VWF-dependent Platelet Adhesion Research Articles

P721Shear stress induced unfolding of von willebrand factor may be involved in the premature development of myocardial infarction

Abstract Background Normally, von Willebrand factor (vWF) becomes highly reactive with platelets upon unfolding into a fibrillar conformation at critical shear rate (more than 5000 s–1), that may occur in stenotic arteries. At shear rates below critical value (1200–1300 s–1), which occur in intact coronary arteries, normally there is no conformational rearrangement of vWF. Pathologic unfolding of vWF at shear rates below critical value may increase a risk of the development of coronary thrombosis. There is little information on the role of shear stress induced conformational rearrangement of vWF in the development of myocardial infarction in young individuals. Purpose To investigate vWF-dependent platelet adhesion of patients with premature myocardial infarction at shear rates below critical value (1200–1300 s–1). Methods Using a microfluidic system, we measured platelet adhesion to a fibrinogen-coated optical surface at shear rates of 1200–1300 s–1 during 10 minutes. We assessed platelet-rich plasma of 8 male persons 40–52 years old, who had previous myocardial infarction at the age of 34–39. The control group comprised 6 healthy male volunteers 30–55 years old. We compared the intensity of scattered laser light measured in volts (V) at 10th minute. To study vWF-dependent platelet adhesion, we blocked GPIb receptor with monoclonal antibody to inhibit platelet interaction with vWF. To compare the intensity of vWF-dependent platelet adhesion with normally occurring adhesion to fibrinogen, we blocked GPIIb/IIIa receptor with monoclonal antibody. Results The inhibition of GPIb vWF-receptor decreased platelet adhesion to fibrinogen surface at shear rates of 1200–1300 s–1 by 17.8±4.7% in healthy volunteers and by 92±2.8% in persons with premature myocardial infarction (p<0.05). Inhibition of GPIIb/IIIa receptor decreased platelet adhesion by 91.5±3.8% in healthy volunteers and by 97.3±3.2% in persons with premature myocardial infarction. Conclusion Pathologic unfolding of vWF at shear rates below critical value may be involved in the development of premature myocardial infarction. Acknowledgement/Funding This work was supported by the grant of the Russian Science Foundation (project #16-15-10098)

Defective collagen binding and increased bleeding in a murine model of von Willebrand disease affecting collagen IV binding

Essentials Defective binding to collagen IV has been seen in von Willebrand factor (VWF) A1 domain variants. We developed a murine model of defective VWF-collagen IV interactions with VWF variant p.R1399H. p.1399HH homozygous mice had decreased binding to collagen IV and increased bleeding times. p.1399HH homozygous mice had increased time to thrombosis and decreased platelet adhesion. SUMMARY: Background von Willebrand factor (VWF) binding to typeIV collagen occurs via the VWF A1 domain, with p.R1399H being the most common VWF variant affecting this interaction. Objectives We generated a murine model of 1399H VWF to investigate its invivo effects. Methods Mice expressing the murine 1399H variant were generated via gene targeting in embryonic stem cells. VWF antigen and VWF collagen binding were measured with ELISA. Tail bleeding time assays were performed by clipping a 3-mm segment. Ferric chloride-induced thrombosis was measured via ultrasound in the carotid artery. Platelet aggregation in response to collagensI and IV was measured. VWF-dependent platelet adhesion to collagenIV was measured under flow. Results Breeding of heterozygous p.R1399H and homozygous p.1399HH mice was observed to follow normal Mendelian ratios. No spontaneous bleeding was observed for any of the offspring. VWF expression was normal, but VWF binding to collagenIV was decreased in both heterozygous and homozygous offspring. Blood loss following tail resection was increased for p.1399HH mice, and occlusion times following ferric chloride-induced thrombosis were prolonged. Platelet aggregation was unaffected, but platelet adhesion to collagenIV under flow was diminished for p.1399HH mice. Conclusions These results show that a decrease in the ability of 1399H VWF to bind collagenIV under static conditions corresponds to a decrease in binding under flow conditions, an increased bleeding time, and a prolonged time to thrombosis. This study supports the potential for a bleeding phenotype in patients with aberrant VWF-collagenIV binding.

Distribution of Pathogenic Variants in Von Willebrand Disease Type 2A in the American Population: Results from a National Clinical Reference Laboratory

Introductionvon Willebrand disease (VWD) is a common inherited bleeding disorder with a reported incidence ranging from 0.01% to 1%. VWD is classified as quantitative (types 1 and 3) and qualitative (type 2) defects in von Willebrand factor (VWF). Classical VWD type 2A, the most common qualitative defect of VWD, is characterized by decreased VWF-dependent platelet adhesion and a selective deficiency of high molecular weight VWF multimers. The majority of type 2A VWD is inherited in an autosomal dominant fashion. This subtype is caused by missense variants acting through a variety of mechanisms including defects in multimer assembly or an intrinsically increased sensitivity to cleavage by ADAMTS-13. The majority of pathogenic mutations are located in exon 28, predominantly affecting the A2 domain. Missense mutations have also been reported in the D3 and A1 domains (exons 22 and 25-28). Less common missense mutations include those responsible for dimerization (CK domain, exon 52) and multimerization defects (D2 domain, exons 11-17). Our objective was to determine the genomic distribution of mutations underlying type 2A VWD in patients referred to a USA-based clinical reference laboratory.MethodsIn a clinical reference laboratory, BloodCenter of Wisconsin, VWF gene sequence analysis results from January 2009 to July 2018 were reviewed. Molecular testing was performed by polymerase chain reaction (PCR) amplification and bi-directional Sanger sequencing or capture-based enrichment and next-generation sequencing (NGS). Complete coding region and splice junction of VWF gene was compared to the reference sequence (NM_00552.3). Variant interpretation was performed according to American College of Medical Genetics (ACMG) standards and guidelines by utilizing genome databases, population frequency data, variant specific literature search and predictive computational tools. Variant interpretation was reviewed by a team comprising of genetic counselors, molecular experts and experts in coagulation disorders correlating these findings with the patient's clinical phenotype provided.ResultsPathogenic variants were identified in 46 cases received. Of these, 56.5 % (26 of 46) were in exon 28 of the VWF gene.(Figure 1) 21.7% of mutations (10 out of 46) were identified in exon 26, 4.3% were identified in exon 25 (2 of 46) and 6.5% (3 out of 46) were identified in exon 52 of VWF gene. The remaining 10.7% were identified in exons 6,11,12,22 and 27 (1 each). The distribution of these mutations according to VWF domains was also studied. 39% of these mutations were found in the A2 domain, 30.4% were identified in the D3 domain, and 13% were found in the A1 domain. A small number of these mutations were identified in the D1 domain (1/46), D2 domain (2/46) and CK domain (2/46). Phenotypic information was available for most patients evaluated.ConclusionsIn a predominantly Caucasian population with others of mixed ethnicities, the distribution of VWD type 2A mutations was studied in patients evaluated in a clinical reference laboratory. Most mutations were identified in VWF Exon 28 and in the A2 domain. Next generation sequencing platform enables sequencing of large amounts of DNA in parallel thereby allowing all VWF exons to be evaluated at one time. However, in smaller laboratories, without access to a NGS platform, initial evaluation of the VWF Exon 28 for evaluation of VWD type 2A variants is a cost effective measure. Subsequently, sequencing of other less implicated exons may be performed utilizing a reflexive algorithm. Genetic testing can be very useful in diagnosis of type 2 variants, where mutations in specific regions correspond to the defect in VWF function. [Display omitted] DisclosuresFriedman:Shire: Membership on an entity's Board of Directors or advisory committees; CSL Behring: Membership on an entity's Board of Directors or advisory committees.

Oral Bruton tyrosine kinase inhibitors selectively block atherosclerotic plaque–triggered thrombus formation in humans

AbstractInteraction of von Willebrand factor (VWF) with platelet glycoprotein Ib (GPIb) and interaction of collagen with GPVI are essential for thrombus formation on ruptured or eroded atherosclerotic plaques (atherothrombosis). GPIb and GPVI signal through Bruton tyrosine kinase (Btk), which can be blocked irreversibly by oral application of ibrutinib, an established therapy for chronic lymphocytic leukemia (CLL) with long-term safety. We found that ibrutinib and the novel Btk inhibitors acalabrutinib and ONO/GS-4059 block GPVI-dependent static platelet aggregation in blood exposed to human plaque homogenate and collagen but not to ADP or arachidonic acid. Moreover, Btk inhibitors prevented platelet thrombus formation on human atherosclerotic plaque homogenate and plaque tissue sections from arterially flowing blood, whereas integrin α2β1 and VWF-dependent platelet adhesion to collagen, which is important for physiologic hemostasis, was not affected. This plaque-selective platelet inhibition was also observed in CLL patients taking 450 mg of ibrutinib and in volunteers after much lower and intermittent dosing of the drug. We conclude that Btk inhibitors, by targeting GPIb and GPVI signal transduction, suppress platelet thrombus accretion from flowing blood on atherosclerotic plaque but spare hemostatic platelet function. Btk inhibitors hold promise as the first culprit lesion–focused oral antiplatelet drugs and are effective at low doses.

Functional characterization of tissue factor in von Willebrand factor-dependent thrombus formation under whole blood flow conditions.

Von Willebrand factor (VWF) plays an important role in mediating platelet adhesion and aggregation under high shear rate conditions. Such platelet aggregates are strengthened by fibrin-network formation triggered by tissue factor (TF). However, little is known about the role of TF in VWF-dependent thrombus formation under blood flow conditions. We evaluated TF in thrombus formation on immobilized VWF under whole blood flow conditions in an in vitro perfusion chamber system. Surface-immobilized TF amplified intra-thrombus fibrin generation significantly under both low and high shear flow conditions, while TF in sample blood showed no appreciable effects. Furthermore, immobilized TF enhanced VWF-dependent platelet adhesion and aggregation significantly under high shear rates. Neutrophil cathepsin G and elastase increased significantly intra-thrombus fibrin deposition on immobilized VWF-TF complex, suggesting the involvement of leukocyte inflammatory responses in VWF/TF-dependent mural thrombogenesis under these flow conditions. These results reveal a functional link between VWF and TF under whole blood flow conditions, in which surface-immobilized TF and VWF mutually contribute to mural thrombus formation, which is essential for normal hemostasis. By contrast, TF circulating in blood may be involved in systemic hypercoagulability, as seen in sepsis caused by severe microbial infection, in which neutrophil inflammatory responses may be active.

Balance between von Willebrand factor and ADAMTS13 following major partial hepatectomy.

Conventional coagulation tests are frequently prolonged after liver surgery, suggesting a postoperative bleeding tendency. At the same time, thrombotic complications following partial hepatectomy (PH) are not uncommon. Little is known about changes in the platelet adhesive protein von Willebrand factor (VWF) and its cleaving protease a disintegrin and metalloprotease with a thrombospondin type 1 motif, member 13 (ADAMTS13) following a PH. Plasma samples were collected before and after PH and pylorus-preserving pancreaticoduodenectomy (PPPD), and from 24 healthy individuals. Plasma levels of VWF and ADAMTS13, VWF activity and VWF-dependent platelet adhesion were measured, and compared between the groups. Median (i.q.r.) VWF levels increased more after PH (17 patients) than following PPPD (10), reaching the highest level on postoperative day (POD) 3 (570 (473-656) versus 354 (305-476) per cent respectively; P = 0·009). VWF levels remained raised on POD 30. A decrease in median (i.q.r.) ADAMTS13 activity was observed for both patient groups, reaching the lowest level on POD 7 (24 (16-32) versus 38 (23-66) per cent for PH and PPPD respectively; P = 0·049), and levels remained significantly reduced at POD 30. VWF activity was significantly higher on day 7 following PH compared with PPPD (median (i.q.r.) 517 (440-742) versus 385 (322-484) per cent respectively; P = 0·009), and remained increased at POD 30. VWF-dependent platelet adhesion under conditions of flow was increased until POD 30 in patients after PH and PPPD, but was more pronounced in the PH group. There are changes in the balance between VWF and ADAMTS13 levels and activity in patients after both PH and PPPD. Changes in the VWF-ADAMTS13 axis were more pronounced and of longer duration after PH than following PPPD.

Bleeding Phenotype Observed in Murine Defect of VWF-Collagen 4 Interactions

VWF binding to type 4 collagen has recently been shown to occur via the VWF A1 domain, but the clinical implications of this interaction remain to be elucidated. In humans, the R1399H sequence variant (2% of the Caucasian population is heterozygous for this variant) abrogates VWF binding to collagen 4, while select other VWF A1 domain variants decrease VWF binding to collagen 4 to varying degrees. We generated a murine model of 1399H VWF to investigate the in vitro and in vivo effects of this variant more thoroughly.Mice expressing the murine R1399H mutation were generated via gene targeting in ES cells, generation of germline-competent ES cell chimeras, and backcrossing the resultant mutant mice onto a C57Bl6 background. VWF antigen (VWF:Ag) and VWF collagen binding with murine type 4 collagen (VWF:CB4) were measured by ELISA (n≥5 for each group) on wild-type p.R1399 mice (WT), heterozygous p.R1399H mice (R1399H) and homozygous p.H1399 mice (1399HH) and compared to mice lacking VWF entirely (VWF -/-). Tail bleeding time was performed by clipping a 3 mm segment of tail, immersing in warm saline, and monitoring bleeding for a maximum of 10 minutes (n≥4 for each group). Blood loss was also measured during the tail bleeding time assay for each mouse. VWF-dependent platelet adhesion to collagen 4 was measured under flow using the Venaflux system. Channels were coated with murine type 4 collagen overnight. Blood was drawn from the inferior vena cava, anticoagulated with PPACK and heparin, and platelets labeled with mepacrine. Whole blood samples were run over the collagen 4-coated channel at a shear rate of 1111/sec (n≥3 for each group). Images at 180 seconds following the start of flow over the chip were analyzed using ImageJ.Breeding of heterozygous and homozygous 1399H mice was observed to follow normal Mendelian ratios, indicating no deleterious effects of the 1399H variant on viability. No spontaneous bleeding was observed for any of the offspring. VWF expression as measured by VWF:Ag was similar for WT, R1399H, and 1399HH mice, but VWF:CB4/VWF:Ag ratios were decreased for R1399H mice, to an average of 55% of WT and 1399HH mice, to an average of 15% of WT. Blood loss during tail bleeding was increased compared to WT for both R1399H and 1399HH mice by approximately 1.8 fold and 2.8 fold respectively. Bleeding time also increased, with an average bleeding time of 92 seconds in the WT mice, 177 seconds in the R1399H mice, and 235 seconds in the 1399HH mice. Previous studies have demonstrated that platelet adhesion to collagen 4 under flow is dependent on VWF, with VWF -/- mice demonstrating absence of platelet adhesion. Platelet adhesion to collagen 4 was diminished in both heterozygous and homozygous 1399H mice, with maximum platelet binding intensity 61% of WT for the R1399H mice and 28% of WT for the 1388HH mice. These figures closely approximate the static collagen 4 binding as seen by comparable VWF:CB4/VWF:Ag ratios and platelet adhesion under flow for the R1399H heterozygous and 1399HH homozygous mice.These results show that a decrease in VWF's ability to bind collagen 4 under static conditions corresponds to a decrease in binding under flow conditions, at least at the measured shear rate, and that this defect also translates to increased bleeding time and blood loss in a tail clip model. This study supports the potential for a bleeding phenotype in patients with aberrant VWF-collagen 4 binding. Since 2% of the US population is heterozygous for the p.R1399H sequence variation, defective VWF interaction with collagen 4 may be a relatively common cause of clinical bleeding and can be modeled in engineered mice. DisclosuresFlood:CSL Behring: Consultancy; Baxter: Consultancy. Montgomery:Baxter: Consultancy; CSL Behring: Consultancy; Bayer: Consultancy; Octapharma: Consultancy; Grifols: Consultancy; Biogen: Consultancy.

Infusion of DDAVP Does Not Improve Primary Hemostasis in Patients with Cirrhosis

Rationale: Cirrhosis frequently affects multiple components of hemostasis. Reversal of the coagulopathy of these patients is frequently required in case of bleeding episodes, or as prophylaxis before invasive procedures. A simple and cost-effective way to correct a prolonged bleeding time in patients with cirrhosis is administration of 1-deamino-8-D-arginine vasopressin (DDAVP). Although DD AVP is widely used as a pro-hemostatic agent in patients with cirrhosis, it is unclear whether DDAVP truly enhances the hemostatic potential in these patients. We investigated the hemostatic effects of a single bolus dose of DDAVP in patients with cirrhosis.Methods: Ten patients with cirrhosis (n=6 Child-Pugh score B, n=4 Child-Pugh score C) and ten patients with mild hemophilia A (FVIII 0.05-0.40 IU/ml) were included in this study. All patients received an intravenous single bolus of 0.3 microgram/kg DDAVP. Plasma was collected prior to and at 1, 3, 6, and 24 hours after DDAVP administration. Levels of Von Willebrand factor (VWF:Ag), VWF propeptide, Factor VIII (FVIII), and ADAMTS13 were measured in all plasma samples, whereas VWF multimers and functional VWF-dependent platelet adhesion were determined in the samples pre- and 1 hour after DDAVP administration. VWF-dependent platelet adhesion was studied under flow conditions in a reconstituted blood model.Results: The median age of patients with cirrhosis was 55 years (IQR 47-58) and 46 years (IQR 40-55) in patients with haemophilia. Baseline values of coagulation parameters are shown in table 1. Following DDAVP administration, VWF:Ag, FVIII levels and VWF propeptide increased in patients with hemophilia, while patients with cirrhosis only showed a modest increase in VWF propeptide and FVIII levels (figure 1). Only in those patients with low Child-Pugh scores, VWF:Ag slightly increased following DDAVP. The proportion of high molecular weight VWF multimers and VWF-dependent platelet adhesion increased in patients with hemophilia one hour after DDAVP administration, but did not change in the patients with cirrhosis. Levels of ADAMTS13 were unaffected in both patient groups after DDAVP.Conclusion: DDAVP administration in patients with cirrhosis did not have effect on VWF:Ag and ADAMTS13, but VWF propeptide and FVIII did increase. The lack of relevant hemostatic effects of DDAVP in patients with cirrhosis may be the explanation for the limited value in previous clinical studies in these patients.Table 1Baseline levels of VWF:Ag and related proteinsPatient groupHemophilia ACirrhosisp-valueVWF:Ag (IU/dl)114 (94.5-164.3)521 (238-643.8)<0.001ADAMTS13 (%)76.6 (57.3-87.9)93.9 (59.2-130.4)0.247VWF propeptide (IU/dl)108.5 (97.3-121.3)284 (236.5-392.8)<0.001FVIII (IU/dl)20 (12.3-38)152.5 (117.3-193.8)<0.001Data are presented as medians with IQR; VWF:Ag= Von Willebrand factor antigen; ADAMTS13= a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13; FVIII= Factor VIII [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Flow-Dependent Functions Of Soluble Or Immobilized Tissue Factor In Mural Thrombus Formation Mediated By Von Willebrand Factor

Mural thrombus formation at sites of damaged vessel wall, essential for both physiologic hemostasis and pathological thrombosis, is established by platelet adhesion/aggregation and blood coagulation mechanisms. Although tissue factor (TF) is up-regulated upon vessel wall damage and plays a pivotal role in the latter process, its functional relevance under physiologic blood flow conditions is poorly understood. Using an in vitro perfusion chamber system, we have therefore studied the relevant role of TF in thrombus formation mediated by von Willebrand factor (VWF), a distinctive flow-dependent thrombogenic surface, under whole blood flow conditions with varying shear rates. Human recombinant TF (Innobin) were co-coated with purified VWF (100 ug/ml) onto a glass plate to prepare ‘surface-immobilized TF/VWF complex'. Surface density of immobilized TF, evaluated by the ELISA-based assay using an anti-TF monoclonal antibody, was increased in a concentration-dependent and saturated manner by soluble TF (1-100 pM) added on a plate. Citrated whole blood, recalcified with 8 mM CaCl2 prior to perfusion, was perfused over a VWF-surface in the presence or absence of surface-immobilized TF. Platelet adhesion and aggregation was evaluated by the surface coverage of generated thrombi in a defined area after 5-min perfusion. Mural thrombi formed on VWF-surface were also double-stained with fluorescently labeled anti-fibrin and anti-fibrinogen antibodies. Fibrin generation was evaluated by confocal laser scanning microscopy as a ratio of fibrin relative to fibrinogen fluorescence within mural thrombi. As a result, surface-immobilized TF significantly augmented flow-dependent fibrin generation as a function of increasing surface density of TF under both low (250 s-1) and high (1500 s-1) shear rate conditions. In this regard, soluble TF, when added to sample blood, similarly increased intra-thrombus fibrin generation in a dose-dependent manner in the absence of immobilized TF. However, coagula formation in sample blood was enormously amplified by soluble TF during perfusion, as judged by the flow-path occlusion time. In addition to the enhancing effects on fibrin generation, immobilized TF significantly up-regulated VWF-dependent platelet adhesion and aggregation under high shear rate conditions, albeit with no appreciable effects under low shear rate conditions. These results suggest a synergistic functional link between immobilized TF and VWF in mural thrombus formation under high shear rate conditions. Our results clearly illustrate the thrombogenic potentials of two distinct forms (soluble or surface-immobilized) of TF, in which surface-immobilized TF plays a concerted role on VWF-dependent thrombus formation with lesser risk of systemic hypercoagulability which may be induced by circulating soluble TF under high shear rate conditions. Disclosures:No relevant conflicts of interest to declare.

Modulation of the von Willebrand factor-dependent platelet adhesion through alternative proteolytic pathways

IntroductionPlatelet adhesion to collagen under high shear rates depends on the optimal size of the von Willebrand factor (VWF) multimers, which is determined by their limited proteolysis. The present study attempts to identify the role of hemostatic-fibrinolytic enzymes (thrombin, plasmin) and leukocyte-derived proteases (matrix metalloproteinase (MMP)-8, MMP-9, neutrophil elastase) in the cleavage of VWF and to characterize the effect of flow and platelets on this proteolysis and its functional consequences on platelet adhesion.Methods and resultsAccording to VWF immunoblots, plasmin, neutrophil elastase and thrombin at concentrations of in vivo relevance resulted in extensive degradation of VWF within several minutes. Platelets protected VWF against this proteolysis under static conditions, whereas perfusion of the proteases at 3350s-1 shear rate over VWF immobilized on artery cross sections enhanced its degradation and blocked the protective effect of platelets. In parallel with VWF digestion, the examined proteases impaired the VWF-dependent platelet adhesion as reflected in the decreased surface-bound GpIIb/IIIa immunoreactivity following perfusion of collagen-coated surfaces or artery sections with blood and plasmin, neutrophil elastase or thrombin. Within the time frame of minutes no VWF cleavage could be detected under static or flow conditions after exposure to MMP-8 and MMP-9 at concentrations relevant to physiological neutrophil counts. ConclusionOur results indicate a shear- and platelet-dependent role for several proteases in the local modulation of the VWF function.

Flow-based measurements of von Willebrand factor (VWF) function: Binding to collagen and platelet adhesion under physiological shear rate

Introduction VWF circulates in plasma as a series of heterogeneous multimers, mediating platelet tethering, translocation and finally adhesion to areas of injured endothelium under physiological high arterial blood flow. VWF-platelet binding requires conformational changes in VWF, which are induced by immobilization and shear. Because of unavailability of a simple flow-based measurement system, VWF activity assays are generally performed under static conditions. We describe an easily reproducible in vitro flow-chamber model using commercially available flow devices to examine VWF-collagen binding and VWF-mediated platelet adhesion under physiological flow conditions. Methods The collagen surface of the flow-chamber was analyzed by atomic force microscopy. Collagen-bound VWF was characterized by multimer analysis and multi labelling immunofluorescence detection of exposed GPIb binding domains. Platelet adhesion was captured by time-lapse microscopy. Results The described flow-chamber system facilitates multimer analysis of collagen-bound VWF, whereas all VWF multimers bound to collagen under physiological low to high shear rates. Multi labelling immunofluorescence detection exhibited exposed GPIb binding domains co-localized with VWF molecules. VWF-dependent platelet adhesion using time-lapse microscopy showed values comparable to experiments done with whole blood, and platelet adhesion was dependent on the VWF concentration. Conclusions The established flow-chamber model represents an easy-to-set-up and customized tool for the characterization of VWF-binding to collagen as well as the determination of VWF-dependent platelet adhesion under defined flow conditions in real-time.

Development of a Severe von Willebrand Factor/ADAMTS13 Dysbalance During Orthotopic Liver Transplantation

Patients with liver disease show profound changes in their hemostatic system, which may further change during liver transplantation. We previously demonstrated that highly elevated levels of the platelet adhesive protein von Willebrand factor (VWF) in patients with cirrhosis lead to an increased VWF-dependent platelet deposition under flow as compared to healthy controls. In this study we examined VWF parameters during the course of liver transplantation. We collected serial plasma samples from 20 patients undergoing liver transplantation in which we determined plasma levels of VWF and the VWF-cleaving protease ADAMTS13. Furthermore, we performed functional tests of VWF-dependent platelet adhesion. We found persistently elevated levels of VWF during and after liver transplantation. The capacity of VWF to interact with platelets normalized during the course of transplantation, and flow-mediated VWF-dependent platelet adhesion remained at levels far exceeding those observed in healthy individuals during and after transplantation. Plasma levels of ADAMTS13 dropped during transplantation, and in four patients levels below 10% of normal were observed after reperfusion. We observed the development of a hyperreactive primary hemostatic system, as evidenced by high levels of fully functional VWF and a temporary ADAMTS13 deficiency, during liver transplantation, and speculate that these changes contribute to postoperative thrombotic complications.

β2-Glycoprotein I inhibits von Willebrand factor–dependent platelet adhesion and aggregation

AbstractPatients with antiphospholipid syndrome are characterized by the association of thrombosis or pregnancy morbidity and the presence of antiphospholipid autoantibodies. Particularly, anti-β2-glycoprotein (β2 GPI) autoantibodies correlate with thrombosis, suggesting an antibody-induced gain of prothrombotic function and/or an antibody-induced loss of antithrombotic function of β2 GPI. In the search for potential antithrombotic properties of β2 GPI, we found that β2 GPI inhibits von Willebrand factor (VWF)–induced platelet aggregation. In addition, platelet adhesion to a VWF-coated surface was decreased by 50% in the presence of β2 GPI (P < .03). β2 GPI binds to the A1 domain of VWF but preferably when the A1 domain is in its active glycoprotein Ibα-binding conformation. Anti-β2 GPI antibodies isolated from a subset of antiphospholipid syndrome patients neutralized the β2 GPI-VWF interactions and thus the inhibitory activity of β2 GPI. In comparison to healthy individuals, the amounts of active VWF in circulation were increased 1.5-fold (P < .001) in patients positive for lupus anticoagulant (LAC) due to anti-β2 GPI antibodies. Thus, β2 GPI is a biologically relevant inhibitor of VWF function by interfering with VWF-dependent platelet adhesion. Anti-β2 GPI autoantibodies neutralize this inhibitory function and are associated with increased levels of active VWF. This mode of action could contribute to the thrombosis and consumptive thrombocytopenia observed in patients with anti-β2 GPI antibodies.

Beta2-Glycoprotein I Interferes with von Willebrand Factor-Dependent Platelet Adhesion.

Patients with the antiphospholipid syndrome are characterized by the association of thrombosis or pregnancy morbidity with the presence of antibodies against phospholipid-binding proteins, such as beta2-Glycoprotein (beta2-GPI) or prothrombin. In particular, antibodies against beta2-GPI strongly correlate with thrombotic complications. One model that explains this correlation involves an antibody-induced gain-of-function of beta2-GPI, which results in prothrombotic properties of this plasma protein. In an alternative model, beta2-GPI may display anti-thrombotic properties that disappear in an antibody-dependent manner. Of course, both possibilities may occur simultaneously. It should be noted, however, that little is known about the physiological function of beta2-GPI. Several in vitro-based studies have pointed to beta2-GPI as a potential inhibitor of fibrinolysis and/or the intrinsic pathway of coagulation. In the present study, we investigated the hypothesis that beta2-GPI affects platelet function. Addition or immune-depletion of beta2-GPI from platelet-rich plasma had little effect on ADP- or collagen-induced platelet aggregation, whereas it did modulate ristocetin-induced platelet aggregation. In a system employing washed platelets, beta2-GPI completely inhibited platelet aggregation induced by VWF/ristocetin or by a recombinant VWF type 2B mutant (VWF/R1306Q). This suggests that beta2-GPI is directed to the von Willebrand (VWF)-glycoprotein Ib axis. Indeed, beta2-GPI interfered with platelet adhesion to VWF-coated coverslips under conditions of flow as well, resulting in a 2-fold reduction of platelet coverage. In direct binding studies, wt-VWF displayed weak binding to immobilized beta2-GPI. However, conversion of latent VWF into a platelet-binding conformation (either via ristocetin or via a type 2B mutation) induced efficient, dose-dependent and saturable binding of VWF to beta2-GPI. By using a number of recombinant deletion-mutants and individual domains, we found that binding was mediated by the VWF A1 domain. Interestingly, anti-beta2-GPI antibodies isolated from patients with the antiphospholipid syndrome not only interfered with the interaction between beta2-GPI and VWF, but also neutralized the inhibitory effect of beta2-GPI towards VWF-dependent platelet aggregation. Finally, we analysed plasma of antiphospholid-syndrome patients for the presence of VWF that is in its platelet-binding conformation by using a specific nanobody (Hulstein et al ( 2005) Blood 106:3035). Levels of active VWF were increased 1.7-fold in patients compared to healthy individuals or patients that lack beta2-GPI-directed antibodies (p<0.0001). This increase in active VWF is similar to that observed in patients with acquired TTP. In conclusion, we demonstrate that beta2-GPI interacts preferentially with the platelet-binding conformation of VWF, thereby inhibiting VWF-dependent platelet adhesion and aggregation. Anti-beta2-GPI antibodies obtained from antiphospholipid-syndrome patients reverse this anti-thrombotic effect of beta2-GPI. We propose that beta2-GPI functions as a natural first-line barrier that prevents small amounts of active VWF to interact with platelets.

Filamin Binding to Glycoprotein Ibα Signals Talin-Directed α IIbβ 3 Activation.

von Willebrand factor (VWF) binding to platelet glycoprotein (Gp) Ib-IX-V triggers platelet activation. Under conditions of pathologically elevated arterial wall shear stress, VWF-dependent platelet adhesion is coupled to aggregation and thrombus formation principally through GpIbα-induced signaling to α IIbβ 3. To elucidate the mechanism of GpIbα signaling to α IIbβ 3, we have examined molecular interactions involving structural proteins that bind to the cytoplasmic domains of GpIbα and β 3. In CHO cells co-expressing human GpIb-IX and α IIbβ 3, the activation of α IIbβ 3 as reported by monoclonal antibody PAC-1 binding is stimulated by ristocetin (1 mg/ml) + purified human VWF(5 μg/ml). When filamin binding to the cytoplasmic domain of GpIbα is eliminated by deleting GpIbα residues 560-570, PAC-1 binding is eliminated. When human platelets in reconstituted whole blood are treated with a peptide that interferes with filamin binding to GpIbα (as reported in Blood 2003; 102:2122–2129), shear-dependent (1500 sec−1 shear rate or 60 dynes/cm2 shear stress) platelet deposition onto bovine type I collagen is inhibited. In washed resting platelets and platelets activated by 120 dynes/cm2 shear stress, filamin co-immunoprecipitates with both GpIbα and α IIbβ 3; only its association with β 3 is eliminated by DNaseI (1 mg/ml), demonstrating that filamin binds indirectly to α IIbβ 3 through other cytoskeletal elements. One such element is observed to be talin, which co-immunoprecipitates with filamin, α IIbβ 3 and small amounts of GpIbα in resting platelets. When platelets are sheared for two minutes at 120 dynes/cm2, talin's DNaseI-resistant association with filamin is decreased and its DNaseI-sensitive association with α IIbβ 3 is increased. These changes are prevented when shear-dependent VWF binding to GpIb-IX-V is blocked by monoclonal antibody AK2. Shear-dependent VWF binding to GpIb-IX-V also results in the proteolysis of talin, which is considered to be one mechanism by which the N-terminal head domain of talin regulates α IIbβ 3 activation. Shear-dependent talin proteolysis is not affected by blocking VWF binding to α IIbβ 3 with a RGD peptide. These results demonstrate that the cytoplasmic domain of GpIbα transduces signals to activate α IIbβ 3 through its interactions with filamin. These signals depend only on VWF binding to GpIb-IX-V. Under pathologically elevated wall shear stress in vitro, the mechanism of signaling may be the release of talin by filamin, thus permitting the proteolysis of talin and enhancing talin's interaction with α IIbβ 3.

Molecular characterization of an human monoclonal antibody that interacts with a sulfated tyrosine-containing epitope of the GPIb receptor and inhibits platelet functions

Modification of tyrosine residues in extracellular proteins by a sulfate moity plays an important role in many ligand/receptors interactions. In the present work, we describe a unique human monoclonal antibody, termed Y1-scFv, that is specific for a sulfated epitope in the platelat receptor GPIb. The Y1-scFv single chain antibody (scFv) competes with von Willebrand factor (vWF) for binding to human platelets and thus effectively inhibits platelet aggregation. Limited proteolysis of GPIb molecule, using the endoproteases, mocarhagin and cathepsin G, revealed that a seven amino-acid epitope, Tyr-276 to Glu-282, contains the recognition site for Y1-scFv. This GPIb region contains three sulfated tyrosine residues. Binding studies of Y1-scFv to cells and to synthetic peptides in vitro indicated that of the seven residues comprising the epitope only sulfo-Tyr-276 and adjacent Asp-277 are critical for the interaction. To identify the reciprocal sequences in the antibody that recognize the sulfated epitope, we introduced mutations within the complementary-determining region of the heavy chain (CDR3H) of Y1-scFv (MRAPVI). Arginine residue in the second position was critical for the binding. Moreover, a mutant, containing two sequential arginine residues, in the second and third positions of the CDR3H (MR RPVI), showed a nine-fold increased binding to GPIb. This antibody mutant also demonstrated a significant increase in inhibition of vWF-dependent platelet aggregation and adhesion under flow. In conclusion, this unique antibody and mutants, that recognize a sulfated epitope in GP1b receptor, efficiently inhibited platelet adhesion and aggregation, making it a candidate for a new anti-thrombotic agent.

Physical and Enzymatic Perturbation of the Architecture of the Tunica media Destroys its Inherent Thromboresistance

The media layer of the arterial cryo-cross sections, is defective for vWf-dependent platelet adhesion. Exposure of the same layer by stripping off the most inner portions of the vessel wall results in a highly thrombogenic surface. Stripping or balloon dilation was applied to porcine arteries prior to functional assays. Cryosections of treated or untreated arteries were perfused with porcine blood at 3,350 s(-1) and platelet deposition was detected by indirect immunofluorescence. Following balloon dilation, vWf-dependent platelet deposition increased; covering 9.08 +/- 1.36% of the total media surface area, this value for untreated vessels was 0.88 +/- 0.14%. A 10-fold increase was also found in the binding of vWf-coated fluorescent beads to the media. In addition to mechanical procedures, treatment by serine-proteases like trypsin, chymotrypsin and proteinase 3, or by chondroitinase ABC, but not by heparitinase also resulted in a 7-10-fold increase in platelet coverage over the media. Collagen in the media may be complexed with another vessel wall component shielding the vWf-binding sites. Mechanical or biochemical processes unmask these sites, and increase the thrombogenicity of the vessel wall.

Production and Characterization of Saratin, an Inhibitor of von Willebrand Factor-Dependent Platelet Adhesion to Collagen

Platelets tether to collagen in both a von Willebrand factor (vWF)-dependent and a vWF-independent manner. We have recently characterized a recombinant protein, saratin, isolated from the saliva of the leech Hirudo medicinalis, expressed it in Hansenula polymorpha, and studied its effect on direct and indirect platelet-collagen interactions. Saratin dose dependently inhibited the binding of purified human vWF to human type I and III collagens (IC(50)= 0.23 +/- 0.004 and 0.81 +/- 0.04 microg mL(-1), respectively) and to calf skin collagen (IC(50)= 0.44 +/- 0.008 microg mL(-1)). Furthermore, saratin showed a similar inhibitory potency against the binding of human, rodent, and porcine plasma vWF to these collagens. In a flow chamber under conditions of elevated shear (2700 s(-1)), saratin dose dependently and potently inhibited platelet aggregate formation on a collagen-coated surface (IC(50)= 0.96 +/- 0.25 microg mL(-1)), but at reduced shear (1300 s(-1)) a rightward shift in the dose-response curve was noted (IC(50)= 5.2 +/- 1.4 microg mL(-1)). Surface plasmon resonance analysis revealed both high and low affinity binding sites for saratin on human collagen type III (K(d) 5 x 10(-8) M and 2 x 10(-6) M, respectively). Although low concentrations of saratin, which inhibited platelet adhesion under increased shear (i.e., saturation of high-affinity binding sites), had no effect on vWF-independent collagen-induced platelet aggregation, high concentrations (i.e., saturation of low-affinity binding sites) were found to inhibit platelet aggregation. These data demonstrate that saratin is a potent inhibitor of vWF-dependent platelet adhesion to collagen and hence may have therapeutic potential as an antithrombotic agent.

BINDING OF THE VON WILLEBRAND FACTOR A1 DOMAIN TO HISTONE

Activation of the von Willebrand Factor (vWF) A1 domain is a critical factor in regulating the interaction of vWF with its platelet membrane receptor, the glycoprotein (GP) Ib-IX-V complex. This activation controls vWF-dependent platelet adhesion at high shear. The vWF-GP Ib-IX-V interaction is induced in vivo by exposure of platelet-rich plasma to high shear force, or by association of vWF with one or more unidentified components of the subendothelial matrix. In vitro, soluble vWF is activated to bind to platelets by nonphysiological modulators, such as the bacterial glycopeptide, ristocetin, or the snake venom protein, botrocetin, or by removal of negatively-charged sialic acid residues. Analysis of vWF modulators and the very marked charge asymmetry of amino acid sequences within the A1 domain has led to an electrostatic model for vWF modulation. Endothelial membrane/matrix and detergent-soluble fractions of human placenta were screened for the ability to bind vWF by electrophoresis of extracts on SDS-polyacrylamide gels, electrotransferring to nitrocellulose and probing with fluid-phase 125I-labeled vWF or a 39/34-kDa vWF fragment (Leu-480-Gly-718) that encompasses the A1 domain. In the course of these studies, it was found that both vWF and the 39/34-kDa vWF fragment bound strongly to histone. Purified soluble histone also bound vWF since, like ristocetin, it induced vWF flocculation. Histone binding to vWF did not activate or inhibit vWF binding to platelets. While the vWF-histone interaction has no conceivable physiological role, it suggests that binding to the A1 domain of vWF alone is insufficient to modulate vWF adhesive activity. This implies that specific interactions of the vWF A1 domain with either ristocetin or botrocetin are required for GP Ib-IX-V recognition to occur. © 1997 Elsevier Science Ltd