Defective processing of unusually large von Willebrand factor multimers and thrombotic thrombocytopenic purpura

Abstract

The following brief essay is a glance backward at a few early skirmishes with thrombotic thrombocytopenic purpura (TTP), and at studies initiated unexpectedly 25 years ago that continue to the present. Before the 1960s, I had seen little of the world beyond a broad swath of central Texas running approximately from San Antonio to Fort Worth. I then had a surprising opportunity to attend the Johns Hopkins University School of Medicine in Baltimore, Maryland. A terrifying half‐hour during my junior year rotation in Internal Medicine in 1966 was spent presenting the topic, ‘TTP and Other Microangiopathic Hemolytic Anemias’, to a small audience that included snickering classmates and the formidable Osler Professor and Medicine Chairman, A. McGehee Harvey. Dr Harvey was considered by awed students to be a clinical expert on everything. A particular threat to me on that day was his special interest in the incipient field of ‘autoimmune disorders.’ After my tremulous ‘chalk‐talk’ using the blackboard (no slides then), TTP remained almost completely mysterious to both presenter and attendees. Beyond the fact that it was an untreatable and inevitably lethal disorder caused by profound in vivo platelet clumping, little was known. During 1970–3, I was a Hematology Fellow, and then junior faculty member, at the University of Miami School of Medicine. The Chairman of Medicine there was William J. Harrington, who had conducted the death‐defying idiopathic thrombocytopenic purpura (ITP) plasma infusion experiment on himself 20 years earlier that provided the critical clue to the pathogenesis of the disorder. Dr Harrington was an imposing teacher and clinician who mumbled encouragement, witticisms and profundities in a barely intelligible South Boston accent. He was equally disoriented by my residual Texas twang. TTP was occasionally discussed among Fellows and Hematology faculty, but remained resistant to any dogma, bluster or treatment. The Hematology trainees in my group included Drs John Byrnes and Eric Lian. We hoped to unearth some clue to the cause of TTP, as Dr Harrington had done with ITP. Both Drs Byrnes and Lian soon made important observations. In the New England Journal of Medicine in 1977, Byrnes and Khurana [1Byrnes J.J. Khurana M. Treatment of thrombotic thrombocytopenic purpura with plasma.N Engl J Med. 1977; 297: 1386-9Crossref PubMed Scopus (275) Google Scholar] described the effectiveness of fresh or stored plasma, or the cryoprecipitate‐poor fraction of plasma, in inducing remissions in a young woman with relapsing TTP. Dr Lian and his colleagues reported in Blood in 1979 [2Lian E.C. Harkness D.R. Byrnes J.J. Wallach H. Nunez R. Presence of a platelet aggregating factor in the plasma of patients with thrombotic thrombocytopenic purpura (TTP) and its inhibition by normal plasma.Blood. 1979; 53: 333-8Crossref PubMed Google Scholar] and 1981 [3Lian E.C. Savaraj N. Effects of platelet inhibitors on the platelet aggregation induced by plasma from patients with thrombotic thrombocytopenic purpura.Blood. 1981; 58: 354-9Crossref PubMed Google Scholar] that some TTP plasmas induced platelet clumping in vitro. I was at the Boston VA Hospital by 1981, and recall summarizing the findings from the three papers during a discussion of TTP at a weekly hematology conference. It struck me that in vitro aggregation caused by the plasma of some of the TTP patients shared a few characteristics with platelet agglutination induced by ristocetin and human von Willebrand factor (VWF), or bovine VWF alone. For example, the platelet clumping was not inhibited by EDTA or aspirin, and did not require platelet metabolism. Studying TTP was a formidable logistical task in 1981. I was convinced that the disorder was even less common than advertised because I had seen (or recognized) only one or two TTP patients in total during my previous decade as a hematologist. I began to study the mechanism and inhibition of ristocetin‐induced VWF‐mediated platelet agglutination at the new University of Texas Medical School in Houston during the 1970s. My initial instruction in VWF clinical laboratory techniques came from Dr Ronald S. Weinger, who had come to Houston via Chicago, Boston and El Paso to develop a ‘Hemophilia Care Center.’ Dr Weinger and his technician, Christine Rudy, developed an especially porous 0.5% agarose version of the Laurell two‐dimensional immunoelectrophoresis (2‐D IEP) system for the qualitative estimation of the size distribution of plasma VWF. This porous 2‐D IEP method emphasized the evaluation of larger, rather than smaller, VWF multimers. My technician, Joseph Troll, and I were purifying and radioiodinating VWF and ristocetin, and studying their interaction with platelets. Our various methods were brought to the Boston VA Hospital in 1980 when all of us moved from Houston at the invitation of Dr Daniel Deykin, Chairman of the Medical Service. One of the attractions of the Boston VA Hospital hemostasis research laboratory was the opportunity to work with Drs Mark Weinstein and Suchen Hong. Dr Weinstein was developing a porous, unreduced SDS‐1% agarose gel electrophoretic technique for the purpose of displaying optimally the largest plasma VWF multimers. Dr Hong cultured human umbilical vein endothelial cells (HUVECs), and was soon providing endothelial cell supernatants and lysates for the analyses of VWF forms by 0.5% agarose 2‐D IEP and SDS‐1% agarose gel electrophoresis. All of these methods were in place in 1981 when a healthy‐appearing 21‐year‐old woman visited the Boston VA Hospital out‐patient clinic for routine blood work. She had a history of episodes of TTP in previous years that had been treated with plasma infusion/exchange in New York City. In the out‐patient clinic that day, her platelets, hemoglobin and red cell morphology were normal. Within hours of this routine clinic visit she had a minor injury at home, and soon thereafter was brought back to the Boston VA Hospital with a severe, recurrent TTP episode. We had a plasma sample saved from earlier in the day when she was clinically normal, and then collected plasma from her throughout a prolonged course of plasma infusion and exchange procedures. This therapy was, tragically, associated with only a partial response, and eventually she died of the disorder. However, she left us the gift of her plasma samples. Her initial out‐patient plasma sample, obtained when her blood counts and clinical status were normal, contained more slowly migrating, presumably larger, VWF forms compared to normal plasma using the 0.5% agarose 2‐D IEP and SDS‐1% agarose gel methods (Fig. 1A,B). During her prolonged episode, the slowly migrating VWF forms were most obvious at times of partial remission when her platelet counts increased towards normal. We were astonished by the presence of these slowly migrating VWF forms, and by their similar electrophoretic mobility to the VWF forms that we observed in cultured human endothelial cell supernatant. Drs Nachman, Jaffe and Ferris [4Nachman R.L. Jaffe E.A. Ferris B. Multiple molecular forms of endothelial cell factor VIII related antigen.Biochem Biophys Acta. 1981; 667: 361-9Crossref PubMed Scopus (0) Google Scholar] at Cornell Medical College had also recently seen more slowly migrating forms of VWF in HUVEC supernatant using 0.9% agarose 2‐D IEP. As our data accumulated on this patient, I could concentrate on little other than deciphering the perplexing results. During a Friday afternoon drive with my family toward the tiny town of Stockbridge, in the Berkshire Mountains of western Massachusetts, I had an ‘eureka’ (or, at least, a ‘gadzooks’) moment. Was it possible that our unfortunate patient did not process normally the huge VWF multimers escaping from endothelial cells? Were these gigantic VWF forms to blame for the in vivo platelet clumping in TTP? I mentioned to my (skeptical) wife that there was a slight possibility that our drive into the sunshine of a New England afternoon had provoked an instant of insight into a long‐ fathomless disease. During subsequent weeks, I asked colleagues in Houston to send plasma samples from one of their patients who was suspected of having a congenital chronic relapsing TTP‐like illness. She had received regular prophylactic plasma infusions for two decades. In the initial description of her case, a 1960 Blood article by Dr Irving Schulman and colleagues [5Schulman I. Pierce M. Lukens A. Currimbhoy Z. Studies on thrombopoiesis. I. A factor in normal human plasma required for platelet production; chronic thrombocytopenia due to its deficiency.Blood. 1960; 16: 943-57Crossref PubMed Google Scholar] (Chicago), it was suggested that she might be an unique example of ‘thrombopoetin deficiency.’ As the patient reached adulthood, she moved about the country sustained by regular plasma infusions. Her physicians in Cleveland (Dr Oscar Ratnoff) and in Houston (Drs Ronald Weinger and Phillip Cimo) suspected that she actually had some type of recurrent thrombotic microangiopathy. I also requested plasma samples from two patients with relapsing TTP described in separate case reports in the New England Journal of Medicine in 1977 [1Byrnes J.J. Khurana M. Treatment of thrombotic thrombocytopenic purpura with plasma.N Engl J Med. 1977; 297: 1386-9Crossref PubMed Scopus (275) Google Scholar] and 1978 [6Upshaw J.D. Congenital deficiency of a factor in plasma that reverses microangiopathic hemolysis and thrombocytopenia.N Engl J Med. 1978; 298: 1350-2Crossref PubMed Google Scholar]. The samples were soon sent north by the helpful authors, Drs John Byrnes [1Byrnes J.J. Khurana M. Treatment of thrombotic thrombocytopenic purpura with plasma.N Engl J Med. 1977; 297: 1386-9Crossref PubMed Scopus (275) Google Scholar] in Miami, and Jefferson Davis Upshaw [6Upshaw J.D. Congenital deficiency of a factor in plasma that reverses microangiopathic hemolysis and thrombocytopenia.N Engl J Med. 1978; 298: 1350-2Crossref PubMed Google Scholar] in Memphis (‘Upshaw syndrome’). (Curiously, a chronic relapsing ‘TTP‐HUS’‐like illness has recently been given the chronologically inverted eponym, ‘Upshaw–Schulman syndrome’[7Veyradier A. Lavergne J.M. Ribba A.S. Obert B. Loirat C. Meyer D. Girma J.P. Ten candidate mutations in six French families with congenital thrombotic thrombocytopenic purpura (Upshaw–Schulman syndrome).J Thromb Haemost. 2004; 2: 424-9Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar, 8Matsumoto M. Kokame K. Soejima K. Miura M. Hayashi S. Fujii Y. Iwai A. Ito E. Tsuji Y. Takeda‐Shitaka M. Iwadate M. Umeyama H. Yagi H. Ishizashi H. Banno F. Nakagaki T. Miyata T. Fujimura Y. Molecular characterization of ADAMTS13 mutations in Japanese patients with Upshaw‐Schulman syndrome.Blood. 2004; 103: 1305-10Crossref PubMed Scopus (0) Google Scholar]. The members of our laboratory team at the Boston VA Hospital were amazed to find that the plasma samples from the three other chronic relapsing TTP patients also contained the more slowly migrating, presumably larger, VWF multimers (Fig. 1C). I began to refer to these forms as ‘unusually large VWF multimers’, for lack of a more imaginative term. My Boston teammates considered the expression to be an unwieldy mouthful. Nevertheless, ‘ULVWF’ and ‘chronic relapsing TTP’ entered the literature together in the paper I constructed on behalf of our group as a 1982 New England Journal of Medicine‘Medical Intelligence’ report [9Moake J.L. Rudy C.K. Troll J.H. Weinstein M.J. Colannino N.M. Azocar J. Seder R.H. Hong S.L. Deykin D. Unusually large plasma factor VIII. von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura.N Eng J Med. 1982; 307: 1432-5Crossref PubMed Google Scholar]. In addition to my two technical colleagues, Christine Rudy and Joe Troll, the other coauthors included: Dr Mark Weinstein and his technician, Noreen Colannino; Dr Jose Azocar, a Hematology fellow; Dr Richard Seder, our Blood Bank associate; Dr Suchen Hong; and Dr Daniel Deykin. In the manuscript, I proposed that ULVWF multimers induce the pathologic systemic platelet clumping in TTP and are the elusive ‘agglutinative’ substances mentioned in 1924 by Moschcowitz [10Moschcowitz E. Hyaline thrombosis of the terminal arterioles and capillaries: a hitherto undescribed disease.Proc NY Pathol Soc. 1924; 24: 21-4Google Scholar]. The 1982 report concluded that ‘patients with chronic TTP have a defect in the processing of very large vWF multimers after synthesis and secretion by endothelial cells, and that this defect makes the patients susceptible to periodic relapses.’[9Moake J.L. Rudy C.K. Troll J.H. Weinstein M.J. Colannino N.M. Azocar J. Seder R.H. Hong S.L. Deykin D. Unusually large plasma factor VIII. von Willebrand factor multimers in chronic relapsing thrombotic thrombocytopenic purpura.N Eng J Med. 1982; 307: 1432-5Crossref PubMed Google Scholar]. Our group then proceeded to report in Blood in 1985 that the processing of ULVWF multimers could be restored rapidly, but transiently, in the Houston and Miami chronic relapsing TTP patients by infusing normal fresh‐frozen plasma or cryoprecipitate‐poor plasma (cryosupernatant) [11Moake J.L. Byrnes J.J. Troll J.H. Rudy C.K. Hong S.L. Weinstein M.J. Colannino N.M. Effects of fresh frozen plasma and its cryosupernatant fraction on von Willebrand factor multimer forms in chronic relapsing thrombotic thrombocytopenic purpura.Blood. 1985; 65: 1232-6Crossref PubMed Google Scholar] (Fig. 1D). Whatever the precise biochemical identity of the ULVWF ‘processing activity’ was, it was contained in both of these plasma products. After a near‐fatal illness/injury in Boston in 1982–3, I moved back to the Baylor College of Medicine and adjacent Rice University in Houston in 1984. Additional evidence was accumulated there that was compatible with the platelet‐clumping culprits in TTP being ULVWF multimers. Children with congenital chronic relapsing TTP, as well as many adult patients with acquired types of TTP (Fig. 1E) were studied at Baylor, its five associated hospitals in the Texas Medical Center, and Rice University. My adroit technical colleagues during most of the next two decades, Patsy McPherson, Leticia Nolasco, Nancy Turner and Dr Thomas Chow, were indispensable contributors to this work. Our TTP data appeared in dollops during subsequent years [12Moake J.L. McPherson P.D. Abnormalities of von Willebrand factor multimers in thrombotic thrombocytopenic purpura and the hemolytic–uremic syndrome.Am J Med. 1989; 87: 9N-14NAbstract Full Text PDF PubMed Google Scholar, 13Chintagumpala M. Hurwitz R. Moake J. Mahoney D. Steuber C. Chronic relapsing thrombotic thrombocytopenic purpura in infants with large von Willebrand factor multimers during remission.J Pediatr. 1992; 120: 49-53Abstract Full Text PDF PubMed Google Scholar, 14Moake J. Chintagumpala M. Turner N. McPherson P. Nolasco L. Steuber C. Santiago‐Borrero P. Horowitz M. Pehta J. Solvent/detergent‐treated plasma contains the component that reverses von Willebrand factor‐mediated shear‐induced platelet aggregation and prevents episodes of chronic relapsing thrombotic thrombocytopenic purpura.Blood. 1994; 84: 490-7Crossref PubMed Google Scholar, 15Chow T.W. Turner N.A. Chintagumpala M. McPherson P.D. Nolasco L.H. Rice L. Hellums J.D. Moake J.L. Increased von Willebrand factor binding to platelets in single episode and recurrent types of thrombotic thrombocytopenic purpura.Am J Hematol. 1998; 57: 293-302Crossref PubMed Scopus (0) Google Scholar]. Drs John Byrnes (Miami) and Aaron Marcus (Cornell Medical College, New York City) provided early and consistent encouragement and support. The contention that failure to degrade ULVWF multimers is the cause of many cases of TTP remained heretical, however, and was always in danger of being submerged beneath waves of vigorous dissent by other investigators and commentators. One persistent critic wrote in Blood in 1985: ‘VWF is unlikely to play a major role in platelet aggregation induced by [the] majority of [idiopathic] TTP plasmas.’[16Lian E.C. Siddiqui F.A. Investigation of the role of von Willebrand factor in in thrombotic thrombocytopenic purpura.Blood. 1985; 66: 1219-21Crossref PubMed Google Scholar] Ironically, this same investigator later experienced a ‘VWF/TTP epiphany’, and then was a coauthor of influential papers on VWF‐cleaving protease/ADAMTS‐13 defects in idiopathic TTP [17Tsai H.M. Lian E.C. Antibodies of von Willebrand factor cleaving protease in acute thrombotic thrombocytopenic purpura.N Engl J Med. 1998; 339: 1585-94Crossref PubMed Scopus (0) Google Scholar, 18Levy G.G. Nichols W.C. Lian E.C. Foroud T. McClintick J.N. McGee B.M. Yang A.Y. Siemieniak D.R. Stark K.R. Gruppo R. Sarode R. Shurin S.B. Chandrasekaran V. Stabler S.P. Sabio H. Bouhassira E.E. Upshaw Jr, J.D. Ginsburg D. Tsai H.M. Mutations in a member of the ADAMTS gene family cause thrombotic thrombocytopenic purpura.Nature. 2001; 413: 488-94Crossref PubMed Scopus (1430) Google Scholar]. As the controversy simmered and lingered without resolution, I was stunned and excited in 1997–8 to hear, and then to read in Blood and the New England Journal of Medicine, reports of experiments that were compatible with the VWF‐related concept of TTP pathophysiology. In 1997, Miha Furlan and colleagues [19Furlan M. Robles R. Solenthaler M. Wassmer M. Sandoz P. Lammle B. Deficient activity of von Willebrand factor‐cleaving protease in chronic relapsing thrombotic thrombocytopenic purpura.Blood. 1997; 89: 3097-103Crossref PubMed Google Scholar] described four patients with relapsing TTP who had a chronic deficiency of VWF‐cleaving metalloprotease activity in plasma. Because no inhibitor of the enzyme was detected, the deficiency was ascribed to an abnormality in the production, survival or function of the protease. The following year, elegant papers by Furlan et al.[20Furlan M. Robles R. Galbusera M. Remuzzi G. Kyrle P.A. Brenner B. Krause M. Scharrer I. Aumann V. Mittler U. Solenthaler M. Lammle B. von Willebrand factor‐cleaving protease in thrombotic thrombocytopenic purpura and hemolytic–uremic syndrome.N Engl J Med. 1998; 339: 1578-84Crossref PubMed Scopus (0) Google Scholar], and Han‐Mou Tsai and Eric Lian [17Tsai H.M. Lian E.C. Antibodies of von Willebrand factor cleaving protease in acute thrombotic thrombocytopenic purpura.N Engl J Med. 1998; 339: 1585-94Crossref PubMed Scopus (0) Google Scholar] published in the same issue of the New England Journal of Medicine demonstrated that VWF‐cleaving metalloprotease activity was absent, or barely detectable, during acute episodes in the citrate‐plasma of patients with acquired idiopathic TTP. The activity returned to normal as the patients recovered. An IgG autoantibody against the VWF‐cleaving metalloprotease enzyme probably accounted for the lack of protease activity in most of the acquired idiopathic TTP patients reported by Tsai and Lian. In their papers, Furlan et al.[19Furlan M. Robles R. Solenthaler M. Wassmer M. Sandoz P. Lammle B. Deficient activity of von Willebrand factor‐cleaving protease in chronic relapsing thrombotic thrombocytopenic purpura.Blood. 1997; 89: 3097-103Crossref PubMed Google Scholar, 20Furlan M. Robles R. Galbusera M. Remuzzi G. Kyrle P.A. Brenner B. Krause M. Scharrer I. Aumann V. Mittler U. Solenthaler M. Lammle B. von Willebrand factor‐cleaving protease in thrombotic thrombocytopenic purpura and hemolytic–uremic syndrome.N Engl J Med. 1998; 339: 1578-84Crossref PubMed Scopus (0) Google Scholar], and Tsai and Lian [17Tsai H.M. Lian E.C. Antibodies of von Willebrand factor cleaving protease in acute thrombotic thrombocytopenic purpura.N Engl J Med. 1998; 339: 1585-94Crossref PubMed Scopus (0) Google Scholar] independently defined VWF ‘processing activity’ as a specific VWF‐cleaving metalloprotease activity that is absent, or nearly so, from the plasma of many acquired idiopathic TTP patients. The VWF‐cleaving protease degrades large (and ‘unusually large’) VWF multimers in order to prevent their entrance into, or persistence in, the circulation. It was gratifying for me to write the editorial accompanying the wonderful work from the laboratories of Furlan and Tsai [21Moake J.L. Moschcowitz, multimers and metalloprotease.N Eng J Med. 1998; 339: 1629-31https://doi.org/10.1056/NEJM199811263392210Crossref PubMed Scopus (80) Google Scholar]. The unfolding story became even more convincing as the VWF‐cleaving metalloprotease was identified precisely in 2001 as the 13th member of a family of 18 ADAMTS‐type enzymes (a disintegrin and metalloprotease with thrombospondin‐1‐like domains), i.e. ‘ADAMTS‐13’[22Gerritsen H.E. Robles R. Lammle B. Furlan M. Partial amino acid sequence of purified von Willebrand factor‐cleaving protease.Blood. 2001; 98: 1654-61Crossref PubMed Scopus (0) Google Scholar, 23Fujikawa K. Suzuki H. McMullen B. Chung D. Purification of von Willebrand factor‐cleaving protease and its identification as a new member of the metalloproteinase family.Blood. 2001; 98: 1662-6Crossref PubMed Scopus (0) Google Scholar, 24Zheng X. Chung D. Takayama T.K. Majerus E.M. Sadler J.E. Fujikawa K. Structure of von Willebrand factor cleaving protease (ADAMTS13), a metaloprotease involved in thrombotic thrombocytopenic purpura.J Biol Chem. 2001; 276: 41059-63Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. My return to Houston and the Baylor College of Medicine in 1984 reunited me with former collaborators in Chemical Engineering at the adjacent Rice University campus. David Hellums and Larry McIntire had established the Biomedical Engineering Laboratory at Rice, and were investigating the effects of high shear stress on blood cells. Hellums had already described the phenomenon of shear stress‐induced platelet aggregation, and I became part of a small group attempting to elucidate the mechanism. In the mid‐1980s, my Rice technician, Nancy Turner, and I decided to study the effects of elevated levels of shear stress on platelet aggregation using the platelet‐rich‐plasma of an elderly patient of mine with severe type3 von Willebrand's disease (VWD). Both plasma and platelets of the patient lacked detectable VWF. We were surprised to find that the patient's platelets would not aggregate under high shear conditions unless we added large VWF multimers that had been purified from normal human cryoprecipitate. An additional unanticipated finding was that human endothelial cell supernatant containing ULVWF forms induced aggregation even more effectively in high shear fields [25Moake J.L. Turner N.A. Stathopoulos N.A. Nolasco L.H. Hellums J.D. Involvement of large plasma vWF multimers and unusually large vWF forms derived from endothelial cells in shear stress‐induced platelet aggregation.J Clin Invest. 1986; 78: 1456-61Crossref PubMed Google Scholar]. I considered this latter observation to be functional evidence compatible with my by then 5‐year‐old, and still unpopular, hypothesis that unprocessed ULVWF multimers derived from endothelial cells cause systemic aggregation in TTP. As I daily become more decrepit, it is thrilling to watch energetic young investigators with clever new techniques attracted to the study of ULVWF and TTP. A Rice University predoctoral student, Maneesh Arya, and his Bioengineering advisor, Bahman Anvari, used laser optical ‘tweezers’ to demonstrate that ULVWF multimers are, indeed, hyper‐adhesive [26Arya M. Anvari B. Romo G.M. Cruz M.A. Dong J.F. McIntire L.V. Moake J.L. Lopez J.A. Ultra‐large multimers of von Willebrand factor form spontaneous high‐strength bonds with the platelet GP Ib‐IX complex: studies using optical tweezers.Blood. 2002; 99: 3971-7Crossref PubMed Scopus (0) Google Scholar]. Jing‐fei Dong, a brilliant neurosurgeon‐turned molecular biologist at Baylor College of Medicine, observed that long ‘strings’ of unprocessed ULVWF multimers emanate from stimulated endothelial cells and induce platelet adhesion under flowing conditions [27Dong J.F. Moake J.L. Nolasco L. Bernardo A. Arceneaux W. Shrimpton C.N. Schade A.J. McIntire L.V. Fujikawa K. López J.A. ADAMTS‐13 rapidly cleaves newly secreted ultra‐large von Willebrand factor multimers on the endothelial surface under flowing conditions.Blood. 2002; 100: 4033-9Crossref PubMed Scopus (0) Google Scholar](Fig. 2A). Dong and his associates then found that the ULVWF strings are cleaved by normal plasma containing ADAMTS‐13 activity, but not by TTP plasma samples in which ADAMTS‐13 is either absent or inhibited [27Dong J.F. Moake J.L. Nolasco L. Bernardo A. Arceneaux W. Shrimpton C.N. Schade A.J. McIntire L.V. Fujikawa K. López J.A. ADAMTS‐13 rapidly cleaves newly secreted ultra‐large von Willebrand factor multimers on the endothelial surface under flowing conditions.Blood. 2002; 100: 4033-9Crossref PubMed Scopus (0) Google Scholar]. Many other aspects of ULVWF processing are now rapidly coming into focus. These include the involvement in ULVWF secretion, anchorage and cleavage of: cytokines (e.g. TNF‐α, IL‐8, IL‐6 in complex with its receptor) [28Bernardo A. Ball C. Nolasco L. Moake J.L. Dong J.F. Effects of inflammatory cytokines on the release and cleavage of the endothelialcell‐derived ultra‐large von Willebrand factor multimers under flow.Blood. 2004; 104: 100-6Crossref PubMed Scopus (0) Google Scholar]; P‐selectin [29Padilla A. Moake J. Bernardo A. Ball C. Wang Y. Arya M. Nolasco L. Turner N. Berndt M.C. Anvari B. Lopez J. Dong J.F. P‐selectin anchors newly released ultra‐large von Willebrand factor multimers to the endothelial cell surface.Blood. 2004; 103: 2150-6Crossref PubMed Scopus (0) Google Scholar]; the A3 domain (adjacent to the A2 cleavage site) in VWF monomeric subunits [30Dong J.F. Moake J.L. Bernardo A. Fujikawa K. Ball C. Nolasco L. Lopez J.A. Cruz M.A. ADAMTS‐13 metalloprotease interacts with the endothelial cell‐derived ultra‐large von Willebrand factor.J Biol Chem. 2003; 278: 296339Abstract Full Text Full Text PDF Scopus (150) Google Scholar]; and the CUB, thrombospondin, and cysteine‐rich domains of ADAMTS‐13 (Fig. 2B‐D). Various ELISA‐based methods for detecting specific A2 domain cleavage by plasma ADAMTS‐13 have appeared recently, including the technique devised by Miguel Cruz and colleagues at Baylor [31Whitelock J.L. Nolasco L.N. Bernardo A. Moake J.L. Dong J.F. Cruz M.A. ADAMTS‐13 activity in plasma is rapidly measured by a new ELISA method that uses recombinant VWF‐A2 domain as substrate.J Thromb Haemost. 2004; 2: 485-91Abstract Full Text Full Text PDF PubMed Scopus (0) Google Scholar]. This approach may soon allow the rapid detection of ADAMTS‐13‐deficiency in clinical laboratories. My initial interest in TTP was triggered by the intuition, probably shared with many others, that unraveling this most extreme example of in vivo platelet aggregation would catalyze the improved treatment of common arterial thrombotic disorders. It remains to be known if a variety of different types of kinetic disturbances in the ADAMTS‐13/ULVWF‐cleaving mechanism, less dramatic than in TTP, constitute risk factors for heart attack and stroke. These putative disturbances might be the consequence of congenital polymorphisms in the genes encoding domains in either ADAMTS‐13 or ULVWF monomeric subunits involved in docking or cleavage functions. Alternatively, ADAMTS‐13/ULVWF interaction might be impaired on or near the surface of endothelial cells injured by atherosclerosis, inflammation, drugs or chemicals – or perhaps even in secondary thrombotic microangiopathies and the various types of hemolytic‐uremic syndrome. Throughout these years, my research on TTP has been supported by: the NHLBI (NIH); the VA; Baylor College of Medicine and Rice University; The Methodist Hospital of Houston; contributions from the family and friends of Kiera Burroughs; and, most recently, the Mary Rodes Gibson Foundation of College Station, Texas. Mary Gibson was the endearing VWD type3 patient mentioned in the text with severe, life‐long bleeding. She donated her blood samples generously and enthusiastically for many important experiments on shear‐aggregation and TTP. With far‐sighted kindness, she created a foundation that outlives her to promote the continuing investigation of VWF and TTP at Rice University and Baylor College of Medicine. Many admired colleagues and life‐long friends associated with the work on TTP are mentioned by name in this sketch. Other important participants are to be found among the authors listed in the references. I am, in addition, indebted to many inspirational teachers. Among these are: Drs Dorothea Gibson and Lorraine Shirley (Texas Christian University, Ft. Worth, Texas); Drs Barry Wood, Albert Lehninger, Daniel Nathan, Lockard Conley, Albert Owens and Lyle Sensenbrenner (Johns Hopkins University School of Medicine); Drs Leighton Cluff and George Caranasos (Johns Hopkins and the University of Florida Medical Center); Drs Khalil Ahmed and Nicholas Bachur (Baltimore Cancer Research Center/NIH); and Drs Adel Yunis, Donald Harkness and Duane Schultz (University of Miami School of Medicine).