Circulating Iron in Patients with Sickle Cell Disease Mediates the Release of Neutrophil Extracellular Traps.

Introduction: Neutrophils promote chronic inflammation and release neutrophil extracellular traps (NETs) that can drive inflammatory responses. Inflammation influences progression of sickle cell disease (SCD), and a role for NETs has been suggested in the onset of vaso-occlusive crisis (VOC). We aimed to identify factors in the circulation of these patients that provoke NET release, with a focus on triggers associated with hemolysis. Methods: Paired serum and plasma samples during VOC and steady state of 18 SCD patients (HbSS/HbSß0-thal and HbSC/HbSß+-thal) were collected. Cell-free heme, hemopexin, and labile plasma iron have been measured in the plasma samples of the SCD patients. NETs formation by human neutrophils from healthy donors induced by serum of SCD patients was studied using confocal microscopy and staining for extracellular DNA using Sytox, followed by quantification of surface coverage using ImageJ. Results: Eighteen patients paired samples obtained during VOC and steady state were available (11 HbSS/HbSß0-thal and 7 HbSC/HbSß+-thal). We observed high levels of systemic heme and iron, concomitant with low levels of the heme-scavenger hemopexin in sera of patients with SCD, both during VOC and in steady state. In our in vitro experiments, neutrophils released NETs when exposed to sera from SCD patients. The release of NETs was associated with high levels of circulating iron in these sera. Although hemin triggered NET formation in vitro, addition of hemopexin to scavenge heme did not suppress NET release in SCD sera. By contrast, the iron scavengers deferoxamine and apotransferrin attenuated NET formation in a significant proportion of SCD sera. Discussion: Our results suggest that redox-active iron in the circulation of non-transfusion-dependent SCD patients activates neutrophils to release NETs, and hence, exerts a direct pro-inflammatory effect. Thus, we propose that chelation of iron requires further investigation as a therapeutic strategy in SCD.

Impact of N-glycan mediated shielding of ADAMTS-13 on the binding of pathogenic antibodies in immune thrombotic thrombocytopenic purpura.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a rare thrombotic disorder, with 1.5 to 6.0 cases per million per year. The majority of patients with TTP develop inhibitory autoantibodies that predominantly target the spacer domain of ADAMTS-13. ADAMTS-13 is responsible for cleaving von Willebrand factor (VWF) multimers, thereby regulating platelet adhesion at sites of high-vascular shear stress. Inhibition and/or clearance of ADAMTS-13 by pathogenic autoantibodies results in accumulation of VWF multimers that promotes the formation of platelet-rich microthrombi. Previously, we have shown that insertion of a single N-glycan (NGLY) in the spacer domain prevents the binding of antispacer domain antibodies. OBJECTIVES: To explore whether NGLY mediated shielding of the ADAMTS-13 spacer domain effectively prevents binding of pathogenic antispacer autoantibodies in patients with immune-mediated TTP (iTTP). METHODS: We screened 5 NGLY-ADAMTS-13 variants (NGLY3, NGLY7, NGLY8, NGLY3+7, and NGLY3+8) for binding of autoantibodies and for their activity in the presence and absence of 50 samples derived from patients with iTTP. RESULTS: NGLY variants showed greatly reduced antibody binding, down to 27% of wild-type (wt) ADAMTS-13 binding. Moreover, NGLY variants of ADAMTS-13 remained more active in FRETS-VWF73 assay in the presence of the plasma samples from these 50 patients with acute phase iTTP when compared with wtADAMTS-13. On average, wtADAMTS-13 activity was reduced to 37% of regular levels in the presence of plasma, while NGLY3 and NGLY3+7 remained 69% and 81% active, respectively. CONCLUSION: These results reinforce our previous findings that NGLYs shield ADAMTS-13 from antibody binding and hence restore ADAMTS-13 activity in the presence of autoantibodies.

Searching for a Common Thrombo-Inflammatory Basis in Patients With Deep Vein Thrombosis or Peripheral Artery Disease.

Background: Inflammation and hypercoagulability play a pivotal role in venous thromboembolism and atherothrombosis. Since venous thrombosis increases the risk of atherothrombotic events and vice versa, common mechanisms may be involved. Objectives: To elucidate the role of neutrophils and coagulation in the occurrence of atherothrombotic events in patients with a history of deep vein thrombosis (DVT or peripheral artery disease (PAD). Materials and Methods: We studied 115 patients from two cohorts (75 DVT, 40 PAD). From those with PAD, 20 patients had progressive disease; from those with DVT, 25 patients had a recurrent DVT and 25 suffered from post thrombotic syndrome (PTS); patients were age and sex matched to DVT and PAD patients without events. Markers of neutrophil recruitment (p-selectin) and activation [nucleosomes, human neutrophil elastase- α1anti-trypsin (HNE-AT)], an anti-inflammatory marker (Lipoxin A4) and a clotting activity marker (d-dimer), were measured with ELISA. Coagulation potential was analyzed by thrombin generation (CAT method). Results: Higher nucleosome levels were found in DVT patients [11.3 U/mL (7.4-17.7)] compared to PAD patients [7.1 U/mL (5.1-13.8)], lower HNE-AT levels were found in DVT patients [33.4 ng/mL (23.5-40.5)] in comparison to PAD patients [158 ng/mL (88.1-283)]. No difference in nucleosome levels was found between DVT patients with cardiovascular (CV) events [12.6 U/mL (8.2-16.1)], and PAD patients with CV events [6.9 U/mL (4.9-11.2)]. Lipoxin A4 levels appeared to be significantly lower in DVT [2.4 ng/mL (1.7-4.8)] vs. PAD [35.6 ng/mL (16.6-80.1)], with similar results in DVT patients with CV events vs. PAD patients with CV events. Thrombin generation showed higher ETP [160.4% (141.1-215.4)], and peak height [292.1% (177.9-330)] values in DVT patients. D-dimer levels were significantly lower in the DVT cohort [330 ng/mL (220-550)] compared to the PAD cohort [550 ng/mL (369-959)]. Conclusion: In DVT patients, neutrophil activity does not appear to be an important driver of CV events. Although neutrophil activity is more pronounced in PAD, its effect is partly dampened by Lipoxin A4. Moreover, no associations were found between NET products and coagulation activity, suggesting that neutrophil activation does not play a pivotal role in the risk of thrombosis in either DVT or PAD.

Mouse venous thrombosis upon silencing of anticoagulants depends on tissue factor and platelets, not FXII or neutrophils.

Tissue factor, coagulation factor XII, platelets, and neutrophils are implicated as important players in the pathophysiology of (experimental) venous thrombosis (VT). Their role became evident in mouse models in which surgical handlings were required to provoke VT. Combined inhibition of the natural anticoagulants antithrombin (Serpinc1) and protein C (Proc) using small interfering RNA without additional triggers also results in a venous thrombotic phenotype in mice, most notably with vessel occlusion in large veins of the head. VT is fatal but is fully rescued by thrombin inhibition. In the present study, we used this VT mouse model to investigate the involvement of tissue factor, coagulation factor XII, platelets, and neutrophils. Antibody-mediated inhibition of tissue factor reduced the clinical features of VT, the coagulopathy in the head, and fibrin deposition in the liver. In contrast, genetic deficiency in, and small interfering RNA-mediated depletion of, coagulation factor XII did not alter VT onset, severity, or thrombus morphology. Antibody-mediated depletion of platelets fully abrogated coagulopathy in the head and liver fibrin deposition. Although neutrophils were abundant in thrombotic lesions, depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, thrombus morphology, or liver fibrin deposition. In conclusion, VT after inhibition of antithrombin and protein C is dependent on the presence of tissue factor and platelets but not on coagulation factor XII and circulating neutrophils. This study shows that distinct procoagulant pathways operate in mouse VT, dependent on the triggering stimulus.

The damage-associated molecular pattern HMGB1 is released early after clinical hepatic ischemia/reperfusion.

OBJECTIVE AND BACKGROUND: Activation of sterile inflammation after hepatic ischemia/reperfusion (I/R) culminates in liver injury. The route to liver damage starts with mitochondrial oxidative stress and cell death during early reperfusion. The link between mitochondrial oxidative stress, damage-associate molecular pattern (DAMP) release, and sterile immune signaling is incompletely understood and lacks clinical validation. The aim of the study was to validate this relation in a clinical liver I/R cohort and to limit DAMP release using a mitochondria-targeted antioxidant in I/R-subjected mice. METHODS: Plasma levels of the DAMPs high-mobility group box 1 (HMGB1), mitochondrial DNA, and nucleosomes were measured in 39 patients enrolled in an observational study who underwent a major liver resection with (N = 29) or without (N = 13) intraoperative liver ischemia. Circulating cytokine and neutrophil activation markers were also determined. In mice, the mitochondria-targeted antioxidant MitoQ was intravenously infused in an attempt to limit DAMP release, reduce sterile inflammation, and suppress I/R injury. RESULTS: In patients, HMGB1 was elevated following liver resection with I/R compared to liver resection without I/R. HMGB1 levels correlated positively with ischemia duration and peak post-operative transaminase (ALT) levels. There were no differences in mitochondrial DNA, nucleosome, or cytokine levels between the two groups. In mice, MitoQ neutralized hepatic oxidative stress and decreased HMGB1 release by ±50%. MitoQ suppressed transaminase release, hepatocellular necrosis, and cytokine production. Reconstituting disulfide HMGB1 during reperfusion reversed these protective effects. CONCLUSION: HMGB1 seems the most pertinent DAMP in clinical hepatic I/R injury. Neutralizing mitochondrial oxidative stress may limit DAMP release after hepatic I/R and reduce liver damage.

Autoregulation of von Willebrand factor function by a disulfide bond switch.

Force-dependent binding of platelet glycoprotein Ib (GPIb) receptors to plasma von Willebrand factor (VWF) plays a key role in hemostasis and thrombosis. Previous studies have suggested that VWF activation requires force-induced exposure of the GPIb binding site in the A1 domain that is autoinhibited by the neighboring A2 domain. However, the biochemical basis of this "mechanopresentation" remains elusive. From a combination of protein chemical, biophysical, and functional studies, we find that the autoinhibition is controlled by the redox state of an unusual disulfide bond near the carboxyl terminus of the A2 domain that links adjacent cysteine residues to form an eight-membered ring. Only when the bond is cleaved does the A2 domain bind to the A1 domain and block platelet GPIb binding. Molecular dynamics simulations indicate that cleavage of the disulfide bond modifies the structure and molecular stresses of the A2 domain in a long-range allosteric manner, which provides a structural explanation for redox control of the autoinhibition. Significantly, the A2 disulfide bond is cleaved in ~75% of VWF subunits in healthy human donor plasma but in just ~25% of plasma VWF subunits from heart failure patients who have received extracorporeal membrane oxygenation support. This suggests that the majority of plasma VWF binding sites for platelet GPIb are autoinhibited in healthy donors but are mostly available in heart failure patients. These findings demonstrate that a disulfide bond switch regulates mechanopresentation of VWF.

Coagulation factor XI improves host defence during murine pneumonia-derived sepsis independent of factor XII activation.

Bacterial pneumonia, the most common cause of sepsis, is associated with activation of coagulation. Factor XI (FXI), the key component of the intrinsic pathway, can be activated via factor XII (FXII), part of the contact system, or via thrombin. To determine whether intrinsic coagulation is involved in host defence during pneumonia and whether this is dependent on FXII activation, we infected in parallel wild-type (WT), FXI knockout (KO) and FXII KO mice with two different clinically relevant pathogens, the Gram-positive bacterium Streptococcus pneumoniae and the Gram-negative bacterium Klebsiella pneumoniae, via the airways. FXI deficiency worsened survival and enhanced bacterial outgrowth in both pneumonia models. This was accompanied with enhanced inflammatory responses in FXI KO mice. FXII KO mice were comparable with WT mice in Streptococcus pneumoniae pneumonia. On the contrary, FXII deficiency improved survival and reduced bacterial outgrowth following infection with Klebsiella pneumoniae. In both pneumonia models, local coagulation was not impaired in either FXI KO or FXII KO mice. The capacity to phagocytose bacteria was impaired in FXI KO neutrophils and in human neutrophils where activation of FXI was inhibited. Deficiency for FXII or blocking activation of FXI via FXIIa had no effect on phagocytosis. Taken together, these data suggest that FXI protects against sepsis derived from Streptococcus pneumoniae or Klebsiella pneumoniae pneumonia at least in part by enhancing the phagocytic capacity of neutrophils by a mechanism that is independent of activation via FXIIa.

DNA and factor VII-activating protease protect against the cytotoxicity of histones.

Circulating histones have been implicated as major mediators of inflammatory disease because of their strong cytotoxic effects. Histones form the protein core of nucleosomes; however, it is unclear whether histones and nucleosomes are equally cytotoxic. Several plasma proteins that neutralize histones are present in plasma. Importantly, factor VII-activating protease (FSAP) is activated upon contact with histones and subsequently proteolyzes histones. We aimed to determine the effect of FSAP on the cytotoxicity of both histones and nucleosomes. Indeed, FSAP protected against histone-induced cytotoxicity of cultured cells in vitro. Upon incubation of serum with histones, endogenous FSAP was activated and degraded histones, which also prevented cytotoxicity. Notably, histones as part of nucleosome complexes were not cytotoxic, whereas DNA digestion restored cytotoxicity. Histones in nucleosomes were inefficiently cleaved by FSAP, which resulted in limited cleavage of histone H3 and removal of the N-terminal tail. The specific isolation of either circulating nucleosomes or free histones from sera of Escherichia coli challenged baboons or patients with meningococcal sepsis revealed that histone H3 was present in the form of nucleosomes, whereas free histone H3 was not detected. All samples showed signs of FSAP activation. Markedly, we observed that all histone H3 in nucleosomes from the patients with sepsis, and most histone H3 from the baboons, was N-terminally truncated, giving rise to a similarly sized protein fragment as through cleavage by FSAP. Taken together, our results suggest that DNA and FSAP jointly limit histone cytotoxicity and that free histone H3 does not circulate in appreciable concentrations in sepsis.

Extracellular histones, cell-free DNA, or nucleosomes: differences in immunostimulation.

In inflammation, extensive cell death may occur, which results in the release of chromatin components into the extracellular environment. Individually, the purified chromatin components double stranded (ds)DNA and histones have been demonstrated, both in vitro and in vivo, to display various immunostimulatory effects, for example, histones induce cytotoxicity and proinflammatory signaling through toll-like receptor (TLR)2 and 4, while DNA induces signaling through TLR9 and intracellular nucleic acid sensing mechanisms. However, DNA and histones are organized in nucleosomes in the nucleus, and evidence suggests that nucleosomes are released as such in inflammation. The cytotoxicity and proinflammatory signaling induced by nucleosomes have not been studied as extensively as the separate effects brought about by histones and dsDNA, and there appear to be some marked differences. Remarkably, little distinction between the different forms in which histones circulate has been made throughout literature. This is partly due to the limitations of existing techniques to differentiate between histones in their free or DNA-bound form. Here we review the current understanding of immunostimulation induced by extracellular histones, dsDNA and nucleosomes, and discuss the importance of techniques that in their detection differentiate between these different chromatin components.

Role of platelets, neutrophils, and factor XII in spontaneous venous thrombosis in mice.

Recently, platelets, neutrophils, and factor XII (FXII) have been implicated as important players in the pathophysiology of venous thrombosis. Their role became evident in mouse models in which surgical handling was used to provoke thrombosis. Inhibiting anticoagulation in mice by using small interfering RNA (siRNA) targeting Serpinc1 and Proc also results in a thrombotic phenotype, which is spontaneous (no additional triggers) and reproducibly results in clots in the large veins of the head and fibrin deposition in the liver. This thrombotic phenotype is fatal but can be fully rescued by thrombin inhibition. The mouse model was used in this study to investigate the role of platelets, neutrophils, and FXII. After administration of siRNAs targeting Serpinc1 and Proc, antibody-mediated depletion of platelets fully abrogated the clinical features as well as microscopic aspects in the head. This was corroborated by strongly reduced fibrin deposition in the liver. Whereas neutrophils were abundant in siRNA-triggered thrombotic lesions, antibody-mediated depletion of circulating Ly6G-positive neutrophils did not affect onset, severity, or thrombus morphology. In addition, absence of circulating neutrophils did not affect quantitative liver fibrin deposition. Remarkably, siRNA-mediated depletion of plasma FXII accelerated the onset of the clinical phenotype; mice were affected with more severe thrombotic lesions. To summarize, in this study, onset and severity of the thrombotic phenotype are dependent on the presence of platelets but not circulating neutrophils. Unexpectedly, FXII has a protective effect. This study challenges the proposed roles of neutrophils and FXII in venous thrombosis pathophysiology.

FSAP-mediated nucleosome release from late apoptotic cells is inhibited by autoantibodies present in SLE.

Inefficient clearance of apoptotic cells and the subsequent exposure of the immune system to nuclear contents are crucially involved in the pathogenesis of systemic lupus erythematosus (SLE). Factor VII-activating protease (FSAP) is activated in serum upon contact with dead cells, and releases nucleosomes from late apoptotic cells into the extracellular environment. We investigated whether FSAP-mediated nucleosome release from late apoptotic cells is affected in SLE patients. Nucleosome release in sera of 27 SLE patients and 30 healthy controls was investigated by incubating late apoptotic Jurkat cells with serum and analyzing the remaining DNA content by flow cytometry. We found that nucleosome release in sera of SLE patients with high disease activity was significantly decreased when compared with that in SLE sera obtained during low disease activity or from healthy individuals. Upon removal of IgG/IgM antibodies from SLE sera, nucleosome release was restored. Similarly, monoclonal antinuclear antibodies inhibited nucleosome release in healthy donor serum or by plasma-purified FSAP. This inhibition was lost when Fab fragments were used, suggesting that antigen cross-linking is involved. In conclusion, FSAP-mediated nucleosome release from late apoptotic cells is greatly impaired in SLE patient sera, possibly hampering the clearance of these cells and thereby propagating inflammation.

Conformational activation of ADAMTS13.

A disintegrin and metalloprotease with thrombospondin motifs 13 (ADAMTS13) is a metalloprotease that regulates von Willebrand factor (VWF) function. ADAMTS13-mediated proteolysis is determined by conformational changes in VWF, but also may depend on its own conformational activation. Kinetic analysis of WT ADAMTS13 revealed ∼ 2.5-fold reduced activity compared with ADAMTS13 lacking its C-terminal tail (MDTCS) or its CUB1-2 domains (WTΔCUB1-2), suggesting that the CUB domains naturally limit ADAMTS13 function. Consistent with this suggestion, WT ADAMTS13 activity was enhanced ∼ 2.5-fold by preincubation with either an anti-CUB mAb (20E9) or VWF D4CK (the natural binding partner for the CUB domains). Furthermore, the isolated CUB1-2 domains not only bound MDTCS, but also inhibited activity by up to 2.5-fold. Interestingly, a gain-of-function (GoF) ADAMTS13 spacer domain variant (R568K/F592Y/R660K/Y661F/Y665F) was ∼ 2.5-fold more active than WT ADAMTS13, but could not be further activated by 20E9 mAb or VWF D4CK and was unable to bind or to be inhibited by the CUB1-2 domains, suggesting that the inhibitory effects of the CUB domains involve an interaction with the spacer domain that is disrupted in GoF ADAMTS13. Electron microscopy demonstrated a "closed" conformation of WT ADAMTS13 and suggested a more "open" conformation for GoF ADAMTS13. The cryptic spacer domain epitope revealed by conformational unfolding also represents the core antigenic target for autoantibodies in thrombotic thrombocytopenic purpura. We propose that ADAMTS13 circulates in a closed conformation, which is maintained by a CUB-spacer domain binding interaction. ADAMTS13 becomes conformationally activated on demand through interaction of its C-terminal CUB domains with VWF, making it susceptible to immune recognition.

Control of VWF A2 domain stability and ADAMTS13 access to the scissile bond of full-length VWF.

Rheological shear forces in the blood trigger von Willebrand factor (VWF) unfolding which exposes the Y1605-M1606 scissile bond within the VWF A2 domain for cleavage by ADAMTS13. The VWF A2 domain contains 2 structural features that provide it with stability: a vicinal disulphide bond and a Ca(2+)-binding site (CBS). We investigated how these 2 structural features interplay to determine stability and regulate the exposure of the scissile bond in full-length VWF. We have used differential scanning fluorimetry together with site-directed mutagenesis of residues involved in both the vicinal disulphide bond and the CBS to demonstrate that both of these sites contribute to stability against thermal unfolding of the isolated VWF A2 domain. Moreover, we show that the combination of site mutations can result in increased susceptibility of FL-VWF to proteolysis by ADAMTS13, even in the absence of an agent (such as urea) required to induce unfolding. These studies demonstrate that VWF A2 domain stability provided by its 2 structural elements (vicinal disulphide bond and CBS) is a key protective determinant against FL-VWF cleavage by ADAMTS13. They suggest a 2-step mechanism for VWF A2 domain unfolding.

Evidence for a role of anti-ADAMTS13 autoantibodies despite normal ADAMTS13 activity in recurrent thrombotic thrombocytopenic purpura.

BACKGROUND: Severe ADAMTS13 deficiency is a critical component of the pathogenesis of idiopathic thrombotic thrombocytopenic purpura but is found only in about 60% of patients clinically diagnosed with this disease. DESIGN AND METHODS: Over a period of 8 years and six episodes of thrombotic thrombocytopenic purpura we studied the evolution of the anti-ADAMTS13 antibody response in a patient using different ADAMTS13 assays and epitope mapping. RESULTS: Anti-ADAMTS13 autoantibodies were found in all episodes but were inhibitory only in the last two episodes. In a flow-based assay, normal ADAMTS13 activity was found only during the first disease episode, while ADAMTS13 activity was normal using a static assay in episodes 1 and 3, and severely deficient in the last two episodes. Fluorescence evolution in a modified fluorescence resonance energy transfer assay using a von Willebrand factor A2 domain peptide substrate was linear in episodes 1, 5 and 6, but increased exponentially in episodes 3 and 4. Despite the variable functional characteristics of the anti-ADAMTS13 autoantibodies, their principal epitope was the ADAMTS13 spacer domain in all episodes. CONCLUSIONS: The patient is unique as he displayed features of maturation or shaping of the anti-ADAMTS13 autoantibody response during the course of multiple episodes of thrombotic thrombocytopenic purpura. Anti-ADAMTS13 autoantibodies may be important in vivo despite normal ADAMTS13 activity in routine assays. Consequently, treatment decisions should not be based solely on activity assay results.

High VWF, low ADAMTS13, and oral contraceptives increase the risk of ischemic stroke and myocardial infarction in young women.

VWF and ADAMTS13 are major determinants of platelet adhesion after vessel injury. In the present study, we aimed to determine whether VWF or ADAMTS13 plasma antigen levels influence the risks of ischemic stroke (IS) or myocardial infarction (MI) in young women and how these risks are affected by oral contraceptive (OC) use. VWF and ADAMTS13 plasma antigen levels were measured in a frequency-matched case-control study of 1018 young (18-49 years) women including 175 IS patients and 205 MI patients. Increasing levels of VWF and decreasing levels of ADAMTS13 were associated with the risk of IS and MI in a dose-dependent manner. Having both high VWF and low ADAMTS13 resulted in an odds ratio (OR) of 6.9 (95% confidence interval [95% CI], 2.0-23.0) for IS and 11.3 (95% CI, 3.6-35.2) for MI. Use of OCs increased the risk of IS and MI associated with high VWF (OR = 12; 95% CI, 5.5-26.2 and OR = 7.5, 95% CI, 3.6-15.7, respectively) and the risk of IS associated with low ADAMTS13 (OR = 5.8, 95% CI, 2.7-12.4). We conclude that high VWF and low ADAMTS13 plasma levels both increase the risk of IS and MI. The risks associated with high VWF or low ADAMTS13 levels are further increased by the use of OCs.

Unraveling the scissile bond: how ADAMTS13 recognizes and cleaves von Willebrand factor.

von Willebrand factor (VWF) is a large adhesive glycoprotein with established functions in hemostasis. It serves as a carrier for factor VIII and acts as a vascular damage sensor by attracting platelets to sites of vessel injury. VWF size is important for this latter function, with larger multimers being more hemostatically active. Functional imbalance in multimer size can variously cause microvascular thrombosis or bleeding. The regulation of VWF multimeric size and platelet-tethering function is carried out by ADAMTS13, a plasma metalloprotease that is constitutively active. Unusually, protease activity of ADAMTS13 is controlled not by natural inhibitors but by conformational changes in its substrate, which are induced when VWF is subject to elevated rheologic shear forces. This transforms VWF from a globular to an elongated protein. This conformational transformation unfolds the VWF A2 domain and reveals cryptic exosites as well as the scissile bond. To enable VWF proteolysis, ADAMTS13 makes multiple interactions that bring the protease to the substrate and position it to engage with the cleavage site as this becomes exposed by shear. This article reviews recent literature on the interaction between these 2 multidomain proteins and provides a summary model to explain proteolytic regulation of VWF by ADAMTS13.

The importance of vicinal cysteines, C1669 and C1670, for von Willebrand factor A2 domain function.

The von Willebrand factor (VWF) A2 crystal structure has revealed the presence of a rare vicinal disulfide bond between C1669 and C1670, predicted to influence domain unfolding required for proteolysis by ADAMTS13. We prepared VWF A2 domain fragments with (A2-VicCC, residues 1473-1670) and without the vicinal disulfide bond (A2-DeltaCC, residues 1473-1668). Compared with A2-DeltaCC, A2-VicCC exhibited impaired proteolysis and also reduced binding to ADAMTS13. Circular dichroism studies revealed that A2-VicCC was more resistant to thermal unfolding than A2-DeltaCC. Mutagenesis of C1669/C1670 in full-length VWF resulted in markedly increased susceptibility to cleavage by ADAMTS13, confirming the important role of the paired vicinal cysteines in VWF A2 domain stabilization.

Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain.

Protein S has an established role in the protein C anticoagulant pathway, where it enhances the factor Va (FVa) and factor VIIIa (FVIIIa) inactivating property of activated protein C (APC). Despite its physiological role and clinical importance, the molecular basis of its action is not fully understood. To clarify the mechanism of the protein S interaction with APC, we have constructed and expressed a library of composite or point variants of human protein S, with residue substitutions introduced into the Gla, thrombin-sensitive region (TSR), epidermal growth factor 1 (EGF1), and EGF2 domains. Cofactor activity for APC was evaluated by calibrated automated thrombography (CAT) using protein S-deficient plasma. Of 27 variants tested initially, only one, protein S D95A (within the EGF1 domain), was largely devoid of functional APC cofactor activity. Protein S D95A was, however, gamma-carboxylated and bound phospholipids with an apparent dissociation constant (Kd(app)) similar to that of wild-type (WT) protein S. In a purified assay using FVa R506Q/R679Q, purified protein S D95A was shown to have greatly reduced ability to enhance APC-induced cleavage of FVa Arg306. It is concluded that residue Asp95 within EGF1 is critical for APC cofactor function of protein S and could define a principal functional interaction site for APC.

An autoantibody epitope comprising residues R660, Y661, and Y665 in the ADAMTS13 spacer domain identifies a binding site for the A2 domain of VWF.

In the majority of patients with acquired thrombotic thrombocytopenic purpura (TTP), antibodies are directed toward the spacer domain of ADAMTS13. We have previously shown that region Y658-Y665 is involved. We now show that replacement of R660, Y661, or Y665 with alanine in ADAMTS13 reduced/abolished the binding of 2 previously isolated human monoclonal antibodies and polyclonal antibodies derived from plasma of 6 patients with acquired TTP. We investigated whether these residues also influenced cleavage of short von Willebrand factor (VWF) fragment substrate VWF115. An ADAMTS13 variant (R660A/Y661A/Y665A, ADAMTS13-RYY) showed a 12-fold reduced catalytic efficiency (k(cat)/K(m)) arising from greatly reduced (> 25-fold) binding, demonstrated by surface plasmon resonance. The influence of these residue changes on full-length VWF was determined with denaturing and flow assays. ADAMTS13-RYY had reduced activity in both, with proteolysis of VWF unaffected by autoantibody. Binding of ADAMTS13-RYY mutant to VWF was, however, similar to normal. Our results demonstrate that residues within Y658-Y665 of the ADAMTS13 spacer domain that are targeted by autoantibodies in TTP directly interact with a complementary exosite (E1660-R1668) within the VWF A2 domain. Residues R660, Y661, and Y665 are critical for proteolysis of short VWF substrates, but wider domain interactions also make important contributions to cleavage of full-length VWF.