Interaction Of Von Willebrand Factor Research Articles

Characterizing coagulation responses in humans and nonhuman primates following kidney xenotransplantation-A narrative review.

The recent report of the first pig kidney transplant in a living human brings hope to thousands of people with end-stage kidney failure. The scientific community views this early success with caution as kidney xenotransplantation exhibits many challenges and barriers. One of these is coagulation dysregulation. This includes (i) pig von Willebrand Factor (vWF) interaction with human platelets, which can induce abnormal clotting responses, heightening the risk of graft failure, (ii) the inefficiency of pig thrombomodulin in activating human protein C, which emphasizes the species-specific variations that aggravate coagulation challenges, and (iii) the development of thrombotic microangiopathy in the pig grafts and the occurrence of systemic consumptive coagulopathy in the recipients. Indeed, coagulation dysregulation largely results from differences in endothelial cell response and incompatibilities between pig and human coagulation-anticoagulation pathways. These barriers can be resolved by modifications to pig vWF and the expression of human thrombomodulin and endothelial protein C receptors in pig cells, serving as strategic interventions to align the coagulation systems of the two species more closely. These coagulation challenges have clinical implications in how they affect graft survival and patient outcome. Genetic engineering of the organ-source pig and the administration of various drugs have assisted in correcting this coagulation dysregulation. Hence, comprehending and controlling coagulation dysregulation is crucial for progress in xenotransplantation as a viable option for treating patients with terminal kidney disease.

The interaction of von Willebrand factor with GPIb receptors at high shear rates significantly contributes to platelet adhesion in patients with coronary artery disease

The interaction of von Willebrand factor with GPIb receptors at high shear rates significantly contributes to platelet adhesion in patients with coronary artery disease

Complement C1q and von Willebrand factor interaction in atherosclerosis of human carotid artery.

Atherosclerosis is an inflammatory disease of the vessel wall, with cholesterol crystal (CC) deposition being a hallmark of the disease. As evidence for a cross-talk between complement activation and hemostasis on CC surfaces has been limited to in vitro data, the aim of this study was to demonstrate the presence of C1q-vWF complexes in human atherosclerosis ex vivo. We used immunofluorescence staining and a proximity ligation assay (PLA, Duolink®) to examine the presence, localization, and co-localization of C1q and vWF in frozen sections of human carotid arteries with atherosclerosis or without atherosclerotic changes as well as material from thrombendarteriectomy. We observed significantly higher levels of C1q and vWF in healthy tissue compared to diseased material and greater co-localization in the PLA in healthy samples than in diseased samples. In diseased samples, fluorescence signals were highest in locations encompassing atheroma and foam cells. While there was overall reduced signal in areas with CCs, the staining was spotty, and there was evidence of co-localization on individual CCs. Thus, we demonstrate the presence of C1q-vWF complexes in human carotid arteries ex vivo, which was most abundant in healthy endothelial and subendothelial space and reduced in diseased tissue. C1q-vWF interaction can also be demonstrated on the CC surface.

The Variable Heavy Chain Antibody Syn-VWFA1 Inhibits the Interaction of Platelets to Von Willebrand Factor in Solution but Not to Immobilized Von Willebrand Factor Thereby Inhibiting 3-Dimensional but Not 2-Dimensional Thrombus Formation

Neutralizing the interaction between GP1bα and von Willebrand Factor (VWF) is an attractive strategy for treatment and prevention of platelet-VWF agglutinate formation in von Willebrand disease type 2B and thrombotic thrombocytopenic purpura. The aim of this study was to characterize a llama-derived variable heavy chain antibody (VHH) targeting the A1 domain of active VWF (Syn-VWFA1), in terms of its interference with platelet VWF interaction. Syn-VWFA1 (1.5 µM) abolished platelet binding to ristocetin-activated VWF (native and Haemate® P VWF) and R1306W VWF (VWF type 2B) in a flow-cytometric platelet function assay (Figure 1). Moreover, Syn-VWFA1 dose-dependently inhibited ristocetin-activated VWF and R1306W VWF platelet agglutination, but not collagen- and ADP-induced platelet aggregation. Under flow conditions, Syn-VWFA1 (1.8 µM) did not affect adhesion of platelets to collagen at high shear. In the shear- and collagen-dependent PFA-200, Syn-VWFA1 prolonged the closure time, but only at high concentrations (>3 µM), while GPIba binding to immobilized VWF remained unaffected by Syn-VWFA1. Syn-VWFA1 increased the cleavage of VWF multimers by ADAMTS13. Furthermore, by applying deuterium exchange, we found a small region in the β1 to α1 loop (1288-1293) in the A1 domain of VWF, which is likely to constitute the nanobody binding site (Figure 2) and binding of Syn-VWFA1 to the A1 domain results in a conformational change exposing regions in the α2 and α3 helices. In conclusion, Syn-VWFA1 dose-dependently blocks platelet-binding to activated VWF in solution, but not to (collagen-) immobilized active VWF. This implies that immobilization of VWF to collagen affects VWF conformation in such a way (1) that access of Syn-VWFA1 to its epitope in the A1 domain is hindered or (2) that conformational changes following Syn-VWFA1 binding are blocked, while allowing platelet binding. This implies that Syn-VWFA1 has potential treatment opportunities for patients with increased circulating active VWF, to prevent 3-dimensional thrombus formation (pathological platelet-VWF aggregate formation) but not 2-dimensional thrombus formation (leaving physiological hemostasis at sites of vascular injury intact).

Calcium Prevents Enhanced Degradation of Factor VIII in the Condition of Motion.

Background: Hemophilia A and B induce recurrent bleeding episodes, mainly in skeletal muscles and joints that are in intermittent motion. We have previously demonstrated that intermittent motion contributes to increased degradation of factors VIII and IX. Objectives: Given that calcium ions are known to enhance factor VIII-von Willebrand factor (vWF) interaction, the present study has investigated the role of these ions on factors VIII and IX in the condition of motion. Methods: The effects of calcium ions were assessed using purified proteins via Western blot, factor VIII activity, immunocytochemistry, and in Institute of Cancer Research (ICR) mice with no specific genetic background. Results: Calcium was found to prevent degradation of plasma-derived factor VIII but not that of factor IX, during intermittent motion. Calcium levels in the microcirculation of mouse striated muscles were elevated following movement, enabling prevention of factor VIII degradation in normal physiology. Calcium supplementation in drinking water increased factor VIII levels in blood and striated muscles of ICR mice during movement. Conclusions: calcium ions decrease factor VIII degradation in the condition of motion. Further research on the impact of calcium salt oral supplementation on hemophilia patients is warranted.

Coagulation factor VIII regulates von Willebrand factor homeostasis invivo

BackgroundCoagulation factor VIII (FVIII) and von Willebrand factor (VWF) circulate as a noncovalent complex, but each has its distinct functions. Binding of FVIII to VWF results in a prolongation of FVIII’s half-life in circulation and modulates FVIII’s immunogenicity during hemophilia therapy. However, the biological effect of FVIII and VWF interaction on VWF homeostasis is not fully understood. ObjectivesTo determine the effect of FVIII in VWF proteolysis and homeostasis in vivo. MethodsMouse models, recombinant FVIII infusion, and patients with hemophilia A on a high dose FVIII for immune tolerance induction therapy or emicizumab for bleeding symptoms were included to address this question. ResultsAn intravenous infusion of a recombinant B-domain less FVIII (BDD-FVIII) (40 and 160 μg/kg) into wild-type mice significantly reduced plasma VWF multimer sizes and its antigen levels; an infusion of a high but not low dose of BDD-FVIII into Adamts13+/- and Adamts13-/- mice also significantly reduced the size of VWF multimers. However, plasma levels of VWF antigen remained unchanged following administration of any dose BDD-FVIII into Adamts13-/- mice, suggesting partial ADAMTS-13 dependency in FVIII-augmented VWF degradation. Moreover, persistent expression of BDD-FVIII at ∼50 to 250 U/dL via AAV8 vector in hemophilia A mice also resulted in a significant reduction of plasma VWF multimer sizes and antigen levels. Finally, the sizes of plasma VWF multimers were significantly reduced in patients with hemophilia A who received a dose of recombinant or plasma-derived FVIII for immune tolerance induction therapy. ConclusionOur results demonstrate the pivotal role of FVIII as a cofactor regulating VWF proteolysis and homeostasis under various (patho)physiological conditions.

Increased Binding of von Willebrand Factor to Sub-Endothelial Collagen May Facilitate Thrombotic Events Complicating Bothrops lanceolatus Envenomation in Humans.

Consumption coagulopathy and hemorrhagic syndrome exacerbated by blood anticoagulability remain the most important causes of lethality associated with Bothrops snake envenomation. Bothrops venom also engages platelet aggregation on the injured endothelium via von Willebrand factor (vWF) interactions. Besides platelet aggregation, some Bothrops venom toxins may induce qualitative thrombopathy, which has been in part related to the inhibition of vWF activation. We tested whether B. lanceolatus venom impaired vWF to collagen(s) binding (vWF:CB) activity. Experiments were performed with B. lanceolatus crude venom, in the presence or absence of Bothrofav, a monospecific B. lanceolatus antivenom. Venom of B. lanceolatus fully inhibited vWF to collagen type I and III binding, suggesting venom interactions with the vWF A3 domain. In contrast, B. lanceolatus venom increased vWF to collagen type VI binding, suggesting the enhancement of vWF binding to collagen at the vWF A1 domain. Hence, B. lanceolatus venom exhibited contrasting in vitro effects in terms of the adhesive properties of vWF to collagen. On the other hand, the antivenom Bothrofav reversed the inhibitory effects of B. lanceolatus venom on vWF collagen binding activity. In light of the respective distribution of collagen type III and collagen type VI in perivascular connective tissue and the sub-endothelium, a putative association between an increase in vWF:CB activity for collagen type VI and the onset of thrombotic events in human B. lanceolatus envenomation might be considered.

Disulfide bond reduction and exchange in C4 domain of von Willebrand factor undermines platelet binding

BackgroundThe von Willebrand factor (VWF) is a key player in regulating hemostasis through adhesion of platelets to sites of vascular injury. It is a large, multi-domain, mechano-sensitive protein that is stabilized by a net of disulfide bridges. Binding to platelet integrin is achieved by the VWF-C4 domain, which exhibits a fixed fold, even under conditions of severe mechanical stress, but only if critical internal disulfide bonds are closed. ObjectiveTo determine the oxidation state of disulfide bridges in the C4 domain of VWF and implications for VWF’s platelet binding function. MethodsWe combined classical molecular dynamics and quantum mechanical simulations, mass spectrometry, site-directed mutagenesis, and platelet binding assays. ResultsWe show that 2 disulfide bonds in the VWF-C4 domain, namely the 2 major force-bearing ones, are partially reduced in human blood. Reduction leads to pronounced conformational changes within C4 that considerably affect the accessibility of the integrin-binding motif, and thereby impair integrin-mediated platelet binding. We also reveal that reduced species in the C4 domain undergo specific thiol/disulfide exchanges with the remaining disulfide bridges, in a process in which mechanical force may increase the proximity of specific reactant cysteines, further trapping C4 in a state of low integrin-binding propensity. We identify a multitude of redox states in all 6 VWF-C domains, suggesting disulfide bond reduction and swapping to be a general theme. ConclusionsOur data suggests a mechanism in which disulfide bonds dynamically swap cysteine partners and control the interaction of VWF with integrin and potentially other partners, thereby critically influencing its hemostatic function.

Platelet adhesion mediated by von Willebrand factor in patients with premature coronary artery disease

Aim To study platelet adhesion mediated by von Willebrand factor (VWF) in patients with premature ischemic heart disease (IHD).Material and methods This study enrolled 58 patients with stable IHD, including 45 men younger than 55 years with the first manifestation of IHD at the age of <50 years and 13 women younger than 65 years with the first manifestation of IHD at the age of <60 years. The control group consisted of 33 patients, 13 men younger than 55 years and 20 women younger than 65 years without IHD. Platelet adhesion to the collagen surface at the shear rate of 1300 s-1 was studied by evaluating the intensity of scattered laser light from the collagen-coated optical substrate in a flow chamber of a microfluidic device after 15-min circulation of whole blood in the chamber. Decreases in platelet adhesion after addition to the blood of monoclonal antibodies (mAb) to platelet receptors glycoproteins Ib (GPIb) to inhibit the receptor interaction with VWF were compared for patients of both groups.Results In patients with premature IHD, the decrease in platelet adhesion following the platelet GPIb receptor inhibition was significantly less than in patients of the control group (74.8 % (55.6; 82.7) vs. 28.9 % (-9.8; 50,5), p <0.001). For the entire sample, the median decrease in platelet adhesion following the GPIb receptor inhibition was 62.8 % (52.2; 71.2). With an adjustment for traditional risk factors of IHD, a decrease in platelet adhesion of >62.8% after blocking GPIb receptors increased the likelihood of premature IHD (OR=9.84, 95 % CI: 2.80-34.59; p <0.001).Conclusion Blocking the interaction of GPIb receptors with VWF in patients with premature IHD and increased shear rate induced a greater decrease in platelet adhesion than in patients without this disease. This suggested that an excessive interaction of VWF with platelets might contribute to the pathogenesis of premature IHD.

Dual roles of fucoidan-GPIbα interaction in thrombosis and hemostasis: implications for drug development targeting GPIbα

BackgroundPlatelet GPIbα–von Willebrand factor (VWF) interaction initiates platelet adhesion, activation, and thrombus growth, especially under high shear conditions. Therefore, the GPIb–VWF axis has been suggested as a promising target against arterial thrombosis. The polysaccharide fucoidan has been reported to have opposing prothrombotic and antithrombotic effects; however, its binding mechanism with platelets has not been adequately studied. ObjectiveThe objective of this study was to explore the mechanism of fucoidan and its hydrolyzed products in thrombosis and hemostasis. MethodsNatural fucoidan was hydrolyzed by using hydrochloric acid and was characterized by using size-exclusion chromatography, UV-visible spectroscopy, and fluorometry techniques. The effects of natural and hydrolyzed fucoidan on platelet aggregation were examined by using platelets from wild-type, VWF and fibrinogen-deficient, GPIbα-deficient, and IL4Rα/GPIbα-transgenic and αIIb-deficient mice and from human beings. Platelet activation markers (P-selectin expression, PAC-1, and fibrinogen binding) and platelet-VWF A1 interaction were measured by using flow cytometry. GPIbα–VWF A1 interaction was evaluated by using enzyme-linked immunosorbent assay. GPIb-IX–induced signal transduction was detected by using western blot. Heparinized whole blood from healthy donors was used to test thrombus formation and growth in a perfusion chamber. ResultsWe found that GPIbα is critical for fucoidan-induced platelet activation. Fucoidan interacted with the extracellular domain of GPIbα and blocked its interaction with VWF but itself could lead to GPIbα-mediated signal transduction and, subsequently, αIIbβ3 activation and platelet aggregation. Conversely, low-molecular weight fucoidan inhibited GPIb-VWF–mediated platelet aggregation, spreading, and thrombus growth at high shear. ConclusionFucoidan-GPIbα interaction may have unique therapeutic potential against bleeding disorders in its high-molecular weight state and protection against arterial thrombosis by blocking GPIb–VWF interaction after fucoidan is hydrolyzed.

The mechanistic and structural role of von Willebrand factor in endotoxemia-enhanced deep vein thrombosis in mice

BackgroundAlthough the concept of immunothrombosis has established a link between inflammation and thrombosis, the role of inflammation in the pathogenesis of deep vein thrombosis remains to be fully elucidated. Further, although various constituents of venous thrombi have been identified, their localizations and cellular and molecular interactions are yet to be combined in a single, multiplexed analysis. ObjectivesThe objective of this study was to investigate the role of the von Willebrand factor (VWF) in inflammation-associated venous thrombosis. We also performed a proof-of-concept study of imaging mass cytometry to quantitatively and simultaneously analyze the localizations and interactions of 10 venous thrombus constituents. MethodsWe combined the murine inferior vena cava stenosis model of deep vein thrombosis with the lipopolysaccharide model of endotoxemia. We also performed a proof-of-concept study of imaging mass cytometry to assess the feasibility of this approach in analyzing the structural composition of thrombi. ResultsWe found that lipopolysaccharide-treated mice had significantly higher incidences of venous thrombosis, an effect that was mitigated when VWF was inhibited using inhibitory αVWF antibodies. Our detailed structural analysis also showed that most thrombus components are localized in the white thrombus regardless of endotoxemia. Moreover, although endotoxemia modulated the relative representation and interactions of VWF with other thrombus constituents, the scaffolding network, comprised VWF, fibrin, and neutrophil extracellular traps, remained largely unaffected. ConclusionsWe observe a key role for VWF in the pathogenesis of inflammation-associated venous thrombosis while providing a more comprehensive insight into the molecular interactions that constitute the architecture of venous thrombi.

Von Willebrand factor in diagnostics and treatment of cardiovascular disease: Recent advances and prospects.

Von Willebrand factor (VWF) is a large multimeric glycoprotein involved in hemostasis. It is essential for platelet adhesion to the subendothelium of the damaged endothelial layer at high shear rates. Such shear rates occur in small-diameter arteries, especially at stenotic sites. Moreover, VWF carries coagulation factor VIII and protects it from proteolysis in the bloodstream. Deficiency or dysfunction of VWF predisposes to bleeding. In contrast, an increase in the concentration of high molecular weight multimers (HMWM) of VWF is closely associated with arterial thrombotic events. Severe aortic stenosis (AS) or hypertrophic obstructive cardiomyopathy (HOCM) can deplete HMWM of VWF and lead to cryptogenic, gastrointestinal, subcutaneous, and mucosal bleeding. Considering that VWF facilitates primary hemostasis and a local inflammatory response at high shear rates, its dysfunction may contribute to the development of coronary artery disease (CAD) and its complications. However, current diagnostic methods do not allow for an in-depth analysis of this contribution. The development of novel diagnostic techniques, primarily microfluidic, is underway. Such methods can provide physiologically relevant assessments of VWF function at high shear rates; however, they have not been introduced into clinical practice. The development and use of agents targeting VWF interaction with the vessel wall and/or platelets may be reasonable in prevention of CAD and its complications, given the prominent role of VWF in arterial thrombosis.

Polyphosphate Activates von Willebrand Factor Interaction with Glycoprotein Ib in the Absence of Factor VIII In Vitro.

Polyphosphate (polyP), a phosphate polymer released by activated platelets, may modulate various stages of hemostasis by binding to blood proteins. In this context, we previously reported that polyP binds to the von Willebrand factor (VWF). One of the most significant functions of VWF is to bind to and protect the blood circulating Factor VIII (FVIII). Therefore, here, we study the role of polyP in the VWF-FVIII complex in vitro and suggest its biological significance. Surface plasmon resonance and electrophoretic mobility assays indicated that polyP binds dynamically to VWF only in the absence of FVIII. Using the VWF Ristocetin Cofactor assay, the most accepted method for studying VWF in platelet adhesion, we found that polyP activates this role of VWF only at low levels of FVIII, such as in plasmas with chemically depleted FVIII and plasmas from severe hemophilia A patients. Moreover, we demonstrated that FVIII competes with polyP in the activation of VWF. Finally, polyP also increases the binding of VWF to platelets in samples from patients with type 2 and type 3 von Willebrand disease. We propose that polyP may be used in designing new therapies to activate VWF when FVIII cannot be used.

Blockade of Platelet Glycoprotein Ibα Augments Neuroprotection in Orai2-Deficient Mice during Middle Cerebral Artery Occlusion.

During ischemic stroke, infarct growth before recanalization diminishes functional outcome. Hence, adjunct treatment options to protect the ischemic penumbra before recanalization are eagerly awaited. In experimental stroke targeting two different pathways conferred protection from penumbral tissue loss: (1) enhancement of hypoxic tolerance of neurons by deletion of the calcium channel subunit Orai2 and (2) blocking of detrimental lymphocyte–platelet responses. However, until now, no preclinical stroke study has assessed the potential of combining neuroprotective with anti-thrombo-inflammatory interventions to augment therapeutic effects. We induced focal cerebral ischemia in Orai2-deficient (Orai2-/-) mice by middle cerebral artery occlusion (MCAO). Animals were treated with anti-glycoprotein Ib alpha (GPIbα) Fab fragments (p0p/B Fab) blocking GPIbα–von Willebrand factor (vWF) interactions. Rat immunoglobulin G (IgG) Fab was used as the control treatment. The extent of infarct growth before recanalization was assessed at 4 h after MCAO. Moreover, infarct volumes were determined 6 h after recanalization (occlusion time: 4 h). Orai2 deficiency significantly halted cerebral infarct progression under occlusion. Inhibition of platelet GPIbα further reduced primary infarct growth in Orai2-/- mice. During ischemia–reperfusion, upon recanalization, mice were likewise protected. All in all, we show that neuroprotection in Orai2-/- mice can be augmented by targeting thrombo-inflammation. This supports the clinical development of combined neuroprotective/anti-platelet strategies in hyper-acute stroke.

SIPA in 10 milliseconds: VWF tentacles agglomerate and capture platelets under high shear

AbstractShear-induced platelet aggregation (SIPA) occurs under elevated shear rates (10 000 s−1) found in stenotic coronary and carotid arteries. The pathologically high shear environment can lead to occlusive thrombosis by SIPA from the interaction of nonactivated platelets and von Willebrand factor (VWF) via glycoprotein Ib–A1 binding. This process under high shear rates is difficult to visualize experimentally with concurrent molecular- and cellular-resolutions. To understand this fast bonding, we employ a validated multiscale in silico model incorporating measured molecular kinetics and a thrombosis-on-a-chip device to delineate the flow-mediated biophysics of VWF and platelets assembly into mural microthrombi. We show that SIPA begins with VWF elongation, followed by agglomeration of platelets in the flow by soluble VWF entanglement before mural capture of the agglomerate by immobilized VWF. The entire SIPA process occurs on the order of 10 milliseconds with the agglomerate traveling a lag distance of a few hundred microns before capture, matching in vitro results. Increasing soluble VWF concentration by ∼20 times in silico leads to a ∼2 to 3 times increase in SIPA rates, matching the increase in occlusion rates found in vitro. The morphology of mural aggregates is primarily controlled by VWF molecular weight (length), where normal-length VWF leads to cluster or elongated aggregates and ultra-long VWF leads to loose aggregates seen by others' experiments. Finally, we present phase diagrams of SIPA, which provides biomechanistic rationales for a variety of thrombotic and hemostatic events in terms of platelet agglomeration and capture.

TLT-1 Regulates Thrombus Growth Under Non-Inflammatory Conditions

BackgroundThrombus formation is a complex, dynamic and multistep process, based on two crucial steps: platelet adhesion and platelet aggregation that both involve the large multimeric plasma glycoprotein Von Willebrand Factor (VWF). VWF binding to the GPIb/X/V complex initiates platelet adhesion to the vessel wall at high shear stress and triggers platelet activation resulting in the generation of thrombin and activation of integrin αIIbβ3 on the platelet surface. This activation of αIIbβ3 in turn leads to outside-in signalling and promotes binding of αIIbβ3 to fibrinogen and VWF, mediating thrombus growth. Trigging receptor expressed on myeloid cells like transcript-1 (TLT-1) is a transmembrane receptor, which is targeted to α-granules of platelets and megakaryocytes. Thrombin-induced platelet activation rapidly presents TLT-1 on the platelet surface and releases a soluble form (sTLT-1) into the circulation. To date the only known ligand for TLT-1 is fibrinogen and TLT-1 has been implicated in the regulation of inflammation-associated thrombosis. Interestingly, a putative interaction of VWF with TLT-1 was indicated by a screen with known platelet receptors.AimWe aimed to evaluate the effect of TLT-1/VWF interaction on platelet aggregation and thrombus formation.MethodsRecombinant TLT-1 and VWF were purified and the interaction between TLT-1 and VWF was analyzed by surface plasmon resonance. Static interaction was confirmed by an ELISA based binding assay. Flow assays assessed TLT-1 dependent thrombus formation in vitro. The effects of TLT-1 knockout on thrombus formation in vivo were examined via intravital microscopy of the flow restricted inferior vena cava (IVC) and imaging of platelet attachment and fibrin formation over 6 hours. Furthermore, thrombus formation and resolution was followed by high resolution ultrasound imaging after stenosis induction for 28 days. Integrin aIIbb3 activation was analysed by flow cytometry using the JonA antibody in murine platelet rich plasma.ResultsVWF bound to soluble TLT-1 with high affinity in a calcium dependent manner (K D = 1.9 nM). The binding site on VWF was mapped to the A3D4 domains and high molecular weight VWF multimers had the greatest affinity for TLT-1. Moreover, HEK293 cells transfected with TLT-1 bound to VWF and VWF strings formed specifically on TLT-1 expressing cells, confirming the interaction between the two proteins. VWF inhibited the binding of fibrinogen to TLT-1, suggesting that VWF is a preferred binding partner of TLT-1.Human platelets exhibited increased TLT-1 surface expression after TRAP-6 induced platelet activation and TLT-1 was detected throughout thrombi formed under flow. Furthermore, a TLT-1 blocking antibody inhibited the interaction of TLT-1 with VWF and reduced platelet capture to type I collagen under shear stress. Ex vivo perfusion of blood from TLT-1 knock out mice over type I collagen also resulted in reduced thrombus formation compared to blood from wild-type mice. TLT-1 knock-out platelets were activated by thrombin similar to wild-type controls, based on P-selectin expression in platelet rich plasma. However, activation of integrin αIIbβ3 determined by JonA staining was reduced in the absence of TLT-1. This phenotype of reduced integrin αIIbβ3 activation on P-selectin positive platelets was phenocopied by the thrombin platelet response in platelet rich plasma from VWF -/- mice, but not GPIbα-deficient mice, indicating that the TLT-1-VWF interaction on platelets directly influences integrin αIIbβ3 activation. Significantly, thrombus formation was markedly reduced in TLT-1 knockout mice in the IVC model in vivo in comparison to wild-type mice.ConclusionsThis study demonstrates that TLT-1 is a novel platelet ligand for VWF, and that TLT-1 may preferentially bind VWF over fibrinogen. We propose a TLT-1/VWF dependent integrin αIIbβ3 activation mechanism which plays a pivotal role in thrombus formation under non-inflammatory and potentially inflammatory conditions. DisclosuresRuf: ICONIC Therapeutics: Consultancy; MeruVasimmune: Current holder of individual stocks in a privately-held company; ARCA bioscience: Consultancy, Patents & Royalties.

Primary Myeloma Cell Induced Von Willebrand Factor Release from the Endothelium Is Mediated By VEGF and Attenuated By Heparin

IntroductionMultiple Myeloma (MM) remains an incurable disease and is associated with high rates of venous thromboembolism (VTE), the biological basis for which is not fully understood. Critically, VTE is associated with increased mortality in MM. This highlights the clinical importance of understanding cancer-coagulation crosstalk in MM. Accumulating evidence demonstrates that many solid tumours trigger endothelial cell (EC) activation with resultant von Willebrand Factor (VWF) secretion proposed to contribute to both risk of VTE and cancer metastasis. However, the interplay between VWF and MM disease biology remains poorly defined.Methods/Results100 patients with plasma cell disorders were recruited to this study. Significantly elevated plasma VWF antigen (VWF:Ag) levels were observed in patients with newly diagnosed MM (NDMM) compared to those with monoclonal gammopathy of undetermined significance (MGUS) or smouldering MM (SMM) (median 292.7 IU/dL vs 133.1 IU/dL; P<0.0001). Moreover, VWF:Ag levels in those with relapsed/refractory MM (RRMM) were further elevated (median 339 IU/dL; P<0.0001]). VWF:Ag levels also appear to correlate with disease response to therapy, with lower levels seen in patients achieving a complete response/very good partial response compared with those gaining only a partial response or worse (median 154.45 IU/dL vs 338.5 IU/dL; P<0.05).We also observed increased VWF collagen binding activity in NDMM/RRMM compared with MGUS/SMM (median 455.7 IU/dL vs 189.7 IU/dL; P<0.05). This qualitative measurement is sensitive to alterations in haemostatically active high molecular weight multimers of VWF secreted upon EC activation. Supporting this, raised plasma VWF propeptide (VWFpp) levels, a marker of acute EC activation, were seen in those with NDMM/RRMM compared with MGUS/SMM (median 182 IU/dL vs 133.01 IU/dL; P<0.05). Reduced VWFpp/VWF:Ag ratios were seen in those with NDMM/RRMM, indicating decreased circulatory clearance of VWF. We hypothesise that MM may exhibit a ‘double-hit’ in regulation of plasma VWF:Ag with increased secretion of VWF but also simultaneously reduced VWF clearance leading to sustained and markedly elevated systemic plasma VWF levels.Next we measured VWF:Ag levels in bone marrow (BM) samples from 10 patients and found that VWF is released locally within the BM niche (median 190.2 IU/dL). Co-culture of primary human ECs with supernatant from freshly isolated primary MM cells or several Human Myeloma Cell Lines (HMCLs) demonstrated that MM cells stimulate release of VWF from ECs in a rapid manner, suggestive of Weibel-Palade body exocytosis (median 34.9ng/ml vs 9.2ng/ml in untreated EC; P<0.05). Elevated levels of Vascular Endothelial Growth Factor-A (VEGF-A), a potent EC activator, were observed in MM cell supernatants. Pre-treatment of primary MM cells or HMCLs with anti-VEGF antibody bevacizumab abolished EC VWF secretion in a dose dependant manner (32.3ng/ml vs anti-VEGF treated 10.1ng/ml P<0.0001). Furthermore, clinically relevant doses of low molecular weight heparin (LMWH) also attenuated VWF secretion (29.6ng/ml vs LMWH treated 15ng/ml; P<0.05). Measurement of VEGF-A in primary MM cell and HMCL supernatant treated with LMWH or bevacizumab confirmed significant reductions in VEGF-A levels. Together these data indicate that stimulation of VWF release from the endothelium by MM cells is mediated by VEGF-A and directly inhibited by LMWH treatment.Using flow cytometry, we assessed the direct interaction of MM cells and VWF in vitro. Both primary MM cells and HMCLs bound significantly to human recombinant VWF in a dose-dependent manner. Binding was reduced by 50% following treatment with LMWH (P<0.05). Given that the VWF A1 domain contains a heparin binding motif, we speculate that MM cell adhesion to VWF may be at least in part mediated by this domain and future work focuses on elucidating the receptors involved.ConclusionThese data suggest that MM is not only associated with a marked quantitative increase in plasma VWF:Ag, but also impacts VWF functional activity. Our novel data help define the biological mechanisms underpinning elevated VWF levels in MM, with a key contribution of VEGF-A secreted directly from MM cells in the BM microenvironment. Collectively, our findings provide insights into cancer-coagulation crosstalk in MM and may help identify novel therapeutic targets to reduce VTE risk and disease progression. DisclosuresGlavey: Celgene and BMS company: Research Funding; Janssen: Honoraria, Research Funding; Abbvie: Research Funding; Amgen: Honoraria, Research Funding. Quinn: Takeda: Honoraria. O'Sullivan: Leo Pharma: Research Funding.

A p.Arg127Gln variant in GPIbα LRR5 allosterically enhances affinity for VWF: a novel form of platelet-type VWD

AbstractGain-of-function (GOF) variants in GP1BA cause platelet-type von Willebrand disease (PT-VWD), a rare inherited autosomal dominant bleeding disorder characterized by enhanced platelet GPIbα to von Willebrand factor (VWF) interaction, and thrombocytopenia. To date, only 6 variants causing PT-VWD have been described, 5 in the C-terminal disulfide loop of the VWF-binding domain of GPIbα and 1 in the macroglycopeptide. GOF GP1BA variants generate a high-affinity conformation of the C-terminal disulfide loop with a consequent allosteric conformational change on another region of GPIbα, the leucine-rich-repeat (LRR) domain. We identified a novel GP1BA variant (p.Arg127Gln) affecting the LRR5 domain of GPIbα in a boy with easy bruising and laboratory test results suggestive of PT-VWD. We thus aimed to investigate the impact of the p.Arg127Gln variant on GPIbα affinity for VWF and GPIbα structure. Chinese hamster ovary cells expressing p.Arg127Gln GPIbα showed increased binding of VWF induced by ristocetin and enhanced tethering on immobilized VWF as compared with cells expressing wild-type GPIbα. Surface plasmon resonance confirmed that p.Arg127Gln enhances the binding affinity of GPIbα for VWF. Hydrogen‐deuterium exchange mass spectrometry showed that p.Arg127Gln of LRR, while having little effect on the dynamics of the LRR locally, enhances the conformational dynamics of the GPIbα C-terminal disulfide loop structure. Our data demonstrate for the first time that GOF variants outside the GPIbα C-terminal disulfide loop may be pathogenic and that aminoacidic changes in the LRR may cause allosterically conformational changes in the C-terminal disulfide loop of GPIbα, inducing a conformation with high affinity for VWF.

Critical Extracellular Matrix Proteins Besides Collagen Bind Von Willebrand Factor

Introduction: Vascular endothelial injury exposes blood to many extracellular matrix proteins including collagen. Von Willebrand Factor (VWF) is a coagulation factor that binds platelets to collagen in the process of normal hemostasis. Collagen IV has been shown to bind VWF through the A1 domain. Many adhesive glycoproteins found in the extracellular matrix of vascular endothelium have been shown to induce hemostasis by binding coagulation factors. We hypothesized that in addition to collagen, other extracellular matrix proteins can bind VWF as well. We investigated VWF's interactions with four other extracellular matrix (ECM) proteins: laminin, fibronectin, thrombospondin, and vitronectin.Methods: Enzyme linked immunoassay (ELISA) was performed to measure the ability of laminin, fibronectin, thrombospondin, and vitronectin to bind to VWF. Each ECM protein was coated individually on a maleic anhydride plate. Detection of VWF:ECM complexes was performed using a combination of two biotinylated monoclonal anti-VWF antibodies. Inhibition of VWF:ECM interactions was tested using monoclonal and polyclonal antibodies against VWF and the individual ECM proteins. We also tested inhibition of VWF:ECM interactions by competing with collagen IV, laminin, fibronectin, thrombospondin, and vitronectin. ELISA was also used to test laminin, fibronectin, thrombospondin, and vitronectin binding to recombinant VWF (rVWF) constructs to determine a specific binding region and to determine the requirement for high molecular weight multimers with each ECM protein. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed on all ECM proteins to investigate purity.Results: Results for the four ECM proteins generally paralleled each other and will be referred to together unless otherwise specified. SDS-PAGE, however, showed independent bands for each ECM protein preparation, confirming that these interactions were with the individual proteins and not with a single protein contaminating all preparations. ELISA showed binding of laminin, fibronectin, thrombospondin, and vitronectin to wild type (WT) rVWF and plasma from heathy individuals with normal VWF. No binding was detected for individuals with type 3 VWD. Polyclonal anti-VWF (DAKO) inhibited all VWF:ECM interactions (average 99% inhibition), indicating that binding is VWF specific. Monoclonal anti-VWF A1 domain (AVW-3) also inhibited all VWF:ECM interactions (average 98% inhibition), confirming that the A1 domain of VWF is crucial for this interaction. Several rVWF variants were used to test the importance of VWF functional domains. Compared to WT rVWF, VWF:ECM binding was reduced or undetectable with all ECM proteins for 1395A (undetectable) and 1399H (average 4%) but normal for 1786D, 1392A and 1402A, which were shown to abrogate VWF binding to collagen III and collagen IV, respectively. An 11 amino acid deletion from 1392 to 1402 also showed undetectable binding. Reduced binding with these constructs indicate the critical nature of the 1395 to 1399 amino acid region of the A1 domain to the VWF:ECM interactions. Compared to WT rVWF, binding was decreased or undetectable on all ECM proteins for 1597W (average 26%), 2773R (undetectable) and Y87S (undetectable) rVWF constructs. The 1597W, 2773R, and Y87S rVWF constructs contain defects in domains of VWF crucial to multimerization. Reduced binding with these constructs indicate that the VWF:ECM interactions are multimer dependent. All VWF:ECM interactions were significantly inhibited by collagen IV (90% inhibition for thrombospondin, complete inhibition for all other ECMs). The VWF:ECM interactions were also partially inhibited by thrombospondin (average 46% inhibition) and vitronectin (average 62% inhibition). Laminin and fibronectin did not noticeably inhibit any VWF:ECM interactions.Conclusions: In addition to collagen, VWF binds other ECM proteins found in vascular endothelium including laminin, fibronectin, thrombospondin, and vitronectin. These four ECM proteins appear to bind to VWF in a restricted region of the A1 domain as competitive binding suggests differences between collagen IV and other ECM proteins. The role and importance of VWF's interaction with these ECM proteins offer alternative binding partners that may represent a unique contribution to hemostasis. DisclosuresFlood:CSL Behring: Consultancy; Shire: Consultancy.

Platelet Function in Thrombocytopenic Patients with Chronic Liver Disease

Background: Thrombocytopenia, the most common hematologic abnormality encountered in patients with chronic liver disease (CLD), results from multiple factors including splenic sequestration, relatively reduced production of thrombopoietin for the degree of thrombocytopenia, consumption of platelets secondary to autoantibodies directed against platelets, and bone marrow suppression. Elevated plasma levels of von Willebrand factor (VWF) in CLD patients may partly compensate for the reduced platelet count via interaction of VWF with the platelet receptors glycoprotein (GP) Ib-IX-V and GPIIb-IIIa (integrin αIIβ3), to promote platelet adhesion and aggregation following vessel injury. Platelets in patients with CLD are reported to have reduced function as measured by light transmission platelet aggregation (LTA) and other assays. However, LTA and most other platelet function tests are unreliable in thrombocytopenic samples. In contrast, platelet function evaluated by whole blood flow cytometry is accurate in samples with low platelet counts. Therefore, in the present study we used whole blood flow cytometry to determine whether platelet function as measured by platelet surface P-selectin, activated GPIIb-IIIa and GPIb on in vivo -circulating and in vitro -stimulated platelets is altered in thrombocytopenic CLD patients compared to healthy controls.Methods: Platelet function end points were evaluated in untreated CLD patients with thrombocytopenia (n = 21) participating at U.S. sites in the Phase 3 E5501-G000-310 or E5501-G000-311 studies of a novel thrombopoietin receptor agonist, avatrombopag, compared to healthy controls (n=10). IRB approval was obtained at each study site and written informed consent was obtained from each subject. CLD patient samples analyzed in the present study were collected prior to the administration of any study drug. No patient received a platelet transfusion within 10 days of the blood draw for this study. Platelet surface P-selectin, activated GPIIb-IIIa and GPIb, without or with in vitro stimulation (0.5 μM adenosine diphosphate [ADP], 20 μM ADP, 1.5 μM thrombin receptor activating peptide [TRAP] or 20 μM TRAP) were measured by whole blood flow cytometry. To explore whether the differences in these markers between CLD patients and healthy subjects were related to the difference in platelet count rather than to differences in platelet activation or reactivity, whole blood from healthy controls was diluted with platelet-poor plasma to adjust the platelet counts to those of the thrombocytopenic CLD patients.Results: Circulating platelets (i.e., no in vitro stimulation) from thrombocytopenic CLD patients showed low levels of platelet surface P-selectin and activated GPIIb-IIIa, similar to the healthy subject controls, demonstrating minimal in vivo platelet activation in thrombocytopenic CLD patients. However, the platelets of thrombocytopenic CLD patients had higher platelet surface GPIb mean fluorescence intensity (MFI) than either diluted or undiluted circulating platelets from healthy controls (Figure). In vitro stimulation of CLD patient platelets by ADP or TRAP resulted in lower levels of platelet surface P-selectin and activated GPIIb-IIIa compared to healthy controls, except for the levels of surface P-selectin after stimulation with 20 μM TRAP. The results for the diluted healthy subject controls were unaffected by the platelet count, except for the samples activated with 1.5 μM TRAP in which dilution resulted in reduced platelet surface P-selectin and activated GPIIb-IIIa.Conclusions: 1) Circulating platelets in thrombocytopenic CLD patients are not activated. 2) Agonist-stimulated platelet surface expression of P-selectin and activated GPIIb-IIIa is reduced in thrombocytopenic CLD patients compared to healthy controls with the same degree of thrombocytopenia, which likely contributes to the excessive bleeding in thrombocytopenic CLD patients. 3) Thrombocytopenic CLD patients have a higher expression of GPIb on circulating platelets compared with healthy controls with the same degree of thrombocytopenia. This finding is consistent with the presence of an increased proportion of large immature platelets. The high levels of platelet surface GPIb, together with the previously reported high levels of circulating VWF in these patients, may contribute to vascular integrity at lower platelet counts. [Display omitted] DisclosuresFrelinger:Sysmex: Research Funding; Pfizer: Research Funding; Ironwood: Research Funding; Baxalta: Research Funding; GLSynthesis: Research Funding; Ionis: Research Funding. Michelson:GLSynthesis: Research Funding; Eisai: Research Funding; Ironwood: Research Funding; Sysmex: Research Funding; Ionis: Research Funding; Pfizer: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; Elsevier: Patents & Royalties; Baxalta: Research Funding; Janssen: Membership on an entity's Board of Directors or advisory committees; Instrumentation Laboratory: Consultancy.