Brain microvascular endothelial cells exhibit lower activation of the alternative complement pathway than glomerular microvascular endothelial cells.

Atypical hemolytic uremic syndrome (aHUS) and bone marrow transplantation-associated thrombotic microangiopathy (TA-TMA) are associated with excessive activation of the alternative complement pathway (AP) and with severe renal, but rarely cerebral, microvascular damage. Here, we compared AP activation and regulation in human glomerular and brain microvascular endothelial cells (GMVECs and BMVECs, respectively) unstimulated or stimulated by the proinflammatory cytokine, tumor necrosis factor (TNF). Compared with GMVECs and under both experimental conditions, BMVECs had increased gene expression of the AP-related genes C3, CFB, and C5 and decreased expression of CFD This was associated with increased expression in BMVECs (relative to GMVECs) of the genes for surface and soluble regulatory molecules (CD46, THBD, CD55, CFI, and CFH) suppressing formation of the AP C3 and C5 convertases. Of note, unlike GMVECs, BMVECs generated extremely low levels of C3a and C5a and displayed decreased activation of the AP (as measured by a lower percentage of Ba generation than GMVECs). Moreover, BMVECs exhibited increased function of CD141, mediating activation of the natural anticoagulant protein C, compared with GMVECs. We also found that the C3a receptor (C3aR) is present on both cell types and that TNF greatly increases C3AR1 expression in GMVECs, but only slightly in BMVECs. Higher AP activation and C3a generation in GMVECs than in BMVECs, coupled with an increase in C3aR production in TNF-stimulated GMVECs, provides a possible explanation for the predominance of renal damage, and the absence of cerebral injury, in individuals with episodes of aHUS and TA-TMA.

Acquired von Willebrand syndrome associated with left ventricular assist device.

Left ventricular assist devices (LVAD) provide cardiac support for patients with end-stage heart disease as either bridge or destination therapy, and have significantly improved the survival of these patients. Whereas earlier models were designed to mimic the human heart by producing a pulsatile flow in parallel with the patient's heart, newer devices, which are smaller and more durable, provide continuous blood flow along an axial path using an internal rotor in the blood. However, device-related hemostatic complications remain common and have negatively affected patients' recovery and quality of life. In most patients, the von Willebrand factor (VWF) rapidly loses large multimers and binds poorly to platelets and subendothelial collagen upon LVAD implantation, leading to the term acquired von Willebrand syndrome (AVWS). These changes in VWF structure and adhesive activity recover quickly upon LVAD explantation and are not observed in patients with heart transplant. The VWF defects are believed to be caused by excessive cleavage of large VWF multimers by the metalloprotease ADAMTS-13 in an LVAD-driven circulation. However, evidence that this mechanism could be the primary cause for the loss of large VWF multimers and LVAD-associated bleeding remains circumstantial. This review discusses changes in VWF reactivity found in patients on LVAD support. It specifically focuses on impacts of LVAD-related mechanical stress on VWF structural stability and adhesive reactivity in exploring multiple causes of AVWS and LVAD-associated hemostatic complications.

TNF Regulates Essential Alternative Complement Pathway Components and Impairs Activation of Protein C in Human Glomerular Endothelial Cells.

Atypical hemolytic uremic syndrome (aHUS) is a thrombotic microangiopathy with severe renal injury secondary to an overactive alternative complement pathway (AP). aHUS episodes are often initiated or recur during inflammation. We investigated gene expression of the surface complement regulatory proteins (CD55, CD59, CD46, and CD141 [thrombomodulin]) and AP components in human glomerular microvascular endothelial cells (GMVECs) and in HUVECs, a frequently used investigational model of endothelial cells. Surface complement regulatory proteins were also quantified by flow cytometry. All experiments were done with and without exposure to IL-1ß or TNF. Without cytokine stimulation, we found that GMVECs had greater AP activation than did HUVECs. With TNF stimulation, THBD gene expression and corresponding CD141 surface presence in HUVECs and GMVECs were reduced, and gene expression of complement components C3 (C3) and factor B (CFB) was increased. Consequently, AP activation, measured by Ba production, was increased, and conversion of protein C (PC) to activated PC by CD141-bound thrombin was decreased, in GMVECs and HUVECs exposed to TNF. IL-1ß had similar, albeit lesser, effects on HUVEC gene expression, and it only slightly affected GMVEC gene expression. To our knowledge, this is the first detailed study of the expression/display of AP components and surface regulatory proteins in GMVECs with and without cytokine stimulation. In aHUS patients with an underlying overactive AP, additional stimulation of the AP and inhibition of activated PC-mediated anticoagulation in GMVECs by the inflammatory cytokine TNF are likely to provoke episodes of renal failure.

Interaction of Shiga toxin with the A-domains and multimers of von Willebrand Factor.

Shiga toxin (Stx) produced by enterohemorrhagic Escherichia coli causes diarrhea-associated hemolytic-uremic syndrome (DHUS), a severe renal thrombotic microangiopathy. We investigated the interaction between Stx and von Willebrand Factor (VWF), a multimeric plasma glycoprotein that mediates platelet adhesion, activation, and aggregation. Stx bound to ultra-large VWF (ULVWF) secreted from and anchored to stimulated human umbilical vein endothelial cells, as well as to immobilized VWF-rich human umbilical vein endothelial cell supernatant. This Stx binding was localized to the A1 and A2 domain of VWF monomeric subunits and reduced the rate of ADAMTS-13-mediated cleavage of the Tyr(1605)-Met(1606) peptide bond in the A2 domain. Stx-VWF interaction and the associated delay in ADAMTS-13-mediated cleavage of VWF may contribute to the pathophysiology of DHUS.

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

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

Treatment of refractory thrombotic thrombocytopenic purpura with N-acetylcysteine: a case report.

BACKGROUND: Thrombotic thrombocytopenic purpura (TTP) is a life-threatening disease resulting in systemic microvascular thrombosis. The disease is caused by excessive platelet (PLT) adhesion to ultra-large (UL) von Willebrand factor (VWF) multimers inadequately cleaved by the processing enzyme ADAMTS-13. While many cases respond to plasma exchange performed with or without concurrent corticosteroids, treatment of the 10% to 20% of patients with refractory disease is difficult. Experimental studies demonstrating that N-acetylcysteine (NAC) inhibits PLT binding to endothelial cell-secreted and anchored UL VWF multimers suggest that NAC may be useful in the treatment of TTP. CASE REPORT: A 44-year-old woman presented with malaise, confusion, chest and abdominal pain, and transient visual loss. Laboratory results and peripheral blood smear were consistent with TTP. The patient was begun on plasma exchange and corticosteroid treatment, but after 10 days the PLT count was still less than 10.0 × 10(9) /L and she developed a fever. Rituximab was initiated, but the patient's condition worsened and she became comatose. Antibiotics were initiated, but cultures remained sterile. After 3 days of coma and further clinical deterioration, treatment with NAC was begun. The patient received a loading dose of 150 mg/kg NAC intravenously (IV) over 1 hour. Within 18 hours the patient awakened abruptly and began communicating with medical personnel. Plasma exchange, corticosteroids, rituximab, and NAC infusion (150 mg/kg IV over 17 hr daily × 10 days) were continued and by Day 17 the PLT count was more than 50 × 10(9) /L. The patient fully recovered and was discharged on Day 31. CONCLUSION: This is the first complete report of a TTP patient treated with NAC. NAC was a safe and effective supplementary treatment for refractory TTP in this patient.

Human complement factor H is a reductase for large soluble von Willebrand factor multimers--brief report.

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.

Mechanical activation of a multimeric adhesive protein through domain conformational change.

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 the multimeric VWF. We found that shear-activated PVWF multimers are more resistant to mechanical unfolding than nonsheared PVWF multimers, as indicated in the higher peak unfolding force. These results provide insight into the mechanism of shear-induced activation of PVWF multimers.

Heat-inactivated Factor B inhibits alternative pathway fluid-phase activation and convertase formation on endothelial cell-secreted ultra-large von Willebrand factor strings.

Defective regulation of the alternative complement pathway (AP) causes excessive activation and promotes the inflammation and renal injury observed in atypical hemolytic-uremic syndrome (aHUS). The usefulness of heat-inactivated Factor B (HFB) in reducing AP activation was evaluated in: fluid-phase reactions, using purified complement proteins and Factor H (FH)-depleted serum; and in surface-activated reactions using human endothelial cells (ECs). C3a and Ba levels, measured by quantitative Western blots, determined the extent of fluid-phase activation. In reactions using C3, FB, and Factor D proteins, HFB addition (2.5-fold FB levels), reduced C3a levels by 60% and Ba levels by 45%. In reactions using FH-depleted serum (supplemented with FH at 12.5% normal levels), Ba levels were reduced by 40% with HFB added at 3.5-fold FB levels. The effectiveness of HFB in limiting AP convertase formation on activated surfaces was evaluated using stimulated ECs. Fluorescent microscopy was used to quantify endogenously released C3, FB, and C5 attached to EC-secreted ultra-large VWF strings. HFB addition reduced attachment of C3b by 2.7-fold, FB by 1.5-fold and C5 by fourfold. Our data indicate that HFB may be of therapeutic value in preventing AP-mediated generation of C3a and C5a, and the associated inflammation caused by an overactive AP.

Generation and breakdown of soluble ultralarge von Willebrand factor multimers.

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.

Endothelial cell ADAMTS-13 and VWF: production, release, and VWF string cleavage.

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 Ca(2+)/Zn(2+)-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 Y(1605)-M(1606) 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.

Human endothelial cells and fibroblasts express and produce the coagulation proteins necessary for thrombin generation.

In a previous study, we reported that human endothelial cells (ECs) express and produce their own coagulation factors (F) that can activate cell surface FX without the additions of external proteins or phospholipids. We now describe experiments that detail the expression and production in ECs and fibroblasts of the clotting proteins necessary for formation of active prothrombinase (FV-FX) complexes to produce thrombin on EC and fibroblast surfaces. EC and fibroblast thrombin generation was identified by measuring: thrombin activity; thrombin-antithrombin complexes; and the prothrombin fragment 1.2 (PF1.2), which is produced by the prothrombinase cleavage of prothrombin (FII) to thrombin. In ECs, the prothrombinase complex uses surface-attached FV and γ-carboxyl-glutamate residues of FX and FII to attach to EC surfaces. FV is also on fibroblast surfaces; however, lower fibroblast expression of the gene for γ-glutamyl carboxylase (GGCX) results in production of vitamin K-dependent coagulation proteins (FII and FX) with reduced surface binding. This is evident by the minimal surface binding of PF1.2, following FII activation, of fibroblasts compared to ECs. We conclude that human ECs and fibroblasts both generate thrombin without exogenous addition of coagulation proteins or phospholipids. The two cell types assemble distinct forms of prothrombinase to generate thrombin.

Production and control of coagulation proteins for factor X activation in human endothelial cells and fibroblasts.

Human endothelial cells (ECs) synthesize, store, and secrete von Willebrand factor multimeric strings and coagulation factor (F) VIII. It is not currently known if ECs produce other coagulation factors for active participation in coagulation. We found that 3 different types of human ECs in primary culture produce clotting factors necessary for FX activation via the intrinsic (FVIII-FIX) and extrinsic (tissue factor [TF]-FVII) coagulation pathways, as well as prothrombin. Human dermal fibroblasts were used as comparator cells. TF, FVII, FIX, FX, and prothrombin were detected in ECs, and TF, FVII, FIX, and FX were detected in fibroblasts. In addition, FVII, FIX, FX, and prothrombin were detected by fluorescent microscopy in EC cytoplasm (associated with endoplasmic reticulum and Golgi proteins). FX activation occurred on human umbilical vein EC surfaces without the addition of external coagulation proteins, proteolytic enzymes, or phospholipids. Tumour necrosis factor, which suppresses the generation of activated protein C and increases TF, augmented FX activation. Fibroblasts also produced TF, but (in contrast to ECs) were incapable of activating FX without the exogenous addition of FX and had a marked increase in FX activation following the addition of both FX and FVII. We conclude that human ECs produce their own coagulation factors that can activate cell surface FX without the addition of exogenous proteins or phospholipids.

Erratum: Deficiency of complement factor H-related proteins and autoantibody-positive hemolytic uremic syndrome in an infant with combined partial deficiencies and autoantibodies to complement factor H and ADAMTS13.

[This corrects the article DOI: 10.1093/ckj/sfy010.].

Ticlopidine- and clopidogrel-associated thrombotic thrombocytopenic purpura (TTP): review of clinical, laboratory, epidemiological, and pharmacovigilance findings (1989-2008).

Thrombotic thrombocytopenic purpura (TTP) is a fulminant disease characterized by platelet aggregates, thrombocytopenia, renal insufficiency, neurologic changes, and mechanical injury to erythrocytes. Most idiopathic cases of TTP are characterized by a deficiency of ADAMTS13 (a disintegrin and metalloprotease, with thrombospondin-1-like domains) metalloprotease activity. Ironically, use of anti-platelet agents, the thienopyridine derivates clopidogrel and ticlopidine, is associated with drug induced TTP. Data were abstracted from a systematic review of English-language literature for thienopyridine-associated TTP identified in MEDLINE, EMBASE, the public website of the Food and Drug Administration, and abstracts from national scientific conferences from 1991 to April 2008. Ticlopidine and clopidogrel are the two most common drugs associated with TTP in FDA safety databases. Epidemiological studies identify recent initiation of anti-platelet agents as the most common risk factor associated with risks of developing TTP. Laboratory studies indicate that most cases of thienopyridine-associated TTP involve an antibody to ADAMTS13 metalloprotease, present with severe thrombocytopenia, and respond to therapeutic plasma exchange (TPE); a minority of thienopyridine-associated TTP presents with severe renal insufficiency, involves direct endothelial cell damage, and is less responsive to TPE. The evaluation of this potentially fatal drug toxicity can serve as a template for future efforts to comprehensively characterize other severe adverse drug reactions.

Conformational stability and domain unfolding of the Von Willebrand factor A domains.

Von Willebrand factor (VWF), a multimeric multidomain glycoprotein secreted into the blood from vascular endothelial cells, initiates platelet adhesion at sites of vascular injury. This process requires the binding of platelet glycoprotein Ib-IX-V to the A1 domain of VWF monomeric subunits under fluid shear stress. The A2 domain of VWF monomers contains a proteolytic site specific for a circulating plasma VWF metalloprotease, A Disintegrin and Metalloprotease with Thrombospondin motifs, member #13 of the ADAMTS enzyme family (ADAMTS-13), that functions to reduce adhesiveness of newly released, unusually large (UL), hyperactive forms of VWF. Shear stress assists ADAMTS-13 proteolysis of ULVWF multimers allowing ADAMTS-13 cleavage of an exposed peptide bond in the A2 domain. Shear stress may induce conformational changes in VWF, and even unfold regions of VWF monomeric subunits. We used urea as a surrogate for shear to study denaturation of the individual VWF recombinant A domains, A1, A2, and A3, and the domain triplet, A1-A2-A3. Denaturation was evaluated as a function of the urea concentration, and the intrinsic thermodynamic stability of the domains against unfolding was determined. The A1 domain unfolded in a 3-state manner through a stable intermediate. Domains A2 and A3 unfolded in a 2-state manner from native to denatured. The A1-A2-A3 triple domain unfolded in a 6-state manner through four partially folded intermediates between the native and denatured states. Urea denaturation of A1-A2-A3 was characterized by two major unfolding transitions: the first corresponding to the simultaneous complete unfolding of A2 and partial unfolding of A1 to the intermediate state; and the second corresponding to the complete unfolding of A3 followed by gradual unfolding of the intermediate state of A1 at high urea concentration. The A2 domain containing the cleavage site for ADAMTS-13 was the least stable of the three domains and was the most susceptible to unfolding. The low stability of the A2 domain is likely to be important in regulating the exposure of the A2 domain cleavage site in response to shear stress, ULVWF platelet adherence, and the attachment of ADAMTS-13 to ULVWF.

Complement Component C3 Binds to the A3 Domain of von Willebrand Factor.

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.

Correlating Conformational Dynamics with the Von Willebrand Factor Reductase Activity of Factor H Using Single Molecule Force Measurements.

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.