The antiplatelet drug clopidogrel is a new thienopyridine derivative whose mechanism of action and chemical structure are similar to those of ticlopidine. The estimated incidence of ...ticlopidine-associated thrombotic thrombocytopenic purpura is 1 per 1600 to 5000 patients treated, whereas no clopidogrel-associated cases were observed among 20,000 closely monitored patients treated in phase 3 clinical trials and cohort studies. Because of the association between ticlopidine use and thrombotic thrombocytopenic purpura and other adverse effects, clopidogrel has largely replaced ticlopidine in clinical practice. More than 3 million patients have received clopidogrel. We report the clinical and laboratory findings in 11 patients in whom thrombotic thrombocytopenic purpura developed during or soon after treatment with clopidogrel.
The 11 patients were identified by active surveillance by the medical directors of blood banks (3 patients), hematologists (6), and the manufacturer of clopidogrel (2).
Ten of the 11 patients received clopidogrel for 14 days or less before the onset of thrombotic thrombocytopenic purpura. Although 10 of the 11 patients had a response to plasma exchange, 2 required 20 or more exchanges before clinical improvement occurred, and 2 had relapses while not receiving clopidogrel. One patient died despite undergoing plasma exchange soon after diagnosis.
Thrombotic thrombocytopenic purpura can occur after the initiation of clopidogrel therapy, often within the first two weeks of treatment. Physicians should be aware of the possibility of this syndrome when initiating clopidogrel treatment.
There are no widely accepted criteria for the definition of hematopoietic stem cell transplant -associated microangiopathy (TAM). An International Working Group was formed to develop a consensus ...formulation of criteria for diagnosing clinically significant TAM.
The participants proposed a list of candidate criteria, selected those considered necessary, and ranked those considered optional to identify a core set of criteria. Three obligatory criteria and four optional criteria that ranked highest formed a core set. In an appropriateness panel process, the participants scored the diagnosis of 16 patient profiles as appropriate or not appropriate for TAM. Using the experts' ratings on the patient profiles as a gold standard, the sensitivity and specificity of 24 candidate definitions of the disorder developed from the core set of criteria were evaluated. A nominal group technique was used to facilitate consensus formation. The definition of TAM with the highest score formed the final
The Working Group proposes that the diagnosis of TAM requires fulfilment of all of the following criteria: (i) >4% schistocytes in blood; (ii) de novo, prolonged or progressive thrombocytopenia (platelet count <50 x 109/L or 50% or greater reduction from previous counts); (iii) sudden and persistent increase in lactate dehydrogenase concentration; (iv) decrease in hemoglobin concentration or increased transfusion requirement; and (v) decrease in serum haptoglobin. The sensitivity and specificity of this definition exceed 80%.
The Working Group recommends that the presented criteria of TAM be adopted in clinical use, especially in scientific trials.
Leukocyte rolling on vascular endothelium is mediated by an interaction between P‐selectin expressed on endothelial cells and P‐selectin glycoprotein ligand‐1 on leukocytes. This interaction reduces ...the velocity of leukocyte movements to allow subsequent firm adhesion and transmigration. However, the interaction has so far been observed only under low venous shear stress and cannot explain the accumulation of monocytes in atherosclerotic plaques found in arteries, where shear stress is much higher. We have previously shown that newly released ultra‐large von Willebrand factor (ULVWF) forms extremely long string‐like structures to which platelets tether. Here, we investigated whether platelets adhered to ULVWF strings are activated and form aggregates. We also determined whether activated platelets on ULVWF strings can support leukocyte tethering and rolling under high shear stresses. We found that platelets adhered to ULVWF expressed P‐selectin and bound PAC‐1, suggesting their rapid activation. We also found that leukocytes tethered to and rolled on these platelet‐decorated ULVWF strings, but not directly on endothelial cells, under high shear stresses of 20 and 40 dyn/cm2 in a P‐selectin dependent manner. These results suggest that the endothelial cell‐bound ULVWF provide an ideal matrix to aggregate platelets and recruit leukocytes to endothelial cells under high shear stress. The observed phenomenon delineates a mechanism for leukocytes to be tethered to arterial endothelial cells under high shear, providing a potential link between inflammation and thrombosis.
See also Lenting PJ, Rastegarlari G. ADAMTS‐13: double trouble for von Willebrand factor. This issue, pp 2775–7.
Summary. Background: von Willebrand factor (VWF) released from endothelial cells is ...rich in ultra‐large (UL) multimers that are intrinsically active in binding platelets, whereas plasma‐type VWF multimers require shear stress to be activated. This functional difference may be attributed to thiols exposed on the surface of plasma‐type VWF multimers, but not on ULVWF multimers. Shear stress induces the exposed thiols to form disulfide bonds between laterally apposed plasma‐type VWF multimers, leading to enhanced VWF binding to platelets. Objectives: We tested a hypothesis that ADAMTS‐13 has a disulfide bond reducing activity that regulates shear‐induced thiol‐disulfide exchange of VWF. Methods: Thiol blocking agents and active thiol bead capturing were used to identify and locate this activity, along with truncated ADAMTS‐13 mutants. Results: ADAMTS‐13 contains a disulfide bond reducing activity that primarily targets disulfide bonds in plasma‐type VWF multimers induced by high shear stress or formed with thiol beads, but not disulfide bonds in native multimeric structures. Cysteine thiols targeted by this activity are in the VWF C‐domain and are known to participate in shear‐induced thiol‐disulfide exchange. ADAMTS‐13 contains cysteine thiols that remain exposed after being subjected to hydrodynamic forces. Blocking these active thiols eliminates this reducing activity and moderately decreases ADAMTS‐13 activity in cleaving ULVWF strings anchored to endothelial cells under flow conditions, but not under static conditions. This activity is located in this C‐terminal region of ADAMTS‐13. Conclusions: This novel disulfide‐bond‐reducing activity of ADAMTS‐13 may prevent covalent lateral association and increased platelet adherence of plasma‐type VWF multimers induced by high fluid shear stress.
Thrombotic thrombocytopenic purpura (TTP) affects 1 in 1600 to 1 in 5000 patients who receive ticlopidine, but little is known about the pathogenesis of this complication.
To investigate whether von ...Willebrand factor (vWF), which has been associated with idiopathic TTP, is involved in the pathogenesis of ticlopidine-associated TTP.
Case series.
Three tertiary care, university-affiliated medical centers.
Seven patients who developed TTP 2 to 7 weeks after initiation of ticlopidine therapy. Controls were 7 consecutive patients without thrombocytopenia who had been receiving ticlopidine for 3 to 5 weeks and 10 randomly selected hospitalized patients.
Platelet-bound vWF in patients' EDTA-anticoagulated whole blood samples; vWF proteinase activity in patients' plasma samples; inhibitory activity of IgG isolated from patients' plasma samples against the proteinase from the controls' plasma samples; and vWF multimeric patterns in patients' EDTA-anticoagulated plasma samples.
Binding of vWF to single platelets was increased in the three patients tested during the most thrombocytopenic phase of TTP episodes. Initial plasma samples from all seven patients lacked the largest vWF multimers and were severely deficient in vWF metalloproteinase. IgG molecules, isolated from plasma samples of five patients, inhibited metalloproteinase in plasma samples from the controls. In patients examined, these abnormalities resolved upon the remission that accompanied plasma exchange and discontinuation of ticlopidine therapy.
In the patients who developed ticlopidine-associated TTP, autoantibodies to the vWF metalloproteinase were formed; this led to the same type of vWF abnormalities observed in patients with idiopathic acute TTP. The findings suggest that failure to process large and unusually large vWF multimers in vivo caused binding of vWF to platelets, systemic platelet thrombosis, and TTP.
Thrombotic thrombocytopenic purpura (TTP) is a devastating thrombotic disorder caused by widespread microvascular thrombi composed of platelets and von Willebrand factor (VWF). The disorder is ...associated with a deficiency of the VWF-cleaving metalloprotease, ADAMTS-13, with consequent accumulation of ultralarge (UL) VWF multimers in the plasma. ULVWF multimers, unlike plasma forms of VWF, attach spontaneously to platelet GP Ibα, a component of the GP Ib-IX-V complex. We have found that ULVWF multimers secreted from stimulated endothelial cells (ECs) remained anchored to the endothelial surface where platelets and Chinese hamster ovary cells expressing the GP Ib-IX-V complex attached to form long beads-on-a-string structures in the presence of fluid shear stresses in both the venous (2.5 dyne/cm2) and arterial (20 and 50 dyne/cm2) ranges. Although measurement of the activity of the ADAMTS-13 VWF-cleaving metalloprotease in vitro requires prolonged incubation of the enzyme with VWF under nonphysiologic conditions, EC-derived ULVWF strings with attached platelets were cleaved within seconds to minutes in the presence of normal plasma (containing approximately 100% ADAMTS-13 activity) or in the presence of partially purified ADAMTS-13. By contrast, the strings persisted for the entire period of perfusion (10 minutes) in the presence of plasma from patients with TTP containing 0% to 10% ADAMTS-13 activity. These results suggest that cleavage of EC-derived ULVWF multimers by ADAMTS-13 is a rapid physiologic process that occurs on endothelial cell surfaces.
Background and Objectives: The adhesion ligand von Willebrand factor (VWF) is a multimeric glycoprotein that mediates platelet adhesion to exposed subendothelium. On endothelial cells, freshly ...released ultra‐large (UL) VWF multimers form long string‐like structures to which platelets adhere. Methods: The formation and elongation of ULVWF strings were studied in the presence of the thiol‐blocking N‐ethylmaleimide (NEM). The presence of thiols in ULVWF and plasma VWF multimers was determined by maleimide‐PEO2‐Biotin labeling and thiol‐chromatography. Finally, covalent re‐multimerization of ULVWF was examined in a cell‐ and enzyme‐free system. Results: We found that purified plasma VWF multimers adhere to and elongate ULVWF strings under flow conditions. The formation and propagation of ULVWF strings were dose‐dependently reduced by blocking thiols on VWF with NEM, indicating that ULVWF strings are formed by the covalent association of perfused VWF to ULVWF anchored to endothelial cells. The association is made possible by the presence of free thiols in VWF multimers and by the ability of (UL) VWF to covalently re‐multimerize. Conclusion: The data provide a mechanism by which the thrombogenic ULVWF strings are formed and elongated on endothelial cells. This mechanism suggests that the thiol‐disulfide state of ULVWF regulates the adhesion properties of strings on endothelial cells.
The adhesion ligand von Willebrand factor (VWF) is synthesized and stored in vascular endothelial cells and megakaryocytes/platelets. As in endothelial cells, platelet VWF also contains ultra‐large ...(UL) multimers that are hyperactive in aggregating platelets. ULVWF in platelet lysates of thrombin‐stimulated platelets was only detected in the presence of EDTA, suggesting that ULVWF is cleaved by a divalent cation‐dependent protease. A recent study shows that platelets contain the VWF‐cleaving metalloprotease ADAMTS‐13, but its activity remains unknown. In this study, we show that platelet lysates cleave endothelial cell‐derived ULVWF under static and flow conditions. This activity is inhibited by EDTA and by an ADAMTS‐13 antibody from the plasma of a patient with acquired TTP. ADAMTS‐13 was detected in platelet lysates and on the platelet surface by four antibodies that bind to different domains of the metalloprotease. Expression of ADAMTS‐13 on the platelet surface increases significantly upon platelet activation by the thrombin receptor‐activating peptide, but not by ADP. These results demonstrate that platelets contain functionally active ADAMTS‐13. This intrinsic activity may be physiologically important to prevent the sudden release of hyperactive ULVWF from platelets and serves as the second pool of ADAMTS‐13 to encounter the increase in ULVWF release from endothelial cells.
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