J. Evan Sadler

Von Willebrand factor, ADAMTS13, and thrombotic thrombocytopenic purpura

Discoveries during the past decade have revolutionized our understanding of idiopathic thrombotic thrombocytopenic purpura (TTP). Most cases in adults are caused by acquired autoantibodies that inhibit ADAMTS13, a metalloprotease that cleaves von Willebrand factor within nascent platelet-rich thrombi to prevent hemolysis, thrombocytopenia, and tissue infarction. Although approximately 80% of patients respond to plasma exchange, which removes autoantibody and replenishes ADAMTS13, one third to one half of survivors develop refractory or relapsing disease. Intensive immunosuppressive therapy with rituximab appears to be effective as salvage therapy, and ongoing clinical trials should determine whether adjuvant rituximab with plasma exchange also is beneficial at first diagnosis. A major unanswered question is whether plasma exchange is effective for the subset of patients with idiopathic TTP who do not have severe ADAMTS13 deficiency.

Induction of specific storage organelles by von Willebrand factor propolypeptide.

Endothelial cells store the multimeric adhesive glycoprotein von Willebrand factor (vWf), which promotes the formation of a platelet plug at the site of vessel injury. To investigate the packaging of vWf into the granules called Weibel-Palade bodies, we expressed pro-vWf cDNA and cDNA lacking the prosequence in a variety of cell lines. Storage granules formed only in cells that contain a regulated pathway of secretion. Furthermore, packaging required the prosequence. Pro-vWf, lacking the C-terminal region involved in interchain disulfide bonding, formed granules. We conclude that the signal for storage is universal in that an adhesive glycoprotein can be stored by a hormone-secreting cell; the storage of vWf is independent of its covalent multimeric structure; the unusual rod shape of Weibel-Palade bodies is due to vWf; and the vWf propolypeptide is necessary for the formation of vWf storage granules.

Pathogenesis of Thrombotic Microangiopathies

Profound thrombocytopenia and microangiopathic hemolytic anemia characterize thrombotic microangiopathy, which includes two major disorders: thrombotic thrombocytopenic purpura (TTP) and hemolytic uremic syndrome (HUS). TTP has at least three types: congenital or familial, idiopathic, and nonidiopathic. The congenital and idiopathic TTP syndromes are caused primarily by deficiency of ADAMTS13, owing to mutations in the ADAMTS13 gene or autoantibodies that inhibit ADAMTS13 activity. HUS is similar to TTP, but is associated with acute renal failure. Diarrhea-associated HUS accounts for more than 90% of cases and is usually caused by infection with Shiga-toxin-producing Escherichia coli (O157:H7). Diarrhea-negative HUS is associated with complement dysregulation in up to 50% of cases, caused by mutations in complement factor H, membrane cofactor protein, factor I or factor B, or by autoantibodies against factor H. The incomplete penetrance of mutations in either ADAMTS13 or complement regulatory genes suggests that precipitating events or triggers may be required to cause thrombotic microangiopathy in many patients.

Functional architecture of Weibel-Palade bodies

Weibel-Palade bodies (WPBs) are elongated secretory organelles specific to endothelial cells that contain von Willebrand factor (VWF) and a variety of other proteins that contribute to inflammation, angiogenesis, and tissue repair. The remarkable architecture of WPBs is because of the unique properties of their major constituent VWF. VWF is stored inside WPBs as tubules, but on its release, forms strikingly long strings that arrest bleeding by recruiting blood platelets to sites of vascular injury. In recent years considerable progress has been made regarding the molecular events that underlie the packaging of VWF multimers into tubules and the processes leading to the formation of elongated WPBs. Mechanisms directing the conversion of tightly packaged VWF tubules into VWF strings on the surface of endothelial cells are starting to be unraveled. Several modes of exocytosis have now been described for WPBs, emphasizing the plasticity of these organelles. WPB exocytosis plays a role in the pathophysiology and treatment of von Willebrand disease and may have impact on common hematologic and cardiovascular disorders. This review summarizes the major advances made on the biogenesis and exocytosis of WPBs and places these recent discoveries in the context of von Willebrand disease.

Remission of Chronic Thrombotic Thrombocytopenic Purpura after Treatment with Cyclophosphamide and Rituximab

Context Many adults with thrombotic thrombocytopenic purpura (TTP) have autoantibodies that promote clotting by inhibiting a von Willebrand factorcleaving protease (ADAMTS13) in plasma. Contribution This 42-year-old woman had chronic relapsing TTP despite plasma exchange; splenectomy; and treatment with vincristine, prednisone, and cyclosporine. She had low to absent ADAMTS13 activity and an IgG inhibitor. After immunosuppressive therapy with rituximab and cyclophosphamide, the TTP remitted, ADAMTS13 levels normalized, and the inhibitor was undetectable. Cautions Intensive immunosuppressive therapy aimed at the autoimmune aspect of TTP should be tested in prospective trials before being used widely. The Editors Most adults with idiopathic thrombotic thrombocytopenic purpura (TTP) have autoantibodies that inhibit a von Willebrand factorcleaving protease in plasma (1-3). This protease recently was cloned and identified as a new member of the ADAMTS metalloproteases, ADAMTS13 (4-8). Plasma exchange therapy has increased survival in patients with TTP from less than 10% to approximately 75% (9). However, plasma exchange does not directly address the underlying autoimmune process, and up to one third of patients have relapse after initially successful treatment (10). Anecdotal data suggest that immunosuppressive therapy can sometimes be beneficial, but this approach has not been tested systematically in patients known to have ADAMTS13 deficiency. We report the case of a woman with chronic relapsing TTP who had undetectable ADAMTS13 activity and elevated ADAMTS13 inhibitor titers. Treatment with rituximab and cyclophosphamide resulted in prompt disappearance of ADAMTS13 antibodies, normalization of ADAMTS13 activity, and remission of thrombotic microangiopathy. Case Report A 42-year-old woman was hospitalized in May 1999 with gait disturbance, disorientation, and lethargy. She had been healthy, with three uncomplicated pregnancies and deliveries and no previous symptoms of autoimmune disease. Computed tomography showed many cerebral infarcts. The platelet count was 60 109 cells/L, the hemoglobin level was 96 g/L, and the fibrinogen level was normal. The serum lactate dehydrogenase level was 1157 U/L. The patient received intravenous immunoglobulin but did not respond. She was transferred to our institution in June 1999 (Figure, day 1). Figure. Platelet count ( top ) and ADAMTS13 activity ( bottom ) in a patient with thrombotic thrombocytopenic purpura during 2.5 years of observation. On admission, hematocrit was 0.231, platelet count was 24 109 cells/L, haptoglobin level was 0.09 g/L (normal range, 0.27 to 2.2 g/L), and serum creatinine concentration was 141 mol/L (1.6 mg/dL) (normal range, 50 to 125 mol/L [0.6 to 1.4 mg/dL]). Prothrombin time and partial thromboplastin time were normal. Results of direct and indirect antiglobulin tests were negative. The peripheral smear contained more than 10 schistocytes per high-power field. Thrombotic thrombocytopenic purpura was diagnosed, and plasma exchange (1.5 volumes daily) was begun (Figure, top). The platelet count normalized but decreased to 34 109 cells/L by day 22. Prednisone (1 mg/kg of body weight per day) and aspirin (325 mg/d) were added without sustained benefit. The platelet count improved transiently after laparoscopic splenectomy on day 36 but decreased to less than 100 109 cells/L by day 43. Two intravenous doses of vincristine, 2 mg each, resulted in gradual improvement. Frequency of plasma exchanges was decreased to every other day, and the patient was discharged on day 85 with a normal platelet count. Plasmapheresis was discontinued on day 97. On day 105, the platelet count was 43 109 cells/L. Plasma exchange was resumed, and cyclosporine, 75 mg twice per day, was started. The platelet count increased, and plasma exchange was tapered over 3 weeks. During the next 4 months, blood counts were relatively stable. The daily prednisone dose was decreased to 40 mg. On day 248, the patient was admitted with cortical blindness and a platelet count of 58 109 cells/L. Plasma exchange was followed by an increase in platelet count but no improvement in vision. The patient was discharged after 12 days, and plasma exchange was tapered over 2 weeks. During the next 11 months, the patient was hospitalized four times for relapsed TTP and each time was treated with plasma exchange. The prednisone dose was tapered to 5 mg/d. On day 572, the patient was hospitalized with deafness and thrombocytopenia. The platelet count increased rapidly with plasma exchange. The serum creatinine concentration increased to 221 mol/L (2.5 mg/dL), and cyclosporine therapy was discontinued. The patient received two doses of intravenous rituximab (375 mg/m2 weekly). The creatinine concentration decreased to 159 mol/L (1.8 mg/dL), the platelet count remained above 100 109 cells/L, and symptoms were stable for 5 months. On day 740, the platelet count was 69 109 cells/L. The patient received daily plasma exchange for 16 days, responded well, and was discharged. She was admitted twice during the next 4 months to receive plasma exchange for relapsing TTP. Beginning on day 849, the patient received one dose of intravenous cyclophosphamide, 1 g/m2, and four doses of intravenous rituximab, 375 mg/m2, every 7 to 14 days. No side effects of rituximab were noted. During 10 months of follow-up, the patient has had a normal platelet count, stable hematocrit, normal lactate dehydrogenase level and creatinine concentration, and rare schistocytes. No other signs or symptoms of TTP have been noted. Her neurologic defects (blindness and deafness) persist unchanged. Methods Informed consent was obtained from the patient and her family. ADAMTS13 was assayed as described elsewhere (11); however, the substrate was 10 g of von Willebrand factor per mL in 5 mmol of TrisHCl per L (pH, 8.0), 1.5 mol of urea per L, and 1 mmol of phenylmethanesulfonyl fluoride per L (Sigma, St. Louis, Missouri). Plasma samples were heat-treated at 56 C for 30 minutes and were serially diluted in phosphate-buffered saline. Diluted plasma (5 L) was added to normal human plasma (5 L) and incubated at 37 C for 30 minutes. Residual ADAMTS13 activity was measured as described earlier. One unit of inhibitor reduces the ADAMTS13 activity of an equal volume of normal plasma by 50%. Recombinant ADAMTS13 (7) truncated after the metalloprotease (residues 1 to 289) was cloned in pcDNA3.1/V5-His TOPO (Invitrogen, Carlsbad, California), expressed in transiently transfected Chinese hamster ovary cells (CHO-S), and purified from conditioned medium on TALON metal affinity resin (BD Biosciences Clontech, Palo Alto, California). ADAMTS13 was immunoprecipitated with IgG adsorbed from plasma samples onto protein A agarose. Proteins were analyzed by using sodium dodecyl sulfatepolyacrylamide gel electrophoresis and Western blot with monoclonal anti-V5 antibody (Invitrogen). Results Early in her disease, the patient had complete deficiency of plasma ADAMTS13 activity and an inhibitor was detected (Figure, bottom). Plasma therapy over the next 19 months resulted in several short periods of remission, even though ADAMTS13 inhibitor titers decreased minimally and plasma ADAMTS13 activity remained low. The inhibitory activity was recovered in IgG purified from the patients plasma and immunoprecipitated the metalloprotease domain of recombinant ADAMTS13 (data not shown). For continuing autoimmune TTP, immunosuppressive therapy with rituximab was initiated. Rituximab is usually given at a dose of 375 mg/m2, repeated weekly for four doses (12, 13). The patient received an abbreviated course of two doses, which was followed by prompt disappearance of ADAMTS13 inhibitor, normalization of ADAMTS13 activity, and a normal platelet count. The patient had the first of three additional clinical relapses 5 months after receiving rituximab. Before the second course of rituximab plus cyclophosphamide, ADAMTS13 activity was 33% of normal and no ADAMTS13 inhibitor was detected. These values were obtained after 9 days of daily plasmapheresis, which may have partially corrected the ADAMTS13 deficiency. Subsequently, symptoms of TTP resolved, platelet count and plasma ADAMTS13 activity increased, and the ADAMTS13 inhibitor remained undetectable. The most recent ADAMTS13 level was 17%, with no inhibitor detected. Discussion Although idiopathic TTP is usually an autoimmune disease (1-3), standard therapy does not address this underlying mechanism. Plasma exchange may increase patient survival because it removes deleterious antibodies and replenishes ADAMTS13 protein. Most patients have self-limited disease that remits after 1 to several weeks of plasma exchange. However, approximately one third of patients have a chronic relapsing course (10). Thus, TTP often behaves as an aggressive autoimmune disease, and some patients might benefit from additional therapy directed at B cells and antibody production. Many immunosuppressive regimens have been tried in TTP, with encouraging but inconclusive results. Corticosteroids are often administered at the time of plasma exchange (14), but their efficacy has not been systematically evaluated (9, 10). Case reports and small series have described sustained responses to splenectomy (15), and responses to cyclosporine (16), vincristine, cyclophosphamide, and azathioprine (17, 18) have also been described. This anecdotal experience provides a rationale for testing additional immunosuppressive strategies in patients with relapsing or refractory TTP. Rituximab is a chimeric anti-CD20 monoclonal antibody developed for treatment of non-Hodgkin lymphoma; CD20 is expressed on B cells. Rituximab has emerged as a promising treatment for autoimmune disorders, including autoimmune hemolytic anemia (12) and idiopathic thrombocytopenic purpura (13). Common side effects observed during the treatment of lymphoma include transient hypotension and fever during the first

Assembly of Weibel–Palade body-like tubules from N-terminal domains of von Willebrand factor

Endothelial cells assemble von Willebrand factor (VWF) multimers into ordered tubules within storage organelles called Weibel–Palade bodies, and tubular packing is necessary for the secretion of VWF filaments that can bind connective tissue and recruit platelets to sites of vascular injury. We now have recreated VWF tubule assembly in vitro, starting with only pure VWF propeptide (domains D1D2) and disulfide-linked dimers of adjacent N-terminal D′D3 domains. Assembly requires low pH and calcium ions and is reversed at neutral pH. Quick-freeze deep-etch electron microscopy and three-dimensional reconstruction of negatively stained images show that tubules contain a repeating unit of one D′D3 dimer and two propeptides arranged in a right-handed helix with 4.2 units per turn. The symmetry and location of interdomain contacts suggest that decreasing pH along the secretory pathway coordinates the disulfide-linked assembly of VWF multimers with their tubular packaging.

Exosite interactions contribute to tension-induced cleavage of von Willebrand factor by the antithrombotic ADAMTS13 metalloprotease

Von Willebrand factor (VWF) is a multimeric protein that mediates platelet adhesion at sites of vascular injury, and ADAMTS13 (a disintegrin and metalloprotease with thrombospondin)is a multidomain metalloprotease that limits platelet adhesion by a feedback mechanism in which fluid shear stress induces proteolysis of VWF and prevents disseminated microvascular thrombosis. Cleavage of the Tyr1605–Met1606 scissile bond in the VWF A2 domain depends on a Glu1660–Arg1668 segment in the same domain and on the noncatalytic spacer domain of ADAMTS13, suggesting that extensive enzyme–substrate interactions facilitate substrate recognition. Based on mutagenesis and kinetic analysis, we find that the ADAMTS13 spacer domain binds to an exosite near the C terminus of the VWF A2 domain. Deleting the spacer domain from ADAMTS13 or deleting the exosite from the VWF substrate reduced the rate of cleavage ≈20-fold. A cleavage product containing the exosite was a hyperbolic mixed-type inhibitor of ADAMTS13 proteolysis of either VWF multimers or model peptide substrates but only if the ADAMTS13 enzyme contained the spacer domain. The specificity of this unique mechanism depends on tension-induced unfolding of the VWF A2 domain, which exposes the scissile bond and exosite for interaction with complementary sites on ADAMTS13.

Integrin αvβ3 on human endothelial cells binds von Willebrand factor strings under fluid shear stress

Acutely secreted von Willebrand factor (VWF) multimers adhere to endothelial cells, support platelet adhesion, and may induce microvascular thrombosis. Immunofluorescence microscopy of live human umbilical vein endothelial cells showed that VWF multimers rapidly formed strings several hundred micrometers long on the cell surface after stimulation with histamine. Unexpectedly, only a subset of VWF strings supported platelet binding, which depended on platelet glycoprotein Ib. Electron microscopy showed that VWF strings often consisted of bundles and networks of VWF multimers, and each string was tethered to the cell surface by a limited number of sites. Several approaches implicated P-selectin and integrin alpha(v)beta(3) in anchoring VWF strings. An RGDS peptide or a function-blocking antibody to integrin alpha(v)beta(3) reduced the number of VWF strings formed. In addition, integrin alpha(v) decorated the VWF strings by immunofluorescence microscopy. Furthermore, lentiviral transduction of shRNA against the alpha(v) subunit reduced the expression of cell-surface integrin alpha(v)beta(3) and impaired the ability of endothelial cells to retain VWF strings. Soluble P-selectin reduced the number of platelet-decorated VWF strings in the absence of Ca(2+) and Mg(2+) but had no effect in the presence of these cations. These results indicate that VWF strings bind specifically to integrin alpha(v)beta(3) on human endothelial cells.

Interaction of von Willebrand Factor Domain A1 with Platelet Glycoprotein Ibα-(1–289) SLOW INTRINSIC BINDING KINETICS MEDIATE RAPID PLATELET ADHESION

We investigated the crucial hemostatic interaction between von Willebrand factor (VWF) and platelet glycoprotein (GP) Ibα. Recombinant VWF A1 domain (residues Glu497-Pro705 of  VWF) bound stoichiometrically to a GPIbα-calmodulin fusion protein (residues His1-Val289 of GPIbα; GPIbα-CaM) immobilized on W-7-agarose with a K d of 3.3 μm. The variant VWF A1(R545A) bound to GPIbα-CaM 20-fold more tightly, mainly because the association rate constantk on increased from 1,100 to 8,800m −1 s−1. The GPIbα mutations G233V and M239V cause platelet-type pseudo-von Willebrand disease, and VWF A1 bound to GPIbα(G233V)-CaM and GPIbα(M239V)-CaM with aK d of 1.0 and 0.63 μm, respectively. The increased affinity of VWF A1 for GPIbα(M239V)-CaM was explained by an increase in k on to 4,500m −1 s−1. GPIbα-CaM bound with similar affinity to recombinant VWF A1, to multimeric plasma VWF, and to a fragment of dispase-digested plasma VWF (residues Leu480/Val481-Gly718). VWF A1 and A1(R545A) bound to platelets with affinities and rate constants similar to those for binding to GPIbα-CaM, and botrocetin had the expected positively cooperative effect on the binding of VWF A1 to GPIbα-CaM. Therefore, allosteric regulation by botrocetin of VWF A1 binding to GPIbα, and the increased binding affinity caused by mutations in VWF or GPIbα, are reproduced by isolated structural domains. The substantial increase in k on caused by mutations in either A1 or GPIbα suggests that productive interaction requires rate-limiting conformational changes in both binding sites. The exceptionally slow k on andk off provide important new constraints on models for rapid platelet tethering at high wall shear rates.

Localization of Disulfide Bonds in the Cystine Knot Domain of Human von Willebrand Factor

von Willebrand factor (VWF) is a multimeric glycoprotein that is required for normal hemostasis. After translocation into the endoplasmic reticulum, proVWF subunits dimerize through disulfide bonds between their C-terminal cystine knot-like (CK) domains. CK domains are characterized by six conserved cysteines. Disulfide bonds between cysteines 2 and 5 and between cysteines 3 and 6 define a ring that is penetrated by a disulfide bond between cysteines 1 and 4. Dimerization often is mediated by additional cysteines that differ among CK domain subfamilies. When expressed in a baculovirus system, recombinant VWF CK domains (residues 1957–2050) were secreted as dimers that were converted to monomers by selective reduction and alkylation of three unconserved cysteine residues: Cys2008, Cys2010, and Cys2048. By partial reduction and alkylation, chemical and proteolytic digestion, mass spectrometry, and amino acid sequencing, the remaining intrachain disulfide bonds were characterized: Cys1961–Cys2011 (1-4), Cys1987–Cys2041 (2-5), Cys1991–Cys2043 (3-6), and Cys1976–Cys2025. The mutation C2008A or C2010A prevented dimerization, whereas the mutation C2048A did not. Symmetry considerations and molecular modeling based on the structure of transforming growth factor-β suggest that one or three of residues Cys2008, Cys2010, and Cys2048 in each subunit mediate the covalent dimerization of proVWF.