Miriam Galbusera

von Willebrand factor-cleaving protease in thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome.

BACKGROUND Thrombotic thrombocytopenic purpura and the hemolytic-uremic syndrome are severe microvascular disorders of platelet clumping with similar signs and symptoms. Unusually large multimers of von Willebrand factor, capable of agglutinating circulating platelets under high shear stress, occur in the two conditions. We investigated the prevalence of von Willebrand factor-cleaving protease deficiency in patients with familial and nonfamilial forms of these disorders. METHODS Plasma samples were obtained from 53 patients with thrombotic thrombocytopenic purpura or hemolytic-uremic syndrome. Von Willebrand factor-cleaving protease was assayed in diluted plasma samples with purified normal von Willebrand factor as the substrate. The extent of the degradation of von Willebrand factor was assessed by electrophoresis in sodium dodecyl sulfate-agarose gels and immunoblotting. To determine whether an inhibitor of von Willebrand factor-cleaving protease was present, we measured the protease activity in normal plasma after incubation with plasma from the patients. RESULTS We examined 30 patients with thrombotic thrombocytopenic purpura and 23 patients with the hemolytic-uremic syndrome. Of 24 patients with nonfamilial thrombotic thrombocytopenic purpura, 20 had severe and 4 had moderate protease deficiency during an acute event. An inhibitor found in 20 of these patients was shown to be IgG in five of five tested plasma samples. Of 13 patients with nonfamilial hemolytic-uremic syndrome, 11 had normal levels of activity of von Willebrand factor-cleaving protease during the acute episode, whereas in 2 patients, the activity was slightly decreased. All 6 patients with familial thrombotic thrombocytopenic purpura lacked von Willebrand factor-cleaving protease activity but had no inhibitor, whereas all 10 patients with familial hemolytic-uremic syndrome had normal protease activity. In vitro proteolytic degradation of von Willebrand factor by the protease was studied in 5 patients with familial and 7 patients with nonfamilial hemolytic-uremic syndrome and was normal in all 12 patients. CONCLUSIONS Nonfamilial thrombotic thrombocytopenic purpura is due to an inhibitor of von Willebrand factor-cleaving protease, whereas the familial form seems to be caused by a constitutional deficiency of the protease. Patients with the hemolyticuremic syndrome do not have a deficiency of von Willebrand factor-cleaving protease or a defect in von Willebrand factor that leads to its resistance to protease.

Heterogeneity of plasma von Willebrand factor multimers resulting from proteolysis of the constituent subunit.

In this report we demonstrate that proteolytic cleavage of the constituent subunit is one of the causes determining the heterogeneous size distribution of plasma von Willebrand factor (vWf) multimers. As shown by two-dimensional nonreduced/reduced agarose/polyacrylamide gel electrophoresis, the structure of circulating vWf molecules may deviate from that represented by assemblage of a variable number of identical subunits. Indeed, even though the largest multimers in normal plasma appear to be composed predominantly of intact 225-kD subunits, those of intermediate and smaller size contain also 189-, 176-, and 140-kD proteolytic fragments. Different subunit composition patterns are repeated regularly in multimers of increasing molecular mass, yielding series of bands with similar structure. One of these series consists of molecules without evidence of proteolytic fragmentation, and its smallest member appears to be a dimer of 225-kD subunits. Type IIA von Willebrand disease, characterized by absence of the largest multimers, displays a pattern wherein the fragments of 176 and 140 kD are relatively increased, that of 189 kD is markedly decreased or absent, but the composition of individual multimers is otherwise similar to that of species seen also in normal plasma. In contrast to those in the circulation, all normal platelet vWf multimers contain only intact subunit. These results suggest that proteolytic cleavage of plasma vWf subunits occurs after release from cellular sites, whereas platelet vWf stored in alpha-granules is protected from proteolysis. These findings provide information that may be relevant for understanding the normal processing of vWf multimers and for elucidating the pathogenesis of some of the congenital and acquired structural abnormalities of this molecule.

Alternative Pathway Activation of Complement by Shiga Toxin Promotes Exuberant C3a Formation That Triggers Microvascular Thrombosis

Shiga toxin (Stx)-producing E.coli O157:H7 has become a global threat to public health; it is a primary cause of diarrhea-associated hemolytic uremic syndrome (HUS), a disorder of thrombocytopenia, microangiopathic hemolytic anemia, and acute renal failure with thrombi occluding renal microcirculation. In this study, we explored whether Stx triggers complement-dependent microvascular thrombosis in in vitro and in vivo experimental settings of HUS. Stx induced on human microvascular endothelial cell surface the expression of P-selectin, which bound and activated C3 via the alternative pathway, leading to thrombus formation under flow. In the search for mechanisms linking complement activation and thrombosis, we found that exuberant complement activation in response to Stx generated an increased amount of C3a that caused further endothelial P-selectin expression, thrombomodulin (TM) loss, and thrombus formation. In a murine model of HUS obtained by coinjection of Stx2 and LPS and characterized by thrombocytopenia and renal dysfunction, upregulation of glomerular endothelial P-selectin was associated with C3 and fibrin(ogen) deposits, platelet clumps, and reduced TM expression. Treatment with anti–P-selectin Ab limited glomerular C3 accumulation. Factor B-deficient mice after Stx2/LPS exhibited less thrombocytopenia and were protected against glomerular abnormalities and renal function impairment, indicating the involvement of complement activation via the alternative pathway in the glomerular thrombotic process in HUS mice. The functional role of C3a was documented by data showing that glomerular fibrin(ogen), platelet clumps, and TM loss were markedly decreased in HUS mice receiving C3aR antagonist. These results identify Stx-induced complement activation, via P-selectin, as a key mechanism of C3a-dependent microvascular thrombosis in diarrhea-associated HUS.

In response to protein load podocytes reorganize cytoskeleton and modulate endothelin-1 gene: Implication for permselective dysfunction of chronic nephropathies

Effacement of podocyte foot processes occurs in many proteinuric nephropathies and is accompanied by rearrangement of the actin cytoskeleton. Here, we studied whether protein overload affects intracellular pathways, leading to cytoskeletal architecture changes and ultimately to podocyte dysfunction. Mouse podocytes bound and endocytosed both albumin and IgG via receptor-specific mechanisms. Protein overload caused redistribution of F-actin fibers instrumental to up-regulation of the prepro-endothelin (ET)-1 gene and production of the corresponding peptide. Increased DNA-binding activity for nuclear factor (NF)-kappaB and Ap-1 nuclear proteins was measured in nuclear extracts of podocytes exposed to excess proteins. Both Y27632, which inhibits Rho kinase-dependent stress fiber formation, and jasplakinolide, an F-actin stabilizer, decreased NF-kappaB and Ap-1 activity and reduced ET-1 expression. This suggested a role for the cytoskeleton, through activated Rho, in the regulation of the ET-1 peptide. Focal adhesion kinase (FAK), an integrin-associated nonreceptor tyrosine kinase, was phosphorylated by albumin treatment via Rho kinase-triggered actin reorganization. FAK activation led to NF-kappaB- and Ap-1-dependent ET-1 expression. These data suggest that reorganization of the actin cytoskeletal network in response to protein load is implicated in modulation of the ET-1 gene via Rho kinase-dependent FAK activation of NF-kappaB and Ap-1 in differentiated podocytes. Increased ET-1 generation might alter glomerular permselectivity and amplify the noxious effect of protein overload on dysfunctional podocytes.

Complement Factor H Mutation in Familial Thrombotic Thrombocytopenic Purpura with ADAMTS13 Deficiency and Renal Involvement

Thrombotic thrombocytopenic purpura is a rare disorder of small vessels that is associated with deficiency of the von Willebrand factor-cleaving protease ADAMTS13, which favors platelet adhesion and aggregation in the microcirculation. The disease manifests mainly with central nervous system symptoms, but cases of renal insufficiency have been reported. Presented are findings of the genetic basis of phenotype heterogeneity in thrombotic thrombocytopenic purpura in two sisters within one family. The patients had ADAMTS13 deficiency as a result of two heterozygous mutations (causing V88M and G1239V changes). In addition, a heterozygous mutation (causing an S890I change) in factor H of complement was found in the patient who developed chronic renal failure but not in her sister, who presented with exclusive neurologic symptoms.

Treatment of Bleeding in Dialysis Patients

Bleeding is a common and potentially serious complication of acute and chronic renal failure. The pathogenesis of bleeding in uremia is multifactorial; however, the major role is played by abnormalities in platelet–platelet and platelet–vessel wall interaction. Platelet dysfunction is partially due to uremic toxins present in circulating blood. Despite decreased platelet function, abnormalities of blood coagulation and fibrinolysis predispose the uremic patients to a hypercoagulable state carrying the risk of cardiovascular and thrombotic complications. Dialysis improves platelet abnormalities and reduces, but does not eliminate, the risk of hemorrhage. Hemodialysis can even contribute to the bleeding through the continuous platelet activation induced by the interaction between blood and artificial surfaces and the use of anticoagulants. Correction of anemia improves hemostasis in uremic patients. Therapeutic management of bleeding in patients with uremia is discussed.

Reversible Activation Defect of the Platelet Glycoprotein IIb-IIIa Complex in Patients With Uremia

Patients with chronic renal failure may experience a bleeding tendency and blood loss after surgical procedures or trauma. Altered platelet function has been indicated as the major cause of uremic bleeding, but its pathogenesis remains to be clarified. In two groups of patients with chronic renal disease of various etiology, the receptor function of glycoprotein (GP) Ib and GP IIb-IIIa complex was studied. Glycoprotein Ib was assessed with both 125I-von Willebrand factor (vWF) and 125I-asialo-vWF binding to platelets. Activation-dependent receptor function of the GP IIb-IIIa complex was studied with 125I-fibrinogen and 125I-vWF binding to washed platelets stimulated with adenosine diphosphate plus epinephrine (10 mumol/L each). Flow cytometric analyses on resting and stimulated platelets were performed using an activation-dependent, anti-GP IIb-IIIa monoclonal antibody (PAC1) as well as an activation-independent antibody (LJ-P1). Binding of PAC1 also was assessed in washed and stimulated platelets and in platelet-rich plasma before and after dialysis. We found that the activation-dependent receptor function of the GP IIb-IIIa complex is defective in uremia, as shown by decreased binding of both vWF and fibrinogen to stimulated platelets. Moreover, binding of the activation-dependent anti-GP IIb-IIIa monoclonal antibody, PAC1, was significantly decreased in uremia compared with that of the activation-independent antibody, LJ-P1. Thus, the number of GP IIb-IIIa receptors expressed on the platelet membrane is normal, but their activation is impaired. In contrast to the functional abnormality of GP IIb-IIIa, the vWF-binding activity of GP Ib was normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a “Kidney Disease: Improving Global Outcomes” (KDIGO) Controversies Conference

In both atypical hemolytic uremic syndrome (aHUS) and C3 glomerulopathy (C3G) complement plays a primary role in disease pathogenesis. Herein we report the outcome of a 2015 Kidney Disease: Improving Global Outcomes (KDIGO) Controversies Conference where key issues in the management of these 2 diseases were considered by a global panel of experts. Areas addressed included renal pathology, clinical phenotype and assessment, genetic drivers of disease, acquired drivers of disease, and treatment strategies. In order to help guide clinicians who are caring for such patients, recommendations for best treatment strategies were discussed at length, providing the evidence base underpinning current treatment options. Knowledge gaps were identified and a prioritized research agenda was proposed to resolve outstanding controversial issues.

Thrombotic Thrombocytopenic Purpura: Evidence That Infusion Rather Than Removal of Plasma Induces Remission of the Disease

Plasma exchange has recently been reported to be more effective than plasma infusion for the treatment of thrombotic thrombocytopenic purpura (TTP). However, in the only available controlled study, the plasma infused during the exchange procedure was three times that given by infusion alone. Here we report the case of a patient with chronic relapsing TTP who had 21 relapsing episodes in the last 3 years. During 18 relapses, infusion of plasma, as infusion alone or in the context of an exchange procedure, invariably induced remission of the disease. By contrast, plasma removal alone (replaced with albumin and saline) was ineffective in three further consecutive relapses so that infusion was eventually necessary to induce remission. We concluded that the effective component of plasma exchange in TTP is infusion, rather than removal of plasma. Unusually large von Willebrand factor (ULvWF) multimers were found during both acute and remission phases, possibly reflecting intravascular leakage from ongoing endothelial cell injury. A relative increase of the 176-kd fragment and a relative decrease of the 225-kd subunit were demonstrated during the acute phase, indicating in vivo proteolytic vWF fragmentation. Since in vitro evidence is available that such fragments of vWF induce platelet aggregation, it is speculated that protease inhibitors of normal plasma help restore normal vWF processing activity in the circulation, which explains remission of the disease associated with the plasma infusion.

Complement activation: the missing link between ADAMTS-13 deficiency and microvascular thrombosis of thrombotic microangiopathies.

Endothelial injury is the central factor in the events leading to thrombotic microangiopathy (TMA); however, the mechanisms involved are not fully understood. Here we investigate the role of neutrophils (PMNs) and of complement activation in inducing microvascular damage and loss of thromboresistance in TMA associated with ADAMTS-13 deficiency. PMNs isolated during the acute phase of the disease released excessive amounts of reactive-oxygen species (ROS), N-derived oxidants and proteinases and induced damage and thromboresistance loss in human microvascular endothelial cell line (HMEC-1) ex vivo. Endothelial cytotoxicity and thromboresistance loss was also induced by TMA serum. Complement-derived products were responsible for the above effects: in fact, TMA serum caused C3 and Membrane Attack Complex (MAC) deposition on HMEC-1 and its cytotoxic effect was abolished by complement inhibition. TMA serum caused surface expression of P-selectin on HMEC-1 which may promote PMN adhesion and resulted in increased PMN cytotoxicity, indicating that complement may have a role in PMN activation. In addition, TMA serum stimulated control PMNs to release ROS and proteinases, and to cause endothelial cell cytotoxicity. All of the above effects were abrogated by complement inactivation. These data document for the first time that complement-initiated PMN activation and endothelial injury may have a crucial role in microvascular thrombosis of TMA associated with ADAMTS-13 deficiency.