Role of transforming growth factor beta1 in microvascular endothelial cell apoptosis associated with thrombotic thrombocytopenic purpura and hemolytic-uremic syndrome.

Primary human microvascular endothelial cells (MVEC) of restricted lineage undergo apoptosis when exposed to plasma from patients with thrombotic thrombocytopenic purpura (TTP) and sporadic hemolytic-uremic syndrome (HUS). This reflects the pathology and tissue distribution of lesions in vivo. As extracellular matrix (ECM) is critical to MVEC survival, and cytokines which regulate ECM, such as transforming growth factor (TGF)-beta1, have been reported anecdotally to be altered in TTP/HUS, we examined the role of TGF-beta1 and two ECM proteins, fibronectin and thrombospondin (TSP), in these disorders. Levels of active TGF-beta1 were elevated in acute but not convalescent phases of TTP/sporadic HUS, as well as TTP associated with human immunodeficiency virus infection and use of the anti-platelet drug ticlopidine. MVEC from tissues susceptible to TTP-mediated apoptosis showed little active TGF-beta1 production when exposed to TTP plasmas. In contrast, pulmonary MVEC and large-vessel EC, which are resistant to TTP-linked pathology, showed marked induction of TGF-beta1 following TTP plasma exposure. Exogenous TGF-beta1 suppressed TTP plasma-mediated apoptosis in susceptible MVEC in association with blockade of cell entry into S phase. Soluble TSP, devoid of detectable bound TGF-beta1, had a similar effect, which paralleled its ability to induce TGF-beta1 production in MVEC. In vivo, TSP deposition was diminished markedly in involved tissues of TTP patients. These data highlight the role of TGF-beta1 and ECM in TTP and suggest that differential production of TGF-beta1 by MVEC may play a role in their sensitivity or resistance to TTP/sporadic HUS-mediated apoptosis in vitro and in vivo.     

Endothelial cell apoptotic genes associated with the pathogenesis of thrombotic microangiopathies: an application of oligonucleotide genechip technology

Idiopathic thrombotic thrombocytopenic purpura (TTP) is a disease characterized by the apoptotic injury of all microvascular endothelial cells (MVEC) except those of pulmonary origin. It notably also spares EC of large vessel origin. It is fatal unless treated with plasma exchange. The EC lineage restriction of the apoptotic lesions in vivo is reproduced in vitro following exposure of primary human MVEC derived from various tissues to TTP plasma. Oligonucleotide genechip technology was used to identify genes that may contribute to the resistance of lung MVEC to apoptosis induced by TTP plasma and to explore the intrinsic genotypic heterogeneity between MVEC of TTP-sensitive (skin) versus resistant (lung) lineage. Exposure of cells to TTP or normal plasma yielded 157 genes that were differentially expressed in primary human lung MVEC. A global change in expression of pro- and anti-apoptotic genes was seen, including increases in caspase 1, Fas, and Bcl-xl, already shown by experimental means to be involved in TTP pathogenesis. Additional differences suggest the importance of pathways related to the death receptor ligand TRAIL, as well as a role for disruption of EC-extracellular matrix interactions in the initiation of apoptosis. Maintenance of specific prosurvival signals at baseline may be a feature of lung MVEC resistance in TTP as suggested by higher expression than skin EC of the TRAIL antagonist, osteoprotegerin, and the vascular endothelial growth factors, VEGF/VPF and VEGF-C, and their receptors, VEGFR-2 (KDR) and VEGFR-3 (Flt4).     

Synergistic interactions between interferon-gamma and TRAIL modulate c-FLIP in endothelial cells, mediating their lineage-specific sensitivity to thrombotic thrombocytopenic purpura plasma-associated apoptosis

Microvascular endothelial cell (MVEC) injury coupled to progression of platelet microthrombi facilitated by ADAMTS13 deficiency is characteristic of idiopathic and HIV-linked thrombotic thrombocytopenic purpura (TTP). Cytokines capable of inducing MVEC apoptosis in vitro are up-regulated in both TTP and HIV infection. However, the concentrations of these cytokines required to elicit EC apoptosis in vitro are 2- to 3-log–fold greater than present in patient plasmas. We report that clinically relevant levels of tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) and interferon (IFN)– act in synergy to induce apoptosis in dermal MVECs, but have no effect on large-vessel ECs or pulmonary MVECs. This reflects the tissue distribution of TTP lesions in vivo. Sensitivity to TTP plasma or TRAIL plus IFN- is paralleled by enhanced ubiquitination of the caspase-8 regulator cellular FLICE-like inhibitory protein (c-FLIP), targeting it for proteasome degradation. c-FLIP silencing with anti-FLIP short interfering RNA (siRNA) in pulmonary MVECs rendered them susceptible to TTP plasma– and cytokine-mediated apoptosis, while up-regulation of c-FLIP by gene transfer partially protected dermal MVECs from such injury. TTP plasma–mediated apoptosis appears to involve cytokine-induced acceleration of c-FLIP degradation, sensitizing cells to TRAIL-mediated caspase-8 activation and cell death. Suppression of TRAIL or modulation of immunoproteasome activity may have therapeutic relevance in TTP.     

Lysophosphatidylcholine regulates human microvascular endothelial cell expression of chemokines

The role of lysophosphatidylcholine (LPC) in the induction of MCP-1, IL-8 and RANTES, which are chemotactic factors to monocytes, neutrophils and lymphocytes, respectively, by human vascular endothelial cells (EC), was examined. LPC induced the expression of MCP-1 and IL-8 in a concentration- and time-dependent manner in microvascular EC (MVEC) and in large vessel EC from aorta, pulmonary artery and umbilical vein. LPC also induced RANTES in MVEC but not in large vessel EC. Signaling pathways responsible for LPC induction of chemokines were examined in MVEC. LPC and TNFalpha, a cytokine secreted in sites of inflammation, additively stimulated RANTES expression. LPC did not augment TNFalpha induction of MCP-1 or IL-8. A platelet-activating factor receptor antagonist (BN52021) failed to block LPC induction of MVEC chemokines, but the G(i)-protein inhibitor pertussis toxin partially blocked LPC induction of RANTES and IL-8. LPC activated multiple kinases in MVEC; it increased the phosphorylation of ERK1/2, AKT and p38 MAP kinase in a time-dependent manner. An inhibitor of the MAPK/ERK pathway, PD98059, blocked the phosphorylation of ERK1/2 and RANTES induction by LPC, but augmented IL-8 induction. LY294002, a specific inhibitor of phosphoinositide 3 kinase (PI3 kinase), blunted the phosphorylation of AKT and inhibited LPC induction of RANTES more strongly than IL-8. Inhibition of p38 MAP kinase pathway by SB202190 also blocked LPC-induced expression of IL-8 and RANTES. Our results suggest that LPC induction of chemokines in MVEC is distinct from that in large vessel EC, and required the activities of MAP kinases and PI3 kinase for the induction of RANTES and IL-8. We speculate that the presence of LPC, a bioactive lipid product of phospholipase A(2) (PLA(2)) and a constituent of oxidized low-density lipoprotein, can differentially influence the chemotaxis of particular leukocyte subpopulations during inflammation.     

Endothelial microparticles released in thrombotic thrombocytopenic purpura express von Willebrand factor and markers of endothelial activation

It has been suggested that endothelial apoptosis is a primary lesion in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). We tested this hypothesis by examining the phenotypic signatures of endothelial microparticles (EMP) in TTP patients. In addition, the effect of TTP plasma on microvascular endothelial cells (MVEC) in culture was further delineated. EMP released by endothelial cells (EC) express markers of the parent EC; EMP released in activation carry predominantly CD54 and CD62E, while those in apoptosis CD31 and CD105. We investigated EMP release in vitro and in TTP patients. Following incubation of MVEC with TTP plasma, EMP and EC were analysed by flow cytometry for the expression of CD31, CD51, CD54, CD62E, CD105, CD106 and von Willebrand factor (VWF) antigen. EMP were also analysed in 12 TTP patients. In both EC and EMP, CD62E and CD54 expression were increased 3- to 10-fold and 8- to 10-fold respectively. However, CD31 and CD105 were reduced 40-60% in EC but increased twofold in EMP. VWF expression was found in 55 +/- 15% of CD62E+ EMP. Markers of apoptosis were negative. In TTP patients, CD62E+ and CD31+/CD42b- EMP were markedly elevated, and preceded and correlated well with a rise in platelet counts and a fall in lactate dehydrogenase. CD62E+ EMP (60 +/- 20%) co-expressed VWF and CD62E. The ratio of CD31+/42b- to CD62E+ EMP exhibited a pattern consistent with activation. In conclusion, our studies indicate endothelial activation in TTP. EMP that co-express VWF and CD62E could play a role in the pathogenesis of TTP.     

Antibody inhibitors to von Willebrand factor metalloproteinase and increased binding of von Willebrand factor to platelets in ticlopidine-associated thrombotic thrombocytopenic purpura

BACKGROUND: 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. OBJECTIVE: To investigate whether von Willebrand factor (vWF), which has been associated with idiopathic TTP, is involved in the pathogenesis of ticlopidine-associated TTP. DESIGN: Case series. SETTING: Three tertiary care, university-affiliated medical centers. PATIENTS: 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. MEASUREMENTS: 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. RESULTS: 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. CONCLUSION: 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.     

Two mechanistic pathways for thienopyridine-associated thrombotic thrombocytopenic purpura: a report from the SERF-TTP Research Group and the RADAR Project.

OBJECTIVES: We sought to describe clinical and laboratory findings for a large cohort of patients with thienopyridine-associated thrombotic thrombocytopenic purpura (TTP). BACKGROUND: The thienopyridine derivatives, ticlopidine and clopidogrel, are the 2 most common drugs associated with TTP in databases maintained by the U.S. Food and Drug Administration (FDA). METHODS: Clinical reports of TTP associated with clopidogrel and ticlopidine were identified from medical records, published case reports, and FDA case reports (n = 128). Duration of thienopyridine exposure, clinical and laboratory findings, and survival were recorded. ADAMTS13 activity (n = 39) and inhibitor (n = 30) were measured for a subset of individuals. RESULTS: Compared with clopidogrel-associated TTP cases (n = 35), ticlopidine-associated TTP cases (n = 93) were more likely to have received more than 2 weeks of drug (90% vs. 26%), to be severely thrombocytopenic (84% vs. 60%), and to have normal renal function (72% vs. 45%) (p < 0.01 for each). Compared with TTP patients with ADAMTS13 activity >15% (n = 13), TTP patients with severely deficient ADAMTS13 activity (n = 26) were more likely to have received ticlopidine (92.3% vs. 46.2%, p < 0.003). Among patients who developed TTP >2 weeks after thienopyridine, therapeutic plasma exchange (TPE) increased likelihood of survival (84% vs. 38%, p < 0.05). Among patients who developed TTP within 2 weeks of starting thienopyridines, survival was 77% with TPE and 78% without. CONCLUSIONS: Thrombotic thrombocytopenic purpura is a rare complication of thienopyridine treatment. This drug toxicity appears to occur by 2 different mechanistic pathways, characterized primarily by time of onset before versus after 2 weeks of thienopyridine administration. If TTP occurs after 2 weeks of ticlopidine or clopidogrel therapy, therapeutic plasma exchange must be promptly instituted to enhance likelihood of survival.     

Pathogenesis of thrombotic thrombocytopenic purpura: ADAMTS13 deficiency and beyond

Thrombotic thrombocytopenic purpura (TTP) is characterized by the systemic deposition of platelet thrombi with abundance of von Willebrand factor (vWF) in the arterioles and capillaries. Recently, vWF protease (ADAMTS13) activity was found to be severely deficient in hereditary TTP as well as acquired idiopathic TTP. Homozygous or compound heterozygous mutations of ADAMTS13 gene were demonstrated in hereditary TTP. Autoantibodies against ADAMTS13 were present in majority of patients with idiopathic TTP and ticlopidine- and clopidogrel-associated TTP. The deficiency of ADAMTS13 leaves unchecked the hyperadhesive vWF unfolded under high shear stress in the microvessels, resulting in the formation of platelet thrombi, which in turn causes TTP. ADAMTS13 activity is usually normal in hemolytic uremic syndrome. Approximately 0 to 67% of idiopathic TTP patients reported may not be severely deficient of ADAMTS13. Therefore, acquired TTP is not caused by ADAMTS13 deficiency alone and may be triggered by certain stimuli that possibly cause autoimmune reactivity to ADAMTS13, and also induce platelet aggregation either dependent on or independent of vWF, and/or vascular injury, to account for variable clinical and laboratory presentations. Plasma samples from TTP patients have been shown to induce endothelial cell apoptosis and activation. Platelet aggregating factors independent of vWF purified from the plasma of a subset of TTP patients have been reported and shown to be inhibited by normal plasma and immunoglobulin G purified from normal plasma. Defective fibrinolysis and abnormal natural coagulation inhibitors may enhance the thrombi formation in the microcirculation.     

Case report: use of thienopyridines in a patient with acquired idiopathic thrombotic thrombocytopenic purpura

Thienopyridines are commonly used anti-platelet drugs that may be associated with the development of secondary, drug-induced thrombotic thrombocytopenic purpura (TTP), a rare but potentially life threatening condition. We report the case of a 70?year-old man with a history of recurrent idiopathic TTP episodes who was treated with clopidogrel and then ticlopidine for thromboprophylaxis after percutaneous coronary intervention. Treatment was successful with no signs of TTP recurrence. Platelet counts and ADAMTS13 activity levels remained normal for months after the initiation of anti-platelet therapy, with no reappearance of anti-ADAMTS13 autoantibodies. This report demonstrates that thienopyridines do not necessarily induce TTP in patients with a history of TTP who are in disease remission.     

Effect of plasma exchange on plasma ADAMTS13 metalloprotease activity, inhibitor level, and clinical outcome in patients with idiopathic and nonidiopathic thrombotic thrombocytopenic purpura

Therapeutic plasma exchange is an effective empiric treatment for thrombotic thrombocytopenic purpura (TTP), but how therapy affects the level of a disintegrin and metalloprotease with thrombospondin type 1 motif 13 (ADAMTS13) or inhibitor has not been reported in many patients. We prospectively analyzed ADAMTS13 activity and inhibitor levels in 37 adults with TTP. ADAMTS13 level at presentation was lower than 5% in 16 of 20 patients with idiopathic TTP and in none of 17 patients with TTP associated with hematopoietic stem cell transplantation, cancer, drugs, or pregnancy (P <.00001). Seven of the 16 patients with ADAMTS13 activity lower than 5% ( approximately 44%) had inhibitors. For 8 patients followed serially with ADAMTS13 activity lower than 5% but no inhibitor at presentation, plasma exchange led to complete clinical remission and a rise in ADAMTS13 level. In contrast, 4 patients with low ADAMTS13 activity but high-titer inhibitor (> 5 units/mL) had neither a rise in ADAMTS13 activity nor a reduction in the inhibitor titer: 3 had recurrent disease and 1 died. Among 17 patients with AD-AMTS13 activity at presentation higher than 25%, 10 died. Mortality rate for idiopathic TTP was 15%, whereas mortality for nonidiopathic TTP was 59% (P <.02). We conclude that assays of ADAMTS13 activity and inhibitors in addition to the clinical categories (idiopathic TTP and nonidiopathic TTP) are predictive of outcome and may be useful to tailor patient treatment.     

von Willebrand factor-cleaving protease (ADAMTS-13) activity determination in the diagnosis of thrombotic microangiopathies: the Swiss experience

Severe deficiency of von Willebrand factor (VWF)-cleaving protease (ADAMTS-13) activity (<5% of normal) is a specific finding for acute idiopathic thrombotic thrombocytopenic purpura (TTP), a disorder that presents as thrombocytopenia, microangiopathic hemolytic anemia, and often organ dysfunction such as neurological disturbances or renal failure, and fever. Between January 2001 and July 2003, ADAMTS-13 activity was determined in plasma samples of 396 consecutive patients referred to our laboratory for diagnostic purposes. Plasma samples with ADAMTS-13 activity less than 5% were in addition tested for the presence of inhibitory antibodies. Patients were assigned to 10 predefined clinical categories according to information provided by the referring clinician: thrombotic microangiopathy (TMA) not further specified; neoplasia- or chemotherapy-associated TMA; TMA following hematopoietic stem cell transplantation; TMA with additional/alternative disorder; idiopathic TTP; hemolytic-uremic syndrome (HUS) not specified; HUS with diarrhea prodrome (D+HUS); atypical HUS; other hematological disorder; and no clinical information available. Severe ADAMTS-13 deficiency was found in 69 (17%) patients, including 42 with acquired idiopathic TTP, either at initial presentation or at relapse, 14 with confirmed or suspected hereditary TTP, 10 with TMA not further specified, two with neoplasia- or chemotherapy-associated TMA, and one in continued clinical remission 3.4 years after splenectomy for plasma-refractory TTP. Forty-three (62%) patients with ADAMTS-13 activity less than 5% displayed inhibitory antibodies. Severe ADAMTS-13 deficiency was found in 60% of patients diagnosed with acute idiopathic TTP, but in none of 130 patients diagnosed with HUS or in any of the 14 patients with hematopoietic stem cell transplantation-associated TMA. Thus, plasma ADAMTS-13 activity less than 5% does not identify all patients clinically diagnosed with TTP, and severe ADAMTS-13 deficiency is not invariably associated with clinical manifestations of microvascular platelet clumping.     

von Willebrand factor, ADAMTS-13, and thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) is a severe, occlusive, microvascular "thrombotic microangiopathy" characterized by systemic platelet aggregation, organ ischemia, profound thrombocytopenia, and erythrocyte fragmentation. Failure to degrade "unusually large" (UL) von Willebrand factor (VWF) multimers as they are secreted from endothelial cells probably causes most cases of familial TTP, acquired idiopathic TTP, thienopyridine-related TTP, and pregnancy-associated TTP. The emphasis in this review is the pathophysiology of familial and acquired idiopathic TTP. In each of these entities, there is a severe defect in the function of a plasma enzyme, VWF-cleaving metalloprotease (ADAMTS-13), that normally cleaves hyper-reactive ULVWF multimers into smaller and less adhesive VWF forms. In familial TTP, mutations in the ADAMTS13 gene cause absent or severely reduced plasma VWF-cleaving metalloprotease activity. Acquired idiopathic TTP, in contrast, is the result in many patients of the production of autoantibodies that inhibit the function of ADAMTS-13. Established, evolving, and some of the unresolved issues in TTP pathophysiology will be summarized.     

Elevated endothelial microparticles in thrombotic thrombocytopenic purpura: findings from brain and renal microvascular cell culture and patients with active disease

Endothelial injury is believed to be a key initiating event in the pathogenesis of thrombotic thrombocytopenic purpura (TTP), leading to platelet activation and formation of platelet-rich thrombi in microvasculature. However, the nature of endothelial injury in TTP is poorly defined and clinical assays to rapidly and reliably monitor endothelial damage are not readily available. Using flow cytometry, we measured endothelial microparticles (EMPs) generated from cultured renal and brain microvascular endothelial cells (MVECs) during activation and apoptosis, and evaluated the effect of TTP plasma on them. EMPs were measured using positivity for monoclonal antibodies (mAbs) CD31 and CD51, and their procoagulant activity was assessed using a Russell viper venom assay. Both cell lines generated procoagulant EMPs when cultured with inducers of activation (tumour necrosis factor alpha; TNF-alpha) or apoptosis (mitomycin C). TTP plasma induced a five- to sixfold increase of EMP generation and a two- to threefold increase of procoagulant activity in cultured brain and renal MVECs. TTP plasma induced a threefold and 13-fold increase of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) expression, respectively, on renal MVECs. Procoagulant activity tended to parallel EMP numbers. The effect of TTP plasma on cell viability was similar to that of TNF-alpha, implying that it induced activation rather than apoptosis. Control plasma and idiopathic thrombocytopenic purpura (ITP) plasma had little effect. In the clinical study, EMP assay of blood from acute TTP patients showed levels markedly elevated compared with normal controls, but values returned to normal in remission. In conclusion, TTP plasma activated and induced injury to MVECs in culture, judged by production of EMP and expression of activation markers. Released procoagulant EMP may play a role in the pathogenesis of TTP. Assay of EMP may be a useful marker of disease activity and endothelial injury in TTP and possibly other thrombotic disorders.     

Pathologic effects of plasma from patients with thrombotic thrombocytopenic purpura on platelets and cultured vascular endothelial cells

The pathologic hallmarks of thrombotic thrombocytopenic purpura (TTP) include endothelial cell proliferation and subendothelial hyalin deposits in the microvasculature leading to symptomatic thrombotic occlusions. Plasma or sera from three consecutive patients with TTP were subjected to multiple analyses to determine whether they induce endothelial injury and/or platelet activation, two pathogenic mechanisms that may account for this disorder. Sera were utilized in a microcytotoxicity assay against cultured human umbilical vein endothelial cells (EC). These cells were assessed ultrastructurally and with immunofluorescence techniques to ascertain the nature of inflicted cell damage. Control plasmas were obtained from healthy volunteers as well as patients with immune complex disease and the adult hemolytic uremic syndrome. In the presence of TTP serum, cell kill of 3H-proline- labeled EC averaged 42% versus 8.6% for control sera. Cytotoxicity induced by an IgG fraction of TTP sera averaged 70% versus 16.8% for control IgG. Removal of IgG by immune precipitation diminished cytotoxicity by 70%. Using indirect immunofluorescence, IgG was detected on EC incubated with TTP serum but not on EC treated with control serum. Ultrastructural changes became apparent within 30 min after exposure of cultured EC to TTP serum. Virtually every cell developed numerous cytoplasmic inclusions rarely seen in EC in the presence of normal serum. Prolonged incubation with the TTP serum led to progressive cytolysis, terminating with complete cytoplasmic and nuclear degeneration. Plasma from all three patients with TTP caused spontaneous aggregation of normal washed platelets as monitored by aggregometry. No spontaneous aggregation occurred in response to control plasmas. These results indicate that the sera of the three TTP patients studied were able to mediate time-dependent immune destruction of human cultured endothelial cells and that their plasmas were capable of causing spontaneous aggregation of normal human platelets in vitro. It would seem likely that these mechanisms are also operative in vivo to produce the endothelial destruction as well as the thrombotic vascular occlusions seen in this disorder.     

The Japanese experience with thrombotic thrombocytopenic purpura-hemolytic uremic syndrome

A total of 290 Japanese patients with thrombotic thrombocytopenic purpura-hemolytic uremic syndrome (TTP-HUS) were analyzed with respect to ADAMTS-13 activity and its inhibitor. Twenty-one patients (17 families) had Upshaw-Schulman syndrome, and 12 patients (six families) had familial HUS of undetermined etiology. The number of patients with acquired HUS and TTP was 44 and 213, respectively. In acquired TTP, patients with severe deficiency of ADAMTS-13 activity secondary to the presence of an inhibitor were high responders to plasma exchange, but others were low responders to plasma exchange. The former patients were associated with "idiopathic" TTP, drugs, and pregnancy, and the latter patients with malignancy and stem cell transplantation. Patients with autoimmune disease-associated TTP fit into both groups.     

Thrombotic thrombocytopenic purpura plasma enhances platelet–leucocyte interaction in vitro

In thrombotic thrombocytopenic purpura (TTP), intravascular platelet aggregation and formation of platelet-rich thrombi impair the microcirculation. TTP plasma has been shown to induce aggregation of normal platelets in vitro . The present study investigates the formation of activated platelet aggregates (aPAg) induced by TTP plasma, with particular attention to their binding to leucocytes (LPAg). Results were compared with the effects of plasmas from normal controls (CTL) and from patients with immune thrombocytopenic purpura (ITP) or thrombosis (THR). Following addition of test plasma to normal whole blood (WB), aPAg and LPAg were assayed by flow cytometry using mAbs against CD41 (platelet marker), CD62p (platelet activation marker) and CD45 (pan-leucocyte marker). Compared to control plasma, TTP plasma was more potent than ITP or THR plasma in increasing aPAg; only TTP plasma significantly promoted leucocyte binding to give increased LPAg. Prior removal of neutrophils (PMN) from WB by beads coated with anti-CD15 mAb largely prevented formation of aPAg and LPAg. However, TTP plasma added to normal platelet-rich plasma significantly increased aPAg, which suggested possible hindrance of aPAg formation by erythrocytes and other leucocytes in PMN-depleted blood. We concluded that TTP plasma was most potent in the induction of aPAg and unique in promoting LPAg formation in WB. Neutrophils, and not other leucocytes, appear to be essential for LPAg formation. Enhanced PMN–platelet interaction in the microcirculation may facilitate platelet adhesion to vessel walls and promote the formation of platelet-rich microthrombi in TTP.     

Role of MAP kinases and their cross-talk in TGF-beta1-induced apoptosis in FaO rat hepatoma cell line

Transforming growth factor (TGF) beta1 is a potent inducer of apoptosis in the liver. During TGF-beta1-induced apoptosis, 3 mitogen-activated protein (MAP) kinases (extracellular signal-regulated kinase [ERK], c-Jun N-terminal kinase [JNK], and p38 kinase) showed simultaneously sustained activation in FaO rat hepatoma cells. TGF-beta1-induced apoptosis was markedly enhanced when ERK activation was selectively inhibited by the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase inhibitor PD98059. In contrast, both interfering with p38 activity by overexpression of the dominant negative (DN) MKK6 mutant and inhibition of the JNK pathway by overexpression of the DN SEK1 mutant resulted in suppression of mitochondrial cytochrome c release, abrogating TGF-beta1-induced apoptosis. In addition, antiapoptotic Bcl-2 blocked mitochondrial cytochrome c release, suppressing TGF-beta1-induced activation of JNK and p38. Inhibition of ERK activity enhanced TGF-beta1-induced p38 and JNK activation. However, inhibition of the JNK pathway suppressed p38 but induced transient ERK activation. Similarly, interfering with the p38 pathway also attenuated JNK activation but generated transient ERK activation in response to TGF-beta1. These results indicate that disrupting one MAP kinase pathway affects the TGF-beta1-induced activation of other MAP kinases, suggesting cross-talk among MAP kinase pathways. In conclusion, we propose that the balance and integration of MAP kinase signaling may regulate commitment to TGF-beta1-induced apoptosis modulating the release of cytochrome c from mitochondria.     

Two cases of refractory thrombotic thrombocytopenic purpura associated with collagen vascular disease were significantly improved by rituximab treatment

Thrombotic thrombocytopenic purpura (TTP) is a rare disorder of small vessels. TTP is associated with deficiency of the von Willebrand factor-cleaving protease, ADAMTS13, and its inhibitor. Low ADAMTS13 activity is present in most of idiopathic TTP patients. The prognosis of TTP was improved by plasma exchange treatment, which replaces the ADAMTS13 and removes ADAMTS13 inhibitor. However, ADAMTS13 activity is normal in some TTP patients. These are found among the secondary TTP patients associated with collagen disease, hematopoietic stem cell transplantation, malignancy, or drugs. In addition, most of them do not respond to plasma exchange. On the other hand, several reports demonstrated that rituximab, which is an anti-CD20 monoclonal antibody, is effective for refractory TTP cases caused by ADAMTS13 deficiency. It is considered that the effect of rituximab is associated with disappearance of ADAMTS13 inhibitor. However, rituximab therapy was effective for the TTP patients with normal ADAMTS13 activity in our cases. We considered another mechanism of rituximab for TTP cases.     

The clinical utility of ADAMTS13 activity, antigen and autoantibody assays in thrombotic thrombocytopenic purpura

Thrombotic thrombocytopenic purpura (TTP) has been linked to a severe deficiency in ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) activity. Since the identification of ADAMTS13, and its target cleavage sequence in von Willebrand factor (VWF), several novel ADAMTS13 activity, antigen and autoantibody assays have been developed. Our aim was to evaluate the potential use of these novel assays. ADAMTS13 activity and inhibitors were measured by overnight incubation of patient plasma with pure VWF followed by multimer or collagen binding analysis. ADAMTS13 activity (Rapid peptide assay), antigen and immunoglobulin G anti-ADAMTS13 were measured by enzyme-linked immunosorbent assay. 118 samples from seven TTP patients (six adult idiopathic, one congenital) were studied longitudinally during episodes of TTP, their treatment and prophylaxis. ADAMTS13 antigen levels varied considerably between patients and sample times, but in new cases of acute TTP, rapid assays of ADAMTS13 antigen, on serial samples, maybe helpful in confirming the diagnosis. The rapid peptide ADAMTS13 activity assay showed good concordance of results with the older activity assay methods. The change in ADAMTS13 activity mirrored the autoantibody level and in 5/6 acquired TTP cases, a fall in antibody appeared to predict a rise in ADAMTS13 activity, potentially allowing modification of patient management based on autoantibody levels.     

Role of caspases 1 and 3 and Bcl-2-related molecules in endothelial cell apoptosis associated with thrombotic microangiopathies

We have defined an in vitro model for the study of microvascular endothelial cell (EC) apoptosis mediated by plasma from patients with various forms of thrombotic thrombocytopenic purpura (TTP) and hemolytic-uremic syndrome (HUS). This system reproduces a variety of histopathologic and ultrastructural features of tissue EC involved in TTP/sporadic HUS, suggesting that apoptotic EC injury is a primary pathophysiologic event in the thrombotic microangiopathies. We now document the ability of tetrapeptide-based inhibitors of interleukin 1β-converting enzyme (ICE)-like caspase 1 and cysteine protease protein (CPP)-32-like caspase 3, two members of a novel class of cysteine proteases involved in final pathways to apoptosis, to block TTP/sporadic HUS plasma-mediated apoptosis. Overexpression of Bcl-X_L via gene transfer suppressed this apoptosis by 70%. Transduction of EC with the Bcl-2 homolog A1 had a more limited protective effect. These findings support a role for apoptosis-linked cysteine proteases in the pathophysiology of TTP and sporadic HUS, and raise the possibility that specific apoptosis inhibitors may have a role in the experimental therapeutics of these syndromes. Am. J. Hematol. 59:279–287, 1998. © 1998 Wiley-Liss, Inc.