ULVWF Strings Research Articles

Factor VIII Is Synthesized in Human Endothelial Cells, Packaged in Weibel-Palade Bodies and Secreted Bound to ULVWF Strings.

The cellular synthesis site and ensuing storage location for human factor VIII (FVIII), the coagulation protein deficient in hemophilia A, has been elusive. FVIII stability and half-life is dependent on non-covalent complex formation with von Willebrand factor (VWF) to avoid proteolysis and clearance. VWF is synthesized in megakaryocytes and endothelial cells, and is stored and secreted from platelet alpha granules and Weibel-Palade bodies of endothelial cells. In this paper we provide direct evidence for FVIII synthesis in 2 types of primary human endothelial cells: glomerular microvascular endothelial cells (GMVECs) and umbilical vein endothelial cells (HUVECs). Gene expression quantified by real time PCR revealed that levels of F8 and VWF are similar in GMVECs and HUVECs. Previous clinical studies have shown that stimulation of vasopressin V2 receptors causes parallel secretion of both proteins. In this study, we found that both endothelial cell types express AVPR2 (vasopressin V2 receptor gene) and that AVPR2 mRNA levels are 5-fold higher in GMVECs than HUVECs. FVIII and VWF proteins were detected by fluorescent microscopy in Weibel-Palade bodies within GMVECs and HUVECs using antibodies proven to be target specific. Visual presence of FVIII and VWF in Weibel-Palade bodies was confirmed by correlation measurements. The high extent of correlation was compared with negative correlation values obtained from FVIII detection with cytoplasmic proteins, β-actin and Factor H. FVIII activity was positive in GMVEC and HUVEC cell lysates. Stimulated GMVECs and HUVECs were found to secrete cell-anchored ultra-large VWF strings covered with bound FVIII.

Anti-β2-GPI Antibodies Induce Von Willebrand Factor Release, Modulate Platelet Binding, and Affect VWF Proteolysis

Background:The antiphospholipid syndrome (APS) is characterized by predisposition to thrombosis. The cause for this pathology is poorly understood but is likely multifactorial, involving activation of blood cells and vasculature. The role that anti-β2-GPI antibodies play in von Willebrand factor (VWF) release from endothelial cells, VWF-platelet binding, and VWF cleavage by ADAMTS13 has not been well characterized in APS. We decided to study the effect of these antibodies on expressed ultra large VWF strings (ULVWF strings) that bind platelets (VWF-PLT strings) under flow to better understand platelet–VWF binding and ADAMTS13 regulation in APS.Hypothesis:We hypothesized that Anti-β2-GPI antibodies could induce VWF release from endothelial cells and modulate VWF’s prothombotic effect through alterations in VWF-Platelet binding and VWF cleavage by ADAMTS13.Methods:Human umbilical vein endothelial cells were seeded in 96-well plates/flow chambers prepared with a collagen Type I substrate for static/flow experiments, respectively.Static assays: Cells were incubated for 1 hr with Anti-β2-GPI or control antibodies and the conditioned media was assayed for VWF by ELISA, normalized to normal plasma.Flow Assay Analysis: After stimulation with agonist and perfusion with a platelet suspension, platelets bound to ULVWF in a string pattern were quantified via brightfield microscopy. Images of chambers were captured and VWF-PLT string-units (defined as a string length of 25μM) per slide were quantified. To minimize bias, image acquisition was standardized and the investigator was blinded at time of image acquisition/analysis.β2-GPI Flow assays: Endothelial cells in flow chambers were stimulated with 50ng/mL of phorbol myristate acetate (PMA), and a solution of fixed platelets with β2-GPI or β2-GPI+Anti-β2-GPI were perfused prior to image acquisition.ADAMTS13 assays: After stimulation with 25ng/mL PMA and perfusion with fixed platelets, images were acquired. Then control/patient plasma was perfused over formed strings. Images taken after plasma perfusion were quantified and compared to images prior to plasma perfusion.Data are shown as mean +/- SEM, and significance was determined as p<0.05 by student’s t-test or Mann-Whitney U Test, when appropriate.Results:Static Assays: Compared to control human IgG (8.28 +/- 3.34 mU/mL), VWF release was increased in the presence of two patient-derived Anti-β2-GPI antibodies, APS25-6 Anti-β2-GPI, 35.73 +/- 7.83 mU/mL (P = 0.008) and APS203-2 Anti-β2-GPI, 34.08 +/- 7.119 mU/mL (P = 0.039). As compared to control rabbit IgG (15.80 +/- 7.12 mU/mL), a rabbit polyclonal Anti-β2-GPI antibody, R24-6, also demonstrated increased soluble VWF (43.16 +/- 9.60 mU/mL, P = 0.013) release.β2GPI Flow Assays:The presence of β2GPI (2µM) reduced String-unit formation from 50.10 +/-5.57 Sting-units/image to 20.98 +/- 2.05 String Units/image (P < 0.0001) as compared to buffer. Addition of goat Anti-β2-GPI antibody (1µM) increased the VWF-PLT string observed as compared to β2GPI (2µM), 30.09 +/- 1.83 String Units to 20.98 +/- 2.05 String Units (P = 0.012) indicating that an Anti-β2-GPI antibody partially reverses the effect of β2GPI on reducing VWF-PLT string formation.ADAMTS13 Assay:Compared to pooled normal plasma (ADAMTS13 Activity 100%) (4.57 +/- 0.60 String Units/image cleaved), there was a significant decrease in the amount of string units/image cleaved in two APS plasmas with Anti-β2-GPI antibodies, APS232-9 (-0.23 +/- 0.98, P = 0.0003) and APS227-9 (2.23 +/- 0.73, P = 0.0009). ADAMTS13 Activity of patient plasma was 98.37% and 83.97%, respectively. These results suggest an inhibitory role of APS plasma on the cleavage of ULVWF strings.Conclusions:Anti-β2-GPI antibodies and antiphospholipid syndrome plasma may contribute to the prothrombotic phenotype observed in APS by three mechanisms: 1) the increased release of VWF from endothelial cells after incubation with Anti-β2-GPI, 2) increased platelet binding to ULVWF strings likely mediated by interfering with β2GPI’s known inhibition of Gp1bα VWF-platelet binding, and 3) a reduced ability to cleave VWF-PLT strings by APS plasma, suggestive of ADAMTS13 inhibition that does not correlate with ADAMTS13 activity. Taken together, our results suggest that VWF and its modulation may contribute to the prothrombotic phenotype observed in the antiphospholipid syndrome. DisclosuresNo relevant conflicts of interest to declare.

A Novel Role for Von Willebrand Factor in the Pathogenesis of Experimental Cerebral Malaria

Cerebral malaria (CM) is a major cause of death in young children in sub-Saharan Africa. Although the pathogenesis of this condition remains poorly understood, Plasmodium falciparum infection results in adhesion of infected erythrocytes (IE) to microvascular endothelium, and acute endothelial cell (EC) activation. Recent studies have shown that plasma VWF:Ag and VWF propeptide levels are markedly elevated in children with severe falciparum malaria. Moreover elevated VWF:Ag levels inversely correlate with clinical outcomes. In addition, circulating ultra-large VWF multimers have also been observed in children with severe malaria. Importantly, platelet-decorated UL-VWF strings have been shown to recruit trophozoite-stage falciparum-IE to EC surfaces in shear-based assays. Collectively, these emerging data suggest that VWF may play a novel role in the pathogenesis of CM. To further investigate this hypothesis, we utilized an established murine model of experimental CM which involves wild type (WT) C57BL/6J mice infected with Plasmodium berghei ANKA.In brief, WT C57Bl/6J mice were inoculated with 2x106P. berghei ANKA via I.P. injection. These mice typically developed CM and died within 6-8 days. In keeping with findings in children with falciparum malaria, acute EC activation was also an early and consistent feature in the murine model of CM. Mean plasma VWF:Ag levels were significantly elevated from Day+3 in infected mice compared to controls (Day +3 1.8 fold increase; Day +5 2.5 fold increase; p<0.01). In addition, plasma angiopoietin-2 and osteoprotegerin levels were also both significantly elevated from Day +3 and Day +5 respectively in mice infected with P. berghei. Interestingly, despite the fact that murine plasma ADAMTS13 activity levels were not significantly reduced, pathological ultra-large VWF multimers (UL-VWF) were also observed in murine plasma from Day +3 following P. berghei inoculation. Cumulatively these findings suggest that early marked elevation in VWF:Ag levels, and the appearance of pathological UL-VWF multimers in the plasma, represent hallmarks of both human and murine severe malaria infection.To determine whether VWF plays a direct role in modulating the pathogenesis of CM in vivo, we further investigated P. berghei infection in VWF-/- C57BL/6J mice. Importantly, although there was no difference in blood parasitaemia levels, overall survival was significantly prolonged in VWF-/- mice compared to wild type mice (6 versus 7.25 days; p=0.0106). Moreover, a significant delay in malaria clinical progression in the VWF-/- mice was also observed using a previously validated clinical scoring algorithm for experimental cerebral malaria. (Amante et al, Am J Path 2007). Recent studies have demonstrated that platelets may play a direct role in modulating malaria parasite killing. Consequently, to investigate potential mechanisms through which VWF-/- mice are protected against experimental CM, daily platelet counts were determined in WT and VWF-/- mice following infection. In keeping with observations in human patients infected with falciparum malaria, significant thrombocytopenia was also a consistent feature in the murine model. For example, by Day +4 following inoculation of WT mice mean platelet count had fallen by 71.6 ± 26 %. Interestingly however, significant thrombocytopenia was also observed in VWF-/- mice infected with P. berghei (84.2 ± 9 %). Despite the significant differences in clinical progression and overall survival no significant differences in platelet counts were observed between VWF-/- and WT mice at any time point. These findings suggest that VWF-/-mice are protected against CM through a platelet-independent mechanism.In conclusion, we demonstrate that early and consistent EC activation is a feature of the murine model of experimental CM similar to previous findings of children infected with P. falciparum. In addition, our findings show that marked elevation of VWF:Ag, and the appearance of pathological UL-VWF multimers in the plasma, both represent hallmarks of human and murine severe malaria infection. Finally, we report that VWF-/- mice exhibit significantly prolonged survival against CM, and that this effect is mediated through a platelet-independent mechanism. Given the significant morbidity and mortality associated with CM, these novel data have direct translational significance. DisclosuresNo relevant conflicts of interest to declare.

Thrombotic microangiopathies and the linkage between von Willebrand factor and the alternative complement pathway.

Molecular linkages between von Willebrand factor (VWF) and the alternative complement pathway (AP) have recently been discovered. Endothelial cell (EC)-anchored ultra-large (UL) VWF multimeric strings function as an activating surface for the AP. C3 (in active C3b form) binds to the EC-anchored ULVWF strings, and promotes the assembly of C3bBb (C3 convertase) and C3bBbC3b (C5 convertase). These linkages may help to explain enigmatic clinical problems related to thrombotic microangiopathies, including some cases of refractory thrombotic thrombocytopenic purpura (TTP), TTP associated with only mild-modest deficiencies of ADAMTS-13, the provocation (or exacerbation) of acute episodes in patients with the atypical hemolytic uremic syndrome, and thrombosis in paroxysmal nocturnal hemoglobinuria. Recent experiments have also demonstrated that complement factor H performs a dual role: participating in regulation of the AP by binding to EC-anchored ULVWF strings; and functioning as a reductase to decrease the size of soluble VWF multimers.

Carboxyl Terminal Domains Of ADAMTS13 Inhibit The Formation Of Ultra Large VWF Strings and Platelet/Leukocyte Aggregates On Endothelial Cells In Murine Mesenteric Venules Via a Disulfide Bond Reduction Mechanism

von Willebrand factor (VWF) undergoes self-association mediated by free cysteine residues between two apposed VWF multimers. This process occurs in solution or between soluble and ultra large (UL) VWF on endothelial cells or matrix under flow. ADAMTS13 is a key regulator for the ULVWF string formation and adhesive function, primarily through its proteolytic cleavage at the Tyr1605-Met1606 bond in the central A2 domain of VWF. The proteolytic cleavage of VWF by ADAMTS13 rapidly removes newly secreted ULVWF on activated endothelial cell surface, thereby preventing excessive platelet aggregation and thrombus formation under physiological conditions. Previous studies have shown that the proteolytic cleavage of both cell-bound and soluble ULVWF by ADAMTS13 depends on its amino terminal MDTCS domains. However, the role of more distal carboxyl-terminal domains is not fully established. Here, we show by N-ethylmaleimide (NEM) labeling and mass spectrometry that TSP1 2-8 repeats and CUB domains of human recombinant ADAMTS13 contain multiple free cysteine residues (-SH). We hypothesize that these free thiols may directly interact with those on the surface of VWF multimers, thereby preventing their lateral association, thereby inhibiting thrombus formation under flow. Using an intravital microscopic imaging technique we demonstrate that even in the absence of circulating ADAMTS13 (Adamts13-/-) the formation and elongation of ULVWF strings on endothelial cells of mesenteric venules after FeCl3 injury are highly dynamic over time (180 seconds), suggesting other proteases or reductases in blood are able cleave or reduce ULVWF strings under flow. However, the average length of ULVWF strings remains relatively constant. Recombinant ADAMTS13 (rA13) at physiological concentration (10 nM), when infused, rapidly cleaves or eliminates preformed ULVWF strings and platelet/leukocyte aggregates on endothelial cells. The efficacy of rA13 in reducing the average length of ULVWF strings and platelet/leukocyte aggregation decreases by ∼50% if rA13 is pretreated with NEM. Moreover, a C-terminal fragment of ADAMTS13 comprising 2-8 TSP1 repeats and two CUB domains (T2C) or containing 5-8 TSP1 repeats and two CUB domains (T5C), when infused, also significantly attenuates the formation and/or elongation of ULVWF strings/bundles and platelet/leukocyte aggregation on endothelial cells under the same conditions. The inhibitory activity of T2C and T5C is dose-dependent. At the concentration of 0.5 µM, the average length of the ULVWF strings and the surface coverage of platelets/leukocytes decrease by ∼50% and ∼40% (p&lt;0.001), respectively. Also, treatment with NEM abolishes the inhibitory effect of T2C and T5C on the ULVWF string formation and platelet/leukocyte aggregation on activated endothelial cells surface (Fig. 1). In conclusion, our results demonstrate for the first time that the distal C-terminal fragments of ADAMTS13 lacking the protease domain are of direct anti-thrombotic activity, which is mediated through its disulfide-bond reduction mechanism. Furthermore, the carboxyl-terminal disulfide bond reducing activity may contribute to the overall anti-thrombotic activity of ADAMTS13 in vivo. Disclosures: No relevant conflicts of interest to declare.

Disulfide bond reduction of von Willebrand factor by ADAMTS-13.

von Willebrand factor (VWF) released from endothelial cells is rich in ultra-large (UL) multimers that are intrinsically active in binding platelets, whereas plasma-type VWF multimers require shear stress to be activated. This functional difference may be attributed to thiols exposed on the surface of plasma-type VWF multimers, but not on ULVWF multimers. Shear stress induces the exposed thiols to form disulfide bonds between laterally apposed plasma-type VWF multimers, leading to enhanced VWF binding to platelets. We tested a hypothesis that ADAMTS-13 has a disulfide bond reducing activity that regulates shear-induced thiol-disulfide exchange of VWF. Thiol blocking agents and active thiol bead capturing were used to identify and locate this activity, along with truncated ADAMTS-13 mutants. ADAMTS-13 contains a disulfide bond reducing activity that primarily targets disulfide bonds in plasma-type VWF multimers induced by high shear stress or formed with thiol beads, but not disulfide bonds in native multimeric structures. Cysteine thiols targeted by this activity are in the VWF C-domain and are known to participate in shear-induced thiol-disulfide exchange. ADAMTS-13 contains cysteine thiols that remain exposed after being subjected to hydrodynamic forces. Blocking these active thiols eliminates this reducing activity and moderately decreases ADAMTS-13 activity in cleaving ULVWF strings anchored to endothelial cells under flow conditions, but not under static conditions. This activity is located in this C-terminal region of ADAMTS-13. This novel disulfide-bond-reducing activity of ADAMTS-13 may prevent covalent lateral association and increased platelet adherence of plasma-type VWF multimers induced by high fluid shear stress.

VH1-69 germline encoded antibodies directed towards ADAMTS13 in patients with acquired thrombotic thrombocytopenic purpura.

Autoantibodies directed towards ADAMTS13 are present in the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP). Analysis of a set of antibodies derived from two patients with acquired TTP revealed frequent use of the VH1-69 heavy chain gene segment for the assembly of anti-ADAMTS13 antibodies. We explored the ability of two VH1-69 germline gene-encoded antibodies to inhibit the von Willebrand factor (VWF)-processing activity of ADAMTS13 under different experimental conditions. Furthermore, the presence of VH1-69 encoded anti-ADAMTS13 antibodies in 40 patients with acquired TTP was monitored using monoclonal antibody G8, which specifically reacts with an idiotype expressed on VH1-69 encoded antibodies. Binding of the two VH1-69 encoded monoclonal antibodies was dependent on the presence of the spacer domain. Both antibodies inhibited ADAMTS13 activity under static conditions, as measured by cleavage of FRETS-VWF73 substrate and cleavage of VWF multimers. The recombinant antibodies were also capable of inhibiting the processing of UL-VWF strings on the surface of endothelial cells. G8-reactive antibodies directed towards ADAMTS13 were present in plasma of all patients containing anti ADAMTS13 antibodies. These results suggest that VH1-69 derived antibodies directed towards ADAMTS13 develop in the majority of patients with acquired TTP.

Proteolytic Cleavage of Endothelial Cell-Bound Von Willebrand Factor Polymers by ADAMTS13 in the Absence of Flow Shear Stress

Degradation of newly released unusually large (UL)-vWF on endothelial cells by plasma ADAMTS13 metalloprotease is considered to be critical for maintaining normal hemostasis. An inability to degrade the cell bound string-like UL-vWF multimers by ADAMTS13 may result in excessive platelet adhesion and aggregation on cell surface, leading to thrombotic thrombocytopenic purpura (TTP). However, the requirement for shear stress and potential cofactors in this process is not fully defined. By immunofluorescent microscopy, we showed that upon histamine stimulation the newly released UL-vWF rapidly formed strings and bundles on human umbilical vein endothelial cells (HUVECs). These strings and bundles were removed within 2~5 minutes by plasma-derived and recombinant ADAMTS13 at concentrations of between 2.5 and 10 nM in 20 mM HEPES buffer, pH 7.5 containing 150 mM NaCl and 1 mM CaCl. This process did not appear to depend upon flow shear stress and coagulation factor VIII that has been shown to markedly increase the proteolytic cleavage of soluble vWF under high shear stress. The removal of the cell-bound UL-vWF strings and bundles correlated with the increases of vWF antigen and proteolytic cleavage fragments (176K and 140K) in the conditioned medium as determined by an enzyme-linked immunoassay and Western blot, respectively. The proteolytic cleavage of the cell bound UL-vWF by ADAMTS13 was time- and concentration-dependent, with a half maximal concentration of ADAMTS13 of approximately 5 nM. No cleavage product was detected when the histamine-stimulated HUVECs were incubated with the buffer alone or plasma from patients with acquired TTP and inhibitors or with heat-inactivated recombinant ADAMTS13. These results suggest that the degradation of cell bound UL-vWF is specific for ADAMTS13. Unexpectedly, the vWF multimers in the conditioned medium of ADAMTS13-treated endothelial cells were quite similar in sizes to those in the endothelial cells treated with histamine in the absence of ADAMTS13, indicating that the proteolytic cleavage of UL-vWF by ADASMTS13 occurs at the site proximal to cell membrane. Scanning electron microscopy clearly demonstrated that the breaking point of UL-vWF strings and bundles by ADAMTS13 was located at approximately 2~5 μm from the cell membrane. Structure-function analysis further demonstrated that the Cys-rich and spacer domains of ADAMTS13 were required for proteolytic cleavage of the cell-bound UL-vWF. The more distal carboxyl-terminal domains including TSP1 2–8 and CUB domains of ADAMTS13 appeared to be dispensable. We conclude that the cell bound UL-vWF polymers adopt an open conformation that is sensitive to ADAMTS13 proteolysis, analogous to those observed under the denaturing conditions. We hypothesize that membrane binding and/or interaction of UL-vWF bundles with certain scaffold proteins may provide an access of ADAMTS13 metalloprotease to the cleavage (Tyr1605-Met1606) bond at the central A2 domain of the vWF proximal to cell surface. These findings provide novel insight into the biological function of ADAMTS13 in processing UL-vWF and help understand the pathogenesis of TTP.

ADAMTS-13 Has a Disulfide Bond Reduction Activity on Von Willebrand Factor

Von Willebrand factor (VWF) multimers tether platelets to subendothelium exposed at the site of vessel injury to initiate the bleeding arrest. Upon synthesized, VWF multimers are either constitutively secreted or packed into the storage granules, where they are enriched in ultra-large (UL) multimers that are active in forming spontaneous high strength bonds with the GP Ib-IX-V complex on platelets. This hyper-reactivity of ULVWF multimers is in contrast to VWF multimers circulating in plasma (pVWF) that need to be activated by modulators or high fluid shear stress to aggregate platelets. The biochemical and structural bases for the functional difference between ULVWF and pVWF multimers are not known. We have recently shown that a portion of pVWF, but not ULVWF multimers contain surface exposed free thiols in the D3 and C domains. High fluid shear stress promotes the formation of new disulfide bonds utilizing the thiols to enhance VWF binding to platelets, suggesting that the shear-induced thiol-disulfide exchange may serve as a mechanism for the shear-induced activation of pVWF multimers. ULVWF freshly secreted from endothelial cells forms string-like structures that can be elongated by pVWF multimers through a covalent means. The different thiol distribution between ULVWF and its plasma counterpart may be caused by the former being cleaved by the zinc metalloprotease ADAMTS-13 at a single peptide bond of Y1065-M1606 in the A2 domain. Here, we provide several lines of evidence to demonstrate that ADAMTS-13 also contains a reductase-like activity that plays a role in cleaving ULVWF strings under flow conditions and maintaining circulating VWF multimers in an inactive (thiol) state. First, more than 90% of pVWF non-specifically adhered to the surface of a cone-plate viscometer when pVWF was exposed to a pathological high shear stress of 100 dyn/cm2 for 3 min at 37°C. The adhesion was prevented by recombinant (r) ADAMTS-13 or a truncation mutant that lacked the catalytic domain. Second, rADAMTS-13 prevented the shear-induced thiol-disulfide exchange so that free thiols remained in pVWF after shear exposure. This activity was not blocked by 5 mM of EDTA and was detectable with the N-terminal truncated mutant, suggesting that it is independent of the VWF-cleaving activity. We further found that rADAMTS-13 was able to reduce disulfide bonds, converting the disulfide forms of sheared pVWF to the thiol forms, suggesting that ADAMTS-13 prevents the thiol-disulfide exchange by disulfide bond reduction, not a steric hindered effect. Third, ADAMTS-13 contains the surface exposed thiol(s) that is necessary for the metalloprotease to attack and break a disulfide bond. Unlike VWF multimers, these thiols remained after the metalloprotease was exposed to a pathological high shear stress of 100 dyn/cm2. Fourth, using a series of N- and C-terminal truncation mutants, we located the thiol(s) potentially involved in VWF reduction to the 2nd to 8th TSP-1 motifs and CUB-1 domain of ADAMTS-13. Finally, ethylmaleimide (NEM), which blocks free thiols, did not inhibit rADAMTS-13 to cleave pVWF multimers under static conditions and in the presence of urea and barium. NEM-treated rADAMTS-13 retained only 27.5±4.9% activity in cleaving ULVWF strings under flow conditions as compared to untreated enzyme. These data characterizes a novel mechanism that plays a regulatory role in cleaving ULVWF strings and maintaining the circulating pVWF multimers in inactive forms.

Shear Stress Induced Increase in the Forced (UL)VWF Domain Unfolding Is Non-Proteolytically Regulated by ADAMTS-13

Hemostasis is initiated by tethering platelets to the site of vessel injury, a process mediated by the interaction of the GP Ib-IX-V complex on platelets and von Willebrand Factor (VWF) in the subendothelial matrix. The adhesion activity of VWF depends on the multimer size: those freshly secreted from activated endothelial cells are ultra-large (UL) and overly adhesive, capable of forming spontaneous high strength bonds with the platelet receptor. In contrast, VWF multimers circulating in blood (pVWF) is required to be activated by high fluid shear stress or modulators in order to bind and aggregate platelets. Shear stress has previously been demonstrated to convert globular shape VWF multimers to elongated rope-like structures, but whether this structural change correlates with VWF adhesion activity remains largely unknown. We have showed that, upon secretion, ULVWF multimers form long string-like structures on activated endothelial cells that can be viewed under a regular light microscope, suggesting laterally association between ULVWF multimers. Furthermore, pVWF multimers can be induced to laterally associate with each other by shear stress, potentially resulting in the formation of VWF fibrils. We hypothesize that by forming laterally associated strings, ULVWF multimers require a greater physical force to unfold as compared to pVWF, which can be induced to form fibrillar structures that become more resistant to force-induced unfolding. Here, we present experimental data to support this hypothesis. First, we found that shear stress covalently aggregated pVWF multimers and the process was prevented rADAMTS-13 or disulfide reducing agents. VWF multimers were not cleaved by ADAMTS-13 under this experimental condition, suggesting a non-proteolytic activity. Second, when pVWF multimers are captured and subjected to physical pulling force on an atomic force microscope, the length of VWF multimers extended sequentially in response to increasing force. Plasma VWF multimers were unfolded at the peak forces of 115 pN, which increased to 153 pN after they were exposed to a pathological level of shear stress (100 dyn/cm2) for 3 min at 37°C. The peak force required to unfold pVWF after shear exposure (153 pN) was very similar to that of ULVWF multimers (152 pN). Third, recombinant (r) ADAMTS-13 did not reduce the peak force for unfolding pVWF multimers (115 pN) before but it lessened the increase in magnitude in the peak force required to unfold pVWF exposed to shear stress. This ADAMTS-13 activity was not inhibited by 5 mM of EDTA. Similarly, the peak force for unfolding ULVWF multimers was reduced from 152 pN to 127 pN when ULVWF was pulled in the presence of an equal molar concentration of rADAMTS-13. Taken together, these data demonstrate that shear stress significantly increases the peak force required to unfold pVWF multimers to the levels similar to ULVWF multimers, likely by promoting the formation of laterally associated VWF fibrils. ADAMTS-13 prevents the shear-induced increase in the peak force for unfolding sheared pVWF multimers, independent of the VWF proteolytic activity of the metalloprotease. The results reveal a mechanism for shear-induced VWF activation. They also suggest that ADAMTS-13 contains a non-proteolytic activity that plays a role in cleaving ULVWF strings and preventing pVWF multimers to be activated by lateral association induced by high fluid shear stresses.

Covalent regulation of ULVWF string formation and elongation on endothelial cells under flow conditions.

The adhesion ligand von Willebrand factor (VWF) is a multimeric glycoprotein that mediates platelet adhesion to exposed subendothelium. On endothelial cells, freshly released ultra-large (UL) VWF multimers form long string-like structures to which platelets adhere. The formation and elongation of ULVWF strings were studied in the presence of the thiol-blocking N-ethylmaleimide (NEM). The presence of thiols in ULVWF and plasma VWF multimers was determined by maleimide-PEO(2)-Biotin labeling and thiol-chromatography. Finally, covalent re-multimerization of ULVWF was examined in a cell- and enzyme-free system. We found that purified plasma VWF multimers adhere to and elongate ULVWF strings under flow conditions. The formation and propagation of ULVWF strings were dose-dependently reduced by blocking thiols on VWF with NEM, indicating that ULVWF strings are formed by the covalent association of perfused VWF to ULVWF anchored to endothelial cells. The association is made possible by the presence of free thiols in VWF multimers and by the ability of (UL) VWF to covalently re-multimerize. The data provide a mechanism by which the thrombogenic ULVWF strings are formed and elongated on endothelial cells. This mechanism suggests that the thiol-disulfide state of ULVWF regulates the adhesion properties of strings on endothelial cells.

Magnesium maintains endothelial integrity, up-regulates proteolysis of ultra-large von Willebrand factor, and reduces platelet aggregation under flow conditions

Mg (++) regulates endothelial functions and has anti-inflammatory effects. Its effects on thrombosis have been demonstrated, but the mechanism remains poorly understood. We investigated the roles of MgSO(4) in regulating the release and cleavage of the prothrombotic ultra-large (UL) von Willebrand factor (VWF) and VWF-mediated platelet adhesion and aggregation. Washed platelets were perfused over cultured endothelial cells from human umbilical cord veins under a shear stress of 2.5 dyn/cm(2). Release and cleavage of ULVWF by ADAMTS-13 was measured in the absence or presence of physiological or therapeutic levels of MgSO(4). Whole blood or plasma-free reconstituted blood was perfused over immobilized collagen to measure the effect of MgSO(4) on platelet adhesion and aggregation. Also studied were the effects of MgSO(4) on ristocetin-induced platelet aggregation andVWF-collagen interaction. Maintenance of endothelial integrity required physiological levels of MgSO(4), but exogenous MgSO(4) showed no additional benefits. Exogenous MgSO(4) significantly enhanced the cleavage of the newly released ULVWF strings by ADAMTS-13 and markedly reduced platelet aggregation on immobilized collagen under flow conditions. This effect is likely to be mediated through VWF as Mg(++) partially inhibited ristocetin-induced platelet aggregation and VWF binding to collagen. MgSO(4) is critical for maintaining endothelial integrity and regulates ULVWF proteolysis and aggregation under flow conditions. These results provide a new insight into additional mechanisms involved with magnesium therapy.

Hemolytic uremic syndrome–associated Shiga toxins promote endothelial-cell secretion and impair ADAMTS13 cleavage of unusually large von Willebrand factor multimers

Shiga toxin 1 (Stx-1) and Stx-2 produced by enterohemorrhagic Escherichia coli cause the diarrhea-associated hemolytic uremic syndrome (HUS). This type of HUS is characterized by obstruction of the glomeruli and renal microvasculature by platelet-fibrin thrombi, acute renal failure, thrombocytopenia, microvascular hemolytic anemia, and plasma levels of von Willebrand factor (VWF)-cleaving protease (ADAMTS13) activity that are within a broad normal range. We investigated the mechanism of initial platelet accumulation on Stx-stimulated endothelial cells. Stx-1 or Stx-2 (1-10 nM) stimulated the rapid secretion of unusually large (UL) VWF multimeric strings from human umbilical vein endothelial cells (HUVECs) or human glomerular microvascular endothelial cells (GMVECs). Perfused normal human platelets immediately adhered to the secreted ULVWF multimeric strings. Nanomolar concentrations (1-10 nM) of the Shiga toxins were as effective in inducing the formation of ULVWF-platelet strings as millimolar concentrations (0.1-20 mM) of histamine. The rate of ULVWF-platelet string cleavage by plasma or recombinant ADAMTS13 was delayed by 3 to 10 minutes (or longer) in the presence of 10 nM Stx-1 or Stx-2 compared with 20 mM histamine. Stx-induced formation of ULVWF strings, and impairment of ULVWF-platelet string cleavage by ADAMTS13, may promote initial platelet adhesion above glomerular endothelial cells. These processes may contribute to the evolution of glomerular occlusion by platelet and fibrin thrombi in diarrhea-associated HUS.

Recombinant CUB-1 domain polypeptide inhibits the cleavage of ULVWF strings by ADAMTS13 under flow conditions

The metalloprotease ADAMTS13 (a disintegrin and metalloprotease with thrombospondin motif) converts the hyperreactive unusually large (UL) forms of von Willebrand factor (VWF) that are newly released from endothelial cells into less active plasma forms by cleaving a peptide bond in the VWF A2 domain. Familial or acquired deficiency of this metalloprotease is associated with thrombotic thrombocytopenic purpura (TTP). ADAMTS13 belongs to the ADAMTS metalloprotease family, but, unlike other members, it also contains 2 C-terminal CUB domains (complement component Clr/Cls, Uegf, and bone morphogenic protein 1). Mutations in the CUB region have been found in congenital TTP, but deletion of the region did not impair enzyme activity in conventional in vitro assays. We investigated the functions of the CUB domain in ADAMTS13 activity under flow conditions. We found that recombinant CUB-1 and CUB-1+2 polypeptides and synthetic peptides derived from CUB-1 partially blocked the cleavage of ULVWF by ADAMTS13 on the surface of endothelial cells under flow. The polypeptide bound immobilized and soluble forms of ULVWF, and blocked the adhesion of ADAMTS13-coated beads to immobilized ULVWF under flow. These results suggest that the CUB-1 domain may serve as the docking site for ADAMTS13 to bind ULVWF under flow, a critical step to initiate ULVWF proteolysis.

Shear stress and the role of high molecular weight von Willebrand factor multimers in thrombus formation

High molecular weight von Willebrand factor (VWF) multimers have an important role in the formation of platelet thrombi under conditions of high shear stress resulting from rapid blood flow. Laboratory studies conducted using an endothelial cell system have shown that ultra-large VWF multimers (ULVWF) attach to the surface of histamine-stimulated endothelial cells and form large string-like structures to which platelets adhere. Platelet attachment is mediated through the interaction of the glycoprotein (Gp) Ibalpha subunit of the platelet GpIb-IX-V complex with the A1 domain of VWF. These platelet-ULVWF strings are rapidly cleaved by the addition of normal human plasma or the purified plasma metalloprotease ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin type I motifs domains). Cleaving activity is absent from the plasma of patients with the congenital or acquired forms of thrombotic thrombocytopenic purpura (TTP) and from mixtures of normal plasma with plasma from patients with acquired TTP. The interaction of VWF with P-selectin is at least partially responsible for the attachment of the VWF strings to the endothelium. Increased tensile stress on ULVWF multimers resulting from the tethering of ULVWF strings to the endothelial surface by P-selectin in flowing blood may expose the cleavage site on the A2 domain of VWF, facilitating multimer degradation by ADAMTS-13. These studies have important implications in bleeding and thrombotic disorders, including von Willebrand disease and TTP.

Human Platelets Contain and Release an Active ADAMTS-13-Like Metalloprotease.

The adhesion ligand von Willebrand factor (VWF) is synthesized and stored in vascular endothelial cells and megakaryocytes/platelets. These cells release VWF in response to similar stimulations. However, a longstanding observation stated that, unlike endothelial cells, platelets do not release the ultra-large form (UL) of VWF upon activation. The lack of ULVWF release may be attributed to the difference in multimerization process in the storage granules or the intrinsic proteolysis of the hyperreactive ULVWF. To examine these possibilities, we analyzed the multimer compositions of VWF stored in and released from platelets. We found that ULVWF was detected by immunobloting of washed platelet lysates in the presence, but not in the absence of 5 mM EDTA. Similarly, when stimulated with 5 μM of thrombin receptor-activating peptide (TRAP), platelets released only VWF that was similar in sizes to plasma VWF, suggesting that platelet ULVWF may have been cleaved before or during its release. In support of this hypothesis, we found that platelets expressed ADAMTS-13-like molecule by western blot of platelet lysates and flow cytometry using two different ADAMTS-13 antibodies. The surface expression of ADAMTS-13-like molecule increased significantly upon platelet activation by TRAP and ADP. Consistent with these observations, the Triton-X-100 lysates of washed platelets and supernatant of washed platelets activated by TRAP contained VWF-cleaving activity, as demonstrated by the cleavage of ULVWF strings formed on histamine-stimulated endothelial cells under fluid shear stress. Our finding is consistent with a previous report, but further showed that this ADAMTS-13-like molecule exists not only in cytoplasma, but also on the surface of platelets. More importantly, the ADAMTS-13-like molecule increased its expression on platelet surface and was released upon platelet activation. The ADAMTS-13-like molecule either in platelets or released upon activation was active in cleaving ULVWF. This intrinsic mechanism for ULVWF proteolysis may be physiologically important. It may prevent the sudden release of hyper-reactive ULVWF from platelets and serve as the second pool of ADAMTS-13 to encounter the increase in ULVWF release from endothelial cells during conditions such as severe infection. The role of the platelet membrane-bound ADAMTS-13 in hemostasis remains to be further determined.

Defective processing of unusually large von Willebrand factor multimers and thrombotic thrombocytopenic purpura

Defective processing of unusually large von Willebrand factor multimers and thrombotic thrombocytopenic purpura

Variations among normal individuals in the cleavage of endothelial‐derived ultra‐large von Willebrand factor under flow

von Willebrand factor (VWF) freshly released from endothelial cells is normally cleaved by the ADAMTS-13 metalloprotease to prevent the direct release of these ultra-large (UL) and hyper-reactive multimers into plasma. The balance of ULVWF proteolysis may be regulated by the amount of ULVWF released and the processing capacity of ADAMTS-13. The former associates with the size of ULVWF storage pool, sensitivity of vascular endothelial cells to stimulation, and the type of agonists, whereas the latter associates with the activity of ADAMTS-13. These parameters may vary significantly among individuals. We have determined the variations of ADAMTS-13 activity in 68 normal individuals by a flow-based assay and a static assay using ULVWF strings and recombinant VWF A2 domain as substrates, respectively. We found that the levels of ADAMTS-13 activity required to cleave the platelet-decorated ULVWF strings under flow is significantly higher than that of static assays. Normal plasma diluted to 25% significantly reduced its ability to cleave ULVWF strings under flow, whereas 2% plasma retained 48% enzyme activity in static assay. ADAMTS-13 activity varied from 33 to 100% among individuals and the variations were greater at shorter incubations of plasma with the substrate. Furthermore, the production of ULVWF from endothelial cells also varied among individuals. These results suggest that the commonly used static assays may underestimate the ADAMTS-13 activity required to cleave newly released ULVWF. They also demonstrated that the proteolysis of ULVWF may vary significantly among individuals, potentially contributing to the individual's vulnerability to thrombosis so that measurement of ADAMTS-13 may serve as a marker for TTP and other thrombotic diseases.

P-selectin anchors newly released ultralarge von Willebrand factor multimers to the endothelial cell surface

Abstractvon Willebrand factor (VWF) released from endothelium is ultralarge (UL) and hyperreactive. If released directly into plasma, it can spontaneously aggregate platelets, resulting in systemic thrombosis. This disastrous consequence is prevented by the ADAMTS13 (ADisintegrin and Metalloprotease with ThromboSpondin motif) cleavage of ULVWF into smaller, less active forms. We previously showed that ULVWF, on release, forms extremely long stringlike structures. ADAMTS13 cleaves these strings under flow significantly faster than it does under static conditions. As ULVWF tethering to endothelium is important for its rapid proteolysis, we investigated 2 molecules for their potential to anchor the ULVWF strings: P-selectin and integrin αvβ3. We demonstrated that P-selectin anchors ULVWF to endothelium by several means. First, Chinese hamster ovary (CHO) cells expressing P-selectin specifically adhered to immobilized ULVWF and ULVWF-coated beads to immobilized P-selectin. Second, an anti-VWF antibody coimmunoprecipitates P-selectin from the histamine-activated endothelial cells. Third, P-selectin antibody or soluble P-selectin, but not a αvβ3 antibody, RGDS peptide, or heparin, blocked the formation of ULVWF strings. Fourth, P-selectin expression was in clusters predominantly along the ULVWF strings. Finally, the strength of the minimal ULVWF–P-selectin bond was measured to be 7.2 pN. We, therefore, conclude that P-selectin may anchor ULVWF strings to endothelial cells and facilitate their cleavage by ADAMTS13.

ADAMTS-13 rapidly cleaves newly secreted ultralarge von Willebrand factor multimers on the endothelial surface under flowing conditions

Thrombotic thrombocytopenic purpura (TTP) is a devastating thrombotic disorder caused by widespread microvascular thrombi composed of platelets and von Willebrand factor (VWF). The disorder is associated with a deficiency of the VWF-cleaving metalloprotease, ADAMTS-13, with consequent accumulation of ultralarge (UL) VWF multimers in the plasma. ULVWF multimers, unlike plasma forms of VWF, attach spontaneously to platelet GP Ibalpha, a component of the GP Ib-IX-V complex. We have found that ULVWF multimers secreted from stimulated endothelial cells (ECs) remained anchored to the endothelial surface where platelets and Chinese hamster ovary cells expressing the GP Ib-IX-V complex attached to form long beads-on-a-string structures in the presence of fluid shear stresses in both the venous (2.5 dyne/cm(2)) and arterial (20 and 50 dyne/cm(2)) ranges. Although measurement of the activity of the ADAMTS-13 VWF-cleaving metalloprotease in vitro requires prolonged incubation of the enzyme with VWF under nonphysiologic conditions, EC-derived ULVWF strings with attached platelets were cleaved within seconds to minutes in the presence of normal plasma (containing approximately 100% ADAMTS-13 activity) or in the presence of partially purified ADAMTS-13. By contrast, the strings persisted for the entire period of perfusion (10 minutes) in the presence of plasma from patients with TTP containing 0% to 10% ADAMTS-13 activity. These results suggest that cleavage of EC-derived ULVWF multimers by ADAMTS-13 is a rapid physiologic process that occurs on endothelial cell surfaces.