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O estudo aqui apresentado traz reflexões sobre moradia e laço social partindo de entrevistas realizadas com pessoas moradoras do bairro Jardim Ângela e de experiências profissionais anteriores. Apesar da moradia ser um dos recursos organizadores da vida social, assim como o acesso à educação, à saúde e ao trabalho, nos deparamos na atualidade com uma situação de vulnerabilidade alarmante em que milhões de famílias sofrem com a falta de serviços básicos no Brasil. Através da análise de discurso foucaultiana e à luz da psicanálise, é desenvolvida a análise sobre laço social, destacando a importância destas relações no debate sobre moradia. Esta investigação centrou-se nos temas que surgiram nas entrevistas, mais especificamente no que se referiam a território, família e trajetória, e apontou a importância da leitura do laço social no entendimento da moradia seja como mercadoria, espaço que segrega, como que gera sentimento de pertencimento e proteção, que remete à lembranças e promove o encontro com si mesmo e o outro. The present study reflects on housing and social bonds bringing up relevant elements to studies on this subject. Those reflections come from interviews taken with people living in Jardim Ângela neighborhood, including some of previous professionals\' experience scenes. Although housing is one of social life organizing resources, as well as the access to education, health and work, we face an alarming vulnerability situation, in which millions of families in Brazil suffer from lack of services as sanitation. Based on a Foucault\'s discourse analysis and from a psychoanalysis point of view, we developed the analysis about social bonds, highlighting the importance of relationships on housing debates. This investigation centered on the themes that emerged during the interviews, specifically those related to territory, family and path. The analysis pointed out the importance of looking into the social bonds in the understanding of housing as a commodity, as a space that segregates, that generates a feeling of belonging and protection, that refers to memories and promotes the encounter with oneself and the other

Activation of proteins often involves conformational transitions, and these switches are often difficult to characterize in multidomain proteins. Full-length factor H (FH), consisting of 20 small consensus repeat domains (150 kD), is a complement control protein that regulates the activity of the alternative complement pathway. Different preparations of FH can also reduce the disulfide bonds linking large Von Willebrand factor (VWF) multimers into smaller, less adhesive forms. In contrast, commercially available purified FH (pFH) has little or no VWF reductase activity unless the pFH is chemically modified by either ethylenediaminetetraacetic acid (EDTA) or urea. We used atomic force microscopy single molecule force measurements to investigate different forms of FH, including recombinant FH and pFH, in the presence or absence of EDTA and urea, and to correlate the conformational changes to its activities. We found that the FH conformation depends on the method used for sample preparation, which affects the VWF reductase activity of FH.

von Willebrand factor (VWF) is a multimeric protein composed of monomeric subunits (∼280 kD) linked by disulfide bonds. During hemostasis and thrombosis, ultralarge (UL) VWF (ULVWF) multimers initiate platelet adhesion. In vitro, human C3 binds to ULVWF multimeric strings secreted by and anchored to human endothelial cell to promote the assembly and activation of C3 convertase (C3bBb) and C5 convertase (C3bBbC3b) of the alternative complement pathway (AP). The purified and soluble C3 avidly binds to recombinant human VWF A1A2A3, as well as the recombinant isolated human VWF A3 domain. Notably, the binding of soluble human ULVWF multimers to purified human C3 was blocked by addition of a monovalent Fab fragment antibody to the VWF A3 domain. We conclude that the A3 domain in VWF/ULVWF contains a docking site for C3. In contrast, purified human C4, an essential component of the classical and lectin complement pathways, binds to soluble, isolated A1, but not to ULVWF strings secreted by and anchored to endothelial cells. Our findings should facilitate the design of new therapeutic agents to suppress the initiation of the AP on ULVWF multimeric strings during thrombotic and inflammatory disorders.

Ultralarge von Willebrand factor (ULVWF) multimeric strings are rapidly secreted by, and anchored to, stimulated endothelial cells (EC), and are hyperadhesive to platelets until cleavage by ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). In ADAMTS-13-deficient familial and autoantibody-mediated thrombotic thrombocytopenic purpura (TTP), there is severely restricted cleavage of EC-anchored ULVWF-platelet strings. The small amount of active enzyme released from their EC cleaves ULVWF strings minimally just above EC surfaces, thus generating soluble ULVWF multimers that are 2.5 to 50 times longer than plasma von Willebrand factor (VWF) forms. Soluble ULVWF multimers (detected in TTP and several other disorders) are also hyperadhesive to platelets and can cause excessive platelet adhesion/aggregation. Without exogenous chemicals or extreme shear stress, soluble ULVWF multimers cannot be cleaved by ADAMTS-13 but can be de-assembled (reduced) in vitro, by a free thiol-containing molecule (>30 kD) present in the cryosupernatant fraction of plasma that is not ADAMTS-13, thrombospondin-1, albumin, cysteine, or glutathione. This reduction may prevent occlusion of the microvasculature by embolic soluble ULVWF multimers (± adherent/aggregated platelets). New inhibitors of platelet adhesion to EC-anchored ULVWF multimeric strings and soluble ULVWF include an aptamer, a nanobody, and N-acetylcysteine.

Summary. Background: Secretion of Weibel–Palade body (WPB) contents is regulated, in part, by the phosphorylation of proteins that constitute the endothelial exocytotic machinery. In comparison to protein kinases, a role for protein phosphatases in regulating endothelial exocytosis is undefined. Objective and method: In this study, we investigated the role of protein phosphatase 2B (PP2B) in the process of endothelial exocytosis using pharmacological and gene knockdown approaches. Results: We show that inhibition of protein phosphatase 2B (PP2B) activity by cyclosporine A (CsA), tacrolimus or a cell-permeable PP2B autoinhibitory peptide promotes the secretion of ultralarge von Willebrand factor (ULVWF) from human umbilical vein endothelial cells (HUVECs) in the absence of any other endothelial cell-stimulating agent. PP2B inhibitor-induced secretion and anchorage of ULVWF strings from HUVECs mediate platelet tethering. In support of a role for PP2B in von Willebrand factor (VWF) secretion, the catalytic subunit of PP2B interacts with the vesicle trafficking protein, Munc18c. Serine phosphorylation of Munc18c, which promotes granule exocytosis in other secretory cells, is increased in CsA-treated HUVECs, suggesting that this process may be involved in CsA-mediated WPB exocytosis. Furthermore, the plasma VWF antigen level is also enhanced in CsA-treated mice, and small interfering RNA-mediated knockdown of the α and β isoforms of the PP2B-A subunit in HUVECs enhanced VWF secretion. Conclusions: These observations suggest that CsA promotes VWF release, in part by inhibition of PP2B activity, and are compatible with the clinically observed association of CsA treatment and increased plasma VWF levels in humans.

Von Willebrand factor (VWF) multimers are large adhesive proteins that are essential to the initiation of hemostatic plugs at sites of vascular injury. The binding of VWF multimers to platelets, as well as VWF proteolysis, is regulated by shear stresses that alter VWF multimeric conformation. We used single molecule manipulation with atomic force microscopy (AFM) to investigate the effect of high fluid shear stress on soluble dimeric and multimeric forms of VWF. VWF dimers are the smallest unit that polymerizes to construct large VWF multimers. The resistance to mechanical unfolding with or without exposure to shear stress was used to evaluate VWF conformational forms. Our data indicate that, unlike recombinant VWF multimers (RVWF), recombinant dimeric VWF (RDVWF) unfolding force is not altered by high shear stress $(100\phantom{\rule{0.28em}{0ex}}{\text{dynes/cm}}^{2}$ for 3 min at ${37}^{\ensuremath{\circ}}\mathrm{C})$. We conclude that under the shear conditions used $(100\phantom{\rule{0.28em}{0ex}}{\text{dynes/cm}}^{2}$ for 3 min at ${37}^{\ensuremath{\circ}}\mathrm{C})$, VWF dimers do not self-associate into a conformation analogous to that attained by sheared large VWF multimers.

Summary. Background: Thrombotic thrombocytopenic purpura (TTP) is associated with congenital and acquired deficiency of ADAMTS-13, a metalloprotease that cleaves von Willebrand factor (VWF) and reduces its adhesive activity. Mutations throughout the ADAMTS13 gene have been identified in congenital TTP patients, most of whom have initial episodes during infancy or in early childhood. Patients and methods: We report the case of an adult male who was diagnosed with idiopathic thrombocytopenic purpura at age 34, and with TTP 14 years later. The patient was compound heterozygous for an 18 bp in-frame deletion (C365del) in the disintegrin domain and a point mutation of R1060W in the seventh thrombospondin domain of the ADAMTS-13 gene. Conclusions:In vitro studies found that C365del and R1060W severely impair ADAMTS-13 synthesis in transfected Hela cells, whereas the deletion mutant also failed to cleave VWF under static and 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.

Summary. The adhesion ligand von Willebrand factor (VWF) is synthesized and stored in vascular endothelial cells and megakaryocytes/platelets. As in endothelial cells, platelet VWF also contains ultra-large (UL) multimers that are hyperactive in aggregating platelets. ULVWF in platelet lysates of thrombin-stimulated platelets was only detected in the presence of EDTA, suggesting that ULVWF is cleaved by a divalent cation-dependent protease. A recent study shows that platelets contain the VWF-cleaving metalloprotease ADAMTS-13, but its activity remains unknown. In this study, we show that platelet lysates cleave endothelial cell-derived ULVWF under static and flow conditions. This activity is inhibited by EDTA and by an ADAMTS-13 antibody from the plasma of a patient with acquired TTP. ADAMTS-13 was detected in platelet lysates and on the platelet surface by four antibodies that bind to different domains of the metalloprotease. Expression of ADAMTS-13 on the platelet surface increases significantly upon platelet activation by the thrombin receptor-activating peptide, but not by ADP. These results demonstrate that platelets contain functionally active ADAMTS-13. This intrinsic activity may be physiologically important to prevent the sudden release of hyperactive ULVWF from platelets and serves as the second pool of ADAMTS-13 to encounter the increase in ULVWF release from endothelial cells.

Leukocyte rolling on vascular endothelium is mediated by an interaction between P-selectin expressed on endothelial cells and P-selectin glycoprotein ligand-1 on leukocytes. This interaction reduces the velocity of leukocyte movements to allow subsequent firm adhesion and transmigration. However, the interaction has so far been observed only under low venous shear stress and cannot explain the accumulation of monocytes in atherosclerotic plaques found in arteries, where shear stress is much higher. We have previously shown that newly released ultra-large von Willebrand factor (ULVWF) forms extremely long string-like structures to which platelets tether. Here, we investigated whether platelets adhered to ULVWF strings are activated and form aggregates. We also determined whether activated platelets on ULVWF strings can support leukocyte tethering and rolling under high shear stresses. We found that platelets adhered to ULVWF expressed P-selectin and bound PAC-1, suggesting their rapid activation. We also found that leukocytes tethered to and rolled on these platelet-decorated ULVWF strings, but not directly on endothelial cells, under high shear stresses of 20 and 40 dyn/cm(2) in a P-selectin dependent manner. These results suggest that the endothelial cell-bound ULVWF provide an ideal matrix to aggregate platelets and recruit leukocytes to endothelial cells under high shear stress. The observed phenomenon delineates a mechanism for leukocytes to be tethered to arterial endothelial cells under high shear, providing a potential link between inflammation and thrombosis.

Thrombotic thrombocytopenic purpura is caused by congenital or acquired deficiency of ADAMTS-13, a metalloprotease that cleaves the endothelium-derived ultra-large multimers of von Willebrand factor (ULVWF). The proteolysis converts hyper-reactive and thrombogenic ULVWF into smaller and less adhesive plasma forms. Activity of ADAMTS-13 is usually measured in a static system under non-physiological conditions that require protein denaturation and prolonged incubation. We have demonstrated previously that ULVWF multimers, upon release from endothelial cells, form platelet-decorated string-like structures that are rapidly cleaved by ADAMTS-13. Here we report the direct interaction between ADAMTS-13 and VWF under both static and flowing conditions. ADAMTS-13-coated beads adhered to both immobilized VWF and ULVWF strings presented by stimulated endothelial cells. These beads adhered to VWF under both venous (2.5 dynes/cm2) and arterial (30 dynes/cm2) shear stresses. We then demonstrated that ADAMTS-13 beads adhered to immobilized recombinant VWF-A1 and -A3 domains, but soluble metalloprotease bound preferentially to the A3 domain, suggesting that the VWF A3 domain may be the primary docking site for the metalloprotease. We suggest that tensile stresses imposed by fluid shear stretch endothelial bound ULVWF multimers to expose binding sites within the A domains for circulating ADAMTS-13. The bound enzyme then cleaves within the A2 domain that lies in close proximity and releases smaller VWF multimers into the plasma. Once released, these cleaved VWF fragments become inaccessible for the metalloprotease to prevent further cleavage.

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 Ibα, 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/cm2) and arterial (20 and 50 dyne/cm2) 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.

Introduction: Thrombocytopenia and thrombotic events are common after TAVR. Changes in shear forces during TAVR and the subsequent platelet activation may explain these phenomena Methods: Flow cytometry with dual staining (PE-anti-CD41 and FITC-anti-vWF) was used to assess vWF binding to platelets

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.

The Methodist Hospital, Houston, TexasExtensive microvascular platelet aggregation is characteristic of thrombotic thrombocy-topenic purpura (TTP). Previous studies have indicated that abnormalities of von Will-ebrand factor (vWf) are often present in TTP patient plasma. There has not been previ-ously any direct evidence linking these abnormalities to the process of intravascularplatelet aggregation in TTP. We used flow cytometry to analyze the binding of vWf tosingle platelets, and the presence of platelet aggregates, in the blood of 4 children withchronic relapsing (CR) TTP and 5 adults with single episode or recurrent TTP. vWf on thesingle platelets of CRTTP patients at all time points studied was significantly increasedcompared to controls, and was increased further as platelet counts decreased to levelsbelow 40,000/µl. The single episode and recurrent adult TTP patients had platelet aggre-gates in the blood, as well as increased vWf on single platelets, before therapy com-menced and thereafter until recovery was in process. In the one unresponsive singleepisode TTP patient, vWf on single platelets remained elevated, and platelet aggregatespersisted, until her death. The platelet a-granular protein, P-selectin, was not increasedon the single platelets of most TTP blood samples, suggesting that it is vWf from plasma(rather than from a-granules) that attaches to platelet surfaces in association with plateletaggregation. These results suggest that vWf-platelet interactions are involved in theplatelet clumping process that characterizes TTP. Am. J. Hematol. 57:293–302, 1998.

Objective— Ultralarge von Willebrand factor (vWF) strings are secreted by, and anchored to, stimulated human endothelial cells. A disintegrin and metalloprotease with thrombospondin domains-type 13 cleaves the ultralarge vWF strings into large soluble vWF multimers. Normal plasma contains a nonproteolytic reducing activity that subsequently rapidly diminishes the size of the large soluble vWF multimers. Approach and Results— The vWF reductase activity was isolated from normal cryoprecipitate-poor plasma by chromatography and identified as the complement regulatory protein, factor H (FH), by mass spectroscopy, SDS-PAGE, and monospecific anti-FH antibody. Removal of FH from partially purified vWF reductase by immunoabsorption eliminated the reducing activity, and the activity was recovered in the eluates. Recombinant human FH reduced large soluble vWF multimers in a free thiol-dependent reaction that was not inhibited by a variety of protease inhibitors. Conclusions— FH contributes to the reduction of large soluble vWF multimers.

In this study it is demonstrated for the first time that shear stress induces the binding of exogenous von Willebrand factor (vWF) multimers to platelets. The vWF preparations used were: 125I-vWF purified from human cryoprecipitate (and including all vWF multimers present in normal plasma); and 35S-cysteine-vWF secreted by human umbilical vein endothelial cells (HUVECs) (and containing unusually large vWF forms, as well as all plasma-type vWF multimers). Direct shear-induced binding to washed platelets (300-360 x 10(3)/microliters) of radiolabeled vWF was maximum at 60-120 dynes/cm2 evaluated at 30 sec and was in extent about one-quarter of the binding stimulated by ristocetin after 3 min of incubation. The shear-induced binding of only a small percentage of added radiolabeled vWF was sufficient to initiate aggregation. Radiolabeled vWF attached to both glycoprotein (GP) Ib and GPIIb-IIIa receptors in the shear field, with complete inhibition of binding occurring with simultaneous blockade of both receptors. Binding was potentiated by ADP released from sheared platelets.

The thrombotic microangiopathies (TMA) are a group of disorders defined by the presence of microangiopathic hemolytic anemia and thrombocytopenia. The most common of these is thrombotic thrombocytopenic purpura (TTP), which is a systemic disorder of microvascular thromboses due to deficiency of ADAMTS-13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). A less common TMA is the atypical hemolytic uremic syndrome (aHUS), which is a renal vascular TMA caused by complement dysregulation. Despite overlapping clinical and pathologic manifestations, TTP and aHUS have distinct etiologies. TTP is often caused by a deficiency of ADAMTS-13 that is the result of gene mutations or acquired autoantibodies (Tsai, 2006). Atypical HUS is caused by defects of regulation and/or excessive activation of the alternative complement pathway (Kavanagh & Goodship, 2010). The mechanism by which complement dysregulation contributes to aHUS is not precisely defined, although complement-mediated glomerular endothelial injury and enhanced complement-mediated platelet activation are probably involved (Stahl et al, 2008). Similarly, triggers and co-factors directing systemic platelet deposition in TTP are not completely understood. Evidence that complement activation might play a role in TTP (Noris et al, 1999; Ruiz-Torres et al, 2005; Reti et al, 2012) raises the possibility of a cross-talk between ADAMTS-13/ultra-large von Willebrand factor (ULVWF) and the complement system. We studied plasma samples of 81 patients diagnosed with TMA according to clinical criteria for functional abnormalities in both ADAMTS-13 and complement regulation. Citrated platelet-poor plasma samples were obtained for testing before the initial plasma infusion or exchange procedures. All patients had microangiopathic hemolytic anemia and thrombocytopenia without an alternative cause, and treated with either plasma infusion or plasma exchanges. None of our patients had acute renal failure. Samples for analysis of DNA were not obtained/stored from this group of patients. All human subject studies were conducted according to the approved institutional review board protocols in the Rice University and University of Texas M.D. Anderson Cancer Center. ADAMTS-13 activity was measured by: (1) the rate of cleavage of a substrate that contains 73 amino acids of the A2 domain of VWF with fluorescence resonance energy transfer (FRET) tags on either side of the cleavage site for ADAMTS-13 (FRETS-VWF73), according to the manufacturer’s protocol (GTi Diagnostics); and (2) cleavage of urea-treated ULVWF multimers (obtained from human umbilical vein endothelial cell supernatant) by citrated patient plasma, followed by VWF multimeric analysis using SDS-1% agarose electrophoresis and Western-blotting with anti-VWF antibody. This is a modification of the method described by Furlan, et al. (Furlan et al, 1998). The presence or absence of ADAMTS-13 inhibitors was determined by measuring cleavage of urea-treated ULVWF multimers before and after mixing normal citrated plasma with an equal volume of patient citrated plasma (Furlan et al, 1998). Complement activity was measured by the hemolysis of sheep erythrocytes after incubation with human serum or plasma according to modified techniques from Sanchez-Corral, et al. (Sanchez-Corral et al, 2004). Factor H-depleted plasma causes complement-induced lysis of sheep erythrocytes with the visually apparent release of hemoglobin. Pooled normal plasma or serum caused 7% and 8% hemolysis of sheep erythrocytes, respectively. Optimal dilution of plasma or serum for the assay was determined to be between 4/100 to 6/100, and optimal incubation time was 10 min. Sixty percent (49/81) of TMA patients had severe ADAMTS-13 deficiency (less than 10% activity). Eighty percent (65/81) of our patients’ plasma samples caused little to no hemolysis of sheep erythrocytes (median of 10%; range of 0–15%). In contrast, 20% (16/81) of the patients’ samples showed significant hemolysis (median of 60% hemolysis; range of 23–89%) (Figure 1). Sixteen percent (8/49) of plasma samples from TTP patients with severe ADAMTS-13 deficiency caused increased hemolysis. Only one of the 8 patients with concurrent excessive complement-induced hemolysis and severe ADAMTS-13 deficiency had detectable antibody (in low titer) against ADAMTS-13 (Table 1). Twenty-five percent (8/32) of plasma samples from patients who did not have severe ADAMTS-13 deficiency also caused increased hemolysis. Figure 1 Sheep erythrocyte hemolysis assay in plasma samples from patients with thrombotic microangiopathy Table 1 Patients with a clinical diagnosis of TTP and decreased complement regulation. Severe deficiency of functional ADAMTS-13 is associated with TTP; however, many patients with a TTP-like syndrome have normal ADAMTS-13 levels, as did 40% (32/81) of the patients in our study. There are several reports of patients with reduced ADAMTS-13 function who either did not develop TTP, or did so later in life (Noris et al, 2005). These observations raise the possibility of the presence of additional factors besides ADAMTS-13 deficiency involved in the pathophysiology of TTP (Ruiz-Torres et al, 2005; Reti et al, 2012; Noris et al, 2005; Chapin et al, 2012). Activation of the complement system in both familial (Noris et al, 1999) and acquired TTP (Reti et al, 2012) has been reported, based on the lower concentration of C3 and elevated levels of complement activation products (C3a and sC5b-9) in the sera of patients with acute TTP, and deposition of C3 and C5b-9 on endothelial cell exposed to TTP sera (Ruiz-Torres et al, 2005). We studied activity of the alternative complement pathway in 81 patients with the clinical diagnosis of TTP-like TMA requiring plasma infusion and/or plasma exchange. Some patients with severe ADAMTS-13 deficiency (8/49; 16%) or TTP-like TMA (8/32; 25%), had elevated plasma complement activity. We did not detect an increased titer of ADAMTS-13 inhibitor in ADAMTS-13 deficient TTP patients with complement dysregulation, and the majority of these patients (5/8; 68%) had a history of familial or recurrent TTP (Table 1). Our data suggest that the complement system may be an important co-factor involved in the pathogenesis of TMA. Excessive alternative pathway activity occurred in a significant number of TTP patients indicating that concurrent defects in ADAMTS-13 and complement regulation may occur more frequently than previously reported (Noris et al, 2005; Chapin et al, 2012). In addition, our findings indicate that excessive alternative pathway activity can be associated with a TTP-like TMA in some patients who do not have severe deficiencies of ADAMTS-13. Further genetic studies of patients with the clinical diagnosis of TTP may be informative.

The mechanical force-induced activation of the adhesive protein von Willebrand Factor (VWF), which experiences high hydrodynamic forces, is essential in initiating platelet adhesion. The importance of the mechanical force-induced functional change is manifested in the multimeric VWF’s crucial role in blood coagulation, when high fluid shear stress activates plasma VWF (pVWF) multimers to bind platelets. Here we showed that a pathological level of high shear stress exposure of pVWF multimers results in domain conformational changes, and the subsequent shifts in the unfolding force allow us to use force as a marker to track the dynamic states of multimeric VWF. We found that shear-activated pVWF multimers (spVWF) are more resistant to mechanical unfolding than non-sheared pVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of pVWF multimers.

Because of the degeneration and thrombosis in artificial heart valve implants, it is important to understand the anti-thrombotic mechanisms of cardiac valve endothelial cells (VECs). These anti-thrombotic mechanisms can be integrated into poly(ethylene glycol) diacrylate (PEGDA) tissue-engineered heart valve (TEHV) design. This work will study the effects of (1) PEGDA hydrogel stiffness and (2) specific extracellular matrix (ECM) adhesive peptides on VEC phenotype and anti-thrombotic mechanisms. PEGDA 10% (w/v) hydrogels of MWs 3.4 and 20kDa were polymerized to apply different substrate rigidities in VEC culture. Thiol-ene reactions were used to covalently bind laminin- and fibronectin- derived peptides to the acrylate groups on PEGDA hydrogel surfaces. Laminin-derived peptide motif RKRLQVQLSIRT (RKR) and fibronectin adhesive peptide RGD were modified to include an additional cysteine at the end of each sequence, introducing a free thiol to undergo the thiol-ene reaction. Thiol-PEG-fluorescein (SH-PEG-FITC) served as a negative adhesive substrate control. Porcine aortic VECs were seeded onto each of the ECM-hydrogel combinations and cultured for 2, 6, and 10 days. Cell phenotype, adhesion, and proliferation were then assessed. Analysis of specific VEC anti-thrombotic protein regulation is in progress. At each time point, samples will be analyzed for maintenance of VEC phenotype and expression of thrombotic (von Willebrand Factor [VWF]) and anti-thrombotic (VWF cleaving enzyme [ADAMTS-13], eNOS, PGI2, tPA) proteins using histochemistry and qRT-PCR. Addition of histamine has been shown to stimulate rapid release of thrombogenic ultra-large VWF (ULVWF) strings by vascular endothelial cells. This method will be used to study VEC ULVWF string production and the associated cleavage activity of ADAMTS-13. Control of the hemostatic process will be quantified via western blot and ELISAs. The 3.4 and 20kDa MW PEGDA hydrogels had compressive moduli of 131±5 and 7.5±2kPa, respectively. Binding different concentrations of SH-PEG-FITC onto the gel surfaces showed that acrylate saturation was achieved for both MW compositions using ~5mM of peptide solution. After 2 days, the VECs on the stiffer 3.4kDa RKR gels appeared spread and elongated, whereas the 3.4kDa RGD seeded VECs had cobblestone morphology. VEC adhesion on the RKR and RGD 20kDa gels was observed, but with limited spreading. The cultured VECs may prefer the stiffer 3.4kDa gels over the softer 20kDa gels. After 10 days, VECs on the 3.4kDa RGD gels had minimal proliferation, while VECs on RKR grew confluent, were cobblestone shaped, and expressed VWF. Results suggest that both substrate rigidity and adhesive substrate greatly influence VEC survival, and likely affects anti-thrombotic regulation. Future studies include the use of basal lamina components collagen IV and perlecan peptides to evaluate changes in VEC behavior.

Two children with congenital chronic relapsing thrombotic thrombocytopenic purpura (TTP) have episodes every 3 weeks. These relapses can be prevented by the infusion of normal fresh-frozen plasma (FFP) without concurrent plasmapheresis. We conducted a study to determine whether the exposure of normal plasma to agents that inactivate human immunodeficiency virus and other viruses destroys the component necessary for the effective treatment of this type of TTP that requires only plasma infusion to prevent or reverse relapses. Clinical responsiveness and von Willebrand factor (vWF)-mediated fluid shear stress-induced platelet aggregation were evaluated before and after the infusion of 10 mL/kg FFP or solvent [tri(n- butyl)phosphate]/detergent (Triton X-100)-treated plasma (S/D plasma). Platelet aggregation at shear stresses of 90 to 180 dyne/cm2 (similar to those in the partially occluded microcirculation) imposed for 30 seconds was excessive using the citrated platelet-rich plasma of both patients, and was associated with the presence of unusually large vWF forms in patient platelet-poor plasma. Infusion with either FFP or S/D plasma at 3-week intervals caused the platelet count to increase to (or above) normal within 1 week (on 12 of 12 occasions); the disappearance or diminution of unusually large vWF forms within 1 hour (on 6 of 10 occasions studied); and the reversal within 1 to 4 hours of excessive shear-induced platelet aggregation (on 8 of 9 occasions studied). We conclude that a component in normal plasma resistant to S/D treatment is responsible for preventing thrombocytopenia and TTP episodes, and for controlling excessive shear-induced aggregation in these patients. Our results suggest that excessive in vivo platelet aggregation in chronic relapsing TTP and excessive in vitro vWF-mediated shear-induced aggregation may be similar phenomena.

Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease characterized by systemic microvascular thrombosis caused by adhesion of platelets to ultra-large vWF (ULVWF) multimers. These multimers accumulate because of a deficiency of the processing enzyme ADAMTS13. vWF protein forms long multimers from homodimers that first form through C-terminal disulfide bonds and then join through their N termini by further disulfide bonding. N-acetylcysteine (NAC) is an FDA-approved drug that has long been used to treat chronic obstructive lung disease and acetaminophen toxicity and is known to function in the former disorder by reducing mucin multimers. Here, we examined whether NAC could reduce vWF multimers, which polymerize in a manner similar to mucins. In vitro, NAC reduced soluble plasma-type vWF multimers in a concentration-dependent manner and rapidly degraded ULVWF multimer strings extruded from activated ECs. The effect was preceded by reduction of the intrachain disulfide bond encompassing the platelet-binding A1 domain. NAC also inhibited vWF-dependent platelet aggregation and collagen binding. Injection of NAC into ADAMTS13-deficient mice led to the rapid resolution of thrombi produced by ionophore treatment of the mesenteric venules and reduced plasma vWF multimers. These results suggest that NAC may be a rapid and effective treatment for patients with TTP.

Human umbilical vein endothelial cell (HUVEC)–released ADAMTS-13 (a disintegrin and metalloprotease with thrombospondin repeats) and HUVEC-secreted von Willebrand factor (VWF) strings were investigated under static conditions that allow the accumulation and analysis of ADAMTS-13. The latter was released constitutively from HUVECs and cleaved the secreted and cell-anchored VWF strings progressively during 15 minutes in Ca2+/Zn2+-containing buffer. HUVEC ADAMTS13 mRNA expression was approximately 1:100 of VWF monomeric subunit expression. In contrast to multimeric VWF stored within Weibel-Palade bodies and secreted rapidly in response to cell stimulation, ADAMTS-13 was released directly from the Golgi to the cell exterior without an organelle storage site. The constitutive release of ADAMTS-13 continued at the same slow rate regardless of the presence or absence of histamine stimulation of HUVECs. Consequently, the percentage of VWF strings cleaved by ADAMTS-13 at VWF Y1605-M1606 decreased as the rate of VWF string secretion was increased by cell stimulation. Blockade of HUVEC ADAMTS-13 activity by antibodies to different ADAMTS-13 domains made it possible to detect the attachment of ADAMTS-13 all along the lengths of HUVEC-secreted VWF strings. Constitutive ADAMTS-13 released from endothelial cells may contribute to the maintenance of cell surfaces free of hyperadhesive VWF multimeric strings.

Summary. Background and Objectives: 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. Methods: 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-PEO2-Biotin labeling and thiol-chromatography. Finally, covalent re-multimerization of ULVWF was examined in a cell- and enzyme-free system. Results: 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. Conclusion: 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.

Endothelial cells synthesize and secrete von Willebrand factor (VWF) multimers, including unusually large forms (ULVWF), which are usually cleaved into smaller multimers found in normal plasma (P-VWF). Thrombotic thrombocytopenic purpura (TTP) is a microangiopathic disorder characterized by systemic attachment of platelets to inadequately cleaved ULVWF multimers. We have compared ULVWF and P-VWF in their capacity to become immobilized onto surfaces in vitro and their ability to mediate platelet adhesion. We have also used functional assays to directly compare ULVWF forms with purified P-VWF in mediating platelet aggregation in solution. At comparable concentrations, ULVWF is more effectively adsorbed onto glass surfaces than P-VWF and supports increased platelet adhesion. ULVWF is also significantly more potent than P-VWF in mediating both shear-induced platelet aggregation and ristocetin-mediated platelet agglutination.

Summary. Hepatic stellate cells have been considered to be a primary source for human plasma ADAMTS-13, the von Willebrand factor (VWF)-cleaving metalloprotease. In this study, ADAMTS-13 antigen was detected by immunofluorescence in both venous (HUVECs) and arterial endothelial cells (HUAECs) using both polyclonal antibodies made against peptides found in various domains of human ADAMTS-13, as well as by a monoclonal antibody against the ADAMTS-13 metalloprotease domain. ADAMTS-13 antigen had an intra-cellular distribution in endothelial cells distinct from the Weibel–Palade body location of VWF, and was released from the cells during 48 h in culture. The mRNA for ADAMTS13 was detected in HUVECs and HUAECs using reverse transcription-polymerase chain reaction (RT-PCR), indicating that the enzyme is synthesized in these cells. The ADAMTS-13 protein was immunoprecipitated from HUVECs and had an approximate Mr of 170 kDa, similar to the molecular mass of recombinant ADAMTS-13. The ADAMTS-13 in HUVEC and HUAEC lysates had enzymatic activity using both static and flow assays. We conclude that ADAMTS-13 is synthesized in human endothelial cells, and released constitutively. The vast number of endothelial cells in the body may be an important source of ADAMTS-13.

ADAMTS13 cleaves ultralarge and hyperreactive von Willebrand factor (ULVWF) freshly released from activated endothelial cells to smaller and less active forms. This process may be affected by the amount of ULVWF released and the processing capacity of ADAMTS13, contributing to the development of thrombotic diseases. We examined the effects of inflammatory cytokines on the release and cleavage of ULVWF to evaluate potential links between inflammation and thrombosis. Human umbilical vein endothelial cells were treated with interleukin 6 (IL-6), IL-8, or tumor necrosis factor α (TNF-α), and the formation of platelet-decorated ULVWF strings was quantitated. IL-8 and TNF-α significantly stimulated the release of ULVWF in a dose-dependent manner. IL-6 induced ULVWF release only when it was in complex with the soluble IL-6 receptor. IL-6, but not IL-8 nor TNF-α, inhibited the cleavage of ULVWF strings by ADAMTS13 under flowing, but not static, conditions. These results suggest that inflammatory cytokines may stimulate the ULVWF release (IL-8 and TNF-α) and inhibit the ULVWF cleavage (IL-6), resulting in the accumulation of hyperreactive ULVWF in plasma and on the surface of endothelial cells to induce platelet aggregation and adhesion on the vascular endothelium. The findings describe a potential linkage between inflammation and thrombosis that may be of therapeutic importance.

The metalloprotease ADAMTS-13 cleaves von Willebrand factor (VWF) at the Y842/M843 peptide bond located in the A2 domain. Measurement of ADAMTS-13 activity is a clinical utility for thrombotic diseases, but the current assays used for diagnostic and clinical research are non-physiological and time consuming. We have expressed in bacteria a recombinant VWF-A2 peptide (aa 718-905) that contains both a 6xHis tag at the N-terminal end and a Tag-100 epitope at the C-terminal end. Diluted plasma was mixed with the VWF-A2 peptide and digestion was allowed to proceed in a Ni2+-coated microtiter well plate for 2 h. The immobilized Ni2+ captures the VWF-A2 peptide by its 6xHis tag and cleavage of the A2 peptide is measured by the removal of the C-terminus fragment of the A2 peptide that contains the Tag-100. The cleavage activity for this assay was defined by the low detection of A2 peptide containing the Tag-100 epitope by the antiTag-100 monoclonal antibody. The assay was completed in

von 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.

The unusually large von Willebrand factor (ULvWF) multimers present within endothelial cells and platelets are larger than the vWF multimers normally found in adult human plasma. Furthermore, ULvWF multimers are cleared rapidly from the circulation if they are released by intense endothelial cell stimulation. The mechanisms by which the ULvWF multimers are processed to large plasma vWF multimers are not known. It has been demonstrated that granulocyte proteases are capable of decreasing vWF multimer size in vitro, and that some patients with myeloproliferative syndromes have a relative absence of large plasma vWF multimers in sodium citrate-anticoagulated plasma samples. In order to assess the influence of granulocyte proteases on vWF multimer size, we evaluated the vWF multimeric patterns in 94 plasma samples from 60 patients with neutrophil counts that were either considerably elevated or extremely reduced. In 83 of 94 plasma samples, the vWF multimeric patterns were normal. No patients with very low neutrophil counts had ULvWF multimers present. These observations suggest that granulocyte proteases are not likely to be involved in vivo in the processing of ULvWF multimers from endothelial cells to the smaller vWF forms in circulation.

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.

Ultra-large von Willebrand factor (ULVWF) multimers secreted from the Weibel-Palade bodies (WPBs) of endothelial cells plays a crucial role in the development of thrombotic microangiopathies. Secretion of WPB contents is regulated, in part, by the phosphorylation of vesicle trafficking proteins that constitutes the endothelial exocytotic machinery. In comparison to agonist-induced, protein kinase-dependent signaling pathways that regulate the exocytosis of WPB contents, a role for protein phosphatases in regulating endothelial exocytosis is currently undefined. In this study, we show that inhibition of serine/threonine protein phosphatase 2B (PP2B) activity by cyclosporine A (CsA), tacrolimus (FK506) or a cell-permeable PP2B autoinhibitory (AI) peptide promotes the secretion of hyper-adhesive ULVWF from human umbilical vein endothelial cells (HUVECs) in the absence of any other endothelial cell-stimulating agent. PP2B inhibitor-induced secretion and anchorage of ULVWF strings from HUVECs induce the tethering of adhesive platelets. In support of a role for PP2B in VWF secretion, we demonstrated that the catalytic subunit of PP2B expressed as a GST fusion protein interacts with the vesicle trafficking protein, Munc18c. Serine phosphorylation of Munc18c, which promotes granule exocytosis in other secretory cells, was significantly increased in CsA-treated HUVECs, suggesting that this process may be involved in CsA-mediated WPB exocytosis and secretion of VWF. Furthermore, plasma VWF antigen levels were also enhanced in CsA-treated mice, and siRNA-mediated knockdown of PP2Bb in HUVECs significantly enhanced VWF secretion. These observations suggest that CsA promotes VWF release, at least in part by inhibition of PP2B activity. These observations are compatible with the clinically observed association of CsA treatment and increased plasma VWF levels in humans, as well as the association between prolonged exposure to CsA and thrombotic microangiopathy in a subset of exposed patients.

Factor H is a plasma protein that regulates activation of the alternative complement system. Mutations in the factor H gene are associated with a rare form of thrombotic microangiopathy, known as atypical hemolytic uremic syndrome. Hemolytic uremic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) are characterized by systemic (TTP) or renal (HUS) microvascular thrombosis. The clinical presentations of TTP and HUS have some common clinical features, including the presence of thrombocytopenia, intravascular hemolysis, and mechanical injury to red cells. We investigated whether factor H has any interaction with proteins involved in the pathogenesis of thrombotic thrombocytopenic purpura, namely von Willebrand Factor (VWF) and ADAMTS-13. We found that factor H binds to the A1 and A2 domains of VWF, and inhibits cleavage of VWF by ADAMTS-13 under static and flowing conditions. Factor H deficient mice had a delayed thrombous formation after vessel wall injury, and showed a lower mortality rate and a higher platelet count after Shiga toxin/lipopolysaccharide challenge compared to their wild-type littermates. We concluded that factor H, in addition to its complement regulatory activity, might regulates cleavage of VWF by ADAMTS-13. Presence of an abnormal factor H that is unable to control complement activity on cell surfaces but still capable of inhibiting cleavage of VWF might contribute to the pathogenesis of thrombotic microangiopathies.

von Willebrand factor (VWF) shares a similar domain structure with many polymeric mucins, including the presence of D domains and a C-terminal cysteine knot, which allows these molecules to form polymeric structures that can reach immense sizes. Precipitation of polymeric lung mucins complicates the clinical course of cystic fibrosis and chronic obstructive lung disease, and in both cases the viscosity of the inspissated mucus can be reduced by treatment with N-acetyl cysteine (NAC), which presumably reduces the size of the mucin polymers by reducing sensitive disulfide bonds. Because of the similarity of VWF and mucin multimers, we examined whether NAC could also reduce VWF multimer size both in vitro and in vivo. In vitro, we incubated NAC at different concentrations and for different times with ultra-large VWF multimers (ULVWF) isolated from endothelial cell supernatant and examined the effect on multimer size using agarose gel electrophoresis. NAC reduced ULVWF size in a time- and concentration-dependent manner, with the peak effect reached at 5 min and at concentration of 0.5 mM. We then examined the effect of NAC on ULVWF/platelet “strings” formed on the surface of histamine-activated endothelial cells by perfusing the strings with NAC solutions. At 1 mM, NAC eliminated almost all of the adherent strings within 5 minutes. We next examined the effect of NAC in vivo by following VWF multimer size with time in C57B/6 injected with NAC either intraperitoneally or intravenously. NAC, at a single dose of 500 mg/kg, induced a sustained reduction in VWF multimer size in the treated mice within 4 hours after injection. The effects lasted up to 8 hours. These results suggest that NAC may be a rapid, safe, and effective treatment for patients suspected of suffering from thrombotic thrombocytopenic purpura, a disorder characterized by a failure to process ULVWF.

Von Willebrand factor (VWF) is synthesized and released from endothelial cells and megakaryocytes/platelets in a form that is ultra-large (UL) in size and hyper-reactive in functions, capable of spontaneously activating and aggregating platelets. Upon its release, ULVWF is cleaved by the metalloprotease ADAMTS-13 at a single peptide bond of Tyr842 and Met843 in the A2 domain of VWF, converting this hyper-reactive ULVWF to smaller and less active form. If it is left uncleaved, ULVWF could activate and aggregate platelets, leading to systemic thrombosis, as often found in patients with thrombotic thrombocytopenic purpura. However, defects in ULVWF proteolysis may also occur in other conditions where ADAMTS-13 activity may be suppressed and ULVWF release from endothelial cells is systemic and persistent. To test this hypothesis, we have measured changes in ADAMTS-13 activity and ULVWF proteolysis in pediatric patients with severe sepsis with one or more organ failure, an extremely severe form of systemic inflammation that is likely to trigger the release of a large amount of ULVWF. Ten patients (and 6 matching controls) were recruited and followed for a period of 7 to 28 days in a pediatric intensive care unit. We found that the mean ADAMTS-13 activity in patients on admission as determined by a flow assay was 60.1±30.4%, significantly lower than 97.7±1.5% found control subjects. Among these patients, 70% presented severe to moderate thrombocytopenia (22,000–150,000/ml) on admission. Three patients (30%) had ADAMTS-13 activity less than 30%, below the threshold level of deficiency predetermined for this assay. Two of these three patients also presented severe thrombocytopenia (platelet counts of 22,000 and 66,000/ml, respectively). During the follow-up period, changes in ADAMTS-13 activity correlated approximately with changes in platelet counts. Patients with little or no improvement in ADAMTS-13 activity often showed persistent thrombocytopenia, whereas improvement in the enzyme activity associated with increase in platelet counts. We have also measured plasma levels of four inflammatory cytokines and found that IL-6 and TNF-α were 98.7±10.8 and 516.2±66.0 pM in patients with severe ADAMTS-13 deficiency (< 30% in activity), significantly higher than those with the enzyme activity greater than 30% (49.2±21.3 and 46.3±19.1 pM for IL-6 and TNF-a, respectively, p < 0.001). In comparison, plasma levels of IL-1β and IL-8 were elevated, but showed no difference between the two groups. The reduction in plasma IL-6 and TNF-α levels in some patients correlated with increase in ADAMTS-13 activity and platelet counts. These results suggest that severe sepsis may result in a transient deficiency in ULVWF proteolysis, likely due to deficiency in ADAMTS-13 activity (consumptive or direct inhibition). The suppression of ULVWF proteolysis may contribute to thrombocytopenia often seen in these patients. The deficiency in ULVWF proteolysis may therefore play a bridge role between inflammation and thrombosis.

Deficiency of the von Willebrand factor (VWF)-cleaving metalloprotease ADAMTS-13 is assumed to be the proximate cause of thrombotic thrombocytopenic purpura (TTP), a potentially devastating microvascular thrombotic disorder. Lack of the enzyme leads to the plasma accumulation of unusually large and extraordinarily adhesive VWF multimers (ULVWF), which clump platelets and precipitate microvascular thrombosis, thrombocytopenia, microangiopathic hemolytic anemia, and systemic tissue infarction. Thus, accurate assessment of ADAMTS-13 activity should aid in differentiating TTP from other similar syndromes, and could help predict which patients will benefit from plasma exchange. We retrospectively reviewed the cases of 38 patients diagnosed with TTP between 1998 and 2004 in The Methodist Hospital in Houston, TX, recording comorbid conditions, response to plasma exchange, and ADAMTS-13 activity. All patients had ADAMTS-13 activity measured and received plasma exchange. ADAMTS-13 activity was determined by the capacity of patient plasma to cleave ULVWF derived from the supernatants of cultured umbilical vein endothelial cells. Response to plasma exchange was assessed by platelet counts and serum LDH levels. Those designated as having responded to therapy achieved platelet counts above 150,000 per ml. Ten of the 38 patients had no response to plasma exchange. Of these, all had associated conditions (e.g., transplantation, drugs, vasculitis) and 8 had normal ADAMTS-13 activity. Of the 28 who responded to therapy, 21 had no associated conditions. ADAMTS-13 activity was absent in 23 and low (6% and 12%) in 2. We assessed ADAMTS-13 activity in response to therapy in the patients divided into three groups: those without detectable activity (Group 1, 25 patients); those with low but detectable activity (Group 2, 2 patients); and those with normal activity (Group 3, 11 patients). In group 1, 65% had an inhibitor of ADAMTS-13 activity. Twenty three (92%) responded to plasma exchange, the other 2 had received bone marrow transplantation. Thirteen of the 23 responders required less than 6 exchanges; the other 10 required more than 6 exchanges, and 9 had fluctuating platelet counts during therapy. In 6 of the 9, the platelets fluctuations correlated with the LDH levels and ADAMTS-13 activities; the other 3 had intercurrent infections and the platelet counts did not correlate with their LDH levels or ADAMTS-13 activities. ADAMTS-13 activity values increased with plasma exchange in 23 of 25 patients. Mortality in group 1 was 8%; 32% of patients relapsed and none had renal failure requiring hemodialysis. Both patients in group 2 responded to fewer than 7 plasma exchanges. All patients in group 3 had associated conditions. Three of 11 (27%) responded to therapy. Mortality was 36% and no patient relapsed. Forty five percent of group 3 patients had renal failure requiring hemodialysis. The median number of plasma exchanges in this group was 15 (range 7–23). We conclude that assay of ADAMTS-13 activity is predictive of response to plasma exchange, and may distinguish patients with true TTP from those with disorders that have similar clinical and laboratory manifestations. Patients with low or absent activity responded to therapy with increasing ADAMTS-13 activity, platelet counts, and declining LDH levels.

Thrombotic thrombocytopenic purpura (TTP) is characterized by severe thrombocytopenia, hemolytic anemia, and diffuse and non-focal neurological findings. Microthrombi found in these patients are predominantly composed of platelets and von Willebrand factor (VWF). Recent studies suggest that the systemic thrombosis in TTP is mostly due to the congenital or acquired deficiency of the VWF-cleaving metalloprotease ADAMTS-13, which cleaves the ultra-large and hyperreactive VWF to smaller and less active form found in plasma. Most congenital cases of TTP have so far been identified in children. Here, we report a 60-year-old Caucasian man with a history of chronic relapsing TTP over thirty years, requiring plasma transfusion every 24 days in recent years. Repeat assays showed no ADAMTS13 activity in patient’s plasma under both static and flow conditions without inhibitors being detected. We therefore examined the possible genetic defects in the ADAMTS-13 gene of this patient. Genomic DNA was extracted from patient’s white blood cells and exons of ADAMTS-13 gene were amplified by polymerase chain reaction. The amplified DNA fragments were then screened for mutations by direct DNA sequencing. We identified a deletion of 18 base pairs from G1095 to G1112 (GTGCTCCAAGGGTCGCTG) in the exon10 of ADAMTS-13 gene, resulting in a deletion of six amino acids (C366 to C371) in the disintegrin domain of the metalloprotease. A point mutation (W365C) occurred immediately before the deletion due to a nucleotide realignment. The patient is heterozygote for the deletion. This is the first report of a deletion mutant (without frame shift and truncations) in the disintegrin region that has previously been demonstrated as critical for the ADAMTS-13 function by in vitro mutagenesis, epitope mapping of autoantibody to the metalloprotease in patients with adult acquired TTP, and identification of natural occurring mutations in patients with congenital TTP. Our ongoing studies are to determine the impact of these two mutations on the synthesis, release, and cleavage of ADAMTS-13.

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.

A fluid shear stress of 180 dyn/cm2 was applied for 0.5 and 5 min to platelets in citrated plasma or blood in a cone and plate viscometer with minimal platelet-surface interactions. Platelets aggregated in the shear field if large von Willebrand Factor (vWF) multimers were present. Aggregation did not require ristocetin, other exogenous agents, or desialation of vWF. Unusually large vWF multimers produced by human endothelial cells were functionally more effective than the largest plasma vWF forms in supporting shear-induced aggregation. Shear-induced aggregation was inhibited by monoclonal antibodies to platelet glycoprotein Ib or the IIb/IIIa complex, but was little affected by the absence of fibrinogen. vWF-dependent platelet aggregation under elevated shear stress in partially occluded vessels of the arterial microcirculation may contribute to thrombosis, especially if unusually large vWF multimers are released locally from stimulated or disrupted endothelial cells.

Fluid shear stress in arteries and arterioles partially obstructed by atherosclerosis or spasm may exceed the normal time-average level of 20 dyne/cm2. In vitro, at fluid shear stresses of 30 to 60 dyne/cm2 applied for 30 seconds, platelet aggregation occurs. At these shear stresses, either large or unusually large von Willebrand factor (vWF) multimers in the suspending fluid exogenous to the platelets mediates aggregation. Adenosine diphosphate (ADP) is also required and, in these experiments, was released from the platelets subjected to shear stress. At 120 dyne/cm2, the release of endogenous platelet vWF multimers can substitute for exogenous large or unusually large vWF forms in mediating aggregation. Endogenous released platelet vWF forms, as well as exogenous large or unusually large vWF multimers, must bind to both glycoproteins Ib and the IIb/IIIa complex to produce aggregation. Shear- induced aggregation is the result of shear stress alteration of platelet surfaces, rather than of shear effects on vWF multimers. It is mediated by either large plasma-type vWF multimers, endogenous released platelet vWF forms, or unusually large vWF multimers derived from endothelial cells, requires ADP, and is not inhibited significantly by aspirin. This type of aggregation may be important in platelet thrombus formation within narrowed arterial vessels, and may explain the limited therapeutic utility of aspirin in arterial thrombosis.

In addition to the von Willebrand factor (vWF) multimers found in normal plasma, cultured human umbilical vein endothelial cells (HUVECs) synthesize and release unusually large vWF multimers (ULvWFM). ULvWFM are more effective than the largest plasma vWF forms in attaching to platelets and promoting platelet aggregation in the presence of elevated fluid shear forces (as in narrowed atherosclerotic vessels), and they may be involved in arterial thrombosis. ULvWFM are produced within HUVECs exposed for 48-60 h either to steady venous-like or pulsatile arterial-like wall shear stresses and are produced and released from HUVECs grown in serum-free as well as in serum (bovine or human)-containing media. An activity of 140,000-200,000 Da in the cryosupernatant fraction of both normal and severe von Willebrand's disease plasma, which is not obviously a protease, prevents specifically the accumulation of ULvWFM in the fluid above HUVEC monolayers but does not impair the release by HUVECs of ULvWFM in the retrograde direction into subendothelial collagen. The ULvWF regulatory activity in cryosupernatant may inhibit inappropriate platelet aggregation and thrombosis by preventing the accumulation of endothelial cell-derived ULvWFM in circulating blood.

Shear stress activated platelets undergo aggregation in the presence of large or unusually large von Willebrand factor (vWF) multimers without the addition of ristocetin or any other exogenous chemical. This phenomenon may be analogous to the platelet aggregation that leads to thrombosis in the narrowed arteries and arterioles of patients with atherosclerosis or vasospasm. A triphenyl-methyl compound, aurin tricarboxylic acid (ATA), inhibits shear-induced, vWF-mediated platelet aggregation in platelet-rich plasma (PRP) in concentrations above 200 mumol/L and in buffer suspensions of washed platelets at a concentration of 0.1 mumol/L. In a concentration-dependent manner, ATA also inhibits ristocetin-induced, vWF-mediated platelet clumping in both fresh and formaldehyde-fixed platelet suspensions. This inhibition can be overcome by increasing the concentration of vWF, following the kinetics of first order competitive inhibition. ATA prevents the attachment to platelets of the largest vWF multimeric forms found in normal plasma and of the unusually large vWF multimers derived from endothelial cells. The rate of aggregation and degree of inhibition by ATA is not accounted for by the binding of ristocetin or calcium. Arachidonic acid- and adenosine diphosphate (ADP)-induced aggregation are not inhibited by ATA. Platelets incubated with ATA can be easily separated from the compound. However, ATA binds to large vWF multimeric forms and inhibits their ristocetin-induced interaction with platelet glycoprotein Ib. Because ATA also inhibits shear-induced, vWF-mediated platelet aggregation in vitro in the absence of ristocetin, it may be a useful prototype compound to impede the development of arterial thrombosis in vivo.