Von Willebrand Factor Self-association Research Articles

Self-Association of Von Willebrand Factor (VWF) into Prothrombotic Fibers Is Most Potently Enhanced By the Highly Atherogenic Small, Dense Fraction of Low-Density Lipoprotein (sd-LDL)

We recently demonstrated that plasma lipoproteins play a major role in regulating the function of the hemostatic system by either inhibiting or promoting the self-association of von Willebrand factor (VWF) into large fibers and bundles that are hyperadhesive for platelets. Such fibers can form on the surface of activated endothelium such as during thrombotic microangiopathies. We showed that high-density lipoprotein (HDL) attenuates VWF self-association and reduces the formation of VWF-platelet thrombi, both in vitro and in mice (PMID:26552698). By contrast, low-density lipoprotein (LDL) enhances VWF self-association and promotes the formation of VWF-platelet thrombi ( Blood, in press). Consequently, wild-type mice with elevated plasma LDL display intense and prolonged VWF-platelet thrombosis in stimulated mesenteric microvessels, exceeding even the thrombosis observed in ADAMTS13-deficient mice with normal LDL levels. All the lipoproteins of low density carry apolipoprotein (apo) B100 and comprise fractions of very low density (VLDL), intermediate density (IDL), and low density (LDL). The LDL fraction comprises two subfractions: small-dense (sd) LDL and large-buoyant (lb) LDL. Epidemiologic studies suggest that sd-LDL is the most atherogenic subfraction (PMID:33586462). Another atherogenic lipoprotein also carries apoB-100, Lp(a), in which the apoB-100 is complexed with apo(a). Here, we sought to determine the effect of LDL subfraction(s) on VWF self-association. We isolated LDL, sd-LDL and lb-LDL from healthy donor plasma by sequential KBr density centrifugation. Lp(a) was purified from patients with high circulating Lp(a) (>50 mg/dL) as described by Mueller et al. (PMID:35650291). We first tested these lipoproteins in microfluidic devices adapted from the design of Herbig and Diamond (PMID:26178390). In these devices, high shear stress and flow acceleration are generated around a 30-um-wide pillar within a 60-um-wide flow channel and induce the attachment and self-association of VWF into fibers on and around the pillar. We quantified VWF fiber accumulation from differential interference contrast (DIC) images. Of all the lipoprotein fractions, sd-LDL was the most potent in accelerating VWF strand accumulation (see figure). The effect of lipoproteins in this device correlated with their impact on microvascular thrombosis in vivo. We infused apoB100-containing lipoproteins into wild-type mice (3-4 weeks old) before inducing VWF-platelet thrombi deposition in the mesenteric microvasculature with ionophore. All the apoB100 lipoproteins enhanced microvascular thrombosis [we did not test Lp(a), which had no effect in the microfluidic channel], but sd-LDL was by far the most potent; the treated mice had twice the number of large thrombi (>30-um diameter) as the mice treated with an equivalent quantity of lb-LDL. The effect also persisted for much longer before returning to baseline with the sd-LDL. We have explored possible reasons why sd-LDL is so much more potent. Compared to the other apoB-containing lipoproteins, sd-LDL is smaller and denser, with a greater surface curvature. Whether this fact changes accessibility to sites on apoB100 is not known. We also compared the lipidomes and proteomes of sd-LDL and lb-LDL for clues to the difference. Compared to lb-LDL, sd-LDL contained a much higher content of cholesterol and cholesterol esters per particle. The proteomes of the two particles were also different, with several proteins being more abundant in lb-LDL, in particular apoC-III, apoE, apoA-IV, and apoC-II. Only apoF was significantly more abundant in sd-LDL. We are currently testing to determine if any of these difference accounts for the differences between the two LDL subfractions in promoting VWF self-association. In summary, we show that the lipoprotein associated with the greatest atherosclerotic burden and most cardiovascular disease is also the one with the most potent effect in promoting a prothrombotic state by enhancing VWF self-association. These findings have important implications for the development of the atherosclerotic lesion, the formation of the thrombus after the lesion ruptures, and for the microvascular dysfunction often observed after revascularization.

High-Density Lipoprotein Inhibits Microvascular and Arterial Thrombosis in Mice By Attenuating VWF Self-Association

Background: Von Willebrand factor (VWF) mediates platelet adhesion and aggregation in primary hemostasis and thrombosis. The thrombogenicity of VWF depends on its self-association into fibers and its ability to efficiently capture platelets under high shear stress. We have shown that high shear promotes self-association of VWF into hyperadhesive strands and fibers, and that this process can be enhanced by low density lipoprotein (LDL) and attenuated by high density lipoprotein (HDL). In a model of thrombotic microangiopathy induced by VWF infusion into ADAMTS13-deficient mice, HDL attenuated the thrombocytopenia induced by the VWF challenge, suggesting that HDL may attenuate other types of thrombosis. Aims: To study the effect of HDL on microvascular and arterial thrombosis in mice by intravital microscopy. Methods: Microvascular thrombosis was induced in ADAMTS13-deficient mice by (1) local stimulation of mesenteric venules with calcium ionophore, and (2) infusion of high doses of recombinant human VWF (rVWF). The effect of HDL on VWF-platelet thrombus formation and deposition in the microvasculature was monitored microscopically. Arterial thrombosis was induced in wild-type (WT) mice by (1) laser-induced cremaster injury, and (2) FeCl 3-induced carotid artery injury. The effect of HDL on arterial thrombosis was also monitored microscopically. Results: When we induced microvascular thrombosis with ionophore, ADAMTS13-deficient mice evinced robust local platelet adhesion, platelet string formation, and thrombus embolization. Time to thrombus resolution was significantly prolonged (about 15 min) compared to that in WT mice (< 5 min). In mice pretreated with HDL (100 mg/kg), platelet deposition was largely prevented, with only occasional single platelets adhering. The differences in the extent of thrombi deposition and time to thrombus resolution between HDL-treated and untreated mice were both statistically significant. In the VWF challenge microvascular thrombosis model, rVWF infusion into ADAMTS13-deficient mice induced platelet adhesion to endothelium-associated VWF fibers in the cremaster microcirculation, forming long strings on the walls of arterioles and venules. Many microvessels became completely occluded. The prothrombotic effect persisted over 90 minutes. In contrast, pretreatment with HDL (100 mg/kg) led to significantly reduced platelet adhesion and string formation. Vessel occlusion was almost completely prevented. When thrombosis was induced in the cremaster arterioles of WT mice, HDL pretreatment resulted in notably decreased platelet accumulation and fibrin deposition in growing thrombi. HDL pretreatment also destabilized the thrombi and led to frequent embolization. In the FeCl 3-induced carotid artery injury model, HDL pretreatment slowed thrombus growth, enhanced thrombus embolization, and delayed vessel occlusion. Conclusion(s): The results demonstrate that VWF self-association is an important mechanism promoting thrombosis in large and small vessels. Our results show that HDL possesses a novel antithrombotic effect by inhibiting VWF self-association, preventing thrombosis in both small and large vessels. These results suggest that increasing the plasma HDL level can be exploited to prevent and treat thrombotic microangiopathies and arterial thrombosis.

Low-density lipoprotein promotes microvascular thrombosis by enhancing von Willebrand factor self-association

Abstractvon Willebrand factor (VWF) mediates primary hemostasis and thrombosis in response to hydrodynamic forces. We previously showed that high shear promoted self-association of VWF into hyperadhesive strands, which can be attenuated by high-density lipoprotein (HDL) and apolipoprotein A-I. In this study, we show that low-density lipoprotein (LDL) binds VWF under shear and enhances self-association. Vortexing VWF in tubes resulted in its loss from the solution and deposition onto tube surfaces, which was prevented by HDL. At a stabilizing HDL concentration of 1.2 mg/mL, increasing concentrations of LDL progressively increased VWF loss, the effect correlating with the LDL-to-HDL ratio and not the absolute concentration of the lipoproteins. Similarly, HDL diminished deposition of VWF in a post-in-channel microfluidic device, whereas LDL increased both the rate and extent of strand deposition, with both purified VWF and plasma. Hypercholesterolemic human plasma also displayed accelerated VWF accumulation in the microfluidic device. The initial rate of accumulation correlated linearly with the LDL-to-HDL ratio. In Adamts13−/− and Adamts13−/−LDLR−/− mice, high LDL levels enhanced VWF and platelet adhesion to the myocardial microvasculature, reducing cardiac perfusion, impairing systolic function, and producing early signs of cardiomyopathy. In wild-type mice, high plasma LDL concentrations also increased the size and persistence of VWF-platelet thrombi in ionophore-treated mesenteric microvessels, exceeding the accumulation seen in similarly treated ADAMTS13-deficient mice that did not receive LDL infusion. We propose that targeting the interaction of VWF with itself and with LDL may improve the course of thrombotic microangiopathies, atherosclerosis, and other disorders with defective microvascular circulation.

LDL and HDL Have Opposing Effects on VWF Self-Association

Von Willebrand Factor (VWF) is a large, multimeric plasma protein with roles in primary hemostasis and thrombosis. VWF unfolds in response to shear stress and self-associates into adhesive fibers that can capture and hold platelets at sites of vascular injury, initiating platelet plug formation. VWF has also been implicated in pathologies associated with microvascular dysfunction and occlusion, and dysregulation of VWF can produce the catastrophic microvascular thrombotic disorder thrombotic thrombocytopenic purpura. VWF self-association is regulated by several factors: it is enhanced by shear stress, it is decreased by regulatory cleavage by ADAMTS13 metalloprotease[Dong et al. Blood, 2002, 100:4033], and our group recently showed that high density lipoprotein (HDL) attenuates VWF self-association [Chung et al. Blood, 2016, 127:637]. High levels of HDL are associated with decreased risk of thrombosis, whereas high levels of low density lipoprotein (LDL) is a strong risk factor. This led us to investigate whether LDL also influences VWF self-association and fiber formation.We studied the effects of lipoproteins on VWF self-association by applying shear stress to VWF solutions by vortexing, which caused VWF loss from solution through a process of adsorption and self-association [Chung et al. Blood, 2016, 127:637]. We inhibited the ADAMTS13 in citrated-plasma with EDTA, sheared the treated plasma in the presence of exogenous HDL or LDL, and measured the VWF remaining in solution by sandwich ELISA. HDL attenuated VWF self-association in plasma, whereas LDL had the opposite effect, enhancing self-association. Using purified components, we sheared recombinant VWF in the presence of a fixed concentration of HDL that stabilized the VWF, and added increasing amounts of LDL to attain LDL/HDL ratios found in physiological and pathophysiological conditions. As the molar ratio of LDL/HDL particles increased from 0.17 to 0.67, VWF self-association also increased. When we repeated these assays with double the concentrations of both lipoproteins, the effect on VWF self-association was comparable. We tested EDTA-treated platelet-poor-plasma with known LDL and HDL concentrations and found that the LDL/HDL ratio regulated VWF self-association in a similar manner, but the effect was less pronounced than with the purified components.To further assess the effect of LDL and HDL on VWF self-association under shear, we perfused purified plasma VWF in a microcolumn impingement device [Herbig & Diamond JTH, 2015, 13:1699] at a wall shear rate of 10,000/s, in the presence of PBS buffer, LDL, HDL, and LDL/HDL molar ratios of 0.17 and 0.67. VWF fiber formation was monitored by differential interference contrast and immunofluorescence microscopy. The width of the bundles of VWF fibers were measured and total fluorescence was quantified using image analysis software. The presence of LDL dramatically enhanced the size of VWF fibers and the rate of formation and deposition on the microcolumn, indicating that LDL interacts directly with VWF to enhance self-association. Perfusing HDL with VWF had the opposite effect and attenuated self-association. When VWF was perfused with LDL and HDL together, the LDL/HDL ratio regulated the thickness and length of the VWF fibers formed. Our findings reveal a novel role by which LDL, by enhancing VWF self-association, may contribute to VWF-mediated thrombosis. DisclosuresNo relevant conflicts of interest to declare.

Von Willebrand factor self-association is regulated by the shear-dependent unfolding of the A2 domain

Abstractvon Willebrand factor (VWF) self-association results in the homotypic binding of VWF upon exposure to fluid shear. The molecular mechanism of this process is not established. In this study, we demonstrate that the shear-dependent unfolding of the VWF A2 domain in the multimeric protein is a major regulator of protein self-association. This mechanism controls self-association on the platelet glycoprotein Ibα receptor, on collagen substrates, and during thrombus growth ex vivo. In support of this, A2-domain mutations that prevent domain unfolding due to disulfide bridging of N- and C-terminal residues (“Lock-VWF”) reduce self-association and platelet activation under various experimental conditions. In contrast, reducing assay calcium concentrations, and 2 mutations that destabilize VWF-A2 conformation by preventing coordination with calcium (D1498A and R1597W VWD type 2A mutation), enhance self-association. Studies using a panel of recombinant proteins that lack the A1 domain (“ΔA1 proteins”) suggest that besides pure homotypic A2 interactions, VWF-A2 may also engage other protein domains to control self-association. Addition of purified high-density lipoprotein and apolipoprotein-A1 partially blocked VWF self-association. Overall, similar conditions facilitate VWF self-association and ADAMTS13-mediated proteolysis, with low calcium and A2 disease mutations enhancing both processes, and locking-A2 blocking them simultaneously. Thus, VWF appears to have evolved 2 balancing molecular functions in a single A2 functional domain to dynamically regulate protein size in circulation: ADAMTS13-mediated proteolysis and VWF self-association. Modulating self-association rates by targeting VWF-A2 may provide novel methods to regulate the rates of thrombosis and hemostasis.

Von Willebrand Factor Adhesive Activity and ADAMTS13 Protease Activity in HIV-1-Infected Men.

Background: Endothelial activation caused by HIV-1 infection leads to release of von Willebrand factor (VWF), which enters the circulation or attaches to vessel walls and self-assembles into strings and fibers, enabling platelet adhesion; this adhesive activity is regulated by the VWF-cleaving protease ADAMTS13. Our objective was to assess VWF adhesive activity and ADAMTS13 protease activity in HIV-1 infection.Methods: We measured levels of VWF antigen, VWF activation factor (a measure of adhesive activity), ADAMTS13 antigen, ADAMTS13 activity, and apolipoprotein A1 (which interferes with VWF self-association) in serum samples from HIV-1-infected men whose infections were acute (n=10), chronic untreated (n=10), or chronic treated (n=10), compared to uninfected controls (n=10). Means across groups were compared using analysis of variance with contrasts, and Pearson correlations were calculated.Results: Plasma viral load was positively correlated with VWF adhesive activity, which was elevated in acute relative to chronic treated HIV-1 infection. ADAMTS13 antigen and activity were both positively correlated with plasma viral load, and ADAMTS13 activity was significantly higher in men with acute HIV infection than in uninfected controls, and in both acute and chronic untreated HIV infection relative to chronic treated infection.Conclusion: These findings suggest that even in the setting of increased ADAMTS13 protease activity, VWF in HIV-1 infection is hyperadhesive, which may favor development of microvascular and arterial thromboses and thereby contribute to increased cardiovascular risk in HIV-1-infected individuals.

Potential Mechanisms for Enhanced Activity of Von Willebrand Factor in Patients with Sickle Cell Disease

Sickle cell disease (SCD) is a hemoglobinopathy characterized by vaso-occlusion and hemolysis. Even at disease baseline, von Willebrand factor (VWF) quantity and activity is markedly increased and correlates with the extent of hemolysis [Chen et al. Blood, 2011, 117:3680].VWF is an adhesive plasma protein capable of binding platelets, and secondarily, erythrocytes, and leukocytes. Its adhesive activity is regulated at several levels: being decreased by proteolysis by the metalloprotease ADAMTS13 [Dong et al. Blood, 2002, 100:4033] or by inhibition of self-association into more adhesive forms by high density lipoprotein (HDL) [Chung et al. Blood, 2016, 127:637], or enhanced by oxidative modification [Chen et al. Blood, 2010, 115:706; Fu et al. Blood, 2011, 118:5283]. Here, we explored several potential mechanisms accounting for the increased total active VWF in SCD patients: endothelial activation, enhanced VWF self-association, and defective cleavage of endogenous VWF because of ADAMTS13 dysfunction and/or VWF resistance to cleavage due to oxidation.Methods: We analyzed plasma collected from 12 adult SCD patients at disease baseline; for 3 of these we also have samples collected during vaso-occlusive crisis. We also analyzed multiple crisis samples from one additional patient with no baseline sample. To determine if SCD plasma could activate endothelial cells, we incubated plasma with human umbilical vein endothelial cells (HUVECs) cultured in parallel-plate flow chambers for 20 min at 37˚C then washed the plasma away before perfusing fixed platelets through the chamber. The number and length of platelet-decorated VWF strings were then quantified as a measure of endothelial activation. The plasma levels of VWF, apolipoprotein A-I (ApoA-I, the major apolipoprotein in HDL), myeloperoxidase (MPO), and ADAMTS13 were measured by sandwich ELISA. The activity of ADAMTS13 was evaluated by 1) cleavage of a small VWF A2 peptide substrate; 2) cleavage of multimeric VWF in patient plasma in 1.5 M urea and examining the VWF multimer pattern over time; 3) cleavage of preformed VWF-platelet strings on activated HUVECs; and 4) examining the extent of VWF cleavage in vivo using mass spectrometry (MS). The oxidation of specific methionine residues in VWF and ADAMTS13 from patient plasma was measured by MS as described previously [Chen et al. Blood, 2010, 115:706; Wang et al. JBC, 2015, 290:1422].Results: 1) HUVECs incubated with SCD plasma were activated and produced 2 to 25 fold more VWF strings than cells treated with pooled normal plasma (PNP). Activation was inhibited if the plasma was pre-incubated with hemopexin (0.5 mg/ml), implicating heme as an activator. However, purified heme alone even at high concentration did not activate HUVECs to secrete VWF strings, indicating that heme is required but not sufficient. 2) The ratio of VWF to ApoA-I was markedly elevated in SCD patients, especially during crisis (2.3 ± 0.7 fold of PNP), indicating predisposition to VWF self-association. 3) ADAMTS13 from SCD plasma was defective in cleaving VWF strings (11% to 57% of PNP) and multimeric VWF, but not the A2 peptide substrate. 4) Almost all of the SCD plasma had elevated MPO (3.8 ± 2.5 and 12.8 ± 7.6 fold of PNP in baseline and crisis samples, respectively). 5) Our previous studies showed that HOCl oxidation of VWF Met1606 at the ADAMTS13 cleavage site rendered the VWF noncleavable, and oxidation at other sites increased its platelet-binding activity. We also found that HOCl can inactivate ADAMTS13 by oxidizing Met249 within the metalloprotease domain. We therefore evaluated the extent of VWF Met1606 and ADAMTS13 Met249 oxidation, and VWF cleavage by ADAMTS13 in vivo in patient plasma using tandem MS. We found that ADAMTS13 in SCD plasma had elevated oxidation at Met249 (1.6 ± 0.2 fold of normal controls); VWF cleavage by ADAMTS13 in vivo was decreased by as much as 40% in several of these patients, and the extent of cleavage correlated negatively with the extent of VWF Met1606 oxidation.Summary: Multiple mechanisms contribute to enhanced VWF activity in SCD patients. These include increased secretion by activated endothelial cells, enhanced VWF self-association, and defective cleavage of VWF by endogenous ADAMTS13, partially due to oxidative modification of both VWF and ADAMTS13. DisclosuresNo relevant conflicts of interest to declare.

Flow and delta-P dictate where thrombin, fibrin, and von Willebrand Factor will be found

Hemostasis occurs in two different topological scenarios: complete severing of a vessel or disruption of the vessel wall. Either to meet the daily rigors of active life or during an acute trauma, hemostasis involves the regulated and self-limiting production of thrombin to stop bleeding. In contrast, arterial and venous thrombosis typically involves the unregulated, intraluminal growth of a clot, in the absence of bleeding. For either hemostasis or thrombosis, the presence of flow and pressure gradients (delta-P, ΔP) dictates when and where thrombin and fibrin are located and in what quantity. For hemostatic clots, fibrin formation helped limit clot growth. We found that γ'-fibrinogen had a role in limiting clot growth via anti-thrombin activity at venous, but not arterial conditions. For hemophilic blood, severe factor deficiency (<1% healthy) led to a defect in both platelet and fibrin deposition under flow. However, moderate deficiency, which is associated with a less severe bleeding phenotype, had normalized platelet function but still lacked fibrin production. We conclude signaling levels of thrombin can be generated during moderate hemophilia to sufficiently activate platelets to achieve primary hemostasis, even if fibrin formation remains defective. Finally, as a clot grows, shear stresses can become sufficiently extreme in diseased arteries to drive von Willebrand Factor self-association into massive fibers, potentially the final burst of clot growth towards full thrombotic occlusion.

High-density lipoprotein modulates thrombosis by preventing von Willebrand factor self-association and subsequent platelet adhesion

AbstractThe ability of von Willebrand factor (VWF) to initiate platelet adhesion depends on the number of monomers in individual VWF multimers and on the self-association of individual VWF multimers into larger structures. VWF self-association is accelerated by shear stress. We observed that VWF self-association occurs during adsorption of VWF onto surfaces, assembly of secreted VWF into hyperadhesive VWF strings on the endothelial surface, and incorporation of fluid-phase VWF into VWF fibers. VWF adsorption under static conditions increased with increased VWF purity and was prevented by a component of plasma. We identified that component as high-density lipoprotein (HDL) and its major apolipoprotein ApoA-I. HDL and ApoA-I also prevented VWF on the endothelium from self-associating into longer strands and inhibited the attachment of fluid-phase VWF onto vessel wall strands. Platelet adhesion to VWF fibers was reduced in proportion to the reduction in self-associated VWF. In a mouse model of thrombotic microangiopathy, HDL also largely prevented the thrombocytopenia induced by injection of high doses of human VWF. Finally, a potential role for ApoA-I in microvascular occlusion associated with thrombotic thrombocytopenic purpura and sepsis was revealed by the inverse relationship between the concentration of ApoA-I and that of hyperadhesive VWF. These results suggest that interference with VWF self-association would be a new approach to treating thrombotic disorders.

ADAMTS13 Synergizes with HDL in Preventing Von Willebrand Factor Self-Association Under Shear Stress

Von Willebrand factor (VWF) is a plasma glycoprotein that mediates platelet adhesion at sites of vessel injury. It is synthesized in megakaryocytes and endothelial cells and is assembled in the endoplasmic reticulum and Golgi into an array of multimers. Upon secretion from microvascular endothelium, VWF multimers can further self-associate under shear stress and form surface-bound fibers of potentially enormous sizes capable of spanning the lumens of vessels up to 300 mm in diameter (Zheng et al. Nature Communications 2015 In press). These structures are normally removed by the plasma metalloprotease ADAMTS13. However, when ADAMTS13 is inactivated or when massive VWF secretion overwhelms the capacity of ADAMTS13 to process VWF, these structures persist in the microcirculation and bind platelets avidly to form occlusive thrombi, a process characteristic of the devastating disease thrombotic thrombocytopenic purpura (TTP). These microvascular VWF-platelet thrombi have also been implicated in the microvascular dysfunction that accompanies malaria, sickle cell disease, and sepsis. We recently identified high density lipoprotein particles (HDL) as being able to prevent VWF self-association into thick strands (Chung et al. Blood 2015 in revision). In these studies, we also studied VWF self-association in citrated human plasma under shear stress in a test tube in the presence of EDTA (to inhibit ADAMTS13). VWF self-associated and adsorbed to the tube surface, a phenomenon prevented by addition of HDL at concentrations above those already present in plasma. When EDTA was not added to the plasma, the majority of the VWF was not cleaved but was nevertheless stabilized in solution. This result suggests that when ADAMTS13 has been progressively inactivated by citrate at 37°C, it is able to prevent VWF self-association. It is not clear why EDTA-inhibited ADAMTS13 did not stabilize VWF to the same extent as citrate-inhibited ADAMTS13. It is possible that EDTA and citrate have different effects on the stabilization function of ADAMTS13. Further, addition of recombinant ADAMTS13 to citrated plasma (final ratio VWF monomer:ADAMTS13 = 1.6:1) did not enhance VWF cleavage under shear, but completely stabilized the VWF multimers. These results demonstrate a new function for ADAMTS13: it regulates VWF adhesive activity by preventing VWF self-association through direct binding instead of cleavage. Therefore, we hypothesize that the relative levels of VWF, HDL, and ADAMTS13 in plasma regulate the propensity of VWF multimers to self-associate under shear stress. While high VWF levels and high shear stress favor VWF self-association, high HDL and ADAMTS13 levels prevent self-association. We tested the hypothesis with plasma from wild-type or knockout mice on the C57BL6 background. In comparison to humans, wild-type C57BL6 mice have low VWF levels, high HDL levels (calculated from HDL-cholesterol levels), and express a truncated version of ADAMTS13. Further, ADAMTS13-deficient C57BL6 mice do not spontaneously develop microvascular occlusion. Unlike human citrated plasma, when citrated plasma from wild-type mice was sheared in the presence of EDTA, the VWF multimers did not self-associate. We attributed this difference from human plasma to the low VWF:HDL ratio in this mouse strain. When the plasma from apolipoprotein (Apo) A-I knockout mice was sheared in the presence of EDTA, the VWF multimers also did not self-associate, which we attributed to the low VWF level and the ability of EDTA-inhibited truncated ADAMTS13 to stabilize VWF. When the plasma of a double knockout of ApoA-I and ADAMTS13 was sheared, the VWF self-associated and adsorbed to the tube surface. Addition of HDL to this double knockout plasma stabilized the VWF. The VWF antigen levels in wild-type, single and double knockout mouse plasma were comparable. Double knockout mice challenged with a bolus injection of VWF developed more severe thrombocytopenia than did mice with either single ApoA-I or ADAMTS13 deficiency. Together, these results suggest that ADAMTS13 synergizes with HDL in stabilizing VWF and dampening its self-association into hyperadhesive forms under shear stress, and that interplay between concentrations of VWF, ADAMTS13, and HDL particles can determine the propensity for developing TTP and its severity once developed. DisclosuresNo relevant conflicts of interest to declare.

Free thiol groups in von Willebrand factor (VWF) are required for its full function under physiological flow conditions

Introductionvon Willebrand factor (VWF) is rich in cysteine; next to important structural disulfide bonds, free thiol groups are present. Free thiols on the surface of plasmatic VWF have been shown to play a role in VWF self-association and in platelet binding under pathologically high levels of shear stress. The present study explores the role of VWF free thiol groups under physiological levels of shear stress and in interactions with collagen and platelet-GPIbα receptor. Materials and methodsFree and accessible thiol groups were blocked with N-ethylmaleimide (NEM) and the derivatized molecule was evaluated in functional assays. Reduced cysteine residues were identified using biotin-linked maleimide (MPB) followed by analysis of multimer and domain incorporation and by analysis of derivatized tryptic peptides by mass spectrometry. ResultsBlockade of free thiol groups significantly reduced VWF-mediated platelet recruitment to collagen under physiological flow conditions. This resulted from inhibition of VWF binding to both collagen and the platelet GPIb receptor. Evaluation of derivatization sites revealed a high level of derivatization in the cysteine-rich N- and C-termini of VWF. 19 MPB-derivatized peptides, 13 of which are described here for the first time, were identified by mass spectrometry. ConclusionsThis study shows a significant contribution of free thiol groups in VWF to the mediation of platelet adhesion under physiological shear stress conditions. The free thiol groups are shown to be involved in VWF binding to both collagen III and platelet GP1b receptor.

High Density Lipoprotein and Apolipoprotein A-I Bind Von Willebrand Factor and Prevent Its Self-Association Into Thick Fibers

Von Willebrand factor (VWF) is a large plasma protein secreted constitutively or upon activation by endothelial cells. On activated endothelium, a portion of the VWF molecules remain attached to the surface, where they self-associate to produce long hyperadhesive strands and fibers capable of capturing platelets and other blood cells if not removed by the plasma metalloprotease ADAMTS13. Failure to clear these fibers underlies the pathophysiology of many microangiopathies, most prominently thrombotic thrombocytopenic purpura, in which accumulation of VWF-platelet aggregates in the microvasculature leads to ischemia and organ failure. VWF self-association also plays a major role in the adsorption of purified VWF to surfaces. We observed that adsorption of VWF to surfaces began with contact and direct binding of VWF to the surface via hydrophobic interactions. After saturating the binding sites on the surface, VWF continued to bind to and associate with the immobilized VWF molecules via hydrophilic VWF-VWF interactions, depositing multiple layers of VWF molecules on the surface until essentially all VWF molecules were depleted from the fluid phase. Using the method of Magnani and coworkers, we eluted VWF molecules that were bound to the surface via VWF-VWF interactions with the ionic detergent SDS, and then eluted VWF molecules that were bound to the surface via VWF-surface interactions with the zwitterion detergent CHAPS. Quantification of the eluted VWF showed that self-association of VWF onto the surface accounted for >80% of the adsorptive loss. Using the disappearance of purified VWF from the fluid phase as a measurement of self-association, we fractionated human plasma by heat and identified that apolipoprotein A-I (ApoAI), the major apolipoprotein component in high density lipoprotein (HDL) particles, which is stable to heat at 100° C, stabilized and prevented VWF surface adsorption by interfering with VWF self-association. Commercial preparations of ApoAI and HDL, prepared without exposure to heat, similarly prevented adsorption of purified VWF to surfaces. Half-maximal stabilization occurred at a molar ratio of eight ApoAI molecules to each VWF subunit. We assessed the role of HDL in VWF self-association that leads to the assembly of ultra-large VWF (ULVWF) strings on the surface of phorbol myristyl acetate-stimulated endothelial cells in flow chambers. We observed that HDL reduced the number and length of platelet-decorated ULVWF strings on the endothelial surface, consistent with the ability of HDL to interfere with VWF self-association and ULVWF assembly. We also studied the role of ApoAI in the recruitment of fluid-phase VWF to hyperadhesive ULVWF fibers in a synthetic microvessel system. We observed that ApoAI directly bound to hyperadhesive transluminal ULVWF fibers under flow, and this binding completely blocked the recruitment of fluid phase VWF molecules to the immobilized ULVWF fibers. These results showed that ApoAI or HDL interacted with hyperadhesive forms of VWF and modified the adhesive properties of the ULVWF strings and fibers. Consistent with its antithrombotic properties, the level of ApoAI in patients with hyperadhesive forms of VWF, such as thrombotic thrombocytopenic purpura and sepsis, was significantly reduced. These results suggest that regulation of VWF self-association may be another mechanism by which HDL protects against cardiovascular disease and extends its protective effects from large arteries to the microvasculature. Disclosures:No relevant conflicts of interest to declare.

Free Thiol Groups In Von Willebrand Factor (VWF) Are Required For Its Proper Function Under Physiological Flow Conditions

BackgroundThe multimeric plasma glycoprotein von Willebrand factor (VWF) is exceptionally rich in cysteine, and its structure is largely determined by inter- and intramolecular disulfide bonding. Additionally, VWF was shown to contain unpaired cysteine residues potentially affecting protein function. The significance of free thiols on the surface of plasmatic VWF has been confirmed previously with respect to platelet binding under pathologically high shear stress. Furthermore their potential involvement in functional VWF self-association occurring at elevated shear stress has been suggested. AimsObjective of the present study was to investigate whether free thiol groups of plasma VWF contribute to the physiological VWF function under high physiological arterial shear stress conditions. Furthermore, we aimed to elucidate possible underlying mechanisms involved in this regulation. MethodsFree and accessible thiol groups of plasma-derived VWF were blocked with N-ethylmaleimide (NEM). Derivatization was followed by detailed structural and functional examination including multimer analysis (MMA) and Fourier transform infrared spectroscopy (FTIR). Functional differences between the NEM-derivatized sample and the control sample were detected using an in vitro flow chamber system with respect to VWF-mediated platelet adhesion to collagen. Interactions with collagen type III and platelet glycoprotein (GP)Ib receptor were investigated using surface plasmon resonance (SPR). Identification of accessible cysteine residues was accomplished using biotin-linked maleimide (MPB) followed by analysis of multimer and domain incorporation as well as mass spectrometry. ResultsBlocking free thiol groups provoked substantial loss of VWF activity with respect to platelet recruitment to collagen type III under flow. The lowered platelet adhesion to collagen type III was shown to be a combined effect of inhibition of (i) the initial VWF binding to collagen type III as well as (ii) VWF-platelet GPIb interaction. Free thiol groups were accessible for derivatization solely on the surface of coiled multimers. Domain incorporation studies revealed a high level of derivatization in VWF N- and C-terminus. This was in accordance with the mass spectrometric analysis, where 19 MPB-derivatized peptides, predominantly located at the N- and C-terminus, could be identified. ConclusionBlocking free thiol groups in VWF significantly impaired mediation of platelet adhesion under physiological shear stress conditions. This result suggests an essential functional role of free thiol groups in VWF regarding binding to subendothelial matrix as well as platelet recruitment. Disclosures:Solecka:Octapharma AG: Employment. Fuchs:Octapharma AG: Employment. Kannicht:Octapharma AG: Employment.

Von Willebrand factor self-association on platelet GpIbα under hydrodynamic shear: effect on shear-induced platelet activation

AbstractThe function of the mechanosensitive, multimeric blood protein von Willebrand factor (VWF) is dependent on its size. We tested the hypothesis that VWF may self-associate on the platelet glycoprotein Ibα (GpIbα) receptor under hydrodynamic shear. Consistent with this proposition, whereas Alexa-488–conjugated VWF (VWF-488) bound platelets at modest levels, addition of unlabeled VWF enhanced the extent of VWF-488 binding. Recombinant VWF lacking the A1-domain was conjugated with Alexa-488 to produce ΔA1-488. Although ΔA1-488 alone did not bind platelets under shear, this protein bound GpIbα on addition of either purified plasma VWF or recombinant full-length VWF. The extent of self-association increased with applied shear stress more than ∼ 60 to 70 dyne/cm2. ΔA1-488 bound platelets in the milieu of plasma. On application of fluid shear to whole blood, half of the activated platelets had ΔA1-488 bound, suggesting that VWF self-association may be necessary for cell activation. Shearing platelets with 6-µm beads bearing either immobilized VWF or anti-GpIbα mAb resulted in cell activation at shear stress down to 2 to 5 dyne/cm2. Taken together, the data suggest that fluid shear in circulation can increase the effective size of VWF bound to platelet GpIbα via protein self-association. This can trigger mechanotransduction and cell activation by enhancing the drag force applied on the cell-surface receptor.

Ristocetin-induced self-aggregation of von Willebrand factor

Von Willebrand factor (VWF) is a large multimeric adhesive glycoprotein, with complex roles in thrombosis and hemostasis, present in circulating blood and in secretory granules of endothelial cells and platelets. High shear stress triggers conformational changes responsible for both binding to the platelet receptor glycoprotein GpIb and its self-association, thus supporting the formation of platelet plug under flow. Ristocetin also promotes the interaction of VWF with GpIb and is able to induce platelet aggregation, and thus is largely used to mimic this effect in vitro. In this research paper, we followed the time course of VWF self-association in solution induced by ristocetin binding by light scattering and at the same time we collected atomic force microscopy images to clarify the nature of the assembly that is formed. In fact, this process evolves initially through the formation of fibrils that subsequently interact to form supramolecular structures whose dimensions would be capable of trapping platelets even in the absence of any degree of shear stress or interaction with external surfaces. This intrinsic property, that is the ability to self-aggregate, may be involved in some pathological settings that have been revealed in clinical practice.

The von Willebrand factor self-association is modulated by a multiple domain interaction

Platelet adhesion and aggregation at sites of vascular injury exposed to rapid blood flow require von Willebrand factor (VWF). VWF becomes immobilized by binding to subendothelial components or by a self-association at the interface of soluble and surface-bound VWF. As this self-association has been demonstrated only under shear conditions, our first goal was to determine whether the same interaction could be observed under static conditions. Furthermore, we wanted to identify VWF domain(s) important for this self-association. Biotinylated VWF (b-VWF) interacted dose-dependently and specifically with immobilized VWF in an enzyme-linked immunosorbent assay (ELISA) assay, showing that shear is not necessary to induce the VWF self-association. Whereas anti-VWF monoclonal antibodies (mAbs) had no effect on the self-association, the proteolytic VWF-fragments SpII(1366-2050) and SpIII(1-1365) inhibited the b-VWF-VWF interaction by 70 and 80%, respectively. Moreover, a specific binding of b-VWF to immobilized Sp-fragments was demonstrated. Finally, both biotinylated SpII and SpIII were able to bind specifically to both immobilized SpII and SpIII. Similar results were observed under flow conditions, which confirmed the functional relevance of our ELISA system. We have developed an ELISA binding assay in which a specific VWF self-association under static conditions can be demonstrated. Our results suggest a multiple domain interaction between immobilized and soluble VWF.

Aspects of hydrodynamic shear regulating shear-induced platelet activation and self-association of von Willebrand factor in suspension

The binding of plasma von Willebrand factor (VWF) to platelet receptor GpIb under high hydrodynamic shear leads to platelet activation and subsequent shear-induced platelet aggregation (SIPA). We quantitatively examined the aspects of fluid flow that regulate platelet activation by subjecting human blood and isolated platelets to well-defined shear conditions in a cone-plate viscometer. We made the following observations. First, Annexin V binding to phosphatidyl serine expressed on activated cells was detectable within 10 seconds of shear application. Second, fluid shear stress rather than shear rate controls platelet activation, and a threshold shear stress of approximately 80 dyn/cm(2) is necessary to induce significant activation. Under these conditions, individual domains of soluble VWF and platelet GpIb are subjected to similar magnitudes of fluid forces on the order of 0.1 pN, whereas GpIb with bound VWF is subjected to 1 pN. Third, cell-cell collisions and time-varying stresses are not essential for platelet activation. Fourth, the mechanism of platelet activation can be resolved in 2 steps based on the contribution of VWF and fluid forces. Fluid shear and VWF are required during the first step, when GpIb-VWF binding likely occurs. Subsequently, high shear forces alone in the absence of VWF in suspension can induce platelet activation. In other experiments, purified VWF was subjected to shear in the viscometer, and VWF morphology was assessed using light scattering. These studies demonstrate, for the first time, the ability of hydrodynamic forces to induce VWF aggregation in suspension. This VWF self-association may be an additional feature involved in controlling cell adhesion rates in circulation.