Efficacy of platelet-inspired hemostatic nanoparticles on bleeding in von Willebrand disease murine models.

The lack of innovation in von Willebrand disease (VWD) originates from many factors including the complexity and heterogeneity of the disease but also from a lack of recognition of the impact of the bleeding symptoms experienced by patients with VWD. Recently, a few research initiatives aiming to move past replacement therapies using plasma-derived or recombinant von Willebrand factor (VWF) concentrates have started to emerge. Here, we report an original approach using synthetic platelet (SP) nanoparticles for the treatment of VWD type 2B (VWD-2B) and severe VWD (type 3 VWD). SP are liposomal nanoparticles decorated with peptides enabling them to concomitantly bind to collagen, VWF, and activated platelets. In vitro, using various microfluidic assays, we show the efficacy of SPs to improve thrombus formation in VWF-deficient condition (with human platelets) or using blood from mice with VWD-2B and deficient VWF (VWF-KO, ie, type 3 VWD). In vivo, using a tail-clip assay, SP treatment reduced blood loss by 35% in mice with VWD-2B and 68% in mice with VWF-KO. Additional studies using nanoparticles decorated with various combinations of peptides demonstrated that the collagen-binding peptide, although not sufficient by itself, was crucial for SP efficacy in VWD-2B; whereas all 3 peptides appeared necessary for mice with VWF-KO. Clot imaging by immunofluorescence and scanning electron microscopy revealed that SP treatment of mice with VWF-KO led to a strong clot, similar to those obtained in wild-type mice. Altogether, our results show that SP could represent an attractive therapeutic alternative for VWD, especially considering their long half-life and stability.

Kinetics of Platelet Adhesion to Protein-Coated Surface in Whole Blood Samples at High Flow Rates.

We studied platelet adhesion to fibrinogen-coated surface in whole blood samples under conditions of high flow rates. The degree of platelet adhesion was evaluated by the intensity of laser light scattered from protein-coated optical surface with adhered platelets. The intensity of adhesion in whole blood samples at high flow rates was by 2.7 (2.4; 4.1) times higher than in platelet-rich plasma samples. Among the factors intensifying platelet adhesion in the whole blood at high flow rates, von Willebrand factor is of utmost importance. At low flow rates, platelet adhesion almost totally depends on platelet-fibrinogen interaction. At high flow rates, the interactions of platelets with both fibrinogen and von Willebrand factor become equally important.

Platelets compensate for poor thrombin generation in type 3 von Willebrand disease.

In type 3 von Willebrand disease (VWD3), the most severe form with absent von Willebrand factor (VWF), the bleeding phenotype is variable. Platelet contribution to the hemostatic defect in VWD3 calls upon further studies. We investigated the contribution of platelets to in vitro thrombin generation (TG) and platelet procoagulant activity in VWD3. TG was assessed by calibrated automated thrombogram (CAT) in platelet-poor (PPP) and -rich plasma (PRP) from 9 patients before and in 6 patients also 30 min after receiving their regular VWF therapy. Responsiveness of PPP to FVIII and protein S was also investigated. TG data were compared with routine laboratory variables, rotational thromboelastometry (ROTEM) and platelet expression of P-selectin and phosphatidylserine in flow cytometry. Compared with healthy controls, TG was markedly decreased in VWD3 PPP (peak thrombin was 16% of normal median), but not in PRP (77% of normal median) (p = 0.002). Six out of nine patients (67%) were high responders in their platelet P-selectin, and 5/9 (56%) in phosphatidylserine expression. Replacement therapy improved TG in PPP, while in PRP TG only modestly increased or was unaffected. In PPP, FVIII levels associated with TG and in vitro FVIII-supplemented TG inclined up to threefold. Conversely, a FVIII inhibitory antibody reduced plasma TG in all, but especially in patients with remnant FVIII levels. Inhibition of protein S improved plasma TG, particularly at low FVIII levels. ROTEM failed to detect VWD3.In VWD3, TG is reduced in PPP and regulated by FVIII and protein S, but TG is close to normal in PRP. VWD3 platelets seem to compensate for the FVIII-associated reduction in TG by their exposure of P-selectin and phosphatidylserine.

Characterization of aberrant splicing of von Willebrand factor in von Willebrand disease: an underrecognized mechanism.

Approximately 10% of von Willebrand factor (VWF) gene mutations are thought to alter messenger RNA (mRNA) splicing through disruption of consensus splice sites. This mechanism is likely underrecognized and affected by mutations outside consensus splice sites. During VWF synthesis, splicing abnormalities lead to qualitative defects or quantitative deficiencies in VWF. This study investigated the pathologic mechanism acting in 3 von Willebrand disease (VWD) families with putative splicing mutations using patient-derived blood outgrowth endothelial cells (BOECs) and a heterologous human embryonic kidney (HEK 293(T)) cell model. The exonic mutation c.3538G>A causes 3 in-frame splicing variants (23del, 26del, and 23/26del) which cannot bind platelets, blood coagulation factor VIII, or collagen, causing VWD through dominant-negative intracellular retention of coexpressed wild-type (WT) VWF, and increased trafficking to lysosomes. Individuals heterozygous for the c.5842+1G>C mutation produce exon 33 skipping, exons 33-34 skipping, and WT VWF transcripts. Pathogenic intracellular retention of VWF lacking exons 33-34 causes their VWD. The branch site mutation c.6599-20A>T causes type 1 VWD through mRNA degradation of exon 38 skipping transcripts. Splicing ratios of aberrant transcripts and coexpressed WT were altered in the BOECs with exposure to shear stress. This study provides evidence of mutations outside consensus splice sites disrupting splicing and introduces the concept that VWF splicing is affected by shear stress on endothelial cells.

Genetic heterogeneity in a large cohort of Indian type 3 von Willebrand disease patients.

BACKGROUND: Though von Willebrand disease (VWD) is a common coagulation disorder, due to the complexity of the molecular analysis of von Willebrand factor gene (VWF), not many reports are available from this country. Large size of the gene, heterogeneous nature of mutations and presence of a highly homologous pseudogene region are the major impediments in the genetic diagnosis of VWD. The study is aimed at unravelling the molecular pathology in a large series of VWD patients from India using an effective strategy. METHOD: We evaluated 85 unrelated Indian type 3 VWD families to identify the molecular defects using a combination of techniques i.e. PCR-RFLP, direct DNA sequencing and multiple ligation probe amplification (MLPA). RESULTS: Mutations could be characterized in 77 unrelated index cases (ICs). 59 different mutations i.e. nonsense 20 (33.9%), missense 13 (22%), splice site 4 (6.8%), gene conversions 6 (10.2%), insertions 2 (3.4%), duplication 1 (1.7%), small deletions 10 (17%) and large deletions 3 (5.1%) were identified, of which 34 were novel. Two common mutations i.e. p.R1779* and p.L970del were identified in our population with founder effect. Development of alloantibodies to VWF was seen in two patients, one with nonsense mutation (p.R2434*) and the other had a large deletion spanning exons 16-52. CONCLUSION: The molecular pathology of a large cohort of Indian VWD patients could be identified using a combination of techniques. A wide heterogeneity was observed in the nature of mutations in Indian VWD patients.

Terminal platelet production is regulated by von Willebrand factor.

It is established that proplatelets are formed from mature megakaryocytes (MK) as intermediates before platelet production. Recently, the presence of proplatelets was described in blood incubated in static conditions. We have previously demonstrated that platelet and proplatelet formation is upregulated by MK exposure to high shear rates (1800 s(-1)) on immobilized von Willebrand factor (VWF). The purpose of the present study was to investigate whether VWF is involved in the regulation of terminal platelet production in blood. To this end, Vwf (-/-) mice, a model of severe von Willebrand disease, were used to create a situation in which blood cells circulate in a vascular tree that is completely devoid of VWF. Murine platelets were isolated from Vwf (-/-) and Vwf (+/+) blood, exposed to VWF at 1800 s(-1) in a microfluidic platform, and examined by means of videomicroscopy, as well as fluorescence and activation studies. Proplatelets became visible within 5 minutes, representing 38% of all platelets after 12 minutes and 46% after 28 min. The proportion of proplatelets was 1.8-fold higher in blood from Vwf(-/-) mice than from Vwf(+/+) mice, suggesting a role of VWF in vivo. Fragmentation of these proplatelets into smaller discoid platelets was also observed in real-time. Platelets remained fully activatable by thrombin. Compensation of plasmatic VWF following hydrodynamic gene transfer in Vwf(-/-) mice reduced the percentage of proplatelets to wild-type levels. A thrombocytopenic mouse model was studied in the flow system, 7 days after a single 5-FU injection. Compared to untreated mouse blood, a 2-fold increase in the percentage of proplatelets was detected following exposure to 1800 s(-1) on VWF of samples from mice treated with 5-FU. In conclusion, VWF and shear stress together appear to upregulate proplatelet reorganization and platelet formation. This suggests a new function for VWF in vivo as regulator of bloodstream thrombopoiesis.

The molecular characterization of von Willebrand disease: good in parts.

Since the cloning of the gene that encodes von Willebrand factor (VWF), 27 years ago, significant progress has been made in our understanding of the molecular basis of the most common inherited bleeding disorder, von Willebrand disease (VWD). The molecular pathology of this condition represents a range of genetic mechanisms, some of which are now very well characterized, and others that are still under investigation. In general, our knowledge of the molecular basis of type 2 and 3 VWD is now well advanced, and in some instances this information is being used to enhance clinical management. In contrast, our understanding of the molecular pathogenesis of the most common form of VWD, type 1 disease, is still at an early stage, with preliminary evidence that this phenotype involves a complex interplay between environmental factors and the influence of genetic variability both within and outside of the VWF locus.

Nonsense mediated decay of VWF mRNA subsequent to c.7674-7675insC mutation in type3 VWD patients.

Von Willebrand disease (VWD), the most common genetic bleeding disorder, is caused by defects in Von Willebrand factor (VWF). Quantitative deficiencies of the protein lead to either VWD type3, the severe form of the disease or VWD type1 with milder clinical manifestation. Null alleles are the most common mutations in VWF gene causing type3. However, some of these mutations are not translated into the protein and are selectively degraded at mRNA level by nonsense-mediated decay (NMD) pathway. Here, we have studied a large VWD type3 pedigree with a premature termination codon (PTC) causing insertion mutation (c.7674-7675insC) in VWF exon 45. We further investigated the impact of the mutation on the VWF mRNA expression using a quantitative Real-time PCR assay and cDNA sequencing. The relative expression of the gene was significantly decreased in the patients' platelets (Mean ratio=0.03 (0.01-0.05), p=0.001) compared to their normal relatives. The heterozygote carriers of the mutation had lower than normal VWF mRNA levels (Mean ratio=0.62 (0.29-0.91), p=0.006). Direct sequencing of exon 45 on the platelet-derived cDNA in the carriers revealed only the wild-type allele confirming the decay of the mutation carrying allele. In conclusion, quantitative analysis of VWF gene expression showed that c.7674-7675insC mutation in VWF gene resulted in degradation of VWF mRNA via NMD. This pathway might play an important role in the pathogenesis of VWD characterized by quantitative deficiency of VWF due to reduced mRNA levels.

Resemblance to vWD types and laboratory diagnosis of obligatory carriers of type 3 von Willebrand disease.

OBJECTIVES: It is important to diagnose obligatory carrier (OC) type 3 von Willebrand Disease (vWD) in countries, such as Turkey, where marriages between relatives is common. However, mild bleeding or no bleeding in such patients complicates the diagnosis of the disease. It is not clear how the diagnosis of OC type 3 vWD will be made based on FVIII:C (Factor VIII activity), vWF:Ag (von Willebrand factor antigen), vWF:RCo (von Willebrand factor ristocetin cofactor activity), and PFA (platelet function analyzer )-100 parameters. Therefore, the purpose of the study is to investigate how OC type 3 vWD diagnoses may be established by studying laboratory phenotypes of close relatives of patients with diagnosed 3 vWD. PATIENTS AND METHODS: 8 patients with type 3 vWD (index cases) and 20 patients who were defined as OCs type 3 vWD were enrolled into the study. RESULT: 10 cases had similarity with mild type VWD, 4 cases had similarity with moderate type 1 vWD, 4 other cases had type 1 or 2 vWD similarities, 1 case had similarity with severe type 1 vWD, and 1 case also had similarity with severe type 1 or type 2 vWD; regarding their laboratory phenotypic characteristics. CONCLUSION: we identified that OC type 3 vWD is similar specifically to type 1 vWD in terms of laboratory phenotypic character, and we suggest that it may be used with PFA-100 as an easy and fast method in screening relatives.