VWF Cleavage Research Articles

Hemolytic Uraemic Syndrome Associated with Pregnancy

Hemolytic uraemic syndrome (HUS) and thrombotic thrombocytopenic purpura (TTP) are described as acute syndromes with multisystem abnormalities and pentad of thrombocytopenia, microangiopathic hemolysis, neurological symptoms, renal impairment and fever. Both diseases were believed to form a continuum of the same disease, but recently it was found, that they were having a different pathophysiology, as TTP patients have a deficiency in von wilbrand factor (vWF) cleavage protease. When renal involvement is severe with little or no neurological manifestation, this microangiopathy is termed as haemolytic-uraemic syndrome. If the hemolytic uraemic syndrome is not associated with diarrhoea, it is called Dnegative or atypical HUS. This subdivision is of etiological and prognostic importance. TTP-HUS is associated with high maternal and fetal morbidity and mortality. Treatment of these syndromes differs from syndrome of hemolysis with elevated liver enzymes (HE LLP syndrome) and acute fatty liver of pregnancy hence accurate diagnosis is important for optimal therapy. Plasma transfusion and plasmapheresis have revolutionized management of TTP and HUS by increasing survival 80% to 90%. Here we are reporting a case of D-negative hemolytic uraemic syndrome associated with pregnancy causing intrauterine fetal death. Diagnosis made on clinical and hematological findings, successfully treated by plasmapheresis with residual maternal renal impairment. We are presenting this case, as it is rare disorder associated with high mortality and morbidity, to increase awareness about disease, its diagnosis and management.

ADAMTS13 Binds VWF Via its C-Terminal CUB2 Domain.

Thrombotic thrombocytopenic purpura (TTP) is a life threatening illness due to a deficiency of the VWF-cleaving protease, ADAMTS13. The ADAMTS13 protein is composed of a propeptide, followed by a typical zinc metalloprotease domain. The C-terminal 2/3 of the molecule contains disintegrin-like, cystine-rich, and spacer domains, as well as a total of eight TSP1 motifs and two CUB domains. The function of this C-terminal portion of the molecule and its composite motifs is unknown, though TSP1 and CUB domains of other proteins have been shown to mediate protein-protein interactions. To further explore the interaction between ADAMTS13 and VWF, we cloned full length human cDNAs for both ADAMTS13 and VWF into the mammalian expression vector pcDNA3.1. These constructs were transiently transfected into 293T cells and COS cells respectively, and conditioned media collected for analysis. Using an anti-myc antibody, myc-tagged VWF co-immunoprecipitated (co-IP) with ADAMTS13, as demonstrated by western blot analysis using antisera raised against a C-terminal peptide derived from the predicted ADAMTS13 sequence. This direct interaction required partial denaturation of VWF in 1M urea, with no co-IP observed in the absence of urea. To map the segment within ADAMTS13 responsible for VWF binding, we cloned a series of overlapping ADAMTS13 fragments into the bacterial expression vector, Pet44b. Fusion proteins were purified by binding of the included His-tag to Ni-NTA beads and incubated with recombinant myc-VWF in the presence of 1M urea. Association with VWF was analyzed by co-IP with anti-myc followed by western blot analysis using an antibody to the C-terminal HSV-tag present in each fusion protein. The CUB2 (Glu1298- Thr1427) fusion protein co-IP'd with full-length VWF and also demonstrated concentration-dependent competition with full-length ADAMTS13 for VWF binding. In summary, we have demonstrated a direct protein-protein interaction between VWF and ADAMTS13. Binding requires partial denaturation of VWF and appears to be mediated primarily through contacts with the ADAMTS13 CUB2 domain. This interaction may account for the previously observed co-purification of VWF and ADAMTS13 from human plasma. Furthermore, the requirement for 1M urea suggests that this interaction may only occur physiologically under conditions of high shear. Though others have shown that the C-terminal domains of ADAMTS13, including CUB2, are not required for VWF cleavage in vitro, our data, together with several C-terminal mutations previously reported in TTP patients, suggest that interactions between VWF and the ADAMTS13 CUB2 domain may be important in vivo.

Increased Clearance Explains the Ultra-Large Multimers in Von Willebrand Disease Type 2M Vicenza; Lessons from Recombinant VWF Vicenza and Modeling of Multimer Catabolism.

The pathogenesis of VWD Vicenza has remained elusive. VWD Vicenza is characterized by low plasma but normal platelet VWF concentration, the presence of ultra-large plasma multimers, and a heterozygous R1205H mutation. VWF Vicenza is reported to have a decreased half-life in the circulation. When we expressed rVWF Vicenza R1205H in 293T cells, it was secreted with wild type efficiency and multimer distribution, suggesting that the primary defect is accelerated clearance. To evaluate this hypothesis, we developed a pharmacokinetic model of VWF multimer catabolism. The initial assumptions are: 1. Secretion occurs at a fixed rate with the initial “ultra-large” multimer distribution seen in platelet alpha granules. 2. Cleavage of multimers occurs with a probability p that increases with increasing multimer size. 3. Clearance occurs with a time constant determined by the plasma half life and is independent of multimer size. Modeled multimer distributions were compared to those determined experimentally for patient plasma samples. The effects of DDAVP infusion also were modeled and compared to published data (Casonato et al, Blood 2002; 99:180). For p = 7.5 x 10−4 min−1 and a half life of 12 h, the modeled multimer patterns were comparable to the observed steady-state distribution of normal VWF. Decreasing the half life to 2 hours produces a low plasma concentration of “ultra-large” multimers typical of VWD Vicenza without a change in any other parameter. Conversely, increasing the probability of cleavage by only thirty percent produces typical VWD 2A multimer distributions. The model also reproduces the triplet patterns of normal and type 2A VWF. Finally, the DDAVP simulations reproduced the time course of VWF plasma concentrations and multimer distributions of DDAVP-treated patients. We conclude that increased clearance alone can explain the ultra-large multimer distribution of VWD Vicenza. Similar modeling should allow the estimation of VWF cleavage and clearance rates in other variants of VWD and in other clinical situations including thrombotic thrombocytopenic purpura. [Display omitted] [Display omitted]

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets

Proteolytic cleavage of von Willebrand factor by ADAMTS‐13 prevents uninvited clumping of blood platelets

Proprotein processing activity and cleavage site selectivity of the Kex2-like endoprotease PACE4

Proprotein processing activity of the Kex2-like mammalian endoprotease PACE4 and its cleavage selectivity for sites with basic amino acid residues were determined. Using a recombinant vaccinia virus-based expression system, PACE4 was expressed in pig kidney PK(15) cells and, like two other Kex2-like endoproteases furin and PC6A, shown to correctly process the precursor of von Willebrand factor (pro-vWF). Furthermore, characteristics of the cleavage site selectivity of PACE4 were compared to those of furin and PC6A using the vWF cleavage site mutants vWFR-lG, vWFK-2A, and vWFR-4A as substrates. Cleavage site selectivity of PACE4 and PC6A appeared to be similar but they differed from that of furin.

Subunit composition of plasma von Willebrand factor in patients with the myeloproliferative syndrome

In order to evaluate the role of proteolysis in acquired von Willebrand's disease (vWD) associated with the myeloproliferative syndrome, we have determined the relative quantity of von Willebrand factor (vWF) fragments as compared with the intact 225 kDa subunit in four patients. The plasma vWF of each individual lacked large multimers; each had a prolonged bleeding time; and both platelet and leukocyte counts were elevated. Plasma was obtained from blood drawn into 1 mmol/L leupeptin, 6 mmol/L N-ethylmaleimide, and 5 mmol/L EDTA to prevent in vitro proteolysis. vWF was isolated from plasma by immunoadsorbent chromatography, reduced, subjected to SDS-5% polyacrylamide gel electrophoresis, and immunoblotted with a mixture of 55 anti-vWF monoclonal antibodies. In three patients with essential thrombocytosis (ET) the 176 and 140 kDa fragments were increased in proportion to the intact 225 kDa subunit indicating increased proteolysis. Treatment of one ET patient with CCNU (Lomustine) decreased the platelet count and, to a lesser extent, the white blood cell count. This was associated with a correction of the bleeding time, a partial correction of the multimeric abnormality, and a lessening of vWF cleavage. In a patient with polycythemia rubra vera (PRV) the proportion of the 176 kDa fragment was increased to the upper limit of normal but there was no definite evidence of increased proteolysis. These studies provide evidence that proteolysis plays a role in the acquired von Willebrand's disease associated with the myeloproliferative syndrome. However, other mechanisms must also be considered.

Subunit Composition of Plasma von Willebrand Factor in Patients With the Myeloproliferative Syndrome

In order to evaluate the role of proteolysis in acquired von Willebrand's disease (vWD) associated with the myeloproliferative syndrome, we have determined the relative quantity of von Willebrand factor (vWF) fragments as compared with the intact 225 kDa subunit in four patients. The plasma vWF of each individual lacked large multimers; each had a prolonged bleeding time; and both platelet and leukocyte counts were elevated. Plasma was obtained from blood drawn into 1 mmol/L leupeptin, 6 mmol/L N-ethylmaleimide, and 5 mmol/L EDTA to prevent in vitro proteolysis. vWF was isolated from plasma by immunoadsorbent chromatography, reduced, subjected to SDS-5% polyacrylamide gel electrophoresis, and immunoblotted with a mixture of 55 anti-vWF monoclonal antibodies. In three patients with essential thrombocytosis (ET) the 176 and 140 kDa fragments were increased in proportion to the intact 225 kDa subunit indicating increased proteolysis. Treatment of one ET patient with CCNU (Lomustine) decreased the platelet count and, to a lesser extent, the white blood cell count. This was associated with a correction of the bleeding time, a partial correction of the multimeric abnormality, and a lessening of vWF cleavage. In a patient with polycythemia rubra vera (PRV) the proportion of the 176 kDa fragment was increased to the upper limit of normal but there was no definite evidence of increased proteolysis. These studies provide evidence that proteolysis plays a role in the acquired von Willebrand's disease associated with the myeloproliferative syndrome. However, other mechanisms must also be considered.