U/mL). Clearance experiments revealed that half-life of human VWF was significantly increased in macLRP1(-) mice. Ubiquitous blocking of LRP1 or additional lipoprotein receptors by overexpressing receptor-associated protein in macLRP1(-) mice did not result in further rise of VWF levels (0.1 ± 0.2 U/mL), in contrast to macLRP1(+) mice (rise in VWF, 0.8 ± 0.4 U/mL). This points to macLRP1 being the only lipoprotein receptor regulating VWF levels. When testing the mechanism(s) involved, we observed that VWF-coated beads adhered efficiently to LRP1 but only when exposed to shear forces exceeding 2.5 dyne/cm(2), implying the existence of shear stress-dependent interactions. Furthermore, a mechanism involving ß2-integrins that binds both VWF and LRP1 also is implicated because inhibition of ß2-integrins led to increased VWF levels in control (rise, 0.19 ± 0.16 U/mL) but not in macLRP1(-) mice (0.08 ± 0.15 U/mL).

The dominant-negative von Willebrand factor gene deletion p.P1127_C1948delinsR: molecular mechanism and modulation.

Understanding molecular mechanisms in the dominant inheritance of von Willebrand disease would improve our knowledge of pathophysiologic processes underlying its prevalence. Cellular models of severe type 2 von Willebrand disease, caused by a heterozygous deletion in the von Willebrand factor (VWF) gene, were produced to investigate the altered biosynthesis. Coexpression of the wild-type and in-frame deleted (p.P1127_C1948delinsR) VWF forms impaired protein secretion, high molecular weight multimer formation and function (VWF collagen-binding 1.9% ± 0.5% of wild-type), which mimicked the patient's phenotype. mRNA, protein, and cellular studies delineated the highly efficient dominant-negative mechanism, based on the key role of heterodimers as multimer terminators. The altered VWF, synthesized in large amounts with the correctly encoded "cysteine knot" domain, formed heterodimers and heterotetramers with wild-type VWF, in addition to deleted homodimers. Impaired multimerization was associated with reduced amounts of VWF in late endosomes. Correction of the dominant-negative effect was explored by siRNAs targeting the mRNA breakpoint, which selectively inhibited the in-frame deleted VWF expression. Although the small amount of the deleted protein synthesized after inhibition still exerted dominant, even though weakened, negative effects, the siRNA treatment restored secretion of large multimers with improved function (VWF collagen-binding 28.0% ± 3.3% of wild-type).